Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag –...

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I Exploring humoral responses during Staphylococcus aureus infection by immunoproteomics Inaugural - Dissertation zur Erlangung des akademischen Grades Doktor der Wissenschaften in der Medizin (Dr. rer. med.) der Universitätsmedizin der Ernst-Moritz-Arndt-Universität Greifswald, 28.02.2017 vorgelegt von Nandakumar, Sundaramoorthy geboren am 26.06.1980 in Cuddalore, Tamilnadu, India.

Transcript of Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag –...

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Exploring humoral responses during Staphylococcus aureus

infection by immunoproteomics

Inaugural - Dissertation

zur

Erlangung des akademischen

Grades

Doktor der Wissenschaften in der Medizin

(Dr. rer. med.)

der

Universitätsmedizin

der

Ernst-Moritz-Arndt-Universität

Greifswald, 28.02.2017

vorgelegt von Nandakumar, Sundaramoorthy

geboren am 26.06.1980

in Cuddalore, Tamilnadu, India.

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Aus der Abteilung für Funktionelle Genomforschung des Interfakultären Instituts für Genetik

und Funktionelle Genomforschung der Universitätsmedizin der Ernst-Moritz-Arndt-Universität

Greifswald

(Leiter Univ. Prof. Dr. Uwe Völker)

Dekan: Prof. Dr. Max P. Baur

1. Gutachter: Prof. Dr. Uwe Völker

2. Gutachter: Prof. Dr. Barbara Bröker

Ort, Raum: Seminarraum J04.33/34, Klinikum DZ 7, Greifswald

Tag der Disputation: 12.07.2017

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Table of contents

Abbreviations ........................................................................................................................... VI

Introduction ............................................................................................................................... 1

Staphylococcus aureus ....................................................................................................................... 1

Human immune system .................................................................................................................... 2

Antibodies - adaptive immune system ........................................................................................ 3

Biological properties of human immunoglobulins ................................................................. 5

S. aureus nasal carriage .................................................................................................................... 6

Determinants of S. aureus nasal carriage ................................................................................... 8

S. aureus bacteraemia (SAB) ........................................................................................................... 9

Pathogenesis pattern of S. aureus – host-pathogen interaction ..................................... 11

S. aureus vaccine candidates ........................................................................................................ 14

Immunoproteomics: From individual sample analysis to high throughput .............. 19

Aim of the study ................................................................................................................................ 21

Thesis graphical abstract .............................................................................................................. 23

Materials and Methods ......................................................................................................... 24

Materials ............................................................................................................................................. 24

Materials required for overexpression ................................................................................... 24

List of chemicals ............................................................................................................................... 24

List of consumables ......................................................................................................................... 26

List of instruments .......................................................................................................................... 26

Media and antibiotics ..................................................................................................................... 26

Buffers and solutions ...................................................................................................................... 27

List of databases ............................................................................................................................... 29

List of software ................................................................................................................................. 29

List of recombinant S. aureus antigens .................................................................................... 30

Overview of study population ..................................................................................................... 30

Methods ............................................................................................................................................... 33

Overexpression of recombinant S. aureus antigens ............................................................ 33

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Mass spectrometry analysis of purified recombinant S. aureus antigens ................... 34

Mass spectrometry data analysis of the purified recombinant proteins .................... 36

Principle of FLEXMAP 3D® and workflow ............................................................................... 36

Covalent conjugation of S. aureus antigens to magnetic beads ....................................... 38

Evaluation of magplex bead-antigen conjugation ................................................................ 39

Pre-adsorption of sera with assay buffer containing E. coli lysate ................................ 39

Serological bead based assay method validation ................................................................. 40

Quantitation of antibodies by serological assay using FLEXMAP 3D® .......................... 40

Data analysis ...................................................................................................................................... 41

Calculating the intensity at half maximum by Clark’s theory .......................................... 41

Scaling of data ................................................................................................................................... 45

Mann-Whitney U test ...................................................................................................................... 45

Statistical analyses .......................................................................................................................... 45

Results ........................................................................................................................................ 46

Method optimization and calibration ....................................................................................... 46

Mass spectrometry based quantification of S. aureus and E. coli proteins ................. 47

Method validation ............................................................................................................................ 48

Stability of antigens immobilised on the magplex ............................................................... 52

Evaluation of magplex-antigen coupling (coupling control) ........................................... 53

Quantitation of antibody responses (antigen coupling) .................................................... 60

Study-1 – Humoral responses during S. aureus nasal colonisation ............................... 60

IgG responses during S. aureus nasal colonisation ............................................................................................ 60 Discrimination efficiency of antigens provoking different immune responses in carriers and

non-carriers ......................................................................................................................................................................... 63 Characteristics of immunogenic antigens .............................................................................................................. 64 Clonal complex influence in anti-TSST-1 IgG responses ................................................................................. 68 Summary of study-1 ........................................................................................................................................................ 71

Study-2 – Humoral responses during onset of S. aureus bacteraemia infection ....... 72 IgG responses during onset of S. aureus bacteraemia ...................................................................................... 72 Discrimination efficiency of antigens provoking different immune responses in control and

sepsis ...................................................................................................................................................................................... 74 Characteristics of immunogenic antigens .............................................................................................................. 78 Summary of study-2 ........................................................................................................................................................ 88

Study-3 – Specific serum IgG at diagnosis of S. aureus bloodstream invasion is

correlated with disease progression ........................................................................................ 89

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IgG responses during S. aureus bacteraemia ........................................................................................................ 89 Validation of antigens provoking significantly different immune responses in patients with

sepsis and without sepsis .............................................................................................................................................. 91 Characteristics of immunogenic antigens .............................................................................................................. 95 Summary of study-3 ........................................................................................................................................................ 96

Discussion ................................................................................................................................. 97

Immunoproteomics – Method establishment and optimisation .................................... 97

Study-1: Immune responses during nasal colonisation ..................................................... 99

The influence of clonal complexes on sample specific IgG levels ............................................................. 101

Study-2: Immune responses during onset of bacteraemia ............................................. 102

Study-3: Immune responses during complicated bacteraemia .................................... 106

Conclusion and Outlook .................................................................................................... 108

Publications .......................................................................................................................... 110

References ............................................................................................................................. 112

Summary ................................................................................................................................ 135

Acknowledgements ............................................................................................................ 137

Appendix ................................................................................................................................ 139

Eidesstattliche Erklärung ................................................................................................. 140

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Abbreviations

1-D PAGE – One dimensional poly acrylamide gel electrophoresis

2-D PAGE – Two dimensional poly acrylamide gel electrophoresis

2-D IB – Two dimensional immunoblotting

Å – Angstrom

ABC – ATP binding cassette

Ab – Antibody

ACN – Acetonitrile

ADCC – Antibody-dependent cell-mediated cytotoxicity

Ag – Antigen

agr – Accessory gene regulator

AIP – Auto inducing peptide

AMP – Anti-microbial peptides

APC – Antigen presenting cell

Atl – Autolysin

Aur –Aureolysin

B cell – Bone marrow cell

BCR – B cell receptor

BSA – Bovine serum albumin

BSI – Bloodstream infection

CA-MRSA – Community-acquired MRSA

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CBS – Carboxy block store buffer

CBB – Coomassie brilliant blue

CC – Clonal complex

CCD – Colloidal coomassie dye

CCS – Colloidal coomassie solution

CD – Cluster of differentiation

CDC – Complement dependent cytotoxicity

CHIPS – Chemotaxis inhibitory protein

ClfA – Clumping factor A

ClfB – Clumping factor B

Cna – Collagen adhesin gene

Coa – Coagulase

CP – Capsular polysaccharides

CXCR – Chemokine receptor

DNA – Deoxyribonucleic acid

DTT – Dithiothreitol

Eap – Extracellular adherence protein

EB – Epidermolysis bullosa

E. coli – Escherichia coli

EDTA – Ethylendiaminotetra acetic acid

EDC – 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride

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Efb – Extracellular fibrinogen binding protein

egc – Enterotoxin gene cluster

e.g. – For example

ELISA – Enzyme-linked immunosorbent assay

E-MRSA – Epidemic-MRSA

EntA – Enterotoxin A

Emp – Extracellular matrix binding protein

Eno – Enolase like protein or phosphopyruvate hydratase

EpiP – Intracellular serine protease

et al. – “Et alia: and others

FadB – 3-hydroxyacyl-CoA dehydrogenase protein

Fab region – Fragment antigen-binding region

Fc region – Fragment crystallisable region

Fb – Fibrinogen

Fbp – Fibrinogen binding protein

FC – Fold change

Fnbp – Fibronectin binding protein

Fn – Fibronectin

FPR – Formyl peptide receptors

FPRL-1 – Formyl peptide receptor-like 1 inhibitory protein

Fur – Ferric uptake regulator

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Geh – Triacylglycerol lipase

GlpQ – Glycerophosphoryldiester-phosphodiesterase

GraB – Truncated secreted von Willebrand factor-binding protein

GreA – Transcription elongation factor

GroEL – Chaperonin GroEL

HA-MRSA – Hospital-acquired MRSA

HCA-MRSA – Healthcare-acquired MRSA

HEK – Human embryonic kidney

HF – Healthy fellows

HIS – Histidine

Hla – Alpha-hemolysin

Hlb – Beta-hemolysin

Hld – Delta-hemolysin

Hlg – Gamma-hemolysin

H3PO4 – Ortho-phosphoric acid

HNP – Human neutrophil peptide

HysA – Hyaluronate lyase

IAA – Iodoacetamide

ICAM – Immunoglobulin-related cell adhesion molecules

i.e. – That is

IE – Infective endocarditis

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IEF – Isoelectric focusing

Ig – Immunoglobulin

IL – Interleukin

IMAC – Immobilised metal affinity chromatography

IPTG – Isopropyl beta-D-thiogalactopyranoside

Isd – Iron surface determinant

Isa – Immunodominant staphylococcal antigen

IVIG – Intravenous immunoglobulin

Ka – Association constant

Kd – Dissociation constant

Keq – Equilibrium constant

kDa – Kilodalton

LB – Luria bertani

LCB – Low cross buffer

LC-MS – Liquid chromatography-mass spectrometry

LtaS – Lipo-teichoic acid synthase

LTQ – Linear trap quadrupole

Luk – Leukotoxin

LytM – Peptidoglycan hydrolase

MAP – Multi analyte profiling

Map-W – MHC class II analog protein

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MES – 2(N-Morpholino) ethanesulfonic acid hydrate

MFI – Median fluorescence intensity

MgCl2 – Magnesium chloride

MHC – Major histocompatibility complex

MntC – Manganese transport protein C

MMP – Matrix metalloproteinase

MODS – Multiple organ dysfunction syndrome

MRSA – Methicillin-resistant Staphylococcus aureus

MSCRAMM – Microbial surface components recognising of adhesive matrix molecules

MsrA2 – Peptide methionine sulfoxide reductase A

MsrB – Peptide methionine sulfoxide reductase B

MS – Mass spectrometry

MSSA – Methicillin susceptible Staphylococcus aureus

NA – Not applicable

NaCl – Sodium chloride

NaOH – Sodium hydroxide

NCTC – National clinical trials consortium

NEAT – Near iron transporters

NHS – N-hydroxysulfosuccinimide

Ni-NTA – Nickel nitrilotriacetic acid

No. – Numero

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Nuc – Thermonuclease

OatA – O-acetyltransferasse

PAGE – Polyacrylamide gel electrophoresis

Pbp2 – Penicillin-binding protein 2

PBS – Phosphate buffered saline

PCA – Principal component analysis

PE – Phycoerythrin

PFGE – Pulsed field gel electrophoresis

pH – Potentia hydrogenii (Latin)

pI – Isoelectric point

Plc – 1-phosphatidylinositol phosphodiesterase

PLS – Partial least square

PMN – Polymorphonuclear neutrophils

PrkC – Serine/threonine-protein kinase

PrsA – Foldase protein A

PSM – Phenol soluble modulins

PurA – Adenylosuccinate synthetase

PVDF – Polyvinylidene fluoride

PVL – Panton-valentine leukocidin

RIA – Radio-immunoassay

ROS – Reactive oxygen species

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RPE – R-phycoerythrin

RPLC – Reverse phase liquid chromatography

Rpm – Revolution per minute

RSD – Relative standard deviation

RT – Room temperature

SAB – S. aureus bacteraemia

SABSI – S. aureus bloodstream infection

SAT – Suspension array technology

Sak – Staphylokinase

SA4Ag – Staphylococcus aureus 4-Antigen

SAg – Super antigen

SasG – Surface protein G

S. aureus – Staphylococcus aureus

SB – Super broth

Sbi – Immunoglobulin binding protein

SCIN – Staphylococcal complement inhibitor

SDS – Sodium dodecyl sulphate

Sdr – Serine-aspartate repeat

SE – Staphylococcal enterotoxin

SERAM – Secretable expanded repertoire adhesive molecules

SERPA – Serological proteome analysis

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SigB – RNA polymerase sigma factor B

SIRS – Systemic inflammatory response syndrome

Sod – Superoxide dismutase

Spa – Staphylococcal protein A

Spl – Serine proteases

SsaA – Staphylococcal secretory antigen

Ssl – Staphylococcal superantigen like protein

SSTI – Skin and soft tissue infection

Ssp – Staphopain

STREP – Streptavidin

T cell – Thymus cell

TCA – Tricarboxylic acid cycle

TCR – T cell receptor

TFA – Trifluoroacetic acid

TH – Helper T cell

Tig – Trigger factor

TNF – Tumor necrosis factor

Tris – Tris (hydroxymethyl) aminomethan

Tsst-1 – Toxic shock syndrome toxin-1

Tuf – Elongation factor

VISA – Vancomycin intermediate Staphylococcus aureus

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VRSA – Vancomycin resistant Staphylococcus aureus

VH – Variable region of heavy chain

VL – Variable region of light chain

Vn – Vitronectin

vWF – Von Willebrand factor

WTA – Wall teichoic acid

(m/v)/ (v/v) – (mass to volume)/ (volume/volume)

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Introduction

Staphylococcus aureus

Staphylococcus (in Greek, staphyle means grapes; coccus means grain or berry) was first

described by the Scottish surgeon Sir Alexander Ogston in 1880 [1,2]. In 1884, the

German physician Friedrich Julius Rosenbach observed two isolates of Staphylococcus

and differentiated the isolates based on the colour of colonies into Staphylococcus aureus

(S. aureus) in Latin aurum, gold and

Staphylococcus albus (S. albus) in Latin albus,

white [3]. Mainly, Staphylococcus has two

species Staphylococcus aureus and

Staphylococcus epidermidis. These organisms

are Gram-positive species, non-motile, non-

spore forming cocci and appear as clusters. S.

aureus is one of the most important pathogens

in humans, has a wide spectrum of clinical

manifestations (Figure 1) and remains a

frequent cause of morbidity and mortality.

S. aureus leads to infections ranging from

relatively mild skin infections and soft tissue

infections, to more severe diseases like

pneumonia, surgery-related and bloodstream

infections [4]. Despite hospital-acquired

infections, S. aureus also causes community-

acquired infections such as osteomyelitis,

septic arthritis and skin infections [5]. S.

aureus has been ranked as the second

pathogen among the nosocomial

hematogenous infections and increases the mortality, morbidity, hospitalisation, and

expenses [6–9]. In the last 50 years, various S. aureus clones were disseminated

worldwide and developed strong resistance to antibiotics. S. aureus has the ability to

Figure 1. Clinical manifestation of S. aureus.

Picture adapted from (Wertheim et al., 2005)

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resist acid stress upon exposure to sub lethal acidic pH 4.5, exhibits thermo tolerance for

up to 48°C, and shows resistance responses to alkaline media [10]. Penicillin was the first

among the antibiotics discovered by Alexander Fleming, and at that time S. aureus had

exquisite susceptibility to it. However, within few years after penicillin discovery,

penicillinase-producing S. aureus clones were found in the 1950s and began spreading

around the world, causing hospital and community-acquired infections [11]. Later, first

semi-synthetic penicillinase-resistant beta-lactam methicillin was introduced in 1959,

methicillin-resistant S. aureus appeared in 1961 [12]. These methicillin-resistant clones

were disseminated in healthcare units throughout Europe until 1970s [13]. Vancomycin, a

glycopeptide type antibiotic is the last resort antibiotic for S. aureus treatment. Later, in

1997 partially vancomycin-resistant S. aureus clones were isolated [14,15]. S. aureus is

highly adaptable and developed strong resistance to all antibiotics developed over the last

decades. S. aureus is exceptional in its ability to develop resistance to any antibiotics due

to spontaneous mutation and horizontal gene transfer [16]. S. aureus possesses two types

of behaviours. Normally, S. aureus leads to commensal asymptomatic stage, which is

present in about 30% of humans, which are carriers of S. aureus in the anterior nares. On

the other hand in an acute stage, S. aureus invades tissue with ensuing pathogenesis

[4,17]. S. aureus nasal carriage is the major risk for the development of S. aureus

infections in various clinical backgrounds [15,18]. Majorly, these infections are of

endogenous origin in which hosts are infected with their own S. aureus isolates [19–21].

Human immune system

The immune system is the basic system among the diverse biological structures of the

human organism/body that protects against disease. The immune system must detect a

wide spectrum of foreign agents known as pathogens, and the events leading to the

development of immunity directed against pathogens are extremely complex. The

immune system can be classified as an innate immune system and an adaptive immune

system that act in concert as well as separately in the development of immunity. The

innate immune system provides the first line of defence against a pathogen. It is non-

specific, rapid, lacks immunologic memory generally of short duration. The adaptive

immune system is highly specific, slower in development, exhibits immunological

memory, and long lasting. Innate and adaptive immune systems are discrete systems but

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interact at several levels to develop a complete defence against invading pathogens. Both

systems follow mechanisms to distinguish self from non-self, therefore in normal

situations they are not directed against the host’s tissues and cells. A variety of

communications occurring between the human immune system and foreign substances

co-amplify the particular response as a result and further the invading pathogen undergos

destruction and eradication [22].

Antibodies - adaptive immune system

The adaptive immune system is a parts of the overall immune system. It is also called

acquired immune system or specific immune system. In contrast to the innate immune

system, the adaptive immune system possesses immunological memory, by using of

which it can block/ prevent the repeated damage by a foreign antigen or substance. The

adaptive immune system has both humoral and cell-mediated immunity components. The

cells that imports adaptive immune response are white blood cells known as

lymphocytes. The adaptive immune system can be divided in a T cell-mediated cellular

component and a B cell-mediated humoral component, which provide unique specificity

for their target antigens by the antigen-specific receptors expressed on their surfaces [23].

T cells can recognise the antigens only when these are presented in the processed form by

the MHC molecules of infected/cancerous cells. Helper T cells and Killer T cells are the

main components of T cells. Helper T cells controls the mobilisation of other cells to

confine the source of infected or malignant cells. Killer T cells can trace and directly kill

the infected host cells by the secretion of cytotoxins that form pores in the membrane of

the affected cells. B cells can recognise the antigens directly by their surface-associated

antibodies, which can bind to the specific antigen without the need of antigen processing.

Each B cell tends to make one specific antibody [23]. The B- and T-lymphocytes antigen-

specific receptors mature by somatic rearrangement of germ-line gene components to

form the TCR genes and the receptor genes of immunoglobulins. This recombination

mechanism is inimitable which has unique antigen receptors. It provides fast and specific

immune responses during the later exposure of the same antigen with specific

immunological memory. B lymphocytes produce immunoglobulins (Ig) to bind

pathogens, and T lymphocytes use T-cell receptors (TCR) to respond to antigen in the

form of processed peptides bound to cell surface proteins referred to as major

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histocompatibility complex (MHC). MHC are the essential surface exposed proteins that

play a vital role in the adaptive immune system which recognise the foreign molecules.

There are three classes of MHC molecules, MHC class I, MHC class II and MHC class

III. The MHC class I interacts with CD8 molecules on the surface of cytotoxic T cells, in

turn destructs the host infected or malignant cells. The MHC class II interacts with CD4

molecules on the surface of the helper T cells (TH), which facilitates the establishment of

specific immunity/adaptive immunity. For T lymphocytes, it requires two molecules to

recognise an antigen. First, the MHC molecule binds to the processed antigen peptides;

second, the TCR binds to the MHC molecule-antigen peptide complex. This exhibition of

MHC molecules complexed with processed antigens on their surface is known as antigen

presentation and mediated by antigen presenting cells (APC).

Co-stimulatory molecules

Long-lived plasma cells

Naive

B cell

CD4

Th2

cell

MHC class IIT cell receptor

Antigen internalization via BCR

Pathogen

Plasma B cell

CD40L CD40

B cell receptor

Maturation

Antigen-specific

antibody

αβ

Figure 2. T cell and B cell collaboration leads to the production of antibodies

Antibody production is controlled by the interaction between CD4+ helper T cells and B

cells that express rearranged antigen-specific immunoglobulin on their cell surface

(Figure 2). Antigen specific antibody production is the primary function of B

lymphocytes against invading pathogens. From the repertoire of a few hundred germ line-

encoded gene elements it is possible to design millions of antigen receptors. Antibodies

are encoded by the heavy (H)- and light (L)-chain immunoglobulin (Ig) genes where VH

(variable), DH (diversity), JH (joining), and CH (constant) are gene elements of heavy

chain and VL, JL, and CL are the gene elements of light chain [24]. Antibodies are

produced by B lymphocytes either extracellularly or on cell surface. Nevertheless, there

are five major classes of immunoglobulins IgM, IgG, IgA, IgD and IgE further, IgG has

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four subclasses and IgA has two subclasses. The subclasses are mainly based on the

isotypes of the H chain and the main classes and subclasses have different functions

which has been listed as Table 1. Each antibody is produced either as a circulating form

or a stationary form and here the later one consist of a hydrophobic transmembrane

domain induces the B-cell membrane to make immunoglobulin to act as a B-cell receptor

[25]. The former one which is a circulating form constitutes a considerable proportion of

serum proteins. About 10–15% of the peripheral blood lymphocyte population are in the

B cell.

During an encounter of a specific pathogen, an antibody response is initiated and the

level of antigen-specific antibodies rises in serum over a short period of time. However,

immunological memory is persevered in the B cell for the immediate clonal expansion

upon re-exposure to the same antigen [26]. Enhancement of protective immunity for a

specific antigen requires cooperation between B and T lymphocytes. T helper cell

interaction is required for the development of high-affinity antibodies and protection. It

requires CD4+ T helper cell stimulation for the naïve B lymphocytes to undergo

proliferation and differentiation in response to antigens. B lymphocytes are also capable

of presenting antigens to T cells through their surface MHC class II protein. Antibodies

identify the tertiary structure of proteins and bind to specific epitopes of the antigen [27].

Upon infection, the immune response is triggered, and immunoglobulin formation

undergoes flipping from one isotype to another, typically from IgM to IgG and IgA or

IgE. During this process, antibodies with higher affinity for the antigens are developed

[28].

Biological properties of human immunoglobulins

The primary function of Ig is to inactivate or eliminate the pathogen and its products via

antigen binding. The structure of the Ig is well adapted to this function. Ig is comprised of

a head (variable region - Fab), which binds to an antigen on the invading pathogen, and its

tail (constant region - Fc), which mediates the effects. Mainly, the Fc region interacts with

complement and or with specific receptors (gamma receptors) on the neutrophil and

monocyte surfaces. Human immunoglobulin-antigen interactions take place between the

paratope, the Ig region at which antigen binds and the epitope which is the region of the

antigen that is bound [29]. The four subclasses of IgG possess unique Fc regions. Based

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on the in vitro studies, IgG2 is less efficient than IgG1 in fixing complement [30]. The Ig

comprised of several domains and these domains are associated with different biological

functions. The biological function of Ig is classified as antigen-dependent and antigen-

independent function listed in the Table 1 [31].

Table 1: Summarised characteristics of biological functions of human immunoglobulins [31]

S. aureus nasal carriage

Based on histological studies, the nasal vestibule has a complex epithelial surface and can

be categorised into five distinct regions. The vestibule region of the nose has a thin

squamous epithelium that is covered at the nostrils by short stiff hairs acting as receptors

for mechanical stimuli and induce sneezing. The primary ecological niche of S. aureus in

Property Antigen

dependence Type of human Ig Domain localisation

Complement fixation

Classical pathway + IgM, IgG CH4 of IgM, CH2 of

IgG

Alternate pathway - IgA, IgE Unknown

Opsonic activity

Neutrophils, monocytes + IgG1, IgG3, IgA Unknown

Mast-cell and basophil binding - IgE Unknown

Macrophage binding - IgG1, IgG3 CH3 of IgG

Lymphocyte binding - IgG1, IgG3, IgM CH3 of IgG

Placental passage regulation - IgG1, IgG2, IgG3, IgG4 Entire Fc required

Intestinal passage regulation - None Unknown

Immunoglobulin catabolism - All Ig classes and its

subclasses CH2 of IgG

Passive cutaneous anaphylaxis - IgG1, IgG3, IgG4 CH3 or entire Fc

Protein A interaction - IgG1, IgG2, IgG4 Unknown

Antigenic target for rheumatoid

factors -

IgG1, IgG3, IgG4, IgM, IgA,

IgE

Most likely Fc

region

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human are the anterior nares, and it seems that the anterior nares are the most consistent

region from which S. aureus can be isolated [32]. However, S. aureus can be isolated

from many sites like throat, axilla and perineum as well [33–36]. Nasal colonisation is a

well-known risk factor for staphylococcal infections in the community and hospital.

Nasal carriage plays a vital role in epidemiology and pathogenesis [37,38]. The human

population can be distinguished into persistent carriers, intermittent carriers and non-

carriers [39]. Based on epidemiological longitudinal studies the following proportions

have been determined: persistent carriers 10% to 35%, intermittent carriers 20% to 75%,

non-carriers 5% to 50% [21,39–46]. Persistent carriers have significantly higher S. aureus

density in the nasal region than intermittent carriers. This observation partially explains

their increased risk of endogenous infections and suggests that the basic determinants of

persistent and intermittent carriage are different [47–50]. Furthermore, persistent carriers

are frequently colonised with a single strain of S. aureus for a longer time, but the

intermittent carriers carry different strains over time [39,41,51–53]. It seems that

persistent carriage has a protective effect on the acquisition of other S. aureus strains

[54]. Upon decolonising of human persistent carriers and subsequent inoculation with

mixtures of S. aureus strains, it was observed that former non-carriers were not colonized

in the study group and persistent carriers chose their original colonising strain from the

strain mixture [55]. A higher rate of S. aureus faecal carriage including MRSA in healthy

infants due to mother-to-infant transfer was observed and suggests that transmission

through breast milk occurs and that S. aureus is able to colonise the gut [56]. Plausibly, S.

aureus may colonise the mother’s nipple, which enhances the transmission of S. aureus

to infants through breastfeeding [57]. Due to frequent contact with livestock such as cats,

dogs, pigs, and horses, a higher incidence rate of nasal carriage of CA-MRSA was

observed. This suggests that animals can be another vector for the spread of CA-MRSA

[58–62]. It seems that the mechanism behind the nasal carriage is multifactorial. Factors

such as bacterial proteins (extracellular and cell-wall anchored proteins) [63,64],

ecological factors (healthcare and crowding) [57,65,66] and host factors (gender, age)

[65,67] play a vital role in the nasal colonisation. Typically, high S. aureus carriage rates

occurred in men, in white people, in dialysis patients, in diabetics, and in acquired

immunodeficiency syndrome (AIDS) patients [68–71]. Due to the presence of potential

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virulence factors and higher S. aureus counts at colonised sites, there is an increased

incidence rate of S. aureus infection in MRSA-colonised patients [9,72].

Determinants of S. aureus nasal carriage

S. aureus nasal colonisation is a well-known risk factor for community- and hospital-

acquired staphylococcal infections. It has been suggested that existence of haemoglobin

in nasal fluids stimulates S. aureus colonisation through the inhibition of the agr system

[73]. The presence of triclosan in the nasal fluids of healthy individuals influences S.

aureus colonisation in the nasal niche [74]. To establish a successful colonisation S.

aureus produces adhesins and immune evasion molecules and in parallel down-regulates

virulence factors and other toxins [75,76]. High levels of human defensins (HNP1 to

HNP3) found in the nasal secretions of persistent carriers point to involvement of

neutrophils in S. aureus colonisation [68]. Proteinaceous and non-proteinaceous adhesins

are the primary factor for colonisation and instigation for infection. The most important

factor in establishing nasal carriage/colonisation is the ability of S. aureus to adhere to

the human nasal epithelial region [77]. S. aureus adhesins can bind to a wide array of

human extracellular matrix proteins such as collagen (cna) or von Willebrand factor

(vWF), fibrinogen (Fb), fibronectin (Fn), vitronectin (Vn) and elastin [78]. S. aureus

adhesins are mostly sortase cell wall-anchored proteins and have been called microbial

surface components recognising adhesive matrix molecules (MSCRAMM). MSCRAMM

protein groups vary between clinical isolates [79]. MSCRAMMs typically have N- and

C-termini with LPXTG-motifs and hydrophobic domains which are involved in

anchoring of proteins to the cell wall [80]. S. aureus supposedly adheres to the mucous

membrane of the nose through several MSCRAMM proteins including fibronectin-

binding protein A and B, Surface protein G (SasG), Clumping factor B (ClfB) in human

[63,81–85]. Wall teichoic acid (WTA), Iron surface determinant A (IsdA) and Clumping

factor B (ClfB) promote nasal colonisation in mouse models [63,83,86]. The key receptor

for WTA is SREC-1 (F-scavenger receptor). WTA binds to EGF-domains 3 and 4 of

SREC-1 in a charge-dependent manner and facilitates the adhesion to nasal epithelial

cells [87]. The target receptor of ClfB is loricrin (cornified cell envelope) on the principle

of “dock, lock and latch”. In vivo studies confirmed that ClfB is the promoter and

mediator for nasal colonisation [84]. SdrC and SdrD (serine-aspartate repeat) play an

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important role in adhesion to nasal desquamated epithelial cells [88,89]. Nevertheless,

Sdr (serine-aspartate repeat) proteins mediate the interactions with human extracellular

matrix proteins [90]. An in vitro study suggested that SdrC, D, E are mainly involved in

platelet adhesion through fibrinogen [91]. The iron surface determinants (Isd) were

regulated by environmental iron sources and by Fur regulator. About one to three NEAT

domains were present in every Isd protein and bound to iron-containing proteins such as

haemoglobin and transferrin [92]. Based on transcript analyses in a cotton rat model

(infected with human S. aureus nose isolates), two important regulators of S. aureus, agr

and sae, were inactive during the onset of nasal colonisation which suggests that S.

aureus in the cotton rat nose lean toward adhesion rather than toward dissemination.

Moreover, tagO and tagK messenger RNA levels were high in vivo, indicating a vital role

of WTA during nasal colonisation. An important gene controlled by the WalKR system is

staphylokinase (sak) which is mainly involved in immunomodulatory functions of S.

aureus. Transcript analysis demonstrated that sak expression is significantly increased

after 24 hours post-inoculation and further increases during nasal colonisation [93].

Another important gene of the walKR regulon is sceD, which was expressed in early

nasal colonisation and whose rate of transcription was higher at 96 hours post infection.

This result supports the influence of cell wall dynamics in the primary stage of nasal

colonisation [93]. SERAM proteins such as MHC class II analog protein (Map/Eap/P70)

and Emp bind to various host components such as Fg, Fn or Vn. Still, Emp expression

level is reduced during nasal colonisation in compared to infected isolates [94]. In

contrast, a emp mutant have shown reduced attachment to Fg and Fn [95]. Preclinical

trials of immunisation with purified IsdA and IsdH proteins in cotton rat models have

shown decreased nasal colonisation [96].

S. aureus bacteraemia (SAB)

S. aureus is the second most-common pathogen causing bloodstream infections (BSI)

worldwide and a leading cause of nosocomial BSIs in Europe, with an incidence rate

within the global population of 15-40 cases per 100,000 individuals [8,97–99]. The

increasing frequency of SAB is due to increased antibiotic resistance of S. aureus isolates

[100–102]. A retrospective analysis between 2000 to 2006 disclosed that CA-MRSA

leads to the onset of bacteraemia from 24% to 49% in the hospital settings [103]. The

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mortality rate for untreated SAB is about 80% [104]. SAB leads to sepsis and

endocarditis and disease under antibiotic therapy is still associated with high mortality

[105,106]. There is a wide range of clinical manifestations of SAB from SIRS (Systemic

inflammatory response syndrome) to sepsis to severe sepsis to septic shock to Multiple

organ dysfunction syndrome (MODS). Sepsis is associated with either fever or low body

temperature, increased respiratory rate, elevated heart rate, and edema [107]. Intravenous

antibiotic therapy for MRSA bacteraemia should be carried out for at least 14 days and 4

to 6 weeks for uncomplicated bacteraemia [108]. The current antibiotic therapy is

insufficient for SAB mainly for MRSA bacteraemia. Apparently, preclinical studies on

prophylactic administration of anti-ClfA monoclonal antibody were protective when

sepsis was induced in mice [109]. S. aureus carriers have a higher probability of

acquiring SAB than non-carriers. Primarily, carriers acquire SAB from their colonising S.

aureus isolate with an enhanced chance of survival [9,21].

Importantly, each stage of infection involves the expression of different virulence factors.

Virulence factors such as surface proteins adhere to host material or cells, help to evade

the antibody-mediated host immune reaction, promote tissue damage, stimulate iron

uptake and further increase the severity of disease [110–112]. E-MRSA 15 and 16 were

predominant in MRSA bacteraemia during 2007 and 2009 in the UK, for instance in 2007

E-MRSA 15 (85%) and E-MRSA 16 (9%), in 2009 E-MRSA 15 (79%) and E-MRSA 16

(6%). This finding suggests that genetic variations among the lineages were partly

attributed to geographical areas dispersed exclusively among the particular lineage CC8,

CC22 and CC30 [113,114]. Specific sequence variation at specific loci throughout the

genome makes up the heterogeneity of S. aureus, and proteins situated at this loci might

play a vital role in the development and dissemination of major genetic lineages [115].

Despite of clinical isolates and gene resemblances by PFGE from SAB patients, the

antibody responses were dissimilar. This antibody response dissimilarity might be due to

the variance in the time of onset of bacteraemia (except catheter-related bacteraemia) and

proposes that each bacteraemia patient develops distinctive immune responses to SAB

[116]. PSM α gene-encoded peptides from CA-MRSA isolates heighten the virulence and

have a significant impact on SAB in animal models [117]. Distinctive patterns of immune

response were found for eleven immunogenic antigens (exoproteins) autolysin,

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bifunctional autolysin, GlpQ, Hla, HlgA, HlgB, HlgC, SspP, Plc, Nuc, and LtaS during

exogenous and endogenous infections. Higher IgG responses were shown for toxins,

especially in samples from exogenous infections [118]. Remarkably, patient stratification

was established based on IgG response patterns specific to eight S. aureus antigens (Plc,

SspB, IsaA, Sem, GlpQ, HlgC, SACOL0444, SACOL0985) in sepsis and no sepsis

patients, which may potentially help during diagnosis of SAB [119]. A further study on S.

aureus infected patients complicated by bacteraemia from 4 hospitals described higher

antibody levels to Hla, Hld, PVL, SEC-1, and PSM α3 may well defend against sepsis in

patients who develop aggressive S. aureus infections. Moreover, higher antibodies titers

to SEB and PVL were protective against sepsis [120].

Pathogenesis pattern of S. aureus – host-pathogen interaction

The main ecological niche and the place where the largest pool of S. aureus in humans

can be found are the anterior nares. Further common habitats of S. aureus are skin [121],

perineum [122], axillae [121,122], vagina [123], and gastrointestinal tract [121]. Nasal

secretory substances contain shield components of the host against microbes. Research

documented that nasal secretions from non-carriers were bacteriostatic, while the nasal

secretions from carriers allowed the progression of S. aureus [124].

For an effective defence reaction, the infected cells and immune cells need to

communicate and these reactions are mediated by molecules called “Cytokines”.

Cytokines are secreted by professional and non-professional phagocytic cells upon

stimulation. S. aureus has the ability to internalise into both professional and non-

professional phagocytic cells, e.g. epithelial cells, endothelial cells, fibroblasts,

osteoblasts, and it survives in the intracellular milieu. This intracellular milieu protects S.

aureus from the host immune system and also from antibiotics [125]. Earlier studies have

shown the impact of S. aureus during the infection of mice and human cell lines.

Likewise, several cytokines displayed increased levels in a S. aureus pneumonia mouse

model 6 h post infection, for instance pro-inflammatory cytokines such as TNF-α, IL-1β,

IL-6 were induced and anti-inflammatory cytokines IL-10, IL-12p70 were not increased

significantly. Furthermore, alterations in the host proteome were observed including

proteins involved in the inflammation reaction and coagulation [126]. In an in vitro study,

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different growth behavior and different expression of metabolic enzymes (threonine

degradation, level of fermentation enzymes and TCA cycle) was found for intracellular S.

aureus especially 6.5 h post infection in two human lung epithelial cell lines (S9 and

A549) and in HEK 293 cells [127]. In another proteomic study investigating host and

pathogen interaction after internalisation of S. aureus into A549 human epithelial cells

physiological adaptation reactions of S. aureus have been studied in detail and reduced

growth rate, induction of the stringent response, adaptation to micro-aerobic conditions

and cell wall stress have been described. The host displayed strong apoptosis signalling,

clathrin-mediated endocytosis signalling, calcium signalling and integrin signalling after

infection with S. aureus HG001 in comparison to the non-infected control cells [128].

Apparently, S. aureus targets the host cell adhesion molecules (cadherin, integrin or

ICAM) for the establishment of contact with host cells and tissues surfaces [129]. MRSA

in livestock’s aid as reservoirs for human colonisation, for instance S. aureus clonal

lineage ST398 from swine [130]. A microarray study revealed that animal lineages are

interrelated to human lineages [131]. S. aureus employs an array of virulence factors that

protect from the host’s immune system and enables crossing of mucosal barriers,

dissemination, and replication in distant organs. The virulence factors include teichoic

acid (non-protein molecules), peptidoglycan (non-protein molecules), capsular

polysaccharides (non-protein molecules), MSCRAMM proteins, super antigens,

enterotoxins, extracellular enzymes, proteases (serine proteases, cysteine proteases, and

metalloproteases), immune-evasion molecules and immunomodulators.

Despite of host evasion, survival of S. aureus within the host is influenced by the nutrient

procurement such as iron and manganese [132–134]. S. aureus produces high-affinity

iron-binding compounds such as aureochelin and staphyloferrin during iron deprivation

[135,136]. Manganese transport protein C (MntC) is a MSCRAMM protein, which

interacts with host factors such as collagen type IV, laminin, fibronectin, fibrinogen, and

plasminogen. MntC digests plasminogen to plasmin, which in turn degrades the host

fibrinogen as like staphylokinase [134]. S. aureus is not susceptible to lysozyme owing to

the cell wall modifying enzyme OatA along with WTA [137]. Clumping factor B (ClfB),

a surface located MSCRAMM protein adheres to the ligand cytokeratin 10 (glycine-

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serine loop), the main component of desquamated human epithelial cells [138]. The other

MSCRAMM protein SdrC binds to β-neurexin which is primarily present in brain tissue

[139]. Staphylococcal protein A (Spa) has five sequential, homologous extracellular Ig-

binding domains, binds to the Fc region of the IgG and helps S. aureus to escape from the

host immune system [140,141]. Antibody-binding to S. aureus via the Fab region

facilitates the opsonisation and enhancing the killing by neutrophils, whereas binding via

Fc region restricts phagocytosis [142]. Staphylokinase (Sak), a serine protease-like

molecule inhibits and neutralises the bactericidal property of α-defensins (HNPs) and

catalyses the cleavage of plasminogen to plasmin, which is capable of digesting human

IgG and complement C3b/C3bi and prevents the opsonisation [143,144]. The immune

evasion clusters (IEC) such as SCIN and CHIPS act as an immune modulators and attach

to human complement proteins C5a and formyl peptide receptors (FPRs). As a result it

inhibits phagocyte recruitment [145–147]. An in vitro study revealed that serine-aspartate

repeat containing protein D (SdrD) enhanced the adhesion to keratinocytes via

Desmosomal cadherin 1 (Dsg1) [148]. Ssl7 are immune evasion molecules, which bind to

IgA and complement C5 and in turn inhibit the generation of C5a [149,150]. S. aureus

produces at least five molecules that prevent the action of serum complement

convertases. These convertases are important for opsonisation of bugs because activated

complement components such as C3b and iC3b stimulate phagocytosis [151]. Recently,

Laarman et al. revealed that the cysteine protease Staphopain A (ScpA/SspA) of S.

aureus cleaves the N-terminus of the CXCR2 and thereby likely prevents neutrophil

triggering and enrolment [152]. S. aureus produces various pore-forming toxins such as

α, β, γ, δ – Hemolysin, Panton-valentine leukocidin (PVL), phenol-soluble modulins

(PSM). These toxins play a vital role in target cell lysis such as erythrocytes, leukocytes

(neutrophils, monocytes and T-cells)[153–156]. The PSM peptides from CA-MRSA

strains were primarily sensed by the innate immune system, acting as a pro-inflammatory

agents, induce cytokines, they have cytotoxic activity towards neutrophils and, thus, lead

to escape from neutrophils [155,157]. It has been found that serum lipoproteins inactivate

PSMs [158]. LukG and H play primary roles in cytolytic activity and specifically lyse

neutrophils by forming an octameric pore [159,160]. PVLs are bi-component toxins

mostly associated with CA-MRSA clinical strains, whose contribution to S. aureus

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pathogenesis are still unclear [161,162]. Figure 3 represents the interactions between host

and S. aureus; their outcome during the interaction.

S. aureus

Neutrophil Complement

T cell

Acquired

InnateAur, Sod,Sak,

Nuc,

Lysozyme &

ROS - AMP

Chips, Sak,

Scin, Efb

Oposonization

SAg, HlgB &C

(Toxins)CD4+

Immunoglobulins

(opsonizing,

neutralizing)

Proteases, spa,

sbi

B cell

Figure 3. Schematic representation of interaction (“Trade-off”) between S. aureus and human

immune system

S. aureus vaccine candidates

In general, bacteria possess less potential to develop resistance to a vaccine than to an

antibiotic, although some microbes tend to antigenic variation to escape from the

vaccine-induced immunity. S. aureus is an encapsulated bacterium, it is a major cause of

community and nosocomial infections. Globally, there is a swift increase in drug

resistance that has added urgency to the development of an effective vaccine [163]. An

effective S. aureus vaccine would provide great potential security and have many

universal benefits [164]. Nevertheless, efforts to generate an S. aureus vaccine have

failed so far [165–167]. Previous vaccine studies indicated that a single antigen approach

seems to provoke a lower likelihood of success than a multi-antigen approach. An

introduction of an effective vaccine for S. aureus will dramatically reduce the burden on

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the health care institutions and will protect the individuals from the risk. Also, it is better

to have a mixture of multivalent antigens for a successful S. aureus vaccine, for example

ClfA, Isd family proteins and MntC, due to the high genetic variability of virulence

factors within staphylococcal isolates. Screening the efficacy of antigen candidates in

multiple preclinical models provides an opportunity for better understanding their

strength and weakness. The summary table of S. aureus active and passive vaccines

under clinical trials are listed in the Table 2 and Table 3. However, serum therapy for S.

aureus infections was less effective than for diphtheria and tetanus due to the complex

pathogenesis and virulence variability [168]. For an efficient vaccine development,

considering bacterial and host-related factors of carriage and characteristics of human`s

response to different episodes of S. aureus encounter is extremely important. The

investigation of humoral immune responses from patients carrying S. aureus will enable

the identification of S. aureus antigens that are immunogenic, and will help us to identify

potential vaccine candidates. Earlier S. aureus vaccines, which failed in clinical trials

were made of single or divalent antigens such as capsular polysaccharides (CP). These

single or bivalent antigen-based vaccines could not manage the multiple virulence

strategies of S. aureus. The CP5 and CP8 strengthen the virulence of S. aureus by

evading phagocytosis [169]. Li et al. revealed that immunisation of rabbits and mice with

ClfA produced functional antibodies which inhibit S. aureus binding to fibrinogen, but

which were not protective in murine models with surgical infections like catheter-related

endocarditis [170]. A prospective study of SA4Ag (Staphylococcus aureus 4-antigen)

vaccine antigen evaluation suggested that MntC genes were more highly expressed than

other genes (CP5, CP8 and ClfA) in nasal and wound samples. In parallel, antigen-

specific antibodies were monitored in serum with the result that most of the patients

generated responses to ClfA and MntC [171]. Active immunisation of S. aureus infected

mice with octavalent antigens (IsaA, LytM, Nuc, Atl and PSM α 1 - 4 peptides) did not

result in protection for mice against S. aureus bacteraemia and skin infections [172].

So far it is still unclear if the number of antigens selected (mono- or multivalent), the

patient diversity or the manufacturing problems are directed to the lack of positive results

[173]. Earlier preclinical evidence strongly suggested that a multi-antigen approach may

lead to a robust immune response against S. aureus in a murine model [174]. For

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developing an ideal S. aureus vaccine, active and passive vaccine approaches should be

followed. Passive immunisation might be used as an adjunct to antibiotics in severe S.

aureus infections.

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Table 2 : Active vaccination (antibody production) clinical trials

Product Composition Status Limitation Reference

StaphVAX™

CP5 and CP8

(Capsule

polysaccharide)

Phase III failed

Single antigen

and limited

efficacy.

[175–177]

V710 IsdB (LPXTG) Phase II/III

terminated

No efficacy, did

not produce

opsonising

antibodies and

single antigen

approach.

NCTC00735839,

[178]

PentaStaph

CP 5 and CP8, Wall

teichoic acid,

Panton-valentine

leukocidin (PVL),

Alpha-hemolysin

(Hla)

Phase I

completed for 2

antigens on 27

April 2011 and

further trails on

other antigens.

Trial not yet

completed. [179]

SA75

Killed S. aureus

(whole cell) mainly

contains GST-Cna

(collagen binding

protein), His-Clf

(clumping factor),

GST-D (fibronectin

binding protein),

and Eap

(extracellular

adherence protein)

Phase I

completed

Immunisation

with whole cell

may not induce

protective

antibodies.

[180]

GSK2392102A

Tetravalent vaccine

adjuvanted with

ASO3B (Hla, ClfA,

CP5 and CP8

Phase I

Higher antibody

levels with no

adjuvant effect.

[181],

NCTC01160172

STEBVax

SEB

(Staphylococcal

enterotoxin B)

Phase I

suspended

Limited SEB

expressing

strains.

NCTC00974935

SA4Ag CP5, CP8, ClfA and

MntC Phase II

Trial not yet

completed

NCTC02388165,

[171]

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Table 3 : Passive vaccination (antibody providing) clinical trials

Product Composition Status Limitation Reference

Pagibaximab Anti-Lipoteichoic

acid antibody

Phase II/III

ongoing

Higher dose

(90mg/kg) enhance

the sepsis protection

for neonates and

less efficacy in

lower dose.

[182]

Tefibazumab

(Aurexis/Inhibitex)

Anti- ClfA

monolconal

antibody

Phase II

completed

Decreased relapses

and complications,

but not lower

mortality. No

efficacy during

bacteraemia

progression.

[183]

Altastaph

Pooled human

antibodies against

CP5 and CP8

Phase II

completed

Higher mortality

rate in neonates than

placebo.

[184,185]

Aurograb

Single chain

antibody variable

fragment against

ABC transporter

component GrfA

Phase III

completed

Due to the safety

measures and lack

of efficacy during

phase II, Novartis

terminated this

clinical trial.

[186–188]

Veronate (INH-

A21)

Pooled human IgG

against Fbp, ClfA,

and SdrG

(IgG derived from

donors with high

titers of antibody to

Fbp, ClfA and

SdrG)

Phase III

completed

Less efficacy and no

difference in

episodes of late-

onset

staphylococcal

sepsis (LOS).

[189,190]

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Immunoproteomics: From individual sample analysis to high throughput

S. aureus as a commensal and opportunistic pathogen has distinct tactics to adapt to

different circumstances from colonisation to bacteraemia related infections. Upon S.

aureus invasion in the host, immune cells mount an immune response or antibody

responses. To decipher the complexity during disease progression, “Omics” technologies

play an important role to study the pathophysiology of S. aureus and unravel mechanism

of host-pathogen interaction. The current emerging tool in the field of proteomics widely

used to identify potential vaccine candidates is “immunoproteomics”, a term coined in

2001 by Jungblut [191]. The immunoassays can be performed using different analytical

technologies and Figure 4 represents the technology evolution during the past six

decades.

Different classical methods are available under immunoproteomics to study the antibody

responses such as agglutination, radio-immunoassay (RIA), enzyme-linked

immunosorbent assay (ELISA), immunochromatographic test (lateral flow test) and 2D-

immunoblotting, but the bottleneck of these techniques is the identification of proteins or

peptides.

The ELISA is a high-throughput method and the advantages are that many serum/plasma

samples can be analysed in parallel, instruments are inexpensive and easy handling in

terms of protocol and instruments. The process can be automated, making this technique

attractive in fully equipped laboratories that test large numbers of samples. However, the

limitations are separate assays for each antigen, requirement for high specificity

antibodies and that time to results still can take hours.

For developing countries with limited access to laboratory resources, a simple

immunochromatographic test would be the preferred method. This method requires only

a visual inspection of the antigen-containing lines and can be performed as a point-of-

care assay without laboratory equipment and this test can be performed within a few

minutes [192].

The other classical proteomic pillar is 2-D PAGE. The first two-dimensional

electrophoresis separation was achieved in human serum to resolve 15 human proteins

and further described by Farrel et al. in 1975 [193,194], this technique is used to separate

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complex mixtures of proteins. The combination of 2-D PAGE with immunoblotting and

MS was used to isolate and identify the immunogenic proteins and this approach is called

SERPA [195]. In principle, the proteins are separated in terms of their mass and charge,

whereby during the first dimension the proteins are separated by its isoelectric point and

in the second dimesion by its molecular weight [196]. The main disadvantage of 2-D

PAGE are: i) cross contamination because single spots can contain several proteins, ii)

limited detection of low-abundance proteins, iii) poor resolution in the low molecular

weight region (< 10kDa) and limit ability of resolution of certain protein classes such as

membrane proteins. Protein array methods are an alternative to ELISA and 2-D PAGE.

Here, immobilisation of immunogenic antigens on the solid-phase slide surface is used in

order to detect the antibodies specific to immunogenic antigens, a variant that is called

reverse phase protein array. The other mode of protein array is inverse of reverse phase,

immobilisation of antibodies from patient samples on the solid-phase slide surface in

order to detect the immunogenic antigens/biomarkers. Protein arrays offer the possibility

to by-pass the limitations of ELISA, 2-D PAGE by allowing multiplexing (simultaneous

detection) and measurement of serum antibodies against multiple antigens.

Besides protein arrays several other gel-free approaches are available to identify

immunogenic antigens for instance particle-based flow assays and expression array. An

exciting emerging new technology platform is “Particle-based flow cytometric assays” or

“Suspension array technology” [197]. To date, Luminex, Becton-Dickinson and Diasorin

are offering this technology. The technology evolution in the field of immunological

methods is depicted in the Figure 4.

In order to further investigate the diagnostic potential of these immunoreactive antigens,

a robust and multiplexed assay system for large scale and high-output biology is needed.

Such assay systems allow screening the serological reactivity of hundreds of samples

against a multitude of antigens. The “particle-based flow cytometric assays” or

“suspension array technology” belongs to such multiplexed based assay systems.

Immunoproteomics is a powerful tool for identifying potential candidates for the vaccine

development. Many vaccine candidates were identified using immunoproteomics

approaches in respiratory pathogens such as Burkholderia pseudomallei, Neisseria

meningitidis, Bordetella pertussis, Streptococcus pneumoniae, Staphylococcus

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epidermidis, Campylobacter concisus, Pseudomonas aeruginosa, Leptospira interrogans,

and Francisella tularensis [198]. Vytvytska et al. revealed 15 S. aureus antigens showing

strong antibody responses in healthy and S. aureus infected individuals using SERPA

(Serological proteome analysis) [199]. Holtfreter et al. observed the strong IgG binding

against Hla, SspB, SspA, Plc, SplB, and SplE in carriers using 2-D immuno-blotting (2-D

IB)[200]. Furthermore, a Staph-toxin-array revealed that IgG responses against IsaA,

SACOL0479 and SACOL0480 were significantly lower in non-carriers than carriers

[201]. With this 2-D immuno-blotting approach several promising S. aureus

immunogenic antigens could be identified [198,202].

Among the immunoproteomics approaches, the most promising technology is Suspension

Array Technology (SAT) – FLEXMAP 3D®

. It is a high throughput technique which can

multiplex up to 500 analytes with a wide dynamic range (105). SAT yields more

informative data in shorter analysis time and with lower sample volume than

conventional ELISA [203,204]. SAT has a high potential and represents a promising tool

for personalised medicine and for delivering details of a patient’s physiological and

pathological conditions.

1950 – 59

• RIA and 2D-

PAGE

1960 – 79

• ELISA

1980 - 89

• Automated ELISA system

1990 -2000

• Miniaturized spot array

and

Suspension array

technology (SAT)

Figure 4. Immunoassay technology milestone from 1950 till 2000. This figure shows the technology

evolution in the field of immunological methods.

Aim of the study

S. aureus is a life-threatening pathogen which leads to various infections such as

pneumonia, bacteraemia, endocarditis, septic arthritis, and osteomyelitis. There are no

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22

prognostic (active vaccine) and diagnostic (passive vaccine) measures to prevent S.

aureus infections. Vaccines (Table 2 and 3) developed in the earlier decades failed during

preclinical and clinical trials. However, earlier immunisation studies in mouse models

revealed that antibodies directed against MSCRAMM proteins, pore forming toxins, CP5

and CP8 can indeed protect against S. aureus colonisation or infection [178–180]. Thus,

it is still unclear which antigen or group of antigens would constitute valid vaccine

candidates. For the antigen screening, several immunological methods are available and

there are several disadvantages in these classical methods due to their technical

limitations. To circumvent this classical technology limitations, new emerging

technology, particle-based flow cytometric assay or suspension array technology has

been introduced.

The first objective of this thesis was the technology transfer from planar microarray

technology and other classical methods (RIA, ELISA etc.) to the multiplexed bead-based

FLEXMAP 3D® (SAT) platform to allow high-throughput screening of clinical samples.

The second objective of this thesis was to elucidate the humoral responses (antibody

responses) during S. aureus colonisation/infection and, thereby, to discover potential

antigen candidates for S. aureus vaccine development as depicted in the Figure 5.

In order to achieve the main objective, we investigated serum and plasma samples from

different human S. aureus infected patients such as nasal carriage, bacteraemia, sepsis,

severe sepsis, and septic shock by using high-throughput suspension array technology.

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23

Thesis graphical abstract

Literature search

Antigens from Immunology

in vitro studies

Antigen stratification

Immobilised antigenIgG – Patient Vs. ControlFluorescent labelled Anti-Human IgG

Antigen stratification Setting-up serological assay

using SAT

Application to clinical cohorts

Stu

dy

-1

Stu

dy

-2

Stu

dy

-3

Humoral

responses during

S. aureus nasal

colonisation

Humoral responses

during S. aureus

bacteraemia onset

Humoral responses

during S. aureus

bacteraemia

Identifying potential S. aureus antigens

Active vaccine

Passive vaccine

Patient stratific-

ation

Figure 5. Schematic representation of thesis abstract.

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24

Materials and Methods

Materials

Materials required for overexpression

Research grade chemicals were purchased from Sigma-Aldrich, Merck, ROTH

and Bacto. Protease inhibitors, lysozyme, IPTG, and DnaseI were procured from

Roche and Thermo. Ni-NTA agarose, polypropylene columns, the penta histidine

and streptavidin tag antibodies were procured from Qiagen. Phycoerythrin anti-

human IgG and anti-mouse IgG antibodies were procured from Dianova. E. coli

expression plasmids for S. aureus antigens were constructed by Protagen AG. The

E. coli expression strain SCS1 carrying the helper plasmid pQE30 was

transformed with the ligation mixtures. The correct expression vectors were

confirmed by colony polymerase chain reaction and further quality controlled by

5’ DNA sequencing.

List of chemicals

Table 4: list of chemicals

Chemical name Grade/Quality Supplier Catalogue No.

1,4-dithiothreitol (DTT) NA Thermo R0862

Acetic acid NA Sigma 537020

Agar powder Molecular

biology

Sigma 5040

Ammonium chloride (NH4Cl) Molecular

biology

Sigma A9434

Ammonium sulphate Molecular

biology

Sigma A4418

Ampicillin sodium salt Molecular

biology

Roth K029.2

Bovine serum albumin (BSA) NA Sigma A7906

Bradford reagent NA Bio-Rad 5000006

Bromophenol blue NA Sigma B0126

Calcium chloride (CaCl2) Anhydrous Sigma 499609

Coomassie brilliant blue G-250 NA Bio-Rad 1610406

Di sodium hydrogen phosphate

dihydrate (Na2HPO4.2H2O)

NA Roth 4984

Dimethyl sulfoxide anhydrous NA Sigma 276855

DNAse I NA Roche 10104159001

EDC NA Sigma 22980

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Ethanol absolute ACS Merck

Millipore

107017

Glucose (C6H12O6) NA Sigma G5400

Glycerol NA Sigma G6279

Guanidine hydrochloride (CH5N3 .

HCl)

Elite Sigma G4505

Imidazole (C3H4N2) Puriss. p.a. Sigma 56750

Isopropyl-beta-D-

thiogalactopyranoside (IPTG)

NA Thermo R0392

Kanamycin sulphate salt Premium Sigma K4000

LCB NA Candor 100050

Luria bertani (LB) broth Molecular

biology

Sigma L0322

Lysozyme NA Sigma L1667

Magnesium chloride hexahydrate

(MgCl2.6H2O)

ACS Sigma M9272

MES hydrate Elite Sigma M8250

Methanol ACS Merck

Millipore

106009

Monobasic sodium phosphate Molecular

biology

Sigma S3139

Nickel-nitrilotriacetic acid (Ni-NTA) NA Qiagen 30210

Phosphate buffered saline (PBS) Molecular

biology

Sigma P5368

Potassium dihydrogen phosphate

(KH2PO4)

ACS Sigma P0662

Proclin 300 Premium Sigma 48912-U

Protease inhibitor cocktail NA Roche 04693116001

Sodium chloride (NaCl) Molecular

biology

Roth 3957

Sodium hydroxide (NaOH) NA Roth 6771

Sulfo-NHS NA Sigma 24510

Tris (hydroxymethyl) aminomethane

hydrochloride

Elite Sigma T5941

Tryptone Molecular

biology

Bacto 211701

Trypsin Sequencing Promega V511A

Tween 20 NA Sigma P7949

Water LC-MS grade Merck

Millipore

115333

Yeast extract Molecular

biology

Sigma 92144

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List of consumables

Table 5: List of consumables

Name Supplier Catalogue No.

96 well half area microplate Greiner 675101

96 well Polystyrene microplate Greiner 655095

Aluminium sealing tape, pierceable Sarstedt 95.1995

Carboxy functionalised standard xMAP

super paramagnetic beads

Luminex MC100xx

C18 Zip Tip columns Merck

Millipore

ZTC18S096

Goat anti-human IgG – RPE Dianova 109-116-098

Goat anti-mouse IgG – RPE Dianova 115-116-146

Magnetic separator unit (96 well) Lifesep 2501008

Micro insert, 0.1 mL MA, USA 548-0020

Micro vials VWR Intl. NSCAC4013-1

Penta His Antibody BSA free Qiagen 34660

nanoACQUITY UPLC 2G V/M trap column Waters, USA 186006527

nanoACQUITY BEH 130Å UPLC column Waters, USA 186003546

Sheath fluid Luminex 40-50000

Strep tag antibody Qiagen 34850

PVDF filters - Pore size 0.45µm and

Diameter 33 mm

Sigma Z355518

List of instruments

Table 6: List of instruments

Instrument Company

Centrifuge, 5810R Eppendorf

Electronic single and multi-

channel pipettes

Rainin – Mettler Toledo

FLEXMAP 3D® Luminex

corporation

LTQ-Orbitrap XL mass

spectrometer

Thermo Fischer Scientific Inc.

UV-Visible spectrophotometer VWR®

Well plate shaker Thermo Fischer Scientific Inc.

Media and antibiotics

LB - Medium

- 1 % (w/v) Tryptone; 0.5 % (m/v) Yeast extract; 0.5 % (m/v) NaCl in double

distilled water.

- Mixed contents was autoclaved.

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27

LB Ampicillin and Kanamycin – Agar

- 1 % (m/v) Tryptone; 0.5 % (m/v) Yeast extract; 0.5 % (m/v) NaCl, 1.5 % (m/v)

agar in double distilled water.

- Mixed contents was autoclaved.

- Agar was cooled down to 45°C, then 1.0 mL of sterile ampicillin (100 mg/mL)

and kanamycin (15 mg/mL) were added.

- Agar plates were poured by transferring about 25 mL of the above mixture to each

agar plate and then plates were stored at 4°C.

SB – Medium

- Solution A: 3.2 % (m/v) Tryptone; 2.0 % (m/v) Yeast extract; 0.5 % (m/v) NaCl;

0.5% (v/v) 1N NaOH in double distilled water.

- Solution B (20x M9 salts): 12 % (m/v) Di sodium hydrogen phosphate dihydrate;

6 % (m/v) potassium di hydrogen phosphate, 2 % (m/v) Ammonium chloride,

0.006 % (m/v) calcium chloride in double distilled water.

- Solutions A and B were autoclaved.

- Autoclaved solution A and B were mixed in a proportion of 95:5, then 0.4 % (v/v)

sterile glucose was added.

Buffers and solutions

Protein overexpression (induction, lysis and purification)

- 1M IPTG sterilised solution: About 238.3 mg of IPTG was transferred in 1.0 mL

of double distilled water and filtered through PVDF 0.45µm filters.

- Protease inhibitor cocktail (25x) EDTA free: A tablet of protease inhibitor

cocktail was dissolved in 2 mL of double distilled water and Store at -20 ° C.

- Cell lysis buffer: 20 mM Di sodium hydrogen phosphate dihydrate, 20 mM

imidazole, 100 μg/mL lysozyme, 100 μg/mL DnaseI, 100 μg/mL MgCl2, 40

μl/mL Protease inhibitor cocktail (25x), 5 mM DTT were mixed and titrated to pH

7.4 with NaOH or H3PO4.

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- IMAC denaturing wash buffer: 20 mM Di sodium hydrogen phosphate dihydrate,

20 mM imidazole, 6 M guanidine-HCl, 1 mM DTT in distilled water and titrated

to pH 7.4 with NaOH or H3PO4.

- IMAC elution buffer (denature): 20 mM Di sodium hydrogen phosphate, 500 mM

imidazole, 6 M guanidine-HCl, 1 mM DTT and titrated to pH 7.4 with NaOH or

H3PO4.

1D – SDS-PAGE

- 2x SDS sample buffer: 0.09 M Tris-HCl (pH 6.8), 2 % SDS, 25 % (v/v) Glycerol,

0.02 % (w/v) Bromophenol blue, 0.1 M DTT.

- Fixation solution: 50 % (v/v) double distilled water, 40 % (v/v) ethanol, 10 %

(v/v) acetic acid.

- Coomassie brilliant blue (CBB): 5.0 g (m/v) of coomassie brilliant blue G-250 in

100 mL of double distilled water.

- Colloidal coomassie dye (CCD): 50 g (m/v) of diammonium sulphate (NH4)2SO4,

6.0 mL of ortho phosphoric acid (85 %), 490 mL of distilled water, 10 mL of

CBB stock solution.

- Colloidal coomassie solution (CCS): 200 mL of CCD stock solution, 50 mL of

methanol were prepared freshly.

- Destaining solution: 20 % (v/v) methanol

Protein concentration determination

- BSA standard: 0.1 mg/mL

- Bradford reagent: Brilliant blue G-250 dye

Buffers and reagents for mass spectrometry analysis

- 20 mM Ammonium bicarbonate solution (ABC): About 0.160 g of ammonium

bicarbonate dissolved in 100 mL of LC-MS grade water.

- 25 mM Dithiothreitol (DTT): 0.375 g of DTT in 10 mL of 20 mM ammonium

bicarbonate solution.

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- 100 mM Iodoacetamide (IAA): 0.18 g of IAA in 10 mL of 20 mM ammonium

bicarbonate solution.

- LC buffer A: 2 mL of acetonitrile in 100 mL of LC-MS grade water containing

0.1 mL of acetic acid.

- LC buffer B: 0.1 mL of acetic acid in 100 mL acetonitrile.

Serological assay

- Activation buffer: 100 mM sodium dihydrogen phosphate, pH 6.2.

- Coupling buffer: 50 mM MES, pH 5.0.

- Wash buffer: PBS, 0.05 % tween20.

- Block store buffer: PBS, 1 % BSA.

- Bead buffer: 50 % CBS, 50 % LCB.

- Assay buffer: 45 % CBS, 45 % LCB, 10 % E. coli lysate.

List of databases

- http://aureowiki.med.uni-greifswald.de/

- http://www.uniprot.org/

- http://www.ncbi.nlm.nih.gov/

- http://www.psort.org/psortb/

- http://wlab.ethz.ch/protter/#

- http://www.kegg.jp/

- http://web.expasy.org/

- http://www.proteinatlas.org/

- http://www.cbs.dtu.dk/services/SignalP/

- https://www.citavi.com/

List of software

- Graphpad prism® 5

- Genedata® (Analyst and Refiner MS)

- MeV : Multi experiment viewer

- R studio®

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List of recombinant S. aureus antigens

The recombinant S. aureus antigens were listed in supplementary pages (supplementary 1

and 2). The S. aureus antigens comprised 12 cytoplasmic, 19 cell wall, 12 membrane, 66

extracellular, 16 unknown antigens and 8 auto-inducing peptides. The antigen constructs

were procured from Protagen AG, Germany. The antigens were expressed in E. coli

strain SCS1 and induced with 1 mM IPTG (final concentration), and purified under

denaturing conditions with Ni-NTA agarose recognising the His-tag. Qualitative analysis

was done by 1-D SDS-PAGE, which is described in the next sections.

Overview of study population

Three different studies were carried out in this thesis. Plasma/serum samples were

obtained from patients infected with S. aureus.

In study-1 (Table 7), plasma samples were obtained from healthy individuals of both

carriers and non-carriers from the residents of western Pomerania, where total 32 plasma

samples including 16 healthy carriers and 16 healthy non-carriers.

Table 7: Study-1

Characteristics of plasma samples from nasal carriage patients [205]

Study area Western Pomerania

Study population Healthy blood donors

Number of subjects 32

Time period February 2005 – February 2006

Mean age (yrs.) (±SD) 33.8 (10.7)

Male sex (%) 81.2

Number of non-carriers (%) 16 (50)

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31

In study-2 (Table 8), serum samples were obtained from 49 S. aureus bacteraemia

patients and 43 control patients collected during routine diagnostics between 2011 - 2014

from the Akershus university hospital in Norway. SABs are defined by the positive blood

culture obtained earlier or later than 48 h after hospitalisation.

Table 8: Study-2

Characteristics of serum samples of bacteraemia patients from Akershus university hospital,

Norway (SABSI) [206]

Sepsis

(n = 49)

Control/No-sepsis

(n = 43)

Study benchmark

First S. aureus positive blood

culture (No prior antibiotic

therapy)

Healthy enough to undergo

elective orthopaedic surgery

and no S. aureus surgical site

infection within 1 year of

surgery.

Gender

Male 33 27

Female 16 16

Time period March 2011 – February 2014 March 2011 – June 2014

Age (yrs.) (±SD) 66.2 (16.2) 63.3 (18.8)

Acquired type (%)

Community acquired (CA) 28.5 NA

Hospital acquired (HA) 32.6 NA

Health-care associated (HCA) 38.8 NA

Nasal carriage (%)

Carriers 30.6 27.9

Non-carriers 69.4 72.0

In study-3 (Table 9), serum samples were obtained from 44 patients diagnosed with S.

aureus infection complicated by bacteremia.This observational study was approved by

the University of Maryland Baltimore institutional review board.

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32

Table 9: Study-3

Characteristics of serum samples from S. aureus infected patients complicated by bacteraemia [207]

Sepsis

(n =19)

No-sepsis

(n = 21)

Odds ratio (CI) p-value

Demographics

Mean age (±) 59 ± 13 57 ± 18 0.75

Gender 0.99 (0.29 – 3.43) 1.0

Female 9 10

Male 10 10

Race 0.63

African

American

11 15

White 7 6

Unanswered 1 2

Earlier S. aureus infections 1.52 (0.34 – 6.76) 0.71

Yes 5 4

No 14 17

Earlier MRSA infection or

colonisation

0.71

Yes 7 6

No 10 11

No data 2 4

Dialysis patient 0.48 (0.12 – 1.81) 0.33

Yes 5 9

No 14 12

Diabetes mellitus patient 0.53 (0.13 – 2.23) 0.49

Yes 4 7

No 15 14

Bacteraemia type 5.23 (0.95 – 28.9) 0.069

Primary 17 13

Secondary 2 8

If secondary bacteraemia 1.0 (0.03 – 33.4) 1.0

SSTI 2 7

UTI 0 1

Nosocomial infection 2.31 (0.56 – 9.47) 0.31)

Yes 15 13

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33

Methods

Overexpression of recombinant S. aureus antigens

E. coli expression clones were inoculated on LB ampicillin and kanamycin agar plates at

37°C. After overnight cultivation, a single colony was selected and transfected into the

baffled flask containing SB media, incubated at 37°C, 300 rpm. Cell density was

monitored at 600 nm every 60 min and 5 µL was sampled for SDS-PAGE every 60 min.

The expression of target genes was induced with 100 µL of 1M IPTG at OD600 = 5.0,

incubation was continued for 2 hours and then cells were harvested. Cells were

sedimented by centrifugation at 8500 rpm for 15 min, 4°C. Pellets were resuspended in

lysis buffer and incubated for 30 min in ice. The cells were disrupted by ultrasonication

with the micro-tip on ice using three 10 s bursts at the amplitude intensity of 60 %. The

disrupted cells were centrifuged at 8500 rpm for 15 min, 4°C and the supernatant

transferred to a new container and 5 µL of lysate sampled for SDS-PAGE. In case of

unsoluble proteins such as membrane proteins, the inclusion bodies were resuspended in

IMAC wash buffer and disrupted by ultrasonication with the micro-tip on ice using three

10 s bursts at the amplitude intensity of 60 %. Then the disrupted inclusion bodies were

centrifuged at 8500 rpm for 15 min at 4°C. The lysate was purified by immobilised metal

affinity chromatography (IMAC). Ni-NTA (Nickel-Nitrilotriaceticacid) agarose slurry

was resuspended and about 4.0 mL of Ni-NTA agarose slurry was filled in a 5.0 mL

polypropylene column. About 2.0 mL of double distilled water was then added to the

column to settle the resin by gravity. After settling of resin the storage solution was

gently aspirated. Columns were then washed with 10 column volumes of double distilled

water followed by 20 column volumes of IMAC wash buffer. Then, lysate was loaded

onto the column and the the flow through collected for SDS-PAGE. Columns were

No 4 8

Current MRSA 0.46 (0.12 – 1.79) 0.32

Yes 11 15

No 8 5

Days since presentation of

symptoms

0.9 ± 0.3

(n = 18)

1.3 ± 0.3 0.35

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34

washed with 10 column volumes of IMAC wash buffer and the wash fractions collected.

Bound protein was eluted with IMAC elution buffer as four elution fractions (E1 to E4).

Total protein concentration was determined for the elution fractions by Bradford method

using BSA as standard. BSA standard concentrations ranged from 0.2 µg to 20 µg. For

determination of the protein content the elution fractions were diluted 2 to 10-fold based

on the absorbance range in the standard curve. Based on the concentration of protein

from Bradford assay, elution samples were prepared for 1D SDS-PAGE. About 2 µg to 5

µg of protein in elution fraction, flow-through, wash fractions and lysate fractions were

mixed with 2x SDS sample buffer. This mixture was incubated at 70°C for 10 min. The

SDS-PAGE was performed using NuPAGE®

4 - 12% Bis-Tris precast gels with a

constant voltage of 180 V, with MES running buffer. Gels were fixed with fixation buffer

for 2 x 15 min, stained with CCS solution overnight, destained with destaining solution

for 2 x 15 min and washed with water for 2 x 60 min.

Mass spectrometry analysis of purified recombinant S. aureus antigens

(Sample preparation was done by Mrs. Kirsten Bartels and sample measurements

by MS was performed by Dr. Vishnu Dhople)

The purified S. aureus antigens/proteins were analysed by mass spectrometry in detail to

study the composition and purity of the purified proteins. About 2 µg purified proteins

were taken for trypsin digestion. In order to dilute the guanidine HCl in the purified

protein sample, 20 mM ammonium bicarbonate was added to the samples to reach a final

volume of 20 µL. Dithiothreitol (DTT) was added to the final concentration of 2.5 mM,

and incubated for 60 min at 60°C. Subsequently, the volume was adjusted to 25 µL with

20 mM ammonium bicarbonate. To avoid the reformation of disulphide bridges,

iodoacetamide (IAA) was added to a final concentration of 10 mM and incubated for 30

min at 37°C under darkness. Further, trypsin was added in a ratio of 1: 25 (trypsin:

protein) and incubated overnight (16 – 18 h) at 37°C. The digestion was quenched with

acetic acid with the final concentration of 1%. Further, the peptides were purified and

enriched using zip-tips with C18 resin. For the digested peptides, a tip capacity of 2 µg

was used for the peptide purification and enrichment. As a first step in the peptide

purification and enrichment process, the column was stepwise equilibrated with 100%

acetonitrile (ACN), 80% (v/v) ACN solution, 50% (v/v) ACN solution, 30% (v/v) ACN

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35

solution and 1% (v/v) acetic acid. The sample was loaded onto the column by pipetting

up and down 20 times. The column was washed with 50 µL of 1% (v/v) acetic acid. The

peptides were eluted first with 50% (v/v) ACN solution by pipetting up and down 10

times and then with 80% (v/v) ACN solution. The eluates were pooled in a micro vial and

then dried to remove the ACN in a speed vacuum centrifuge. The dried purified and

enriched peptides were dissolved in 10 µL of LC buffer A (2% acetonitrile in water with

0.1% acetic acid). In order to study the protein composition, the trypsinised peptides from

purified proteins were analysed using liquid chromatography-mass spectrometry (LC-

MS). The LC-MS analysis was performed on a nanoACQUITY UPLC (Waters

Corporation, USA) coupled to LTQ-Orbitrap XL mass spectrometer (Thermo electron

corporation, Germany) equipped with a nano-ESI source and installed with Picotip

Emmitter (New objective, USA). For LC separation, the digested peptides were first

enriched on a nanoACQUITY UPLC 2G-V/Mtrap symmetry C18 pre-column (2 cm

length, 180 µm inner diameter and 5 µm particle size) from waters corporation and

resolved the peptides using nanoACQUITY BEH 130Å C18 column (10 cm length, 100

µm inner diameter and 1.7 µm particle size from Waters corporation, USA). Peptides

were separated using gradient elution mode. The separation was achieved with the

formation of linear gradient of 36 minutes containing LC buffer A (2% acetonitrile in

water with 0.1% acetic acid) and LC buffer B (acetonitrile with 0.1% acetic acid),

gradient-1-5% buffer B in 2 min, 5-60% B in 25 min, 60-99% B in 28 min, 99-1% B in

36 min). The peptides were eluted at a flow rate of 400 nL/ min and analysed using a

mass spectrometer with a nano ESI source. The electrospray voltage was 1.54 kV and

without sheath and auxiliary gas flow. The eluted peptides were measured in positive

polarity mode with m/z range of 300 -1700, and the MS in profile mode at resolution of

60000 FWHM in Orbitrap with a target value of 1 * 106. Further, the MS/MS was

performed in centroid mode by CID fragmentation at 35% normalised energy in LTQ

with an isolation width of 2 Da triggering with a threshold count of 2000 with a target

value of 1 * 104 or with a maximum injection time of 100 ms activated for 30 ms. The

target ions already selected for MS/MS were dynamically excluded for 30 s. Only doubly

and triply charged ions were triggered for tandem ms analysis.

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36

Mass spectrometry data analysis of the purified recombinant proteins

LTQ-Orbitrap XL mass spectrometer raw data was imported to Genedata Refiner MS. In

general, default parameters were used for each activity of Refiner MS. Firstly, the raw

data were binned into the Refiner MS workflow. After loading the raw data, the retention

time (RT) range was restricted with the RT minimum of 10 min and RT maximum was

not defined. The data was cleaned by subtracting the background noise by enabling the

chromatogram smoothing of 3 scans and disabled chemical noise smoothing. Further,

intensity thresholding, RT and m/z structure removal were done with default parameters.

Each individual time resolved data set was then independently aligned in retention time

direction by the pairwise alignment based tree scheme. Intensity thresholding, peak

detection, isotope clustering and MS/MS consolidation were done before MS/MS search.

MS/MS search was performed with a S. aureus_8325_iRT_BSA and E. coli_K12

specific FASTA protein database using the MASCOT search engine. The settings applied

for the MASCOT search engine were a peptide tolerance 10 ppm, a peptide isotope error

of 1, an ion fragment (MS/MS) tolerance of 0.2 Da, an ion charge of 2+ and 3+,

calculated the monoisotopic mass and performed decoy search. Oxidation of methionine

and carbamidomethylation of cysteine were defined as variable modifications. Further,

peptides were filtered based on a maximum false discovery rate (FDR) of 1% with a

deterministic target decoy method. Then, peaks were annotated with a m/z window of

0.01 Da and a RT window of 0.2 min. Further, identification and quantification of protein

was performed using protein inference with the deterministic method and Hi3

quantification with minimum peptide count of 2. Finally, intensities of the ranked

peptides with more hits were summed up and exported to Genedata analyst. The purity of

proteins was calculated and expressed in percentage.

Principle of FLEXMAP 3D®

and workflow

The FLEXMAP 3D® is basically as a flow cytometry designed by Luminex. The key

feature of this innovative technology is multiplexing of up to 500 different proteins in a

single well, and with wide dynamic range (~ 4.5 logs), fast analysis time, and low

amount of samples (~ 4.0 µL). In this study, we developed S. aureus multiplex assays

using FLEXMAP 3D®. The carboxylated magnetic beads (magplex) are 6.5 µm super

paramagnetic beads, with similar excitation and unique emission profiles which provide

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37

unique spectral characteristics within individual magnetic regions and are internally

encoded with three spectrally different fluorochromes (red, infra-red, orange-red) [208].

The magplex beads are identical in size and differ in the stoichiometry of the internal

classification dyes. Each magplex bead region has similar excitation, but unique emission

spectra. Magnetic beads are interrogated by two lasers; red and green. The red laser

excites the internal fluorochromes at 635 nm and the emission from the internal dye is

detected by two avalanche photo diodes. Then, a green laser excites the surface attached

reporter phycoerythrin at 532 nm and emission is detected by a photo multiplier tube.

The internal dye emission is deconvoluted as region events by a high-speed digital signal

processor and quantitated as the reporter signal bound to protein-magnetic beads. The

resulting data is denoted by median fluorescence intensity (MFI). Batch setup and data

analysis were done using software xPONENT®

4.2. The Figure 6 represents the

workflow of a serological assay using a indirect detection method.

Antigen conjugation to magnetic beads

Generation of bead mix

Multiplexing in well plates

Serum/Plasma binding to bead

mix

Addition of phycoerythrin detection antibodyData acquisition in FlexMAP3D®

Data analysis and validation

Bead regions

MF

I

Figure 6. Immunoproteomics workflow

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38

Covalent conjugation of S. aureus antigens to magnetic beads

The carbodiimide chemistry is a simple two-step process during which the carboxyl

groups of magnetic beads are first activated with EDC (1-Ethyl -3-[3-

dimethylaminopropyl] carbodiimide hydrochloride) reagent in the presence of Sulfo-NHS

(N-hydroxysulfosuccinimide) to form a sulfo-NHS-ester intermediates (Figure 7). The

reactive intermediate is then replaced by reacting with the primary amine of the coupling

molecule (antibody, protein, linker or peptide) to form a covalent amide bond.

Primary amines on purified antigens were covalently coupled to carboxylated

functionalised magplex beads with unique bead addresses. Briefly, 6.25 * 105 of the stock

magnetic sphere was transferred to a 96 well half area microplate and allowed to

equilibrate at room temperature. During the whole workflow, beads were protected from

light to avoid photobleaching. The plate was placed on a magnetic separator for 3 min

and the magnetic beads collected. Activation of beads was carried out with 150 µL of 100

mM monobasic sodium phosphate (pH 6.2). Subsequently, carboxy group activation was

initiated with EDC – Sulfo-NHS crosslinking master mix containing 15 µL of 50 mg/mL

sulfo-NHS in water free DMSO, 15 µL of 50 mg/mL EDC in activation buffer and 120

µL of activation buffer, incubated for 20 min, shaking at 900 rpm at RT. Activated beads

were washed thrice with 50 mM MES buffer (pH 5.0). About 125 µL of recombinant S.

aureus antigen (100 µg/mL) was covalently coupled to the activated beads, and incubated

for 2 hours, shaking 900 rpm at RT. The antigen coupled magnetic beads were washed

thrice with 100µL of wash buffer (PBS, 0.05% (v/v) tween20 pH 7.4). Finally, the

concentration of magnetic spheres was adjusted to 125 beads/µL with blocking-storage

buffer (PBS, 1 % (w/v) BSA, 0.05 % (v/v) proclin300 pH 7.4) and the magnetic

microspheres were stored in amber eppendorf tubes and stored at 4°C.

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39

Step - 1 Step - 2

Figure 7. Reaction mechanism of carbodiimide coupling. It’s a two-step process, (step 1)

carboxylated magnetic beads coupled with Sulfo-NHS and EDC, form a semi-stable amine reactive

intermediate ester. In the step 2, the reactive ester intermediate is replaced by the primary amine of

protein.

Evaluation of magplex bead-antigen conjugation

To ensure the efficiency of bead-antigen conjugation, antigen coupled beads were

verified with the antibody directed against the recombinant tagged antigen. Briefly,

antigen coupled magnetic beads were vortexed and sonicated for 3 min, 50 µL of antigen

coupled bead master mix was transferred to well plates and co-incubated with 50 µL of

Penta HIS/Anti-STREP tag antibody (final concentration 10 µg/mL) for 45 min, shaking

900 rpm at RT. Penta HIS/Anti-STREP tag coupled beads were washed thrice with

100µL of wash buffer (PBS, 0.05 % (v/v) tween20 pH 7.4). The beads were resuspended

in 50µL RPE-conjugated goat anti-mouse IgG (final concentration 5 µg/mL), incubated

for 30 min, shaking 900 rpm at RT and the unbound substrates were washed thrice with

100 µL of wash buffer (PBS, 0.05 % (v/v) tween20 pH 7.4). The beads were resuspended

in 100 µL of sheath fluid. Measurements were performed on the FLEXMAP 3D®

analyser system with the following conditions: a sample size of 80 µL, sample timeout 60

s, bead count 10000, and gate settings 7500-15000 under standard PMT. The assay was

performed twice; MFI values reflected the binding efficiency of antigen coupled beads.

After the successful conjugation of magplex-antigen, all the beads were pooled to

generate a master mix (multiplex).

Pre-adsorption of sera with assay buffer containing E. coli lysate

Pre-adsorption of E. coli lysate with sera reduces the cross-reactivity of E. coli protein

specific antibodies. For the preparation of E. coli lysate, a BL21 E. coli strain was

obtained from the Department of Microbiology, University of Greifswald. Overnight

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40

cultures of E. coli (BL21) were grown in Luria-bertani medium at 30°C with shaking at

180rpm. After centrifugation the pellet was resuspended with TE lysis buffer (10mM

Tris, 1mM EDTA, pH 8.0). Resuspended cells were lysed by ultrasonication with the

micro-tip on ice using five 10 s bursts at the amplitude intensity of 60 %. Serum/plasma

samples were prepared subsequently in seven dilutions (50, 500, 1000, 10000, 50000,

100000, 200000 folds) using assay buffer containing E. coli lysate (10% (v/v), 45% PBS,

1% (m/v) bovine serum albumin pH 7.4, 45% (v/v) LCB).

Serological bead based assay method validation

To study the influence of E. coli lysate, magplex-antigen beads were bound to the pooled

human serum (PHS) diluted with assay buffer containing two different compositions

(panel 1 and 2). The composition 1 (panel 1) contains 10% E. coli lysate (v/v), 45% PBS,

1% (m/v) bovine serum albumin pH 7.4, 45% (v/v) LCB and the composition 2 (panel 2)

contains 50% PBS, 1% (m/v) bovine serum albumin pH 7.4, 50% (v/v) LCB. The PHS

samples were prepared subsequently in seven dilutions (50, 500, 1000, 10000, 50000,

100000, 200000 folds) using two different compositions (panel 1 and 2).

For the complete study, a negative control sample has been used. We collected the serum

samples from two healthy fellows (HF). The HF samples were prepared subsequently in

seven dilutions (1:50, 500, 1000, 10000, 50000, 100000, 200000) using assay buffer

containing E. coli (10% E. coli lysate (v/v), 45% PBS, 1% (m/v) bovine serum albumin

pH 7.4, 45% (v/v) LCB).

Quantitation of antibodies by serological assay using FLEXMAP 3D®

A serological bead-based array by indirect detection was developed for the detection of

human antibodies against S. aureus antigens. The suspension array technology (SAT) by

FLEXMAP 3D® is a miniaturised and high throughput technology which allows the

parallel sample processing during the assay. Serum/plasma samples were stored at -80°C

and then brought to room temperature for sample preparation. 50µL of diluted sera

samples were co-incubated with 125µL of antigen-coupled master mix beads (125 beads

per region) and this mixture was incubated for 18 to 20 h (overnight), orbital shaking 900

rpm at 4°C. The plates were washed thrice with 100µL of wash buffer (PBS, 0.05% (v/v)

tween20 pH 7.4). To visualise the bound human antibodies, the beads were resuspended

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41

in 50µL of RPE-conjugated goat anti-human IgG (final concentration 5 µg/mL),

incubated for 60 min on the orbital shaker (900 rpm). The unbound substrates were

washed thrice with 100µL of wash buffer (PBS, 0.05% (v/v) tween20 pH 7.4) and finally

resuspended in 100µL of sheath fluid, vortex for 2 min. The measurements were

performed on FLEXMAP 3D® with the following conditions: sample volume of 80 µL,

sample timeout 60 s, bead count 100, and gate settings 7500 – 15,000 under standard

PMT. The assay was performed in duplicate.

Data analysis

All measurements were background corrected by subtracting the MFIs of sample beads

(incubated with serum/plasma) from MFIs of control beads (incubated with assay buffer).

The resulting background normalised MFI values were used for calculating the intensity

at half maximum using Clark´s theory interaction model, a non-least square algorithm (R

script for calculating the intensity at half maximal was developed by Dr. Stephan

Michalik). The product of the half-maximal MFI and the corresponding serum/plasma

dilution was calculated as this reflects the antigen binding intensity of antibodies

contained in each serum/plasma sample. Calculations were performed using a R package

(R 3.0.1).

Calculating the intensity at half maximum by Clark’s theory

Antigen-Antibody relations are similar to enzyme-substrate interaction. For any chemical

reaction, the reaction proceeds predominantly in one direction, but the reverse rate

steadily increases until the forward and reverse speed are identical. At this stage, the

reaction is said to have reached its equilibrium. The association between an antigen and

antibody involves various noncovalent forces between the antigenic determinant or an

epitope of the antigen and the variable region of the antibody (VH/VL). The non-covalent

forces behind the antigen-antibody interaction are as follows: hydrogen bonds, ionic

bonds, hydrophobic interactions, van der Waals forces. The noncovalent interactions

between antigen-antibody are weak compared to covalent interactions. These interactions

happen within the short distance of about 1 angstrom (Å). The combined strength of the

noncovalent interactions between a single antigen-binding site on an antibody and a

single epitope is the affinity of the antibody for that epitope. Antibodies with low affinity

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42

bind the antigen weakly and they dissociate readily. Antibodies with high affinity bind

the antigen strongly and bind for longer time and this antibody affinity is a quantitative

measure of binding strength. The association between the antibody (Ab) and an antigen

(Ag) can be described in the following equation 1.

(Equation 1)

Where the rate constant for the forward reaction (association) is Ka and the rate constant

for the reverse reaction (dissociation) is Kd. The ratio of Ka/Kd is equal to the

equilibrium constant Keq and can be written as Ka/Kd = Keq, measure of affinity. Keq is

the equilibrium constant for the above reaction and can be calculated from the ratio of the

molar concentration of bound Ag-Ab complex to the molar concentration of unbound

antigen and antibody during equilibrium and equation 2 can be written as follows [209].

(Equation 2)

Further the above equation 2 can be rewritten as follows

(Equation 3)

Where AB free and AG free are free antibody and antigen concentration, B is the bound

antigen: antibody complex concentration and the above equation 3 can be rewritten as

follows.

(Equation 4)

(Equation 5)

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43

The equation 5 is the equation for a rectangular hyperbola with horizontal asymptote

corresponding to 100% saturation of AGtotal, such that [bound] = [antigen]. Figure 8

depicts classical hyperbolic saturation curve. The ratio B/AGtotal is also referred to as F

(fractional occupancy). Keq is defined as the concentration of free antibody at which 50%

of the antigens are occupied (i.e., fractional occupancy = 0.5). The equation 5 can be

rewritten as follows (Equation 6). In order to derive the intensity at half maximum, the

equation 6 can be rewritten as equation 7.

Antibody freeKeq

B

AG total F=1

Figure 8. The classical hyperbolic binding curve, expressed at the fractional occupancy (f) of the

antigens.

(Equation 6)

(Equation 7)

Where MFI = B = Antigen-Antibody complex, MFImax = AGtotal = Saturated antigen-

antibody complex, Dil. at MFImax/2 = Kd = concentration of free antibody at which 50%

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44

of the antigen are occupied. For instance; the below Figure 9 depicts the hyperbolic

model for seven serial dilutions which leads to saturation in our experimental setup.

Dilution

MFI

1:2

00000

1:1

00000

1:5

0000

1:1

000

0

1:1

000

1:5

00

1:5

0

Antibody concentration

Figure 9. Hyperbolic model in our experimental setup.

Figure 10 represents the saturation curve for one antigen (LukF-PV) in two different

samples. The ratio between two different samples is about 4.25 fold. Based on this

mathematical model (Clark`s theory), intensities at half maximum were calculated.

Figure 10. Hyperbolic curve obtained from our mathematical model using Clark`s theory, for one

antigen (LukF-PV) in two different sample. Red dotted lines represent the concentration at half

maximum.

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45

Scaling of data

Data scaling involves the transformation of axis in such a way as to underline the features

or trends in the data that might be obscured by dominant signals/responses. To represent

the data in different graphical methods, the raw MFI value (calculated intensity) of each

antigen was log10 transformed to generate data values.

Mann-Whitney U test

The Mann-Whitney U test is also known as Wilcoxon rank sum test, tests for difference

between two groups on a single, ordinal variable with no specific distribution [210]. The

non-parametric Mann-Whitney U test can be used for testing the null hypothesis that two

samples come from the same population. Although non-parametric, the Mann-Whitney U

test still assumes that the distributions of the datasets to be compared are similar in shape.

The major difference between the Mann-Whitney U test and Student T test is that the

latter involves the concept of normal distribution.

Statistical analyses

Statistical analyses were performed using the Genedata AG - Analyst™

and

GraphPadPrism5 packages. The resulting antigen binding intensity (calculated intensity

at half maximum) values based on clack`s theory were imported into Genedata analyst™

.

The raw MFI values (calculated intensity) were transformed to log10 and normalised with

central tendency median and exceptional in study-1, i.e. only transformed to log10. We

performed the significance test in order to determine the probability of obtaining the

value of a test static assumed that the null hypothesis is true. From the p-value, it suggests

that the probability of rejecting the null hypothesis although it is true. When the p-value

is below the cutoff range, then it is termed as statistically significant. In clinical studies,

the significant cutoff is 0.05 and lower p-value, the more acceptable is the null

hypothesis. Further statistical elements such as Wilcoxon test, clustering or classification

using principal component analysis (PCA), partial least square (PLS) and other prediction

analysis were accomplished using Genedata analyst™

.

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46

Results

Method optimization and calibration

Serological assay is performed to study antigen–antibody responses and as a starting

point S. aureus antigen library was generated. For that, recombinant S.aureus antigens

were overexpressed using E. coli vectors in LB media and further the overexpressed

antigens were purified using NiNTA affinity chromatography. The purity was evaluated

by running SDS PAGE.

Figure 11. SDS-PAGE-based qualitative analysis of the purified S. aureus antigens. Ten µg of

purified antigens were loaded on the lanes and it corresponds to ClfA (1), Cell wall hydrolase (2),

Cna domain 2 (3), Chips (4), IsdA (5), IsdH (6), LukF (7), Atl (8), Fbp related (9), Cna domain 1 (10),

Enolase (11), SceD (12), HysA (13), LukE (14), LukS (15), Plc (16), SasG (17), uncharacterised

protein – SAOUHSC00106 (18), LytM (19), SCIN (20), SdrD domain 2 (21), Ssl7 (22), Spa (23), Ssl1

(24), EpiP (25), Ssl3 (26), IsdB (27), SdrD domain 1 (28). The molecular weight of marker proteins is

indicated (in kDa). The gel and the figure were provided by Protagen AG.

The purity of the antigens was defined based on the coomassie staining of the SDS gels.:

The above Figure 11 depicts the purity of the isolated antigens in the respective lanes

against marker proteins (M). The obtained yield for clumping factor A (lane 1) was less

compared to other antigens. Secondly, multiple bands were identified for the antigens

IsdH (lane 6) and HysA (lane 13).In principle, histidine tagging had been used during the

plasmid construction so that the targeted antigens were expected to be eluted for sure.

The multiple bands detected for these antigens were unexpected and needed to be further

investigated by mass spectrometry.

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47

Mass spectrometry based quantification of S. aureus and E. coli proteins

In order to distinguish if the additional bands are fragments of the target protein or host

cell proteins, we performed an identification of the proteins contained in the sample and

MS based Hi3 quantification for the subset of proteins. The detected peptides were

searched against a mascot database containing S. aureus_8325 and E. coli_K12 and

quantified based on the Hi3 method using area normalisation. The purity of the proteins

was expressed in percentage (%). The MS results of the purified proteins confirmed

purity greater than 90.0 % in spite of the presence of degradation bands in the SDS

PAGE. For instance, hyaluronate lyase (HysA) and intracellular serine protease (EpiP)

have a purity of above 95% as shown in Figures 12 and 13 (mass spectrometry). Thus,

most of the additional bands likely contain degradation products of the proteins produced

from E. coli. In the Figure 13, two lanes of the 1-D SDS PAGE belongs to the marker

(M) and purified recombinant EpiP and the MS results also confirms the purity of EpiP

HysAMkDa

Mass spectrometry1-D SDS PAGE

96.11

1.050.660.070.800.350.270.220.170.29

% P

urit

y

Identified proteins

Hyaluronate lyase/HysA

Elongation factor/Tu

Peptidyl-prolyl cis-trans

isomerase

Pyruvate formate lyase

Tryptophanase

Acyl carrier protein

Chaperone protein DnaK

Protein disaggregation

chaperone

4-deoxy-L-threo-5-

hexosulose-uronate ketol-

isomerase

Figure 12. Recombinant hyaluronate lyase (HysA) overexpressed in E. coli and its analysis by 1-D

SDS PAGE and mass spectrometry.

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48

kDa M EpiP

1-D SDS PAGE Mass spectrometry

98.64

0.14 1.040.04 0.02 0.06

% P

urit

y

Identified proteins

Intracellular serine

protease/EpiP

Elongation factor/Tu

Tryptophanase

Chaperone protein DnaK

Protein disaggregation

chaperone

4-deoxy-L-threo-5-

hexosulose-uronate ketol-

isomerase

Figure 13. Recombinant Intracellular serine protease (EpiP) overexpressed in E. coli and its analysis

by 1-D SDS PAGE and mass spectrometry.

Method validation

The serological method was validated with pooled human serum samples. In this

validation study, about 115 multiplexed antigens were incorporated and they were

conjugated to the magplex-beads.

To study the influence of E. coli lysate in the reduction of cross binding with Anti-E. coli

antibodies, human samples were pooled and prepared seven serial dilutions using assay

buffer containing two different buffer constituents (constituent 1 and constituent 2). The

constituent 1 contains 45% (v/v) LCB, 45% (v/v) CBS with 10% (v/v) of E. coli lysate

and the constituent 2 contains 50% (v/v) LCB, 50% (v/v) CBS. The prepared samples

were incubated with 115 antigen coupled magplex and then detected using PE anti human

IgG. The samples were analysed with FLEXMAP 3D® and the MFIs from seven serial

dilutions were further calculated for intensity at half maximum using Clark’s theory. The

calculated intensities were further evaluated between the two different conditions and the

ratios (constituent 1/constituent 2) were calculated and depicted in Figure 14. For

instance, Tuf, Antitoxin MazE, SigB, Autolysin – amidase domain, Seo, MsrA2, LytN,

Eno, MsrB, GroEL, alpha-hemolysin (Hla), trigger factor (Tig) shows high signal for the

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49

constituent 2 and their ratios were less than 0.5. The signals detected by human IgG were

significantly reduced by the presence of E. coli lysate in the assay buffer, indicating the

potential interference of E. coli proteins, probably because they contain similar epitopes

as the corresponding S. aureus proteins (e.g. Tuf).

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50

0 0.2 0.4 0.6 0.8 1 1.2 1.4

Tuf

Antitoxin MazE

SigB

PknB

Atl - AM domain

SsaA2

Seo

MsrA2

LytN

Eno

MsrB

GroEL

Hla

AIP - Typ1 - Thiolacton

Tig

Pbp2

HysA

Atl

HlgA

FadB

Fbp related

GraB

SAOUHSC_00106

Ssl2

IsaA

Aur

PurA

EssC - Domain 1

EntA

SACOL0480

CitC

Luk F PV

SAOUHSC_02241

HlgB

EssC - Domain 1

LukD

Sak

HlgC

GreA

SspB

SACOL1788

SplB

Sod

AIP - Typ4

Flr

IsdB

AIP - Typ4 - Thiolacton

EsxA

SplA

ClfB

EpiP

Coa

GlpQ

SACOL0479

IsaB

Ssl1

Ssl10

SdrD Domain 2

SACOL1802

Seb

Map-W

Sec

LukE

Sel

LukS

Scn

Sem

Nuc

Sec

Tsst-1

SdrF Domain 2

SACOL0444

Ssl7

SACOL2197

Hld

AIP - Typ2 - Thiolacton

SplD

Sek

Emp

Ear

Geh

SplC

IsdH

Plc

SdrE

Atl - GL domain

AIP - Typ1

SdrD Domain 1

Chips

IsdA

SceD

PrsA

AIP - Typ3

Hlb

Seq

SdrC

sdrG

SasG Domain 2

LytM

Truncated Map-W

Ssl11

Cna dm1

Sei

Lip

Cna Domain 2

SACOL0985

Set15

SdrF Domain 1

SasG Domain 1

Ssl3

AIP - Typ2

Efb

Ssl5

Ratio

An

tigen

s

Ratio (With E. coli lysate/Without lysate)

Figure 14. Influence of E. coli lysate in the reduction of binding with anti-E. coli antibodies using

human pooled serum samples. The ratio between the MFIs from two different buffer constituents

were calculated.

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51

In the validation part range has been determined using the serum samples from healthy

fellows. Serum dilution range was optimised to eliminate the false positive or negative

results. The serum samples from healthy individuals were obtained and they were serially

diluted to 22 successive dilutions (2, 4, 8, 10, 20, 40, 50, 100, 200, 300, 400, 500, 600,

700, 800, 900, 1000, 5000, 10000, 50000, 100000, 200000 fold) using assay buffer

containing (10% (v/v) E. coli lysate, 45% (v/v) CBS, 45% (v/v) LCB). The diluted

samples were incubated with 121 multiplexed antigens, PE anti human IgG was used to

detect the bound antibodies. As an example, Figure 15 and 16 shows the range (curve) of

serum samples of 22 dilutions for two antigens FPRL-1 inhibitory protein and foldase

protein (PrsA). In the Figure 15 and 16, X-axis represents the inverse of dilution (1/x

fold) and Y-axis represents the MFI. In the Figure 15, X axis scale has been transformed

to log10 scale and in the Figure 16, both X and Y axis are in the linear scale. The

correlation (r2) for the dilution range C1 which includes 22 dilutions excluding raw serum

for the FPRL-1 inhibitory protein is -0.3745 whereas for the foldase protein PrsA is

0.9720. In general, a linear dilution trend is seen between 40fold and 200000fold (C6).

There is a loss of correlation (r2) when lower fold dilutions were included in the series (2,

4, 8, 10, 20 folds) and this correlation is highly antigen dependent. Figure 17 shows the

overlay of seven correlations (C1, C2, C3, C4, C5, C6 and C7) for 121 antigens. On

average, the correlation of C6 (40 fold to 200000 fold) is about 0.9121 and the correlation

of C7 (50 fold to 200000 fold) is about 0.8189 for 121 antigens.

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0.000001 0.00001 0.0001 0.001 0.01 0.1 1

MF

I

Serum dilution

FPRL-1 inhibitory protein

Figure 15. Serum dilution range for FPRL-1 inhibitory protein. For better visualisation, X axis scale

has been transformed to log10. The correlation for 22 dilutions (C1) excluding raw sample is -0.3745.

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52

0

10000

20000

30000

40000

50000

60000

70000

80000

0 0.1 0.2 0.3 0.4 0.5 0.6

MF

I

Serum dilution

Foldase protein (PrsA)

Figure 16. Serum dilution range optimisation for foldase protein (PrsA). The correlation for 22

dilutions (C1) excluding raw sample is 0.9720.

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

-1 19 39 59 79 99 119CO

RR

EL

AT

ION

ANTIGENS

C1 C2 C3 C4 C5 C6 C7

Figure 17. Serum dilution optimisation in healthy individuals and its correlation C1, C2, C3, C4, C5,

C6 and C7; where the C1 – Correlation of serum dilution excluding raw serum, C2 - Correlation of

serum dilution excluding raw serum and 2 fold, C3 - Correlation of serum dilution excluding raw

serum, 2 and 4 fold, C4 - Correlation of serum dilution excluding raw serum, 2, 4 and 8 fold, C5 -

Correlation of serum dilution excluding raw serum, 2, 4, 8 and 10fold, C6 - Correlation of serum

dilution excluding raw serum, 2, 4, 8, 10 and 20 fold, C7 - Correlation of serum dilution excluding

raw serum, 2, 4, 8, 10, 20 and 40 fold.

Stability of antigens immobilised on the magplex

For a large set of samples, a bulk quantity of magplex-antigen bead mix is required. To

study the stability of magplex coupled with antigen, stability of the magplex-antigen

beads was monitored over a time period of 28 months in order to attain comparable

results between different sample batches. The histidine/streptavidin tag of 72

recombinant antigens immobilised on the magplex-beads was assayed with anti-His/Strep

mouse IgG and further conjugation with a RPE labelled anti-mouse antibody for the

detection. The intensities obtained from the first measurement (T0) were taken as a

reference. In the Figure 18 X-axis represents the antigens and Y-axis represents the

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53

relative intensities (%) of 72 antigens in different time points like T1 (8months), T2

(18months), T3 (22months), and T4 (28months). We observed some variations in the %

relative intensity between the time intervals for proteins like MsrA2 and this is due to the

technical variations. We sorted the recombinant antigens based on the tags. Among the

72 antigens; 40 carry histidine and 32 streptavidin tags, respectively. For histidine tag

antigens, the range of relative intensities was 9% - 122% (mean 63%), for the streptavidin

tag antigens, the range of relative intensities was 30% - 102% (mean 77%).

1.00

10.00

100.00

0 10 20 30 40 50 60 70

% R

EL

AT

IVE

IN

TE

NS

ITY

ANTIGENS

T1 (8 months) T2 (18 months) T3 (22 months) T4 (28 months)

Histidine tag Streptavidin tag

Figure 18. Stability monitoring of antigen immobilised magplex beads during the different time

intervals.

Evaluation of magplex-antigen coupling (coupling control)

The magplex-antigen coupling was evaluated using mouse monoclonal anti-histidine/

anti-streptavidin antibodies. Here, we determined the background intensity of magplex-

antigen complex (without mouse monoclonal anti-histidine/anti-streptavidin antibodies).

In this experiment, 72 antigens were incorporated to study the background MFI. The

background signal was minimised by the use of magnetic microspheres and various

formulated buffers such as low cross buffer (LCB) in combination with carboxy block

store buffer (CBS). This cocktail buffer significantly reduced the background signal in

magplex-antigen complex suspensions (Figure 19). Low cross buffer composition was

not disclosed by the manufacturer (Candor) and carboxy block store buffer contains PBS,

pH 7.4 + 1.0% BSA. A majority of magplex-antigen complex suspension have shown a

low background intensity with an average MFI value of about 25 median fluorescence

intensity (MFI) (Figure 18). Higher background intensity of about 400 MFI was observed

for Sle1 (N acetylmyl-L-alanine amidase) (Figure 19). Further, size of the protein (Da)

was correlated with the MFI of the background, and it is depicted in the Figure 20.

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54

1 10 100 1000

Cna Domain1

LukEv

EpiP

LytN

MsbA

Ssl7

SdrF Domain2

Aureolysin

SplE

Hld

SdrH

SigB

SbnG

SdrD Domain2

SceD

GlpQ

Bbp Domain2

Ssl2

LytM

SdrG

SAOUHSC00106

Scin

LukDv

SdrF Domain1

EC Fbp

HlgA

SdrC

SplF

IsdE

SdrE

LukS

ClfB

GraB

Ssl5

Spa

SdrD Domain1

EftB

Flr

EftA

ClfA

FnBP A

Hyaluronate lyase

LukF

IsdB

Ssl10

SspA

SspB2

FnBP B

Fibrinogen binding protein related

Protein 2160 esterase

Surface proteinG Domain1

Ferrichrome BP

Ssl12

Surface proteinG Domain2

Bbp Domain1

Autolysin

GapDH

IsdC

Ssl3

Cna Domain2

Hlb

LukS PV

HlgA

Enterotoxin type A

Eno

Scc

IsdH

MazE

IsdA

LukF PV

3 oxoacyl ACP reductase

Sle1

MFI

An

tig

ens

Mean of Background (MFI)

Figure 19. Magplex-antigen complex incubated with PE-Anti mouse IgG and its mean of background

MFI.

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55

0

1

10

100

1000

0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000

MF

I

Protein size (Da)

Correlation - Protein size vs. MFI

Figure 20. Correlation of protein size (Da) with background MFI for 72 antigens.

To study the influence of low cross buffer (LCB) in the reduction of background MFIs,

two experiment panels were designed. In the first panel, magplex-antigen complex in the

presence of LCB and CBS, and in the second panel magplex-antigen complex in the CBS

only. This experiment was performed with three replicates per panel and Figure 21

represents the calculated ratio (LCB+CBS/CBS) of mean MFIs for each antigen. Most of

the antigen shows the ratio of 1.0 indicating that the background MFIs of the two panels

were similar.

The very most important factor for the accuracy of the data are the bead counts. Based on

the luminex expert’s comments, the minimum number of beads required to measure the

stable MFI is 35 bead counts.

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56

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

PVL SAOUHSC01381

EC Fbp

LytM

SdrD Domain1

3 oxoacyl ACP reductase

SdrF Domain1

Protein 2160 esterase

SspA

Bbp Domain1

ClfA

PVL SAOUHSC01382

SdrE

SAOUHSC00106

SdrF Domain2

Gamma hemolysin comp A

EftB

Surface proteinG Domain2

ClfB

Glutamyl endopeptidase

Ferrichrome BP

FnBP B

SAOUHSC00384

IsdA

Autolysin

Hld

EftA

Eno

Hyaluronate lyase

Enterotoxin type A

Fibrinogen binding proien related

Spl IC

SplE

SAOUHSC01124

IsdB

SceD

IsdE

IgG binding proteinA

Bbp Domain2

HlgA

ATP bind protein GrfA

Aureolysin

Surface proteinG Domain1

SdrD Domain2

GlpQ

PhospholipaseC

SdrG

LukDv

SAOUHSC02243

SplF

FnBP A

SAOUHSC00386

IsdC

VWbp

FPRL1

SAOUHSC02241

LytN

Cna Domain1

IsdH

LukEv

Scin

SdrC

Fbp precursor SAOUHSC01115

SdrH

SAOUHSC00390

GapDH

SAOUHSC00395

Cna Domain2

MazE

SAOUHSC00392

RNA polymerase sigma factor

SbnG

N acetamylLalanine amidase

Ratio

An

tig

ens

Ratio (LCB + CBS/CBS)

Figure 21. Influence of low cross buffer in the reduction of background MFIs between two panels,

Panel 1 – LCB + CBS and Panel 2 – CBS only. About 72 antigens were included in this experiment

and the ratio between two panels were depicted thru dot plots.

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57

0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0%

LytM

PVL SAOUHSC01381

Eno

SAOUHSC00386

SdrF Domain1

IgG binding proteinA

Gamma hemolysin comp A

MazE

SbnG

Aureolysin

HlgA

SdrC

3 oxoacyl ACP reductase

IsdC

SAOUHSC00392

ClfB

RNA polymerase sigma factor

SdrF Domain2

Scin

EftB

Fbp precursor SAOUHSC01115

N acetamylLalanine amidase

SdrG

PVL SAOUHSC01382

SspA

GapDH

SAOUHSC00106

Protein 2160 esterase

Fibrinogen binding proien related

SAOUHSC01124

SAOUHSC00395

ATP bind protein GrfA

IsdA

Cna Domain2

Ferrichrome BP

SAOUHSC02241

SAOUHSC02243

SdrD Domain1

Surface proteinG Domain2

VWbp

SAOUHSC00384

SplE

Glutamyl endopeptidase

Enterotoxin type A

Cna Domain1

FPRL1

GlpQ

Bbp Domain2

FnBP B

PhospholipaseC

SdrE

FnBP A

SAOUHSC00390

Spl IC

EC Fbp

LukEv

SceD

Hld

IsdH

SdrD Domain2

EftA

Bbp Domain1

LukDv

IsdE

ClfA

LytN

SplF

Hyaluronate lyase

SdrH

Autolysin

Surface proteinG Domain1

IsdB

Bead counts

An

tig

ens

Bead counts - Background Vs Anti-histidine

Background

Anti-histidine

Figure 22. Evaluation of bead consistency throughout the background and control coupling

measurements during 72 antigen multiplexing. The bar plot represents the mean amount of beads

during the analysis of background (green bar) and anti-histidine (blue bar).

To verify the consistency of bead counts throughout the assay, bead counts were

monitored. Bead counts were measured in background and control coupling

measurements in order to verify the loss the beads. The Figure 22 represents the amount

of beads encountered during background without anti-tag (green bar) and with anti-tag

(blue bar) measurement. Here, on average 200 beads were observed for every antigen

which enhance the quality of data set.

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58

When the histidine tag recombinant antigens were bound to the magplex-beads, a master

mix of about 72 antigens were multiplexed. The coupling efficiency was evaluated using

monoclonal anti-histidine antibodies from mouse and further conjugation with

phycoerythrin anti-mouse detection antibody. The mean MFIs of the amount of antigens

bound to the magplex-beads (control coupling) are depicted in the Figure 23. Antigens

such as Serine protease E, formyl peptide receptor-like 1 inhibitory protein (FPRL-1),

SdrF, Glutamyl endopeptidase were shown less than 5000 MFI. We established that the

antigens with MFI above 10000 were considered for further serological assay and 35

bead counts as the minimum requirement for the data accuracy and the method was

validated. The median coefficient of variation (% CV) for coupling efficiency was 1.17

ranging from 0.30 to 4.80 and the overall %CV for intra-assay was below 5.0%. During

the data analysis, the amount of antigen bound to beads was always considered by

antigen-specific normalisation of the sample (serum/plasma), serial dilution MFI values

to their corresponding coupling control values.

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59

0 5000 10000 15000 20000 25000 30000 35000 40000 45000

SplEFPRL1

SdrF Domain2Glutamyl endopeptidase

SplFSAOUHSC01124

Hyaluronate lyaseFnBP A

SAOUHSC00392SdrD Domain2

Surface proteinG Domain1FnBP B

ClfBBbp Domain2

EftAATP bind protein GrfA

SdrCAureolysin

SdrGFbp precursor SAOUHSC01115

SbnGClfAIsdA

Cna Domain2SdrE

HldIsdH

PhospholipaseCCna Domain1

SAOUHSC00395Autolysin

SdrHIsdC

SAOUHSC00106Surface proteinG Domain2

SAOUHSC00390IgG binding proteinA

IsdESAOUHSC00384

Bbp Domain1N acetamylLalanine amidase

GlpQVWbpSpl IC

SAOUHSC02241SAOUHSC00386

EC FbpScin

SceDLukDvLukEv

IsdBEnterotoxin type A

Ferrichrome BPEftB

HlgALytN

RNA polymerase sigma factorSAOUHSC02243

SdrD Domain1GapDH

SdrF Domain1Gamma hemolysin comp A

Fibrinogen binding proien relatedProtein 2160 esterase

PVL SAOUHSC01382PVL SAOUHSC01381

SspAMazE

Eno3 oxoacyl ACP reductase

LytM

MFI

An

tig

ens

Control coupling

Figure 23. Amount of antigens bound to the magplex beads. This bar plot plotted against MFI on the

X axis and the antigens on the Y axis.

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60

Quantitation of antibody responses (antigen coupling)

The serum/plasma samples were diluted and coupled to master mix (magplex-antigen

beads) and the antibody responses were measured by a serological indirect assay with

FLEXMAP 3D® (refer materials and methods for sample preparation, method

parameters). Intensity at half maximum was calculated based on the Clark`s theory

mathematical model and the hyperbolic curve for all sample sets was obtained using R

studio (R script for calculating the intensity at half maximal was developed by Dr.

Stephan Michalik). The final outcome was calculated intensity (intensity at half

maximum) which represents the antibody responses of each sample to every antigen. The

obtained datasets were further evaluated with statistical analysis using Genedata –

Analyst™

and Graph pad. Based on the above strategies, the humoral responses (antibody

responses) during S. aureus infection was investigated.

Study-1 – Humoral responses during S. aureus nasal colonisation

IgG responses during S. aureus nasal colonisation

A total of 32 plasma samples was obtained from healthy individuals from western

Pomerania with defined clinical characteristics (Table 7). Among the 32 healthy

individuals, 16 were identified as carriers (twice positive) and 16 were identified as non-

carriers (without S. aureus) based on a earlier study [211].

The aim of this study was exploring the knowledge of humoral responses (antibody

responses) and correlation of clonal lineages during S. aureus nasal colonisation in well-

defined persistent carriers and non-carriers against 12 cytoplasmic antigens, 12

membrane antigens, 19 cell wall antigens, 66 extracellular antigens, 16 conserved

antigens, 8 auto inducing peptides respectively (list of antigens in Supplement 1). These

localisations were predicted based on psortb [212].

The IgG responses of carriers and non-carriers of S. aureus for 133 antigens are depicted

in Figure 24 thru a box-whisker plot. This plot represents the distribution of IgG levels in

carriers and non-carriers. The line in each box plot represents the median of the

individual. Each dot belongs to one antigen. The IgG against S. aureus antigens was

heterogeneous and their levels largely varied from individual to individual. The bar plots

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61

were plotted with log10 transformed and non-normalised values of calculated intensity

(Figure 24).

Non-carrierCarrier

Lo

g1

0 c

alc

ula

ted

in

ten

sity

Figure 24. IgG responses of healthy individuals to S. aureus antigens, carriers (n=16), non-carriers

(n=16). Red line in the box whiskers plot represents the median (4.02) of all carriers healthy

individuals and blue line represents the median (3.88) of all non-carriers healthy individuals.

To unravel the complexity of data sets, we carried out a Mann-Whitney U test

(nonparametric method) to identify significantly antigen-specific IgG levels

discriminating between carriers and non-carriers (Figure 25). To identify the significant

antigens, fold change (log2) and p-values (-log10) were calculated and they were plotted

against log2 (fold change) and –log10 (p-value) thru volcano plot. Antigens depicted in red

colour elicited a higher IgG response in carriers than in non-carriers and antigens

depicted in blue colour elicited a higher IgG response in non-carriers than in carriers. The

horizontal dotted lines in the volcano plot represent the p-value threshold of 0.05. For 16

S. aureus antigens, a significantly different immune response (p < 0.05) was detected

between carriers and non-carriers: Geh (triacylglycerol lipase), esterase protein, Hld

(delta-hemolysin), IsdB (iron-regulated surface determinant B), LytM (peptidoglycan

hydrolase), SACOL0480, IsdA (iron-regulated surface determinant A), HlgA (gamma-

hemolysin A), HlgB (gamma-hemolysin B), HlgC (gamma-hemolysin B), SigB (RNA

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62

polymerase sigma factor), SspA (glutamyl endopeptidase), Ssp (extracellular matrix

binding protein), auto inducing peptide thiolacton form 4, ClfB (clumping factor B) and

Ssl12 (staphylococcal superantigen like protein 12). Antigen-specific responses to foldase

protein (PrsA) and coagulase (Coa) were observed with high fold changes but without

passing the significance threshold of 0.05.

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

-3 -2 -1 0 1 2 3

P. V

alu

e (-

log10)

Fold change (log2)

Geh

P = 0.05

Esterase like protein

HldIsdB

SACOL0480 LytM

IsdA

HlgAAIP Thiol acton4 HlgB

HlgCSigB

SspASspClfB

Ssl12

Higher in carrierHigher in non-carrier

Figure 25. A Wilcoxon test was performed to evaluate the reactivity of antibodies against antigens in

carriers and non-carriers.

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63

Discrimination efficiency of antigens provoking different immune responses in

carriers and non-carriers

To explore the efficiency of discrimination, a principal component analysis (PCA) was

performed. At first, all 133 antigens were used for the PCA (Figure 26). Later, the 16

antigens provoking significantly different immune responses in carriers and non-carriers

resulting from the volcano plot have been used for PCA analysis and the discrimination

efficiency between carriers and non-carriers was shown in Figure 27.

Non-carrier

Carrier

Figure 26. Principal component analysis of antibody responses between carriers (n=16) and non-

carriers (n=16) for 133 antigens.

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64

Non-carrier

Carrier

Figure 27. Principal component analysis of antibody responses provoking significantly different

immune responses between carriers (n=16) and noncarriers (n=16) for 16 antigens.

Characteristics of immunogenic antigens

The immunoproteome was investigated based on the subcellular localisation of antigens

during S. aureus nasal colonisation. It is a key property of S. aureus that its proteins vary

in their accessibility by the immune system and that subsequently the antibody response

and function are affected. Antigens eliciting distinct IgG responses were represented by

dot plots. The levels of IgG specific to these antigens were significantly different in

carriers and non-carriers based on volcano plot (Figure 25). The dot plots represent the

calculated intensities (without log-transformation and normalisation) and they have been

sorted based on their intensity range in comparison between the carriers and non-carriers.

The more frequently reacting antigens that were associated with carriers were

predominantly extracellular and cell wall components of S. aureus (Figure 28), and the

reactivity that was associated with non-carriers were directed against clumping factor B

(ClfB) and staphylococcal super-antigen like protein Ssl12 (Figure 29). In carriers, LytM

with log2 fold change (FC) of 1.60, SACOL0480 with FC 1.40, esterase like protein with

FC 1.03, IsdA with FC 1.50, SspA with FC 1.33, AIP-type 4-thiolacton with FC 0.54,

triacylglycerol lipase (Geh) with FC 1.63, HlgA with FC 0.68, HlgB with FC 1.02, IsdB

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65

with FC 0.90, Hld with FC 1.52, Ssp (extracellular matrix binding protein/plasma binding

protein) with FC 0.66, HlgC with FC 1.01 and SigB with FC 1.12 were shown to result in

significantly higher IgG responses (Figure 28). On the other hand, significantly higher

IgG responses were found in non-carriers against clumping factor B with FC -0.44 and

staphylococcal super-antigen like protein Ssl12 with FC -0.81 (Figure 30).

Other antigens such as truncated MHC class II analog protein (Map-W) with FC -1.31,

lipase with FC -1.02, peptide methionine sulfoxide reductase B (MsrB) with FC -0.56

were shown higher IgG responses in non-carriers than in carriers but did not pass the

significance threshold of 0.05.

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66

SACOL0480

Carrier Noncarrier

100

1000

10000

100000 **

Peptidoglycan hydrolase LytM

Carrier Noncarrier

100

1000

10000

100000

1000000 **

IsdA

Carrier Noncarrier

1000

10000

100000

1000000 *

Glutamyl endopeptidase

Carrier Noncarrier

1000

10000

100000

1000000 *

LytM SACOL0480

Glutamyl endopeptidase (SspA)IsdA

Esterase like protein

Carrier Noncarrier

100

1000

10000 **

AIP - Typ4 - Thiolacton

Carrier Noncarrier

1000

10000

100000 *

Esterase like protein

AIP – Type4 – Thiolacton

Ca

lcu

late

d i

nte

nsi

tyC

alc

ula

ted

in

ten

sity

F.C 1.60 F.C 1.40 F.C 1.03

F.C 0.54F.C 1.33F.C 1.50

Triaclyglycerol lipase (Geh)

Carrier Noncarrier

1000

10000

100000

1000000 **

HlgA

Carrier Noncarrier

1000

10000

100000

1000000 *

HlgB

Carrier Noncarrier

1000

10000

100000

1000000 *

Geh HlgA HlgB

IsdB

Carrier Noncarrier

104

105

106

107 **

Delta-hemolysin

Carrier Noncarrier

104

105

106

107 **

Ssp (Extracellular matrix binding protein)

Carrier Noncarrier

104

105

106

107 *

IsdB Hld Ssp (ECM)

Calc

ula

ted

in

ten

sity

Calc

ula

ted

in

ten

sity

F.C 1.63 F.C 0.68 F.C 1.02

F.C 0.66F.C 1.52F.C 0.90

HlgC

Carrier Noncarrier

10000

100000

1000000 *

SigB

Carrier Noncarrier

10

100

1000

10000 *

HlgC SigB

Calc

ula

ted

in

ten

sity

F.C 1.01 F.C 1.12

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67

Figure 28. Reactivity of immunogenic antigens associated with carriers. A Mann-Whitney

(nonparametric), two sided test was performed for these data sets. Red coloured are data of carriers

and green coloured are data of non-carriers. Lines were drawn at the median. Log2 median fold

changes were calculated and represented as FC. Level of significance is represented by asterisks (* -

p-value less than 0.05; ** - p-value less than 0.01; *** - p-value less than 0.001; **** - p-value less

than 0.0001).

Antigens such as foldase protein (PrsA) and coagulase were statistically insignificant, but

they have shown higher log2 fold change. PrsA is membrane protein and it plays a vital

role in virulence of S. aureus. The PrsA-specific IgG levels were higher in carriers with

FC 2.65. Coagulase is an extracellular protein and an important virulence factor in S.

aureus infections. The coagulase-specific IgG were higher in non-carriers with FC -2.60.

Though antibody responses to these two antigens (PrsA and Coa) have shown higher fold

changes, they were statistically insignificant (Figure 30).

Clumping factor B

Carrier Noncarrier

1000

10000

100000 *

ClfB

Ca

lcu

late

d i

nte

nsi

ty

F.C -0.44

Superantigen-like protein_Ssl12

Carrier Noncarrier

10000

100000

1000000 *

Ssl12

F.C -0.81

Figure 29. Reactivity of immunogenic antigens associated with non-carriers. Level of significance is

represented by asterisks (* - p-value less than 0.05; ** - p-value less than 0.01; *** - p-value less than

0.001; **** - p-value less than 0.0001) and log2 median fold change were calculated and represented

as FC.

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68

PrsA

Carrier Noncarrier

100

1000

10000

100000

1000000ns

Coagulase

Carrier Noncarrier

103

104

105

106

107

108 ns

PrsA

F.C 2.65

Coagulase

F.C -2.60

Ca

lcu

late

d i

nte

nsi

ty

Figure 30. Immunogenic insignificant antigens foldase protein (PrsA) associated with carriers and

coagulase (Coa) associated with non-carriers. Level of significance is represented by asterisks (* - p-

value less than 0.05; ** - p-value less than 0.01; *** - p-value less than 0.001; **** - p-value less than

0.0001) and log2 median fold change were calculated and represented as FC.

Clonal complex influence in anti-TSST-1 IgG responses

In this study, there are five different clonal complexes of S. aureus genotypes (CC5, CC8,

CC15, CC25, and CC30) present among the carriers. The strains of the 16 carriers

displayed the following distribution among clonal complexes: CC30 (50%), CC5

(12.5%), CC8 (12.5%), CC15 (12.5%), CC25 (12.5%). The IgG responses to S. aureus

antigens expressed in different clonal complexes were investigated during nasal

colonisation. Very interestingly, anti-TSST-1 IgG have shown differences in the antibody

responses upon classifying the clonal complexes. Figure 31 shows the IgG responses

against TSST-1 from the three groups such as carriers with CC30, carriers with non-

CC30 (other clonal complexes CC5, CC8, CC15, CC25) and non-carriers. Here, carriers

with CC30 clones have shown pronounced IgG responses against TSST-1. While

performing a Mann -Whitney U test, it shows significant difference between CC30 and

non-carriers with log2 FC of 1.41 and it shows insignificant difference between carriers

with non-CC30 (CC5, CC8, CC15, and CC25) and non-carriers with higher IgG response

against TSST-1 in non-carriers.

Besides CC30 of S. aureus, carriers with CC15 clone type have shown higher IgG

responses to antigens such as PrsA, IsdA, IsdH (heme binding proteins), SceD

(transglycosylase), SACOL0480 and pore forming toxins HlgA, HlgB, HlgC and Hld.

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69

Though carriers with CC15 clone have shown higher IgG responses to these antigens, the

group size of CC15-clone carriers was restricted (12.5% - 2/16). Among 16 carriers, the

CC15 clone type was present in 2 healthy individuals and, therefore, statistical analysis

could not be performed for this group. As a simplistic visualisation of clonal complex

influence on the IgG responses against S. aureus antigens, a heat map of log2 transformed

average values of clonal complexes for 132 antigens was created and red square denotes

the higher IgG responses against the antigens in CC15 (Figure 32).

TSST-1

Ca

lcu

late

d i

nte

nsi

ty

F.C – 1.41

Tsst-1

Carrier CC30 Carrier Non CC30 Noncarrier

103

104

105

106

107*

ns

Figure 31. Clonal complex 30 influence on the Anti-TSST-1 IgG responses. Level of significance is

represented by asterisks (* - p-value less than 0.05; ** - p-value less than 0.01; *** - p-value less than

0.001; **** - p-value less than 0.0001) and log2 median fold change were calculated and represented

as FC.

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70

Figure 32. Simplistic visualisation of

clonal complex influence on the IgG

responses against 132 antigens. log2

mean values of carriers group with

different clonal complexes are depicted

in the heat map and red squared regions

show higher IgG responses in CC15.

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71

Summary of study-1

- In this study, we investigated the humoral responses (antibody responses) and

clonal complex correlation of S. aureus nasal colonisation using high-throughput

suspension array technology.

- The overall IgG responses against 133 antigens are highly heterogeneous between

the carriers and non-carriers.

- Overall in trend, carriers shows higher IgG responses than non-carriers.

- About 16 antigens were identified as candidates displaying different IgG

responses between the two groups based on the volcano plot and they further used

in the principal component analysis to test the discrimination efficiency.

- Among the 16 antigens, 14 antigens show significantly higher IgG responses in

carriers and 2 antigens revealed significantly higher IgG responses in non-

carriers.

- Most of the 16 candidate antigens belong to the extracellular and cell wall

component of S. aureus.

- Moreover, few antigens show higher FC but are not statistically significantly

different in level.

- In the correlation of clonal complex with IgG responses, mainly we observed

CC30 based higher IgG responses against TSST-1, whereas non-CC30 (CC5,

CC8, CC25, CC30) and non-carriers shows lower IgG responses against TSST-1.

- Besides CC30; CC15 associated S. aureus nasal colonisation shows pronounced

IgG responses against foldase protein (PrsA), delta-hemolysin (Hld), gamma-

hemolysin A, C (HlgA, HlgC), GroEL, IsdC, esterase like protein, and

staphylococcal secretory antigen (SsaA) than other clonal complexes (CC5, CC8,

CC25, CC30).

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Study-2 – Humoral responses during onset of S. aureus bacteraemia infection

IgG responses during onset of S. aureus bacteraemia

The aim of this study was to explore the humoral responses during S. aureus bloodstream

infection using high-throughput suspension array technology. This study was conducted

as a 3 year population based prospective study between 2011 and 2014. About 303

patients were involved in this study at Akershus university hospital, Oslo, Norway. The

clinical characteristics of bacteraemia patients are listed in the Table 8. In this study, 4

dominant clonal lineages (CC5, CC15, CC30 and CC45) have been observed in

bacteraemia patients. Among the 303 patients, serum samples were obtained from 92

individuals comprising 43 controls (without S. aureus) and 49 sepsis samples (first

episode of S. aureus or one blood culture positive for S. aureus and without pre-antibiotic

therapy).

The IgG responses were studied against 12 cytoplasmic antigens, 12 membrane antigens,

19 cell wall antigens, 66 extracellular antigens, 16 conserved antigens, 8 auto inducing

peptides (list of antigens in Supplement 1) and their localisations were predicted based on

psortb [212].

The IgG distribution among the two groups of control and sepsis samples against 133 S.

aureus antigens are represented by a box whiskers plot in Figure 33. The calculated

intensities were transformed to log10 and non-normalised. The log10 transformed

calculated intensities were used for plotting and each dot belongs to the IgG response

against one antigen. The horizontal lines represent the median values of each individual.

Overall median values of each group were represented by green and red lines. The green

line represents the median of the control patients and the red line represents the median of

the sepsis patients. The log10 median value of the control patients is 3.93 and the log10

median value of the sepsis patients is 3.67.

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Log10 C

alc

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Control Sepsis

Figure 33. Log10 transformed and non-normalised IgG responses of control and bacteraemia patients,

control (n=43), sepsis (n=49). The green line in the box whiskers plot represents the median (3.93) of

the control patients and the red line represents the median (3.67) of the sepsis patients.

To identify candidate antigens which discriminate between control and sepsis, a Mann-

Whitney U test/Wilcoxon test (non-parametric method) was performed. The log2 fold

change and –log10 p-values were calculated and the significant immune responses to

antigens were represented by the volcano plot in the Figure 34. The volcano plot

represents the significant reactivity of antibodies against antigens in control and sepsis

patients. About 72 antigens were statistically significant based on the Wilcoxon test.

Most of the antigens have shown higher antibody responses in the control. The Table 10

shows the list of 72 antigens against which significantly different immune responses were

observed between the control and the sepsis group. They have been sorted in ascending

order based on their level of significance.

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-0.5

1.5

3.5

5.5

7.5

9.5

11.5

13.5

15.5

17.5

-3.5 -2.5 -1.5 -0.5 0.5 1.5 2.5 3.5

P. V

alu

e (-

log

10

)

Fold change (log2)

P = 0.05

Higher in ControlHigher in Sepsis

Figure 34. A Wilcoxon test was performed to evaluate the reactivity of antibodies against antigens in

control and sepsis patients. The dotted line represents the p-value threshold of 0.05. Antibodies

against antigens below the p-value 0.05 threshold were statistically significant. Lower fold changes

represent the higher antibody responses in sepsis and higher fold changes represents the higher

antibody responses in control.

Discrimination efficiency of antigens provoking different immune responses in

control and sepsis

In order to display the discrimination efficiency, a principal component analysis (PCA)

was performed. At first, all 133 antigens were used in the PCA (Figure 35) to test the

discrimination efficiency. Further, the 72 candidate antigens provoking significantly

different immune responses in control and sepsis resulting from volcano plot have been

used for PCA analysis and the discrimination efficiency was shown in the Figure 36.

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75

Control

Sepsis

Figure 35. Principal component analysis (PCA) of antibody responses for control (n=43) and sepsis

(n=49) patients against 133 S. aureus antigens to visualize the discrimination efficiency.

Control

Sepsis

Figure 36 Principal component analysis (PCA) of antibody responses for control (n=43) and sepsis

(n=49) patients against 72 candidate S. aureus antigens to visualize the discrimination efficiency.

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76

Table 10. List of antigens against which significantly different immune responses were observed

between control and sepsis patients. Level of significance was calculated by Wilcoxon non parametric

method and they have sorted in descending order based on the -log10 p-value.

Antigens Log2 Median FC - Log10 (p-value)

AIP - Typ1 3.2449 15.7084

AIP - Typ3 3.0820 15.7084

AIP - Typ2 3.3014 15.6518

AIP - Typ4 2.2328 14.9505

ESAT-6 family virulence protein 1.9402 10.1719

Staphopain B 1.5431 9.9689

3-hydroxyacyl-CoA dehydrogenase protein 1.7635 9.8570

AIP - Typ4 – Thiolacton 1.2560 9.8347

Penicillin-binding protein 2 3.1232 9.5033

Peptide methionine sulfoxide reductase A 2.1288 9.3723

Immunoglobulin-binding protein -0.9142 9.0896

Peptide methionine sulfoxide reductase B 1.9226 8.6481

Uncharacterised protein_SACOL1788 2.1032 7.1565

Trigger factor 1.7263 6.7833

Transcription elongation factor (GreA) 1.6822 5.9297

Enterotoxin O 1.6051 5.6919

Super antigen-like protein_Ssl11 2.1449 5.6751

Serine/threonine-protein kinase 1.8191 5.0876

Chaperonin GroEL 2.3555 5.0086

Superoxide dismutase 1.3648 4.8371

Hyaluronate lyase 2.0106 4.8371

Clumping factor B 1.2790 4.5631

Adenylosuccinate synthetase 1.1525 4.5481

Serine protease B 1.2908 4.4440

Immunodominant staphylococcal antigen B 1.8972 4.3950

AIP - Typ1 – Thiolacton 1.1256 4.3355

Truncated secreted von Willebrand factor-binding protein 1.9616 4.2683

Collagen adhesion 1.3154 4.1106

DNA segregation ATPase and related proteins 1.5016 3.8590

Elongation factor 0.9143 3.7498

Uncharacterised protein_SACOL0908 1.5543 3.6555

Isocitrate dehydrogenase 1.2604 3.5231

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Enterotoxin type A 1.5605 3.2405

Fibrinogen-binding protein like protein -1.3434 3.1904

Uncharacterised protein_SACOL1802 0.9962 3.1532

Serine-aspartate repeat-containing protein D 0.6045 3.1408

Autolysin 0.9982 3.0670

Phosphopyruvate hydratase 1.6840 2.9701

Staphopain A/staphopain thiol proteinase 0.8806 2.9581

Super antigen-like protein_Ssl3 0.7279 2.7810

Enterotoxin C 0.8485 2.7462

Antitoxin MazE 0.7483 2.6774

Foldase protein A 1.3744 2.6660

Super antigen-like protein_Ssl2 0.6288 2.6320

AIP - Typ2 – Thiolacton 0.6467 2.5647

Serine-aspartate repeat protein F 0.7427 2.4218

Aldolase_hypothetical aldolase family protein 0.7074 2.2309

3-oxoacyl-[acyl-carrier-protein] reductase 0.7319 2.1080

Peptidoglycan hydrolase LytM 0.7248 2.0281

Zinc metalloproteinase aureolysin 1.6922 2.0182

Thermonuclease 1.4447 1.9497

Super antigen-like protein_Ssl1 1.0246 1.9303

1-phosphatidylinositol phosphodiesterase 1.0620 1.8728

MHC class II analog protein 0.8209 1.8162

Coagulase 0.9041 1.7604

Serine-aspartate repeat-containing protein C 0.7820 1.7604

Truncated MHC class II analog protein 1.3417 1.7512

Staphylococcal secretory antigen 0.6978 1.7420

RNA polymerase sigma factor SigB 0.9325 1.7055

Super antigen-like protein_Ssl10 0.3806 1.6248

Super antigen-like protein_Set15 1.3410 1.6072

Super antigen-like protein_Ssl5 0.6174 1.6072

Phospholipase C/truncated beta-hemolysin 0.6693 1.5984

Fibronectin binding protein -0.4094 1.5547

Esterase-like protein 0.5388 1.5202

Surface protein G 1.2161 1.4608

Fibronectin Binding protein B -0.4561 1.4524

Intracellular serine protease 1.0975 1.4271

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Glycerophosphoryl diester phosphodiesterase 0.5743 1.4025

Extracellular enterotoxin L 1.1430 1.4025

Iron-regulated surface determinant protein H 0.8575 1.3293

Uncharacterised protein/fibrinogen-binding protein precursor -1.0462 1.3133

The differences in the immune responses against the listed antigens (Table 10) were

highly significant in this study. 72 antigens caused significantly different immune

responses in control and sepsis patients. Among the 72 significant antigens, 67 antigens

have shown higher antibody responses in control and 5 antigens have shown higher

antibody responses in sepsis patients. These 72 antigens belong to the different cellular

components of S. aureus; 11 belong to the cytoplasm, 6 belong to membrane, 10 belong

to cell wall, 38 belong to extracellular space fraction and 7 were of unknown localisation.

Mainly, extracellular components were significantly different in antibody response

comprising quorum sensing molecules such as auto inducing peptides including thiol-

Acton and linear form, super antigens (Ssl2, Ssl3, Ssl5, Ssl10, Ssl11), extracellular

enzymes (Coa, Nuc, Plc, GlpQ, SspB, SspA, Atl) and toxins. In the case of cell wall

components, MSCRAAM proteins were significant such as IsdH, Cna, SasG, SdrC,

SdrD, SdrF, ClfB, FnbpB, and FnbpA.

Characteristics of immunogenic antigens

The immunogenic antigens obtained from this study are listed in the Table 10. About 72

antigens were identified as candidate antigens in this study. The antibody responses

against the immunogenic antigens were depicted through dot plots; antibody responses

were plotted between patients and its calculated intensity; they were sorted based on its

cellular components (cytoplasmic, membrane, cell wall, extracellular and unknown) and

its characteristics.

Figure 37 represents the antibody responses against staphylococcal super antigens (Ssl1,

Ssl2, Ssl3, Ssl5, Ssl10, Ssl11, and Set15). They have been plotted with calculated

intensities in log10 scale; asterisks denote the level of significance, calculated using

Wilcoxon test. The antibody responses against super antigens were significantly higher in

control than in sepsis patients. The log2 median fold change (FC) values were calculated,

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79

for instance 2.14 for Ssl11, 1.34 for Set 15, 1.02 for Ssl1, 0.72 for Ssl3, 0.62 for Ssl2,

0.61 for Ssl5, and 0.38 for Ssl10.

Superantigen-like protein_Ssl1

Control Sepsis

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Ssl1

Superantigen-like protein_Ssl2

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Ssl2

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Ssl3

Superantigen-like protein_Ssl5

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Ssl5

Superantigen-like protein_Ssl10

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Superantigen-like protein_Ssl11

Control Sepsis

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Super antigen-like protein_Set15

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Set15

F.C 1.02 F.C 0.62 F.C 0.72

F.C 0.61 F.C 0.38 F.C 2.14

F.C 1.34

Figure 37. Antibody responses against S. aureus super antigens comparing control and sepsis

patients. Level of significance is represented by asterisks (* - p-value less than 0.05; ** - p-value less

than 0.01; *** - p-value less than 0.001; **** - p-value less than 0.0001) and log2 median fold change

were calculated and represented as FC.

Figure 38 represents the antibody responses against staphylococcal enterotoxins (Sec,

Seo, Sea, and Sel). They have been plotted with calculated intensities in log10 scale;

asterisks denote the level of significance, calculated using Wilcoxon test. The antibody

responses against enterotoxins were significantly higher in control than in sepsis patients.

The log2 median fold change values were calculated; for instance 1.60 for enterotoxin O

(Seo), 1.56 for enterotoxin A (Sea), 1.14 for enterotoxin L (Sel), and 0.84 for enterotoxin

C (Sec).

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Enterotoxin C

Control Sepsis

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Enterotoxin O

Control Sepsis

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Enterotoxin type A

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Extracellular enterotoxin L

Control Sepsis

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Enterotoxin C

Ca

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d i

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Enterotoxin O

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Enterotoxin A Enterotoxin L

F.C 0.84 F.C 1.60

F.C 1.56 F.C 1.14

Figure 38. Antibody responses against S. aureus enterotoxins comparing control and sepsis patients.

Level of significance is represented by asterisks (* - p-value less than 0.05; ** - p-value less than 0.01;

*** - p-value less than 0.001; **** - p-value less than 0.0001) and log2 median fold change were

calculated and represented as FC.

Figure 39 represents the antibody responses against extracellular antigens such as Plc,

Atl, GlpQ, Antitoxin MazE, Coa, Esat-6, HysA, IsaB, LytM, SspA, SspB, Hlb, SplB,

EpiP, SsaA, Sod, GraB – Truncated Willebrand binding protein, Aur, NucI. Dot plots

were plotted with calculated intensities in log10 scale; asterisks denote the level of

significance calculated using Wilcoxon test. The antibody responses against the

extracellular antigens were significantly higher in control than in sepsis patients. The log2

median fold change values were calculated, for instance 1.06 for Plc, 0.99 for Atl, 0.57

for GlpQ, 0.74 for Antitoxin MazE, 0.90 for Coa, 1.94 for Esat-6, 2.01 for HysA, 1.89 for

IsaB, 0.72 for LytM, 0.88 for SspA, 1.54 for SspB, 0.66 for Hlb, 1.29 for SplB, 1.09 for

EpiP, 0.69 for SsaA, 1.36 for Sod, 1.96 for truncated Willebrand binding protein (GraB),

1.69 for Aur, 1.44 for NucI.

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81

Glycerophosphoryl diester phosphodiesterase

Control Sepsis

1000

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1000000 *

1-phosphatidylinositol phosphodiesterase

Control Sepsis

100

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100000 *

Antitoxin MazE

Control Sepsis

10

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Coagulase

Control Sepsis

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1000000 *

Autolysin

Control Sepsis

10

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ESAT-6 family virulence protein

Control Sepsis

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Calc

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Plc

F.C 1.06

Autolysin

F.C 0.99

Calc

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GlpQ

F.C 0.57

Antitoxin MazE

F.C 0.74

Coagulase

F.C 0.90

ESAT-6

F.C 1.94

Hyaluronate lyase

Control Sepsis

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Immunodominant staphylococcal antigen B

Control Sepsis

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Peptidoglycan hydrolase LytM

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Calc

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HysA

F.C 2.01

IsaB

F.C 1.89

LytM

F.C 0.72

Intracellular serine protease

Control Sepsis

10

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Phospholipase C/truncated beta hemolysin

Control Sepsis

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Serine protease B

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Staphopain B

Control Sepsis

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Staphopain A/staphopain thiol proteinase

Control Sepsis

10

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Staphylococcal secretory antigen

Control Sepsis

100

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Superoxide dismutase

Control Sepsis

10

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Truncated secreted von Willebrand factor-binding protein (Coagulase) VWbp

Control Sepsis

100

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Zinc metalloproteinase aureolysin

Control Sepsis

100

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Thermonuclease

Control Sepsis

10

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SspA

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F.C 0.88

SspB

F.C 1.54

Hlb

F.C 0.66

SplB

F.C 1.29

EpiP (IC Spl)

F.C 1.09

SsaA

F.C 0.69

Sod

F.C 1.36

GraB (VWbp)

F.C 1.96

Aur

F.C 1.69

NucI

F.C 1.44

Figure 39. Antibody responses against S. aureus extracellular antigens between control and sepsis.

Level of significance was represented by asterisks (* - p-value less than 0.05; ** - p-value less than

0.01; *** - p-value less than 0.001; **** - p-value less than 0.0001) and log2 median fold change were

calculated and represented as FC.

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Figure 40 represents the antibody responses against linear and thio-lactone forms of auto

inducing peptides (AIP) such as AIP – Type I – linear and thio-lactone, AIP – Type II –

linear and thio-lactone, AIP – Type IV – linear and thio-lactone, and AIP – Type III –

linear form. Dot plots were plotted with calculated intensities in log10 scale; asterisks

denote the level of significance calculated using Wilcoxon test. The antibody responses

against auto inducing peptides were significantly higher in control than in sepsis patients.

Majorly linear forms of AIP have shown higher antibody responses in control patients.

The log2 median fold change were calculated, for instance 3.24 for linear AIP – type I,

1.12 for thio-lactone form of AIP – type I, 3.30 for linear AIP – type II, 0.64 for thio-

lactone form of AIP – type II, 2.23 for linear AIP – type IV, 1.25 for thio-lactone form of

AIP – type IV, and 3.08 for linear AIP – type III. Significantly higher antibody responses

were found to linear AIPs than to thio-lactone form were observed.

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83

AIP - Typ1 - Thiolacton

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AIP - Typ1

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AIP - Typ2

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AIP - Typ2 - Thiolacton

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AIP - Typ3

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AIP - Typ4

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AIP - Typ4 - Thiolacton

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AIP – Type 1 (Linear)

F.C 3.24

AIP – Type 1 (Thio-lactone)

F.C 1.12

AIP – Type 2 (Linear)

F.C 3.30

AIP – Type 2 (Thio-lactone)

F.C 0.64

AIP – Type 4 (Linear)

F.C 2.23

AIP – Type 4 (Thio-lactone)

F.C 1.25

AIP – Type 3 (Linear)

F.C 3.08

Figure 40. Antibody responses against S. aureus linear and thio-lactone forms of auto inducing

peptides between control and sepsis. Level of significance was represented by asterisks (* - p-value

less than 0.05; ** - p-value less than 0.01; *** - p-value less than 0.001; **** - p-value less than

0.0001) and log2 median fold change were calculated and represented as FC.

Figure 41 represents the antibody responses against cell wall antigens such as clumping

factor B (ClfB), collagen adhesin (Cna), iron surface determinant protein H (IsdH),

serine-aspartate repeat containing protein C, D, F (SdrC, SdrD, SdrF), surface protein G

(SasG), and SACOL1788. Dot plots were plotted with calculated intensities in log10

scale; asterisks denotes the level of significance calculated using Wilcoxon test. The

antibody responses against the cell wall antigens were significantly higher in control than

in sepsis patients. The log2 median fold change values were calculated, for instance 1.27

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84

for ClfB, 1.31 for Cna, 0.85 for IsdH, 0.78 for SdrC and SdrD, 0.74 for SdrF, 1.21 for

SasG, and 2.10 for SACOL1788.

Clumping factor B

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Collagen adhesin

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Iron-regulated surface determinant protein H

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SdrC

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SdrD

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SdrF

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Surface protein G

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Uncharacterized protein_SACOL1788

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ClfB

F.C 1.27

Cna

F.C 1.31

IsdH

F.C 0.85C

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SdrC

F.C 0.78

SdrD

F.C 0.78

SdrF

F.C 0.74

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SasG

F.C 1.21

SACOL1788

F.C 2.10

Figure 41. Antibody responses against S. aureus cell wall antigens between control and sepsis. Level

of significance was represented by asterisks (* - p-value less than 0.05; ** - p-value less than 0.01; ***

- p-value less than 0.001; **** - p-value less than 0.0001) and log2 median fold change were calculated

and represented as FC.

Figure 42 represents the antibody responses against S. aureus cytoplasmic antigens such

as adenylosuccinate synthetase (PurA), hypothetical aldolase family protein (SbnG),

GroEL protein, elongation factor (Tuf), esterase like protein, isocitrate dehydrogenase

(CitC), peptide methionine sulfoxide reductase B (MsrB), Phosphopyruvate hydratase

(Eno), RNA polymerase sigma factor (SigB), transcription elongation factor (GreA),

trigger factor (Tig). Dot plots were plotted with calculated intensities in log10 scale;

asterisks denotes the level of significance calculated using Wilcoxon test. The antibody

responses against the cytoplasmic antigen were significantly higher in control than in

sepsis patients. The log2 median fold change values were calculated, for instance 1.15 for

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PurA, 0.70 for SbnG, 2.35 for GroEL, 0.91 for Tuf, 0.53 for esterase like protein, 1.26 for

CitC, 1.92 for MsrB, 1.68 for Eno, 0.93 for SigB, 1.68 for GreA, and 1.72 for Tig.

Adenylosuccinate synthetase

Control Sepsis

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1000

10000 ****

Aldolase_hypothetical aldolase family protein

Control Sepsis

10

100

1000

10000 **

Chaperonin GroEL

Control Sepsis

10

100

1000

10000

100000 ****

Elongation factor Tu

Control Sepsis

100

1000

10000

100000 ***

Esterase-like protein

Control Sepsis

100

1000

10000

100000 *

Isocitrate dehydrogenase

Control Sepsis

10

100

1000

10000

100000 ***

Peptide methionine sulfoxide reductase B

Control Sepsis

10

100

1000

10000

100000 ****

Phosphopyruvate hydratase/Enolase

Control Sepsis

10

100

1000

10000

100000 **

RNA polymerase sigma factor sigB

Control Sepsis

10

100

1000

10000 *

Transcription elongation factor GreA

Control Sepsis

10

100

1000

10000

100000 ****

Trigger factor

Control Sepsis

10

100

1000

10000

100000 ****

Calc

ula

ted

in

ten

sity

Calc

ula

ted

in

ten

sity

Calc

ula

ted

in

ten

sity

Calc

ula

ted

in

ten

sity

PurA

F.C 1.15

SbnG

F.C 0.70 F.C 2.35

GroEL

Tuf

F.C 0.91

Esterase like protein

F.C 0.53

CitC

F.C 1.26

MsrB

F.C 1.92

Eno

F.C 1.68

SigB

F.C 0.93

GreA

F.C 1.68

Tig

F.C 1.72

Figure 42. Antibody responses against S. aureus cytoplasmic antigens between control and sepsis.

Level of significance was represented by asterisks (* - p-value less than 0.05; ** - p-value less than

0.01; *** - p-value less than 0.001; **** - p-value less than 0.0001) and log2 median fold change were

calculated and represented as FC.

Figure 43 represents the antibody responses against S. aureus membrane antigens such as

DNA segregation related proteins (EssC), foldase protein (PrsA), MHC class II analog

protein (Truncated Map-W), penicillin binding protein 2 (Pbp2), Serine/threonine-protein

kinase (PrkC), and truncated MHC class II analog protein (Map-W). Dot plots were

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86

plotted with calculated intensities in log10 scale; asterisks denote the level of significance

calculated using Wilcoxon test. The antibody responses against the membrane antigen

were significantly higher in control than in sepsis patients. The log2 median fold change

were calculated, for instance 1.50 for EssC, 1.37 for PrsA, 0.82 for Truncated Map-W,

3.12 for Pbp2, 1.81 for PrkC, 1.34 for Map-W.

DNA segregation ATPase and related proteins

Control Sepsis

100

1000

10000

100000 ***

Foldase protein A

Control Sepsis

10

100

1000

10000

100000

1000000 **

MHC class II analog protein

Control Sepsis

104

105

106

107

108 *

Penicillin-binding protein 2

Control Sepsis

100

1000

10000

100000

1000000 ****

Serine/threonine-protein kinase

Control Sepsis

100

1000

10000

100000

1000000 ****

Truncated MHC class II analog protein

Control Sepsis

100

1000

10000

100000

1000000 *

Calc

ula

ted

in

ten

sity

EssC

F.C 1.50

PrsA

F.C 1.37

Truncated Map-W

F.C 0.82

Calc

ula

ted

in

ten

sity

Pbp2

F.C 3.12

PrkC

F.C 1.81

Map-W

F.C 1.34

Figure 43. Antibody responses for S. aureus membrane antigens between control and sepsis. Level of

significance was represented by asterisks (* - p-value less than 0.05; ** - p-value less than 0.01; *** -

p-value less than 0.001; **** - p-value less than 0.0001) and log2 median fold change were calculated

and represented as FC.

Figure 44 represents the antibody responses against unknown localisation S. aureus

antigens such as 3-hydroxyacyl-CoA dehydrogenase protein (FadB), 3-oxoacyl-[acyl-

carrier-protein] reductase (FabG), peptide methionine sulfoxide reductase A (MsrA),

SACOL0908, and SACOL1802. Dot plots were plotted with calculated intensities in

log10 scale; asterisks denote the level of significance calculated using Wilcoxon test. The

antibody responses against this set of unknown antigens were significantly higher in

control than in sepsis patients. The log2 median fold change values were calculated, for

instance 1.76 for FadB, 0.73 for FabG, 2.12 for MsrA, 1.55 for SACOL0908, 0.99 for

SACOL1802.

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87

3-hydroxyacyl-CoA dehydrogenase protein

Control Sepsis

100

1000

10000

100000 ****

3-oxoacyl-[acyl-carrier-protein] reductase

Control Sepsis

100

1000

10000

100000 **

Peptide methionine sulfoxide reductase A

Control Sepsis

100

1000

10000

100000

1000000 ****

Uncharacterized protein_SACOL0908

Control Sepsis

10

100

1000

10000

100000 ***

Uncharacterized protein_SACOL1802

Control Sepsis

1

10

100

1000

10000 ***

FadB

Calc

ula

ted

in

ten

sity

F.C 1.76

FabG

F.C 0.73

MsrA

F.C 2.12

Calc

ula

ted

in

ten

sity

SACOL0908

F.C 1.55

SACOL1802

F.C 0.99

Figure 44. Antibody responses for S. aureus unknown antigens between control and sepsis. Level of

significance was represented by asterisks (* - p-value less than 0.05; ** - p-value less than 0.01; *** -

p-value less than 0.001; **** - p-value less than 0.0001) and log2 median fold change were calculated

and represented as FC.

Furthermore, we observed some higher antibody responses in the sepsis patients in

comparison to the control patients. Among the 72 significant antigens, 67 antigen-

specific IgG responses were higher in control patients and 5 antigen-specific IgG

responses were higher in sepsis patients. Figure 45 represents the higher antibody

responses in sepsis patients against fibronectin binding protein A and B (FnbA and

FnbB), immunoglobulin binding protein (Sbi), fibrinogen-binding protein like protein

(Efb-1), and uncharacterised protein or fibrinogen-binding protein precursor (Scc). Dot

plots were plotted with calculated intensities in log10 scale; significant test performed

using Wilcoxon test and the asterisks denotes the level of significance. The log2 median

fold change values were calculated, for instance -0.40 for FnbA, -0.45 for FnbB, -0.91 for

Sbi, -1.34 for Efb-1, and -1.04 for Scc.

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88

Fibronectin Binding protein B

Control Sepsis

103

104

105

106

107 *

Fibronectin binding proteinA

Control Sepsis

103

104

105

106

107 *

Fibrinogen-binding protein like protein

Control Sepsis

100

1000

10000

100000

1000000 ***

Uncharacterized protein/fibrinogen-binding protein precursor

Control Sepsis

103

104

105

106

107 *

Immunoglobulin-binding protein

Control Sepsis

107

108

109

101 0 ****

Calc

ula

ted

in

ten

sity

FnbA

F.C -0.40

FnbB

F.C -0.45

Sbi

F.C -0.91

Calc

ula

ted

in

ten

sity

Efb-1

F.C -1.34

Scc

F.C -1.04

Figure 45. Higher antibody responses for S. aureus antigens in sepsis. Level of significance was

represented by asterisks (* - p-value less than 0.05; ** - p-value less than 0.01; *** - p-value less than

0.001; **** - p-value less than 0.0001) and log2 median fold change were calculated and represented

as FC.

Summary of study-2

- In this study, we investigated the humoral responses during S. aureus bloodstream

infection using high-throughput suspension array technology.

- In terms of overall IgG responses against 133 antigens, control samples generally

display higher IgG responses than those from sepsis patients.

- About 72 antigens were identified as candidates based on the volcano plot and the

discrimination efficiency was tested using principal component analysis.

- Of all the 72 candidates causing significantly different immune responses 50%

belongs to the extracellular components of S. aureus.

- Among the 72 antigens, 67 antigens have shown significantly higher IgG

responses in controls and 5 antigens have shown significantly higher IgG

responses in the sepsis patients.

- Fibrinogen and fibronectin family proteins of S. aureus have shown higher IgG

responses in sepsis patients compared to controls.

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89

Study-3 – Specific serum IgG at diagnosis of S. aureus bloodstream invasion is

correlated with disease progression

IgG responses during S. aureus bacteraemia

The aim of this study was to elucidate the protection efficiency of the adaptive immune

system during S. aureus bloodstream infections (SABSI). A prospective study of 44

patients with S. aureus bloodstream infections was performed. The clinical characteristics

of 44 patients are listed in the Table 9. About 64 recombinant S. aureus antigens were

incorporated into the study of antibody specificities associated with the protection against

sepsis. These 64 S. aureus antigens are listed in the Supplementary Table 2. Antigens

encompassing localisation in extracellular space, cell wall, cytoplasm and with unknown

cellular localisation were used in this study (list of antigens in Supplementary 2).

The IgG responses among these SABSI were depicted in box plots using non-normalised,

log10-transformed intensities (Figure 46). Dots around the box plots represents the single

antigens and their corresponding IgG responses. Thick coloured horizontal line represents

the median of all patients, red horizontal lines belongs to sepsis patients and green

horizontal line belongs to no-sepsis patients. The log10 median values of no-sepsis

patients were 4.21 and the log10 median values of sepsis patients were 3.95. IgG

responses among the groups were heterogeneous and inter-individual responses varied

within the group.

Mainly, patients were grouped into no-sepsis, uncomplicated sepsis, and severe sepsis or

septic shock and results were subjected to a partial least square analysis (PLS) which is

depicted in Figure 47A.

Later, the uncomplicated sepsis, severe sepsis or septic shock groups were grouped

together and the PLS was performed again based on the two groups of bacteraemia

patients developing or not developing sepsis. This resulted in a separation between most

bacteraemia patients without sepsis and with sepsis although some patient data sets were

still visible among the data points of the opposite group (Figure 47B).

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90

No sepsis Sepsis

Log10 C

alc

ula

ted

in

ten

sity

Figure 46. IgG responses of bacteraemia patients without sepsis (n=21) and with sepsis (n=19) against

64 S. aureus antigens. The red line in the box whiskers plot represents the median of all sepsis

patients (3.95) and green line represents the median of all without sepsis (4.21).

Figure 47. Bacteraemia without sepsis, with uncomplicated sepsis and with severe sepsis/septic shock

grouped together in a partial least square analysis (A). Decent discrimination between sepsis

(uncomplicated sepsis, severe sepsis and septic shock) and no-sepsis were shown in PLS (B).

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91

Validation of antigens provoking significantly different immune responses in

patients with sepsis and without sepsis

In order to validate the results, support vector machine was performed. The support

vector machine revealed the best discrimination between patients with and without sepsis

applying Fischer linear discriminant analysis to the calculated intensities of IgG binding

to the top 8 most discriminating antigens listed in the Table 11. These 8 antigens are

phospholipase (Plc), staphopain B (SspB), immunodominant staphylococcal protein A

(IsaA), staphylococcal exotoxin M (SEM), glycerophosphoryl diester phosphodiesterase

(GlpQ), gamma- hemolysin component C (HlgC) and two proteins of unknown function

SACOL0444 and SACOL0985.

Further, to identify antigen candidates, we performed a Wilcoxon test (nonparametric

method). The log2 fold change and –log10 p-value were calculated and they have been

displayed using a volcano plot (Figure 48). About 20 S. aureus antigens have shown

significantly higher IgG responses in no-sepsis patients and they are listed in the Table

11. Most of these antigens belonged to the extracellular components of S. aureus. They

comprise known toxins, other virulence factors and also antigens of unknown functions.

The above results were also tested using principal component analysis (PCA) with serum

IgG binding to the top eight most discriminating antigens. Figure 49 shows the degree of

separation between patients with and without subsequent sepsis.

By using Fischer linear discriminant analysis, sepsis was correctly predicted in 16 of 21

patients and 17 of 23 patients without sepsis were correctly assigned to the non-

complicated group. Thus, the prediction was correct in 75% of the patients (sensitivity

76%; specificity 74%).

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92

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

-3.5 -2.5 -1.5 -0.5 0.5 1.5 2.5 3.5

P. V

alu

e (-

log

10

)

Fold change (log2)

P = 0.05

Higher in nosepsis

Plc

EapGlpQ

SplB

SspBSACOL0444

IsaASemAIP type 4 Thiolacton

CoaSakHlgC

HlgBSplC LukF - PVEfb

SACOL0480 Atl – GL domain

Figure 48. Wilcoxon test was performed to evaluate the reactivity of antibodies against antigens in

bacteraemia patients with and without sepsis. The dotted line represents the p-value threshold of

0.05. Red labelled antigens were significantly (p-value < 0.05) higher in patients without sepsis than

in patients with sepsis.

Figure 49. Patient stratification using principal component analysis (PCA) with eight most

discriminating S. aureus antigens.

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93

Table 11: Top 20 S. aureus antigens predicting the patient stratification (Table

adapted from Stentzel et al. 2015 [207])

Abbrevi

ation Description Gene locus

GI-

Number Protein localisationa

Psortb LocateP

Signal

P

TM

Domain

Plc

1-phosphatidyl-

inositol phosphor-

diesterase

SAUSA300

_0099 87128097 Extracellular

N-terminally

anchored yes 1

SspB Staphopain B SA0900 15926634 Extracellular Secretory yes 1

IsaA Probable

transglycosylase

SAOUHSC

_02887 88196515 Extracellular

N-terminally

anchored yes 0

Uncharacterised

protein

SACOL044

4 57652631

Cytoplasmic

Membrane

Lipid

anchored yes 0

Sem Staphylococcal

enterotoxin M SA1647 15927403 Extracellular

N-terminally

anchored yes 0

Surface protein,

putative

SACOL098

5 57650173

Cytoplasmic

Membrane

N-terminally

anchored yes 0

GlpQ

Glycerophos-

phoryl-diester

phosphor-

diesterase

SAUSA300

_0862 87126873 Extracellular

N-terminally

anchored yes 0

HlgC Gamma-hemolysin

component C

SACOL242

1 57650965 Extracellular Secretory yes 0

SplB Serine protease

SplB

SAOUHSC

_01941 88195635 Extracellular

N-terminally

anchored yes 1

Sak Staphylokinase,

putative

SAOUHSC

_02171 88195848 Extracellular Secretory yes 0

Atl Bifunctional

autolysin

SAOUHSC

_00994b

88194750 Extracellular Secretory yes 0

Efb-c Fibrinogen-binding

protein

SAOUHSC

_01114 88194860 Extracellular

N-terminally

anchored yes 0

Uncharacterised

protein

SACOL048

0 57651321

Cytoplasmic

Membrane

N-terminally

anchored yes 1

GreA Transcription

elongation factor SA1438 15927190 Cytoplasmic Intracellular no 0

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94

a Protein localisation was predicted with different algorithms using the Aureowiki

database and S. aureus COL as reference strain (http://www.protecs.uni-

greifswald.de/aureowiki/Main_Page; November 2014)

b N-terminal part of the protein

LukF.PV LukF-PV SAUSA300

_1381 87126598 - - - -

Tsst-1 Toxic shock

syndrome toxin-1 SA1819 15927587 Extracellular Secretory yes 1

HlgB Gamma-hemolysin

component B

SACOL242

2 57650966 Extracellular Secretory yes 0

Coa Coagulase SAOUHSC

_00192 88194002 Extracellular Secretory yes 0

Lip Triacylglycerol

lipase 1

SAUSA300

_2603 87126156 Extracellular

N-terminally

anchored yes 1

Uncharacterised

protein

SACOL090

8 57651598

Cytoplasmic

Membrane

N-terminally

anchored yes 0

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95

Characteristics of immunogenic antigens

IgG binding to these eight antigens provoked a significantly different immune response in

the groups of bacteraemia patients with and without sepsis. The calculated intensities of

patient groups were depicted through dot plots in Figure 50. Dot plots were plotted with

calculated intensity in log10 scale and the line represents the median. Here, the level of

significance (p-value) were represented with asterisks and its log2 fold change for 8

antigens were shown in the Figure 50. Apparently, higher S. aureus-specific IgG serum

levels were found with patients who never experienced earlier episodes of sepsis. The

immunoproteome signature of eight S. aureus antigens had driven the disease course of

bacteraemia patients with a confidence level of 75% accuracy (76% sensitivity and 74%

specificity).

Plc

Res

po

nse

Nosepsis Sepsis

102

104

106

108 ***

SspB

Res

po

nse

Nosepsis Sepsis

102

104

106

108 **

IsaAR

esp

on

se

Nosepsis Sepsis

102

104

106

108 *

Sem

Res

po

nse

Nosepsis Sepsis

102

104

106

108 *

GlpQ

Res

po

nse

Nosepsis Sepsis

102

104

106

108 **

HlgC

Res

po

nse

Nosepsis Sepsis

102

104

106

108 *

SACOL0444

Res

po

nse

Nosepsis Sepsis

102

104

106

108 **

SACOL0985

Res

po

nse

Nosepsis Sepsis

102

104

106

108 **

Plc

Ca

lcu

late

d i

nte

nsi

ty

SspB IsaA Sem

Ca

lcu

late

d i

nte

nsi

ty

GlpQ HlgC SACOL0444 SACOL0985

F.C 2.34 F.C 1.22 F.C 2.05 F.C 2.18

F.C 1.08 F.C 1.59 F.C 2.10 F.C 1.73

Figure 50. Quantified median IgG binding differences to these eight antigens in comparison between

bacteraemia patients without and with sepsis. Open circles indicate patients without sepsis and black

filled circles indicate patients with sepsis. Lines are drawn at median. Level of significance is

represented by asterisks (* - p-value less than 0.05; ** - p-value less than 0.01; *** - p-value less than

0.001; **** - p-value less than 0.0001) and log2 median fold change were calculated and represented

as FC.

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96

Summary of study-3

- In this study, we investigated the protection efficiency of the adaptive immune

system during SABSI using high-throughput suspension array technology.

- Overall IgG responses against 64 antigens were highly heterogeneous between

no-sepsis and sepsis patients at time of admission already.

- In general, the patients who will not develop sepsis have shown higher IgG

responses than patients later developing sepsis.

- The top 20 S. aureus proteins listed in the table 11 mainly contribute to the

discrimination of no-sepsis and sepsis. As expected, all of these proteins belonged

to the extracellular proteome of S. aureus, and they have shown pronounced IgG

binding.

- About 8 antigens were identified as candidates, and they were used for predicting

the progression of SABSI.

- S. aureus proteins are grouped according to their cellular components. Based on

the normalised median serum IgG binding intensity, extracellular and cell wall

antigens were recognised by the serum antibodies.

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97

Discussion

S. aureus is an important life threatening pathogen, which can lead to soft skin tissue

infection (SSTi), wound infections, pneumonia, and blood stream infections (BSI) in the

community and hospital sites. The S. aureus infection rate due to community and hospital

acquired strains is increasing steadily. S. aureus produces a wide array of virulence

proteins and immune evasion factors that increase the incidence of S. aureus infections.

However, treatment strategies are really challenging due to the increase of multi-drug

resistance of clinical S. aureus isolates. The rapid emergence of S. aureus methicillin and

other antibiotic resistant strains further exaggerates the condition and thereby increases

the mortality rate. To date, the mortality rate due to S. aureus is higher than the mortality

rate due to HIV, viral hepatitis, tuberculosis or influenza [213] and there are no new

classes of antibiotics to treat S. aureus related infections over the past decades too.

However, vaccines would be appropriate preventive measures to treat the S. aureus

related infections. Until now, all S. aureus vaccine trials failed in different phases (see

Table 2 & 3). For an effective vaccine development, it is beneficial to understand the in

vivo behaviour of S. aureus and the host immune responses during disease progression.

The foremost challenging task during vaccine development is the identification of

antigens (single or multi-antigens) that will stimulate the most effective immune

responses against S. aureus. Few antigens have been listed in the literature based on

previous findings using classical methods and in the current thesis suspension array

technology (SAT) has been used to identify additional S. aureus antigens.

Immunoproteomics – Method establishment and optimisation

A serological multiplex assay has been developed for the indirect detection of IgGs in

human serum/plasma samples obtained from S. aureus colonised and infected

individuals. For the indirect detection of specific IgGs through multiplexing, recombinant

S. aureus proteins were required and the purity of these proteins is a very important factor

for this assay in order to avoid any interbacterial cross reactivity during the antibody-

antigen interaction.

Antibodies against E. coli in human serum/plasma samples as well as non-specific

binding and other matrix effects may lead to false positive or false negative results.

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98

Therefore, the serological assay was optimised and validated during the method

development stage. In the first step of method development, the quality of the

recombinant S. aureus proteins was evaluated by 1-D SDS PAGE and LC-MS/MS.

During the purification of S. aureus proteins, some cytoplasmic E. coli proteins were co-

purified and from the 1-D SDS PAGE and in the LC-MS/MS experiments, those proteins

were identified. Nevertheless, the purity of the over-expressed S. aureus proteins was

above 90%. The co-purified E. coli proteins were mainly the peptidyl-prolyl cis-trans

isomerase and the elongation factor, but the level of these two proteins was only in the

range of 2 to 4 % (Figure 12 and 13). The remaining cross-reactivity of E. coli specific

antibodies in the serum/plasma was reduced by using a 10% of E. coli lysate as a pre-

adsorbent to the human serum/plasma samples. This pre-adsorbent decreased the cross

reactivity of E. coli specific antibodies to make sure that the responses are highly specific

to S. aureus antibodies. To obtain consistent, reliable and accurate data, preliminary

validation has been carried out during the method development stage. The basic

validation included determination of the range, linearity and selectivity/specificity. As a

result, pre-adsorption with E. coli lysate improved the specificity/selectivity of the assay.

Furthermore, the serum/plasma sample dilution range was optimised. Hence, 22 different

dilutions were tested for 121 antigens and the optimal range was determined from 40 fold

to 200000 fold dilution. In addition, an assay buffer supplemented with 45% low cross

buffer (LCB), 45% carboxy block store buffer (PBS + 1.0% BSA), 10% E. coli lysate

was applied [214–216]. For these experiments we have used magplex beads throughout

as it has already been shown better sensitivity, with better signal intensity than microplex

beads (non-magnetic beads) and low cross reactivity [217].

Another critical point in the experimental design was the stability of the magplex-antigen

complexes, because the MFI-data from individual experiments, measured at different

time points, were later combined. As a result of these test experiments we could prove

that antigens immobilised on the magplex beads were highly stable over a time period of

18 months. Moreover, streptavidin tag containing antigens in combination with magplex

were highly stable over a time period of 22 months and more stable than the histidine tag

containing antigens. Furthermore, individual characteristics of each antigen seem to play

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99

a critical role. Thus, unstable conjugated antigen-magplex beads should be excluded from

the panel or must be re-coupled (fresh coupling) for an assay.

The serological assay is well suited for the antibody detection against a large set of

recombinant S. aureus antigens in human serum/plasma samples by indirect detection.

This method has the potential to detect 500 proteins/antigens per well in short time (60

s/well), with a small volume of sample (~4.0 µl) and a wide dynamic range (105).

Consequently, this suspension array technology represents a perfect tool for the rapid and

efficient antibody profiling in human body fluids such as serum, plasma and sputum, as

well as samples from throat swabs and nasal polyps.

Study-1: Immune responses during nasal colonisation

In this study-1 we have investigated the antibody responses in S. aureus nasally colonised

healthy individuals to classify immunogenic S. aureus antigens against IgG. Using the

suspension array technology, the antibody responses of persistently nasal colonised

(carriers) and intermittently nasal colonized (non-carriers), healthy individuals were

profiled for the IgG response to 133 S. aureus antigens. The anti-staphylococcal IgG

levels showed divergent responses between carriers and non-carriers, with strong inter-

individual variability within the groups. This potentially indicates a variable number of

previous encounters with S. aureus strains (Figure 24). The overall IgG response of

carriers and non-carriers is represented by median of all carriers and non-carriers

individuals and carriers show a higher median than the non-carriers. Significantly

stronger responses were found for carriers specific IgGs directed against LytM (P <

0.01), SACOL0480 (P < 0.01), esterase like protein (P < 0.01), Geh (P < 0.01), IsdB (P <

0.01), Hld (P < 0.01), IsdA (P < 0.05), SspA (P < 0.05), AIP-type 4 Thiolacton (P <

0.05), HlgA (P < 0.05), HlgB (P < 0.05), Ssp (P < 0.05), HlgC (P < 0.05), SigB (P <

0.05) (Figure 28). These findings are all in line with earlier studies from Kolata et al.,

Holtfreter et al. and Kloppot et al. [218–220].

However, the immune responses are highly strain dependent during colonisation;

USA300, for instance, either as an infecting or colonising strain triggered systemic

immune responses with greater magnitude than other S. aureus strains [221].

Colonisation is a multifactorial process, to which likely many bacterial components

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contribute causing detrimental effects onto the human immune system. Components like

bacterial adhesins, such as cell wall associated factors ClfB, bind to the cell cornified

envelop immobilised protein loricrin and this binding is highly ClfB expression

dependent (in vivo – mouse model) [222]. Interestingly, our study showed significantly

higher IgG response against ClfB (P < 0.05) in non-carriers than carriers (Figure 29).

This result is in line with earlier studies from Dryla et al and Clarke et al., where they

have shown higher IgG levels against autolysin, IssA, Map-W, Hla, IsdH, IsdA and ClfB

in non-carriers as well [223,224]. Furthermore, a study from Verkaik et al. showed the

maternally derived IgG from placenta does not protect against nasal colonisation and

antigens expressed in vivo like CHIPS, Efb, IsdA and IsdB play a major role in nasal

colonisation of young children [225]. Supporting this argument the study of Wertheim et

al. revealed that a ClfB mutant S. aureus strain (DU5997) was not able to persist in the

human nose after 2 weeks. To conclude, ClfB is a major determinant during nasal

colonisation [226] and implies that higher IgG responses against ClfB in non-carriers

could be of protective value against S. aureus bacteraemia.

Staphylococcal super antigens likely modulate nasal colonisation but the mechanism of

this contribution is still unclear. Staphylococcal super antigen 12 (Ssl12) shows

significantly higher IgG responses in non-carriers (Figure 29). Staphylococcal

enterotoxins (SE) and super antigens (SAgs) are acting as potent pro-inflammatory agents

[227]. Holtfreter et al. have already shown that neutralising antibodies against super

antigens, expressed by their colonising strains, enhanced protection against S. aureus

bacteraemia [228]. Perhaps even these non-carriers were transiently in contact with

different S. aureus strains and experienced minor staphylococcal skin infections.

Few antigens showed higher log2 median fold change between carriers and non-carriers,

but they were insignificant. Examples of such antigens are PrsA, which displayed a 2.65

fold higher IgG response in carriers, and vice versa Coa showed a 2.60 fold higher IgG

response in non-carriers (Figure 30). Supporting the importance of antibodies directed

against PrsA, previous animal studies have shown that higher IgG responses against PrsA

increased the survival time during bacteraemia in a mouse model [229].

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The influence of clonal complexes on sample specific IgG levels

Healthy carriers analysed in this study were carrying different clonal complexes (CC) of

S. aureus, such as CC5, CC8, CC15, CC25 and CC30. We investigated the IgG responses

against the S. aureus antigens based on the clonal complexes. Interestingly, we observed

pronounced IgG responses against TSST-1 for the CC30 group carriers compared to non-

CC30 and non-carriers (Figure 31). An earlier study has already shown that individuals

colonized by a TSST-1 producing strains had significantly higher levels of antibody

against TSST-1 than individuals who carried strains without TSST-1 and that repeated

exposure to tst carrying strains triggered specific immunity or immune responses [230].

In the study presented here, 50% (8/16) of the healthy carriers were carrying the CC30

clonal complex with the tst gene and they have shown higher IgG responses against

TSST-1 and this result suggested that humans with higher IgG antibodies against TSST-1

may not develop toxic shock syndrome [225,231,231,232]. Still, we observed minimal

IgG response against TSST-1 in non-carriers and non-CC30 carriers, which indicates that

they might have been exposed to S. aureus during their lifetime and in turn developed an

immune response against TSST-1, which seemed stable. A study from Blomfeldt et al.

shows that the CC30 lineage is associated with a high fatality rate (36.4%), which is

higher in comparison to other clonal complexes [233]. Clonal complexes based genetic

variations of S. aureus have also been observed by Holtfreter et al., where nasal swabs

were obtained from 3891 adults in the population-based study of health in Pomerania

(SHIP). 75% of the nasal swabs were found to contain 7 major clonal complexes (CC7, 8,

15, 22, 25, 30 and 45), thus suggesting that there is a strong association between the

virulence gene pattern and clonal complexes. Moreover, in this study population CC30

was the most common clonal complex where the tst gene was found predominantly [22].

In earlier studies, the CC30 lineage was shown to be associated with nasal colonisation

[234–236], hematogenous infections, infective endocarditis, osteomyelitis and persistent

bacteraemia [237–240]. The S. aureus isolate USA200, a CC30 clone, has a greater

likelihood to cause infective endocarditis (IE), but is less likely to cause lethal sepsis in

rabbit models [241]. Other interesting parts of this study, besides CC30, are further clonal

complex types also influencing the IgG response. For instance CC15 showed higher IgG

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responses compared to other clonal complexes against PrsA, Hld, HlgA, HlgC, GroEL,

IsdC, Esterase like protein and ssaA2 antigens (Figure 32)

As stated before, it is known that carriers have a higher risk of developing nosocomial

related bacteraemia than non-carriers. However, carriers with bacteraemia had a lower

risk of bacteraemia-related death [242]. Passive immunisation by recombinant ClfA, IsdB

and HlgB proteins in mice reduced the risk of bacteraemia [243]. Our results corroborate

these earlier findings and suggest that the carriers have higher level of antibodies against

virulence factor repertoires of S. aureus, which reduces the level of risk and mortality rate

during the bacteraemia compared to non-carriers. The antibodies against TSST-1, IsdA,

IsdB, HlgA, HlgB, HlgC, Hld, SACOL0480, SspA, SspP, SigB, esterase like protein,

Geh and LytM showed higher responses in carriers. These antibodies are likely connected

to the lower risk of death in carriers with bacteraemia than non-carriers with bacteraemia

[242].

Study-2: Immune responses during onset of bacteraemia

In study-2, we have profiled pre-existing antibodies specific to S. aureus during the onset

of S. aureus bloodstream infection (SABSI) by collecting serum samples from

bacteraemia and control patients. Antibody responses were studied against 133 antigens

using suspension array technology. Distinct S. aureus antigen specific immune responses

were observed between control and bacteraemia patients (Figure 33). The distinct

immune responses and the inter individual variability are in line with earlier findings

[244–247]. Diversity in the immune responses occurs due to the following aspects: (1)

genetic diversity of S. aureus isolates, (2) differential S. aureus protein expression and

the selective protein recognition by the immune system in different patients, (3) time

difference in the onset of bacteraemia, (4) colonisation status, condition of preceeding

infections and immune status [248,249].

In this study, we have identified 72 potential candidates reflecting the characteristic of

each group (Table 10). Significantly higher IgG responses against 67 antigens were found

for control patients and only 5 for sepsis patients (Table 10). Higher responses for control

patients were majorly directed against extracellular and cell wall components of S. aureus

(Figure 51) such as GlpQ, Plc, AIP linear form (Type1–4), AIP thiolacton form (Type 1,

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2, 4), MazE, Atl, Coa, SE – A, C, L, O, ESAT-6, esterase like protein, Efb-1, HysA,

IsaB, intracellular serine protease, LytM, Hlb, SplB, SspB2, SspB, SsaA, Set15, Ssl – 1,

2, 3, 5, 10, 11, Sod, Nuc, GraB (Truncated VWbp) and Aur. The biological

characteristics of few of these immunogenic antigens are described below.

Staphopain SspB2 and SspB are cysteine proteinases (with high virulence potential) and

involved in biofilm remodelling, the modification of the clotting cascade, fibrinogen and

collagen damage, the induction of vascular leakage, the blocking of phagocytosis, and the

killing of neutrophils and monocytes [250–253].

14%

14%

54%

8%

10%

Localisation based IgG response

Cell wall

Cytoplasm

Extracellular

Membrane

Unknown

Figure 51. IgG responses against localisation based 72 immunogenic antigen candidates.

The 1-phosphatidylinositol phosphodiesterase (Plc), an extracellular enzyme, promotes

the survival of S. aureus in human whole blood and neutrophils [254]. Hlb (Beta-

hemolysin) a cytolytic toxin, mainly involved in skin colonisation, is cytotoxic for human

keratinocytes [255]. Another extracellular exo-enzyme, hyaluronate lyase (HysA),

degrades hyaluronic acid (HA) in vivo, a major component of intercellular ground

substance of human and animal connective, epithelial and neural tissues. HysA also

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penetrates the tissue in order to increase the dissemination of virulence factors and

increases S. aureus accessibility to deeper tissues [256]. A recent study from Buhren et

al. demonstrated that hyaluronidase has been using as a therapeutic choice for ophthalmic

and dermatosurgery [257]. HysA is controlled by the CodY regulator in S. aureus. The

other virulence factors regulated by CodY are Hla, Hlb, SspA, and SspB [258].

Also the membrane foldase protein A (PrsA) showed higher IgG responses for control

samples, which matches to the earlier study from den Reijer et al. who studied

bacteraemia patients and suggested that this antigen is in vivo expressed mainly in SAB

patients [259].

Also the immune modulation/immune evasion associated antigens showed higher IgG

responses in the control group. For instance staphylococcal super antigens (Ssls), which

are belonging to the non-egc family, are widely involved in the immune evasion by direct

inhibition of immune receptors. Ssls display similar structural features and, occasionally,

share common interaction partners (Ssl3/Ssl4 [260], Ssl5/Ssl11 [261,262] and Ssl1/Ssl5

[263]). Ssl5 & Ssl11 bind to P-selectin glycoprotein ligand 1 (PSGL-1) to inhibit

neutrophil rolling on epithelial cells [262]. Ssl 1 & 5 act as potent neutrophil MMP

(Matrix metalloproteinase) inhibitors, where MMP are important host factors, fine tuning

the immune responses in the defence against pathogens. Ssl 1 & 5 also prevent the

potentiation of important neutrophil IL-8 and restrict the MMP-mediated neutrophil

migration through collagen [263]. Ssl10 inhibits complement activation [264]. Apart

from Ssls, another extracellular protein Efb-1 blocks C3/C5 conversion by convertases

(complement evasion) [265]. A further exo-enzyme is aureolysin (a zinc metallo

protease), which is expressed inside the phagocytic vacuole of S. aureus [266] and

resistant to the anti-microbial peptide LL37 [267]. It is also able to cleave C3 to C3b

[268].

Perhaps higher IgG responses against these immune evasion antigens in control patients

may increase the protection efficiency towards S. aureus bacteraemia. An earlier study

demonstrated that neutralising antibodies against superantigens in nasal carriers increase

the risk of septicemia and the outcome is much better in the case of sepsis with reduced

mortality rate in compare to non-carriers [269].

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The enterotoxins SEA, SEC, SEL and SEO are also superantigens belong to enterotoxin

gene cluster (egc) gene family and polyclonally activate the T cell at pico molar

concentrations. A superantigen binds to both MHC class II and V beta region of T cell

receptor and leads to the activation of both antigen-presenting cells and T lymphocytes.

These interactions lead to the over production of pro-inflammatory cytokines (IL – 2, IFN

– γ, TNF – α) [270]. In the healthy community, there are antibodies against SEA, SEB,

SEC, SED and TSST-1 [271–274], which neutralise egc-encoded SAg and stop their

proliferative effects on T cells [275]. Unexpectedly, neutralising antibodies against SAg

are very unusual [275]. Interestingly, treating streptococcal TSS patients with IVIG from

a pool of human plasma containing anti-SAg antibodies, a reduced cytokine production,

bacterial load, and mortality were observed [276,277].

Significant higher antibody responses against auto inducing peptides (AIP) were found in

control patients for the first time. In general, AIPs are regulated by the accessory gene

regulatory (agr) system, a quorum-sensing regulatory system [278,279]. Agr up-regulates

virulence factors essential for the disease progression in animal models of acute infection

including skin and soft- tissue infections, infective endocarditis, pneumonia, septic

arthritis and osteomyelitis [280–285]. Agr enhances the disease severity by enhancing

expressing of virulence factors such as toxins and degradative exoenzymes [286]. A

study from Shopsin et al. showed that agr functionality is not an essential for the nasal

colonisation in humans [287]. In another study Malachowa et al. demonstrated by

transcriptomic analysis of human blood infected with S. aureus a total lacking of agr

expression in an agr functional strain [288]. A likely reason for this lacking activity is

probably blocking of AIP by apolipoprotein B (apoB) in serum, which might on the other

hand enhance the binding activity with the sensor kinase agrC [289,290]. Due to lack of

agr activity, the expression of immune evasion molecules immunoglobulin protein A

(Spa) and FnBPs is promoted to disseminate into the bloodstream [288,291,292].

Our study on SpA, FnBPs, Sbi and Efb-1 showed stronger IgG responses for sepsis

patients which is in line with an earlier study from Edwards et al.. In this study FnBPs

were associated with systemic inflammation, weight loss, and mortality in murine model

[292,293]. There is clear evidence that FnbA is adequate to trigger S. aureus invasion of

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106

cells via Fn bridging to α5β1 integrins [294,295]. Another study revealed a strong

antibody binding to FnBP complexed with Fn compare to non-complexed FnBP, i.e.

without Fn [296].

In summary, this study confirms the inverse correlation between specific antibodies and

severity of infection which matches to earlier findings from Stentzel et al., Adhikari et

al., and Kolata et al.. In our study a total of 72 antigen candidates have been identified,

which may have potential in patient stratification based on S. aureus-specific antibodies

during the diagnosis of S. aureus bacteraemia. Technically, compared to the classical

methods, suspension array high-throughput technology provided further insights of

humoral responses during the S. aureus bacteraemia progression.

Study-3: Immune responses during complicated bacteraemia

In this study, we have elucidated the protection efficiency of the adaptive immune system

during S. aureus bloodstream infections (SABSI). The IgG antibody responses were

studied against 64 S. aureus antigens and seem to be inversely proportional to the risk of

sepsis development (Figure 46). Eight immunogenic antigens can be used to estimate the

progression of disease in S. aureus bacteraemia patients with 75% accuracy (76%

sensitivity and 74% specificity). These immunogenic proteins comprised conserved

extracellular S. aureus proteins except SEM (superantigen) and all these proteins are

belong to the core genome of S. aureus [297]. Among these eight immunogenic antigens,

HlgC a bicomponent pore forming toxin, shows higher IgG responses in no-sepsis

patients. HlgC corresponds to the Hlg genes and was highly up regulated in USA300

during culture in human blood [298]. There is a high degree of sequence similarity

between PVL and Hlg that leads to the neutralisation of Hlg by anti-PVL antibodies

further contributing to the reduced severity of the infection [299]. Another exoprotein

regulated by the agr quorum sensing system among the immunogenic antigens is Plc (1-

phosphatidylinositol phosphodiesterase). Plc promotes the survival of S. aureus in human

blood and in PMNs [300]. Another known immunogenic antigen is IsaA, an extracellular

component of S. aureus, which showed higher IgG responses in uncomplicated sepsis

patients. A study on passive vaccination of anti-IsaA (1D9) showed an effective

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prophylaxis treatment of S. aureus MSSA related bacteraemia in a mouse model

[301,302].

Another study from Holtfreter et al. has demonstrated experimentally that nasally

colonised volunteers showed higher antibody responses against Hla, SspB, Plc, SplB,

SplE, and SspA [303]. These results corroborates earlier findings of S. aureus carriers,

which are showing higher levels of antibodies than non-carriers, but lower mortality

[304–306]. Similarly, risk of sepsis is significantly lower in patients with higher IgG

levels against Hla, Hld, PVL, SEC-1, and PSM-α3 [299]. Furthermore, epidermolysis

bullosa (EB) patients colonised with S. aureus showed higher anti-staphylococcal IgG

against IsdA, SasG, IsaA, SCIN, Nuc, LytM, SEM,SEN, SEO than the controls and

further did not develop systemic S. aureus infection [307]. On the other hand, children

with low pre-existing IgG levels against Hla and PVL are more prone to invasive S.

aureus disease [308].

In summary, the current study extends the knowledge of humoral responses during

SABSI and provided first hints for potential diagnostic patient stratification, based on S.

aureus-specific antibody screening. In general uncomplicated sepsis patients showed very

strong S. aureus-specific antibody binding, compared to complicated sepsis patients.

Besides the patient stratification according to their risk of sepsis, this study also provides

new and promising candidates for vaccination.

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Conclusion and Outlook

- In this thesis, serological assays using suspension array multiplexed technology

were developed, optimised, validated and established for the detection of human

antibodies in serum/plasma of controls and patients with S. aureus infection.

- This methodology further explores the in vivo progression of human adaptive

immune system during S. aureus colonisation and infection.

- In this novel high-throughput method, the assay can be performed in a fast way

with low volume of sample, with reliable and reproducible results. The method is

suitable for detecting the antibody levels against wide range of S. aureus proteins

by multiplexing.

- For the reliability of the developed assay, a control sample is always required.

Here we used pools of serum/plasma samples as the quality control for the better

reliability.

- Proteins coupled to magnetic beads (magplex) are highly stable over a period of

18 months.

- Screening of hundreds of human serum/plasma samples, revealed a group of

antigens which was able to discriminate between the groups from various

episodes of S. aureus infection.

- From the S. aureus nasal colonisation study, it was suggested that IgG against the

16 immunogenic potential antigen candidates may aid for the development of

vaccines.

- For the first time, we observed clonal complex (CC) based IgG responses and

mainly for CC30 that IgG responses against TSST-1 (p-value < 0.05) were higher

than the other CCs. This suggest that antibodies against TSST-1 may protect the

individuals from the toxic shock related infections. Moreover, CC30 are majorly

found in the hematogenous/bloodstream related infections.

- The other interesting result in this study is that for CC15 were shown stronger IgG

responses against extracellular proteins of S. aureus were shown . Due to the

restricted number of CC15 in the healthy carriers group, we were not able to

perform the statistical analysis. Further studies need to be done on clonal complex

based IgG responses.

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109

- From the SABSI study (study-2), in total 72 antigens were significantly different

in IgG-responses between sepsis and control patients. Among the 72 antigens,

IgG responses against 67 antigens were higher in control and IgG responses

against 5 antigens were higher in sepsis patients. This suggests that antibodies

against these 67 antigens may protect the individuals from S. aureus associated

bacteraemia.

- The group of antigens identified from this studies can potentially be used for

patient stratification. For instance the mortality rate of S. aureus bacteraemia is

high and this patient stratification may reduce the mortality rate of S. aureus

bacteraemia patients.

- The most prominent proteins expressed during colonisation and infection (in vivo)

which elicit protective immune responses in human are derived from the

extracellular and cell wall components of S. aureus. Further studies are required

for the antigen stratification (single or multivalent antigen) for the development of

active/passive vaccines.

- The antibodies against this set of antigens might be associated with the risk of

developing S. aureus infections. The humoral responses provoked for this set of

antigens might be responsible for the lower risk of mortality observed in S. aureus

carriers with bacteraemia than in S. aureus non-carriers with bacteraemia.

- Our findings are expected to facilitate the future immunoproteomics analysis with

the inclusion of a higher number of immunogenic antigens in the assay panel.

These findings might aid for the development of new generation vaccines against

S aureus.

- The assay described here can be used as excellent research tool to gain further

insight into the endemic and demographic properties of the disease.

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110

Publications

1. Specific serum IgG at diagnosis of Staphylococcus aureus bloodstream invasion

is correlated with disease progression. (Pubmed ID: 26155744, DOI:

10.1016/j.jprot.2015.06.018)

Sebastian Stentzel, Nandakumar Sundaramoorthy, Stephan Michalik, Maria

Nordengrün, Sarah Schulz, Julia Kolata, Peggy Kloppot, Susanne Engelmann, Leif Steil,

Michael Hecker, Frank Schmidt, Uwe Völker, Mary-Claire Roghmann , and Barbara M.

Bröker.

2. Characterization of human and Staphylococcus aureus proteins in respiratory

mucosa by in vivo- and immunoproteomics. (Pubmed ID: 28099884, DOI:

10.1016/j.jprot.2017.01.008)

Frank Schmidt, Tanja Meyer, Nandakumar Sundaramoorthy, Stephan Michalik,

Kristin Surmann, Maren Depke, Vishnu Dhople, Manuela Gesell Salazar, Gabriele

Holtappels, Nan Zhang, Barbara M Bröker, Claus Bachert, Uwe Völker.

3. Omics approaches for the study of adaptive immunity to Staphylococcus aureus

and the selection of vaccine candidates. (Pubmed ID: 28248221,

DOI:10.3390/proteomes4010011)

Silva Holtfreter, Julia Kolata, Sebastian Stentzel, Stephanie Bauerfeind, Frank Schmidt,

Nandakumar Sundaramoorthy and Barbara M. Bröker.

4. Laboratory mice are frequently colonized with host-adapted S. aureus and mount

a systemic immune response – note of caution for in vivo infection experiments. (Pubmed

ID: 28512627, DOI: 10.3389/fcimb.2017.00152)

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111

Daniel Schulz, Dorothee Grumann, Patricia Trübe, Kathleen Pritchett-Corning, Sarah

Johnson, Kevin Reppschläger, Janine Gumz, Nandakumar Sundaramoorthy, Stephan

Michalik, Sabine Berg, Jens van den Brandt, Richard Fister, Stefan Monecke, Benedict

Uy, Frank Schmidt, Barbara M. Bröker, Siouxsie Wiles and Silva Holtfreter

5. Proteomics and immunoproteomics profiling of serum proteins and antibodies

from patients with Staphylococcus aureus induced bloodstream infection - Manuscript

under progress.

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112

References

1 Alex ogston Micro organisms in surgical diseases (1881).

2 Alexander Ogston (1844-1929) Classics in infectious diseases. "On abscesses".. Reviews of

infectious diseases. 6(1), 122–128 (1984).

3 Rosenbach FJ. Mikro-organismen bei den Wund-Infections-Krankheiten des Menschen J.F.

Bergman, Wiesbaden (1884).

4 Lowy M.D Staphylococcus aureus Infections (1998).

5 Rubin RJ, Harrington CA, Poon A, Dietrich K, Greene JA, Moiduddin A. The economic impact

of Staphylococcus aureus infection in New York City hospitals. Emerging infectious diseases.

5(1), 9–17 (1999).

6 Pittet D, Wenzel RP. Nosocomial bloodstream infections. Secular trends in rates, mortality,

and contribution to total hospital deaths. Archives of internal medicine. 155(11), 1177–1184

(1995).

7 Richards MJ, Edwards JR, Culver DH, Gaynes RP. Nosocomial infections in combined medical-

surgical intensive care units in the United States. Infection control and hospital epidemiology.

21(8), 510–515 (2000).

8 Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial

bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective

nationwide surveillance study. Clinical infectious diseases : an official publication of the

Infectious Diseases Society of America. 39(3), 309–317 (2004).

9 Wertheim HFL, Vos MC, Ott A et al. Risk and outcome of nosocomial Staphylococcus aureus

bacteraemia in nasal carriers versus non-carriers. Lancet (London, England). 364(9435), 703–

705 (2004).

10 Cebrian G, Sagarzazu N, Pagan R, Condon S, Manas P. Development of stress resistance in

Staphylococcus aureus after exposure to sublethal environmental conditions. International

journal of food microbiology. 140(1), 26–33 (2010).

11 Robinson DA, Kearns AM, Holmes A et al. Re-emergence of early pandemic Staphylococcus

aureus as a community-acquired meticillin-resistant clone. The Lancet. 365(9466), 1256–

1258 (2005).

12 Jevons M, Coe AW, Parker MT. Methicillin resistance in Staphylococci. The Lancet. 281(7287),

904–907.

13 Crisóstomo MI, Westh H, Tomasz A, Chung M, Oliveira DC, Lencastre H de. The evolution of

methicillin resistance in Staphylococcus aureus: Similarity of genetic backgrounds in

historically early methicillin-susceptible and -resistant isolates and contemporary epidemic

clones. Proceedings of the National Academy of Sciences of the United States of America.

98(17), 9865–9870 (2001).

Page 129: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

113

14 Hiramatsu K, Aritaka N, Hanaki H et al. Dissemination in Japanese hospitals of strains of

Staphylococcus aureus heterogeneously resistant to vancomycin. The Lancet. 350(9092),

1670–1673 (1997).

15 Smith TL, Pearson ML, Wilcox KR et al. Emergence of Vancomycin Resistance in

Staphylococcus aureus. N Engl J Med. 340(7), 493–501 (1999).

16 Guinane CM, Penades JR, Fitzgerald JR. The role of horizontal gene transfer in Staphylococcus

aureus host adaptation. Virulence. 2(3), 241–243 (2011).

17 Kluytmans J, van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus:

epidemiology, underlying mechanisms, and associated risks. Clinical Microbiology Reviews.

10(3), 505–520 (1997).

18 Weinstein HJ. The Relation between the Nasal-Staphylococcal-Carrier State and the Incidence

of Postoperative Complications. N Engl J Med. 260(26), 1303–1308 (1959).

19 Luzar MA, Coles GA, Faller B et al. Staphylococcus aureus Nasal Carriage and Infection in

Patients on Continuous Ambulatory Peritoneal Dialysis. N Engl J Med. 322(8), 505–509

(1990).

20 Yu VL, Goetz A, Wagener M et al. Staphylococcus aureus Nasal Carriage and Infection in

Patients on Hemodialysis. N Engl J Med. 315(2), 91–96 (1986).

21 von Eiff Christof, Becker Karsten, Machka Konstanze, Stammer Holger, Peters Georg. Nasal

Carriage as a Source of Staphylococcus aureus Bacteremia (2001).

22 Beck G, Habicht GS. Immunity and the invertebrates. Scientific American. 275(5), 60-3, 66

(1996).

23 Purcell AW, Gorman JJ. Immunoproteomics: Mass spectrometry-based methods to study the

targets of the immune response. Molecular & cellular proteomics : MCP. 3(3), 193–208

(2004).

24 Litman GW, Rast JP, Shamblott MJ et al. Phylogenetic diversification of immunoglobulin

genes and the antibody repertoire. Molecular biology and evolution. 10(1), 60–72 (1993).

25 Delves PJ, Im Roitt. The immune system. First of two parts. The New England journal of

medicine. 343(1), 37–49 (2000).

26 Cooper MD, Alder MN. The evolution of adaptive immune systems. Cell. 124(4), 815–822

(2006).

27 Delves PJ, Roitt IM. The Immune System. N Engl J Med. 343(1), 37–49 (2000).

28 Donnenberg AD, O'Gorman MRG. Handbook of human immunology Taylor & Francis, Boca

Raton (2008).

29 Schroeder HW, Cavacini L. Structure and Function of Immunoglobulins. The Journal of allergy

and clinical immunology. 125(2 0 2), S41-S52 (2010).

30 Maguire GA, Kumararatne DS, Joyce HJ. Are there any clinical indications for measuring IgG

subclasses? Annals of clinical biochemistry. 39(Pt 4), 374–377 (2002).

31 Litman GW, Good RA. Immunoglobulins Springer US, Boston, MA (1978).

Page 130: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

114

32 Nishijima S. S aureus in the anterior nares and subungal spaces. Journal of Int. Med. Res.

(1997).

33 Noble W.C. Carriage of SA in random samples of normal popln. Journal of Hygiene (1967).

34 Williams R.E.O. Healthy carriage of Staphylococcus aureus carriage its prevalence and imp.

Bacterial Rev. (1963).

35 Casewell MW, Hill RL. The carrier state: methicillin-resistant Staphylococcus aureus. The

Journal of antimicrobial chemotherapy. 18 Suppl A, 1–12 (1986).

36 Moss B, Squire J, Topley E, Johnston C. Nose and skin carriage of Staphylococcus aureus in

patients receiving penicillin. The Lancet, Issue 6496. 251(6496), 320–325 (1948).

37 Peacock SJ, Silva I de, Lowy FD. What determines nasal carriage of Staphylococcus aureus?

Trends in microbiology. 9(12), 605–610 (2001).

38 Kluytmans J. Nasal Carriage of Staphylococcus aureus Epidemiology, underlying mechanisms

and associated risks_Clinical Microbiology Rev. (1997).

39 Eriksen NH, Espersen F, Rosdahl VT, Jensen K. Carriage of Staphylococcus aureus among 104

healthy persons during a 19-month period. Epidemiology and infection. 115(1), 51–60 (1995).

40 Gould J.C. The carriage of Staphylococcus pyogenes var. aureus in the human nose. Journal of

Hygiene (1954).

41 Hu L. Typing of Staphylococcus Aureus Colonising Human Nasal Carriers by Pulsed-Field Gel

Electrophoresis (1995).

42 Maxwell JG, Ford CR, Peterson DE, Mitchell CR. Long-term study of nasal staphylococci

among hospital personnel. American journal of surgery. 118(6), 849–854 (1969).

43 Noble W.C. Carriage of Staphylococcus aureus in random samples of a normal population

Cambridge University Press (1967).

44 Armstrong-Esther CA. Carriage Patterns of Staphylococcus Aureus in a Healthy Non-Hospital

Population of Adults and Children (1976).

45 Hoffler U, Bulanda M, Heczko PB, Pulverer G. A comparison of staphylococcal nasal carrier

rates in Germany and Poland. Medical microbiology and immunology. 164(4), 285–290

(1978).

46 Kluytmans, J A J W, Wertheim HFL. Nasal carriage of Staphylococcus aureus and prevention

of nosocomial infections. Infection. 33(1), 3–8 (2005).

47 White A. Quantitative studies of nasal carriers of staphylococci among hospitalized patients

Journal of Clinical Inv. (1961).

48 Calia FM, Wolinsky E, Mortimer EA, JR, Abrams JS, Rammelkamp CH, JR. Importance of the

carrier state as a source of Staphylococcus aureus in wound sepsis. The Journal of hygiene.

67(1), 49–57 (1969).

49 Bruun JN. Post-operative wound infection. Predisposing factors and the effect of a reduction

in the dissemination of staphylococci. Acta medica Scandinavica. Supplementum. 514, 3–89

(1970).

Page 131: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

115

50 White A. Increased infection rates in heavy nasal carriers of coagulase-positive

staphylococci. Antimicrobial agents and chemotherapy. 161, 667–670 (1963).

51 Gould JC, McKillop EJ. The carriage of Staphylococcus pyogenes var. aureus in the human

nose. The Journal of hygiene. 52(3), 304–310 (1954).

52 van Belkum A, Riewarts Eriksen NH, Sijmons M et al. Coagulase and protein A polymorphisms

do not contribute to persistence of nasal colonisation by Staphylococcus aureus. Journal of

medical microbiology. 46(3), 222–232 (1997).

53 VandenBergh MF, Yzerman EP, van Belkum A, Boelens HA, Sijmons M, Verbrugh HA. Follow-

up of Staphylococcus aureus nasal carriage after 8 years: redefining the persistent carrier

state. Journal of clinical microbiology. 37(10), 3133–3140 (1999).

54 Noble WC, Williams RE, Jevons MP, Shooter RA. Some aspects of nasal carriage of

staphylococci. Journal of clinical pathology. 17, 79–83 (1964).

55 Nouwen J, Boelens H, van Belkum A, Verbrugh H. Human Factor in Staphylococcus aureus

Nasal Carriage. Infection and immunity. 72(11), 6685–6688 (2004).

56 Benito D, Lozano C, Jimenez E et al. Characterization of Staphylococcus aureus strains

isolated from faeces of healthy neonates and potential mother-to-infant microbial

transmission through breastfeeding. FEMS microbiology ecology. 91(3) (2015).

57 Peacock SJ, Justice A, Griffiths D et al. Determinants of acquisition and carriage of

Staphylococcus aureus in infancy. Journal of clinical microbiology. 41(12), 5718–5725 (2003).

58 Hanselman BA, Kruth SA, Rousseau J et al. Methicillin-resistant Staphylococcus aureus

colonization in veterinary personnel. Emerging infectious diseases. 12(12), 1933–1938 (2006).

59 Huijsdens XW, van Dijke BJ, Spalburg E et al. Community-acquired MRSA and pig-farming.

Annals of clinical microbiology and antimicrobials. 5, 26 (2006).

60 Weese JS, Dick H, Willey BM et al. Suspected transmission of methicillin-resistant

Staphylococcus aureus between domestic pets and humans in veterinary clinics and in the

household. Veterinary microbiology. 115(1-3), 148–155 (2006).

61 Weese JS, Lefebvre SL. Risk factors for methicillin-resistant Staphylococcus aureus

colonization in horses admitted to a veterinary teaching hospital. The Canadian veterinary

journal. La revue veterinaire canadienne. 48(9), 921–926 (2007).

62 Murthy R, Bearman G, Brown S et al. Animals in healthcare facilities: recommendations to

minimize potential risks. Infection control and hospital epidemiology. 36(5), 495–516 (2015).

63 Weidenmaier C, Kokai-Kun JF, Kristian SA et al. Role of teichoic acids in Staphylococcus

aureus nasal colonization, a major risk factor in nosocomial infections. Nature medicine.

10(3), 243–245 (2004).

64 Lowy FD. Staphylococcus aureus infections. The New England journal of medicine. 339(8),

520–532 (1998).

65 Bogaert D, van Belkum A, Sluijter M et al. Colonisation by Streptococcus pneumoniae and

Staphylococcus aureus in healthy children. Lancet (London, England). 363(9424), 1871–1872

(2004).

Page 132: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

116

66 Goslings WR, Buchli K. Nasal carrier rate of antibiotic-resistant staphylococci; influence of

hospitalization on carrier rate in patients, and their household contacts. A.M.A. archives of

internal medicine. 102(5), 691–715 (1958).

67 Williams RE. Healthy carriage of Staphylococcus aureus: its prevalence and importance.

Bacteriological reviews. 27, 56–71 (1963).

68 Cole AM, Tahk S, Oren A et al. Determinants of Staphylococcus aureus nasal carriage. Clinical

and diagnostic laboratory immunology. 8(6), 1064–1069 (2001).

69 Lipsky BA, Pecoraro RE, Chen MS, Koepsell TD. Factors affecting staphylococcal colonization

among NIDDM outpatients. Diabetes care. 10(4), 483–486 (1987).

70 Nguyen MH, Kauffman CA, Goodman RP et al. Nasal carriage of and infection with

Staphylococcus aureus in HIV-infected patients. Annals of internal medicine. 130(3), 221–225

(1999).

71 Armstrong-Esther CA. Carriage patterns of Staphylococcus aureus in a healthy non-hospital

population of adults and children. Annals of human biology. 3(3), 221–227 (1976).

72 Ellis MW, Hospenthal DR, Dooley DP, Gray PJ, Murray CK. Natural history of community-

acquired methicillin-resistant Staphylococcus aureus colonization and infection in soldiers.

Clinical infectious diseases : an official publication of the Infectious Diseases Society of

America. 39(7), 971–979 (2004).

73 Pynnonen M, Stephenson RE, Schwartz K, Hernandez M, Boles BR. Hemoglobin promotes

Staphylococcus aureus nasal colonization. PLoS pathogens. 7(7), e1002104 (2011).

74 Syed AK, Ghosh S, Love NG, Boles BR. Triclosan promotes Staphylococcus aureus nasal

colonization. mBio. 5(2), e01015 (2014).

75 Burian M, Wolz C, Goerke C. Regulatory adaptation of Staphylococcus aureus during nasal

colonization of humans. PloS one. 5(4), e10040 (2010).

76 Muthukrishnan G, Quinn GA, Lamers RP et al. Exoproteome of Staphylococcus aureus reveals

putative determinants of nasal carriage. Journal of proteome research. 10(4), 2064–2078

(2011).

77 O'Brien LM, Walsh EJ, Massey RC, Peacock SJ, Foster TJ. Staphylococcus aureus clumping

factor B (ClfB) promotes adherence to human type I cytokeratin 10: implications for nasal

colonization. Cellular microbiology. 4(11), 759–770 (2002).

78 Heilmann C. Adhesion mechanisms of staphylococci. Advances in experimental medicine and

biology. 715, 105–123 (2011).

79 McCarthy AJ, Lindsay JA. Genetic variation in Staphylococcus aureus surface and immune

evasion genes is lineage associated: implications for vaccine design and host-pathogen

interactions. BMC microbiology. 10, 173 (2010).

80 Schneewind O, Mihaylova-Petkov D, Model P. Cell wall sorting signals in surface proteins of

gram-positive bacteria. The EMBO journal. 12(12), 4803–4811 (1993).

Page 133: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

117

81 Corrigan RM, Rigby D, Handley P, Foster TJ. The role of Staphylococcus aureus surface

protein SasG in adherence and biofilm formation. Microbiology (Reading, England). 153(Pt 8),

2435–2446 (2007).

82 Mongodin E, Bajolet O, Cutrona J et al. Fibronectin-binding proteins of Staphylococcus

aureus are involved in adherence to human airway epithelium. Infection and immunity. 70(2),

620–630 (2002).

83 Schaffer AC, Solinga RM, Cocchiaro J et al. Immunization with Staphylococcus aureus

clumping factor B, a major determinant in nasal carriage, reduces nasal colonization in a

murine model. Infection and immunity. 74(4), 2145–2153 (2006).

84 Mulcahy ME, Geoghegan JA, Monk IR et al. Nasal colonisation by Staphylococcus aureus

depends upon clumping factor B binding to the squamous epithelial cell envelope protein

loricrin. PLoS pathogens. 8(12), e1003092 (2012).

85 Wertheim HFL, Walsh E, Choudhurry R et al. Key role for clumping factor B in Staphylococcus

aureus nasal colonization of humans. PLoS medicine. 5(1), e17 (2008).

86 Clarke SR, Wiltshire MD, Foster SJ. IsdA of Staphylococcus aureus is a broad spectrum, iron-

regulated adhesin. Molecular microbiology. 51(5), 1509–1519 (2004).

87 Baur S, Rautenberg M, Faulstich M et al. A nasal epithelial receptor for Staphylococcus

aureus WTA governs adhesion to epithelial cells and modulates nasal colonization. PLoS

pathogens. 10(5), e1004089 (2014).

88 O'Brien L, Kerrigan SW, Kaw G et al. Multiple mechanisms for the activation of human

platelet aggregation by Staphylococcus aureus: roles for the clumping factors ClfA and ClfB,

the serine-aspartate repeat protein SdrE and protein A. Molecular microbiology. 44(4), 1033–

1044 (2002).

89 Corrigan RM, Miajlovic H, Foster TJ. Surface proteins that promote adherence of

Staphylococcus aureus to human desquamated nasal epithelial cells. BMC microbiology. 9, 22

(2009).

90 Josefsson E, O'Connell D, Foster TJ, Durussel I, Cox JA. The binding of calcium to the B-repeat

segment of SdrD, a cell surface protein of Staphylococcus aureus. The Journal of biological

chemistry. 273(47), 31145–31152 (1998).

91 George NPE, Wei Q, Shin PK, Konstantopoulos K, Ross JM. Staphylococcus aureus adhesion

via Spa, ClfA, and SdrCDE to immobilized platelets demonstrates shear-dependent behavior.

Arteriosclerosis, thrombosis, and vascular biology. 26(10), 2394–2400 (2006).

92 Clarke SR, Foster SJ. Surface adhesins of Staphylococcus aureus. Advances in microbial

physiology. 51, 187–224 (2006).

93 Burian M, Rautenberg M, Kohler T et al. Temporal expression of adhesion factors and activity

of global regulators during establishment of Staphylococcus aureus nasal colonization. The

Journal of infectious diseases. 201(9), 1414–1421 (2010).

94 Kalinka J, Hachmeister M, Geraci J et al. Staphylococcus aureus isolates from chronic

osteomyelitis are characterized by high host cell invasion and intracellular adaptation, but

Page 134: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

118

still induce inflammation. International journal of medical microbiology : IJMM. 304(8), 1038–

1049 (2014).

95 Chavakis T, Wiechmann K, Preissner KT, Herrmann M. Staphylococcus aureus interactions

with the endothelium: the role of bacterial "secretable expanded repertoire adhesive

molecules" (SERAM) in disturbing host defense systems. Thrombosis and haemostasis. 94(2),

278–285 (2005).

96 Clarke SR, Brummell KJ, Horsburgh MJ et al. Identification of in vivo-expressed antigens of

Staphylococcus aureus and their use in vaccinations for protection against nasal carriage. The

Journal of infectious diseases. 193(8), 1098–1108 (2006).

97 de Kraker, M E A, Jarlier V, Monen JCM, Heuer OE, van de Sande N, Grundmann H. The

changing epidemiology of bacteraemias in Europe: trends from the European Antimicrobial

Resistance Surveillance System. Clinical microbiology and infection : the official publication of

the European Society of Clinical Microbiology and Infectious Diseases. 19(9), 860–868 (2013).

98 Laupland KB, Lyytikainen O, Sogaard M et al. The changing epidemiology of Staphylococcus

aureus bloodstream infection: a multinational population-based surveillance study. Clinical

microbiology and infection : the official publication of the European Society of Clinical

Microbiology and Infectious Diseases. 19(5), 465–471 (2013).

99 Kaasch AJ, Barlow G, Edgeworth JD et al. Staphylococcus aureus bloodstream infection: a

pooled analysis of five prospective, observational studies. The Journal of infection. 68(3),

242–251 (2014).

100 Calfee DP, Durbin LJ, Germanson TP, Toney DM, Smith EB, Farr BM. Spread of

methicillin-resistant Staphylococcus aureus (MRSA) among household contacts of individuals

with nosocomially acquired MRSA. Infection control and hospital epidemiology. 24(6), 422–

426 (2003).

101 Enright MC, Robinson DA, Randle G, Feil EJ, Grundmann H, Spratt BG. The evolutionary

history of methicillin-resistant Staphylococcus aureus (MRSA). Proceedings of the National

Academy of Sciences of the United States of America. 99(11), 7687–7692 (2002).

102 Herold BC, Immergluck LC, Maranan MC et al. Community-acquired methicillin-resistant

Staphylococcus aureus in children with no identified predisposing risk. JAMA. 279(8), 593–

598 (1998).

103 Popovich KJ, Weinstein RA, Hota B. Are community-associated methicillin-resistant

Staphylococcus aureus (MRSA) strains replacing traditional nosocomial MRSA strains? Clinical

infectious diseases : an official publication of the Infectious Diseases Society of America.

46(6), 787–794 (2008).

104 Skinner D, Keefer Cs. Significance of bacteremia caused by staphylococcus aureus: A

study of one hundred and twenty-two cases and a review of the literature concerned with

experimental infection in animals. Archives of internal medicine. 68(5), 851–875 (1941).

105 Klevens RM, Morrison MA, Nadle J et al. Invasive methicillin-resistant Staphylococcus

aureus infections in the United States. JAMA. 298(15), 1763–1771 (2007).

Page 135: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

119

106 Corey GR. Staphylococcus aureus Bloodstream Infections: Definitions and Treatment.

Clinical Infectious Diseases. 48(Supplement 4), S254-S259 (2009).

107 Levy MM, Fink MP, Marshall JC et al. 2001 SCCM/ESICM/ACCP/ATS/SIS International

Sepsis Definitions Conference. Critical care medicine. 31(4), 1250–1256 (2003).

108 Nolan CM, Beaty HN. Staphylococcus aureus bacteremia. Current clinical patterns. The

American journal of medicine. 60(4), 495–500 (1976).

109 Hall AE, Domanski PJ, Patel PR et al. Characterization of a protective monoclonal

antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A. Infection

and immunity. 71(12), 6864–6870 (2003).

110 DeLeo FR, Diep BA, Otto M. Host defense and pathogenesis in Staphylococcus aureus

infections. Infectious disease clinics of North America. 23(1), 17–34 (2009).

111 Foster TJ, Hook M. Surface protein adhesins of Staphylococcus aureus. Trends in

microbiology. 6(12), 484–488 (1998).

112 Mazmanian SK, Skaar EP, Gaspar AH et al. Passage of heme-iron across the envelope of

Staphylococcus aureus. Science (New York, N.Y.). 299(5608), 906–909 (2003).

113 Ellington MJ, Hope R, Livermore DM et al. Decline of EMRSA-16 amongst methicillin-

resistant Staphylococcus aureus causing bacteraemias in the UK between 2001 and 2007. The

Journal of antimicrobial chemotherapy. 65(3), 446–448 (2010).

114 Hope R, Livermore DM, Brick G, Lillie M, Reynolds R. Non-susceptibility trends among

staphylococci from bacteraemias in the UK and Ireland, 2001-06. The Journal of antimicrobial

chemotherapy. 62 Suppl 2, ii65-74 (2008).

115 Patel D, Ellington MJ, Hope R, Reynolds R, Arnold C, Desai M. Identification of genetic

variation exclusive to specific lineages associated with Staphylococcus aureus bacteraemia.

The Journal of hospital infection. 91(2), 136–145 (2015).

116 Verkaik NJ, Boelens HA, Vogel CP de et al. Heterogeneity of the humoral immune

response following Staphylococcus aureus bacteremia. European journal of clinical

microbiology & infectious diseases : official publication of the European Society of Clinical

Microbiology. 29(5), 509–518 (2010).

117 Wang R, Braughton KR, Kretschmer D et al. Identification of novel cytolytic peptides as

key virulence determinants for community-associated MRSA. Nature medicine. 13(12), 1510–

1514 (2007).

118 Kolata J, Bode LGM, Holtfreter S et al. Distinctive patterns in the human antibody

response to Staphylococcus aureus bacteremia in carriers and non-carriers. Proteomics.

11(19), 3914–3927 (2011).

119 Stentzel S, Sundaramoorthy N, Michalik S et al. Specific serum IgG at diagnosis of

Staphylococcus aureus bloodstream invasion is correlated with disease progression. Journal

of proteomics. 128, 1–7 (2015).

Page 136: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

120

120 Adhikari RP, Ajao AO, Aman MJ et al. Lower antibody levels to Staphylococcus aureus

exotoxins are associated with sepsis in hospitalized adults with invasive S. aureus infections.

The Journal of infectious diseases. 206(6), 915–923 (2012).

121 Williams RE. Healthy carriage of Staphylococcus aureus: its prevalence and importance.

Bacteriological reviews. 27, 56–71 (1963).

122 Ridley M. Perineal carriage of Staph. aureus. British medical journal. 1(5117), 270–273

(1959).

123 Guinan ME, Dan BB, Guidotti RJ et al. Vaginal colonization with Staphylococcus aureus in

healthy women: a review of four studies. Annals of internal medicine. 96(6 Pt 2), 944–947

(1982).

124 Cole AM, Dewan P, Ganz T. Innate antimicrobial activity of nasal secretions. Infection

and immunity. 67(7), 3267–3275 (1999).

125 Garzoni C, Kelley WL. Staphylococcus aureus: new evidence for intracellular persistence.

Trends in microbiology. 17(2), 59–65 (2009).

126 Ventura CL, Higdon R, Hohmann L et al. Staphylococcus aureus Elicits Marked

Alterations in the Airway Proteome during Early Pneumonia. Infection and immunity. 76(12),

5862–5872 (2008).

127 Surmann K, Michalik S, Hildebrandt P et al. Comparative proteome analysis reveals

conserved and specific adaptation patterns of Staphylococcus aureus after internalization by

different types of human non-professional phagocytic host cells. Frontiers in microbiology. 5,

392 (2014).

128 Surmann K, Simon M, Hildebrandt P et al. A proteomic perspective of the interplay of

Staphylococcus aureus and human alveolar epithelial cells during infection. Journal of

proteomics. 128, 203–217 (2015).

129 Hauck CR, Agerer F, Muenzner P, Schmitter T. Cellular adhesion molecules as targets for

bacterial infection. European journal of cell biology. 85(3-4), 235–242 (2006).

130 Weese JS. Methicillin-resistant Staphylococcus aureus in animals. ILAR journal / National

Research Council, Institute of Laboratory Animal Resources. 51(3), 233–244 (2010).

131 Sung JM, Lloyd DH, Lindsay JA. Staphylococcus aureus host specificity: comparative

genomics of human versus animal isolates by multi-strain microarray. Microbiology (Reading,

England). 154(Pt 7), 1949–1959 (2008).

132 Maresso AW, Schneewind O. Iron acquisition and transport in Staphylococcus aureus.

Biometals : an international journal on the role of metal ions in biology, biochemistry, and

medicine. 19(2), 193–203 (2006).

133 Horsburgh MJ, Wharton SJ, Cox AG, Ingham E, Peacock S, Foster SJ. MntR modulates

expression of the PerR regulon and superoxide resistance in Staphylococcus aureus through

control of manganese uptake. Molecular microbiology. 44(5), 1269–1286 (2002).

Page 137: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

121

134 Salazar N, Castiblanco-Valencia MM, da Silva LB et al. Staphylococcus aureus manganese

transport protein C (MntC) is an extracellular matrix- and plasminogen-binding protein. PloS

one. 9(11), e112730 (2014).

135 Drechsel H, Freund S, Nicholson G et al. Purification and chemical characterization of

staphyloferrin B, a hydrophilic siderophore from staphylococci. Biometals : an international

journal on the role of metal ions in biology, biochemistry, and medicine. 6(3), 185–192 (1993).

136 Courcol RJ, Trivier D, Bissinger MC, Martin GR, Brown MR. Siderophore production by

Staphylococcus aureus and identification of iron-regulated proteins. Infection and immunity.

65(5), 1944–1948 (1997).

137 Bera A, Biswas R, Herbert S et al. Influence of wall teichoic acid on lysozyme resistance

in Staphylococcus aureus. Journal of bacteriology. 189(1), 280–283 (2007).

138 Walsh EJ, O'Brien LM, Liang X, Hook M, Foster TJ. Clumping factor B, a fibrinogen-

binding MSCRAMM (microbial surface components recognizing adhesive matrix molecules)

adhesin of Staphylococcus aureus, also binds to the tail region of type I cytokeratin 10. The

Journal of biological chemistry. 279(49), 50691–50699 (2004).

139 Barbu EM, Ganesh VK, Gurusiddappa S et al. β-Neurexin Is a Ligand for the

Staphylococcus aureus MSCRAMM SdrC. PLoS pathogens. 6(1), e1000726 (2009).

140 Moks T, Abrahmsen L, Nilsson B, Hellman U, Sjoquist J, Uhlen M. Staphylococcal protein

A consists of five IgG-binding domains. European journal of biochemistry / FEBS. 156(3), 637–

643 (1986).

141 Graille M, Stura EA, Corper AL et al. Crystal structure of a Staphylococcus aureus protein

A domain complexed with the Fab fragment of a human IgM antibody: structural basis for

recognition of B-cell receptors and superantigen activity. Proceedings of the National

Academy of Sciences of the United States of America. 97(10), 5399–5404 (2000).

142 Nordenfelt P, Waldemarson S, Linder A et al. Antibody orientation at bacterial surfaces

is related to invasive infection. The Journal of experimental medicine. 209(13), 2367–2381

(2012).

143 Jin T, Bokarewa M, Foster T, Mitchell J, Higgins J, Tarkowski A. Staphylococcus aureus

resists human defensins by production of staphylokinase, a novel bacterial evasion

mechanism. Journal of immunology (Baltimore, Md. : 1950). 172(2), 1169–1176 (2004).

144 Rooijakkers SHM, van Wamel, W J B, Ruyken M, van Kessel, K P M, van Strijp, J A G. Anti-

opsonic properties of staphylokinase. Microbes and infection / Institut Pasteur. 7(3), 476–484

(2005).

145 van Wamel, Willem J B, Rooijakkers SHM, Ruyken M, van Kessel, Kok P M, van Strijp, Jos

A G. The innate immune modulators staphylococcal complement inhibitor and chemotaxis

inhibitory protein of Staphylococcus aureus are located on beta-hemolysin-converting

bacteriophages. Journal of bacteriology. 188(4), 1310–1315 (2006).

146 Wagner PL, Waldor MK. Bacteriophage control of bacterial virulence. Infection and

immunity. 70(8), 3985–3993 (2002).

Page 138: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

122

147 Postma B, Poppelier MJ, van Galen JC et al. Chemotaxis inhibitory protein of

Staphylococcus aureus binds specifically to the C5a and formylated peptide receptor. Journal

of immunology (Baltimore, Md. : 1950). 172(11), 6994–7001 (2004).

148 Askarian F, Ajayi C, Hanssen A et al. The interaction between Staphylococcus aureus

SdrD and desmoglein 1 is important for adhesion to host cells. Scientific reports. 6, 22134

(2016).

149 Bestebroer J, Aerts PC, Rooijakkers SHM et al. Functional basis for complement evasion

by staphylococcal superantigen-like 7. Cellular microbiology. 12(10), 1506–1516 (2010).

150 Langley R, Wines B, Willoughby N, Basu I, Proft T, Fraser JD. The staphylococcal

superantigen-like protein 7 binds IgA and complement C5 and inhibits IgA-Fc alpha RI binding

and serum killing of bacteria. Journal of immunology (Baltimore, Md. : 1950). 174(5), 2926–

2933 (2005).

151 McGuinness WA, Kobayashi SD, DeLeo FR. Evasion of Neutrophil Killing by

Staphylococcus aureus. Pathogens (Basel, Switzerland). 5(1) (2016).

152 Laarman AJ, Mijnheer G, Mootz JM et al. Staphylococcus aureus Staphopain A inhibits

CXCR2-dependent neutrophil activation and chemotaxis. The EMBO journal. 31(17), 3607–

3619 (2012).

153 Kaneko J, Kamio Y. Bacterial two-component and hetero-heptameric pore-forming

cytolytic toxins: structures, pore-forming mechanism, and organization of the genes.

Bioscience, biotechnology, and biochemistry. 68(5), 981–1003 (2004).

154 Menestrina G, Dalla Serra M, Comai M et al. Ion channels and bacterial infection: the

case of beta-barrel pore-forming protein toxins of Staphylococcus aureus. FEBS letters.

552(1), 54–60 (2003).

155 Kretschmer D, Gleske A, Rautenberg M et al. Human formyl peptide receptor 2 senses

highly pathogenic Staphylococcus aureus. Cell host & microbe. 7(6), 463–473 (2010).

156 Hongo I, Baba T, Oishi K, Morimoto Y, Ito T, Hiramatsu K. Phenol-soluble modulin alpha

3 enhances the human neutrophil lysis mediated by Panton-Valentine leukocidin. The Journal

of infectious diseases. 200(5), 715–723 (2009).

157 Surewaard BGJ, de Haas, C J C, Vervoort F et al. Staphylococcal alpha-phenol soluble

modulins contribute to neutrophil lysis after phagocytosis. Cellular microbiology. 15(8),

1427–1437 (2013).

158 Surewaard BGJ, Nijland R, Spaan AN, Kruijtzer JAW, de Haas, Carla J C, van Strijp, Jos A

G. Inactivation of staphylococcal phenol soluble modulins by serum lipoprotein particles.

PLoS pathogens. 8(3), e1002606 (2012).

159 Ventura CL, Malachowa N, Hammer CH et al. Identification of a novel Staphylococcus

aureus two-component leukotoxin using cell surface proteomics. PloS one. 5(7), e11634

(2010).

160 Yamashita K, Kawai Y, Tanaka Y et al. Crystal structure of the octameric pore of

staphylococcal gamma-hemolysin reveals the beta-barrel pore formation mechanism by two

Page 139: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

123

components. Proceedings of the National Academy of Sciences of the United States of

America. 108(42), 17314–17319 (2011).

161 Labandeira-Rey M, Couzon F, Boisset S et al. Staphylococcus aureus Panton-Valentine

leukocidin causes necrotizing pneumonia. Science (New York, N.Y.). 315(5815), 1130–1133

(2007).

162 Kobayashi SD, Malachowa N, Whitney AR et al. Comparative analysis of USA300

virulence determinants in a rabbit model of skin and soft tissue infection. The Journal of

infectious diseases. 204(6), 937–941 (2011).

163 Laxminarayan R, Duse A, Wattal C et al. Antibiotic resistance-the need for global

solutions. The Lancet. Infectious diseases. 13(12), 1057–1098 (2013).

164 Lucero CA, Hageman J, Zell ER et al. Evaluating the potential public health impact of a

Staphylococcus aureus vaccine through use of population-based surveillance for invasive

methicillin-resistant S. aureus disease in the United States. Vaccine. 27(37), 5061–5068

(2009).

165 Daum RS, Spellberg B. Progress toward a Staphylococcus aureus vaccine. Clinical

infectious diseases : an official publication of the Infectious Diseases Society of America.

54(4), 560–567 (2012).

166 Proctor RA. Challenges for a universal Staphylococcus aureus vaccine. Clinical infectious

diseases : an official publication of the Infectious Diseases Society of America. 54(8), 1179–

1186 (2012).

167 Fowler VG, Proctor RA. Where does a Staphylococcus aureus vaccine stand? Clinical

microbiology and infection : the official publication of the European Society of Clinical

Microbiology and Infectious Diseases. 20 Suppl 5, 66–75 (2014).

168 Parish H.J, Cannon D.A. Staphylococcal infection: Antitoxic immunity. British Medical

Journal (1960).

169 Nanra JS, Buitrago SM, Crawford S et al. Capsular polysaccharides are an important

immune evasion mechanism for Staphylococcus aureus. Human vaccines &

immunotherapeutics. 9(3), 480–487 (2013).

170 Li X, Wang X, Thompson CD, Park S, Park WB, Lee JC. Preclinical Efficacy of Clumping

Factor A in Prevention of Staphylococcus aureus Infection. mBio. 7(1) (2016).

171 Rozemeijer W, Fink P, Rojas E et al. Evaluation of approaches to monitor Staphylococcus

aureus virulence factor expression during human disease. PloS one. 10(2), e0116945 (2015).

172 van den Berg S, Koedijk, Dennis G A M, Back JW et al. Active immunization with an octa-

valent Staphylococcus aureus antigen mixture in models of S. aureus bacteremia and skin

infection in mice. PloS one. 10(2), e0116847 (2015).

173 Jansen KU, Girgenti DQ, Scully IL, Anderson AS. Vaccine review: "Staphyloccocus aureus

vaccines: problems and prospects". Vaccine. 31(25), 2723–2730 (2013).

Page 140: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

124

174 Stranger-Jones YK, Bae T, Schneewind O. Vaccine assembly from surface proteins of

Staphylococcus aureus. Proceedings of the National Academy of Sciences of the United States

of America. 103(45), 16942–16947 (2006).

175 Creech CB, Johnson BG, Alsentzer AR, Hohenboken M, Edwards KM, Talbot TR.

Vaccination as infection control: a pilot study to determine the impact of Staphylococcus

aureus vaccination on nasal carriage. Vaccine. 28(1), 256–260 (2009).

176 Shinefield H, Black S, Fattom A et al. Use of a Staphylococcus aureus conjugate vaccine

in patients receiving hemodialysis. The New England journal of medicine. 346(7), 491–496

(2002).

177 Fattom A, Matalon A, Buerkert J, Taylor K, Damaso S, Boutriau D. Efficacy profile of a

bivalent Staphylococcus aureus glycoconjugated vaccine in adults on hemodialysis: Phase III

randomized study. Human vaccines & immunotherapeutics. 11(3), 632–641 (2015).

178 Fowler VG, Allen KB, Moreira ED et al. Effect of an investigational vaccine for preventing

Staphylococcus aureus infections after cardiothoracic surgery: a randomized trial. JAMA.

309(13), 1368–1378 (2013).

179 Yeaman MR, Filler SG, Schmidt CS, Ibrahim AS, Edwards JE, Hennessey JP. Applying

Convergent Immunity to Innovative Vaccines Targeting Staphylococcus aureus. Frontiers in

immunology. 5, 463 (2014).

180 Proctor RA. Is there a future for a Staphylococcus aureus vaccine? Vaccine. 30(19),

2921–2927 (2012).

181 Levy J, Licini L, Haelterman E et al. Safety and immunogenicity of an investigational 4-

component Staphylococcus aureus vaccine with or without AS03B adjuvant: Results of a

randomized phase I trial. Human vaccines & immunotherapeutics. 11(3), 620–631 (2015).

182 Weisman LE, Thackray HM, Steinhorn RH et al. A randomized study of a monoclonal

antibody (pagibaximab) to prevent staphylococcal sepsis. Pediatrics. 128(2), 271–279 (2011).

183 Weems JJ, Steinberg JP, Filler S et al. Phase II, randomized, double-blind, multicenter

study comparing the safety and pharmacokinetics of tefibazumab to placebo for treatment of

Staphylococcus aureus bacteremia. Antimicrobial agents and chemotherapy. 50(8), 2751–

2755 (2006).

184 Benjamin DK, Schelonka R, White R et al. A blinded, randomized, multicenter study of an

intravenous Staphylococcus aureus immune globulin. Journal of perinatology : official journal

of the California Perinatal Association. 26(5), 290–295 (2006).

185 Rupp ME, Holley HP, Lutz J et al. Phase II, randomized, multicenter, double-blind,

placebo-controlled trial of a polyclonal anti-Staphylococcus aureus capsular polysaccharide

immune globulin in treatment of Staphylococcus aureus bacteremia. Antimicrobial agents

and chemotherapy. 51(12), 4249–4254 (2007).

186 García-Lara J, Foster SJ. Anti-Staphylococcus aureus immunotherapy: current status and

prospects. Anti-infectives/New technologies. 9(5), 552–557 (2009).

Page 141: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

125

187 Garmory HS, Titball RW. ATP-binding cassette transporters are targets for the

development of antibacterial vaccines and therapies. Infection and immunity. 72(12), 6757–

6763 (2004).

188 Burnie JP, Matthews RC, Carter T et al. Identification of an immunodominant ABC

transporter in methicillin-resistant Staphylococcus aureus infections. Infection and immunity.

68(6), 3200–3209 (2000).

189 DeJonge M, Burchfield D, Bloom B et al. Clinical trial of safety and efficacy of INH-A21

for the prevention of nosocomial staphylococcal bloodstream infection in premature infants.

The Journal of pediatrics. 151(3), 260-5, 265.e1 (2007).

190 Bloom BT. INH-A21: a donor-selected Staphylococcal human immune globulin for the

prevention of late-onset neonatal Staphylococcal infection. Expert opinion on investigational

drugs. 15(6), 703–707 (2006).

191 Jungblut PR. Proteome analysis of bacterial pathogens. Microbes and infection / Institut

Pasteur. 3(10), 831–840 (2001).

192 Yetisen AK, Akram MS, Lowe CR. Paper-based microfluidic point-of-care diagnostic

devices. Lab Chip. 13(12), 2210–2251 (2013).

193 O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. The Journal of

biological chemistry. 250(10), 4007–4021 (1975).

194 Smithies O, Poulik MD. Two-dimensional electrophoresis of serum proteins. Nature.

177(4518), 1033 (1956).

195 Klade CS, Voss T, Krystek E et al. Identification of tumor antigens in renal cell carcinoma

by serological proteome analysis. Proteomics. 1(7), 890–898 (2001).

196 Laemmli UK. Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature. 227(5259), 680–685 (1970).

197 Vignali DA. Multiplexed particle-based flow cytometric assays. Journal of Immunological

Methods. 243(1-2), 243–255 (2000).

198 Dennehy R, McClean S. Immunoproteomics: the key to discovery of new vaccine

antigens against bacterial respiratory infections. Current protein & peptide science. 13(8),

807–815 (2012).

199 Vytvytska O, Nagy E, Bluggel M et al. Identification of vaccine candidate antigens of

Staphylococcus aureus by serological proteome analysis. Proteomics. 2(5), 580–590 (2002).

200 Holtfreter S, Nguyen TTH, Wertheim H et al. Human immune proteome in experimental

colonization with Staphylococcus aureus. Clinical and vaccine immunology : CVI. 16(11),

1607–1614 (2009).

201 Kloppot P, Selle M, Kohler C et al. Microarray-based identification of human antibodies

against Staphylococcus aureus antigens. Proteomics. Clinical applications. 9(11-12), 1003–

1011 (2015).

Page 142: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

126

202 Holtfreter S, Kolata J, Broker BM. Towards the immune proteome of Staphylococcus

aureus - The anti-S. aureus antibody response. International journal of medical microbiology :

IJMM. 300(2-3), 176–192 (2010).

203 Verkaik NJ, Vogel CP de, Boelens HA et al. Anti-staphylococcal humoral immune

response in persistent nasal carriers and noncarriers of Staphylococcus aureus. The Journal of

infectious diseases. 199(5), 625–632 (2009).

204 Fulton RJ, McDade RL, Smith PL, Kienker LJ, Kettman JR, JR. Advanced multiplexed

analysis with the FlowMetrix system. Clinical chemistry. 43(9), 1749–1756 (1997).

205 Holtfreter S, Grumann D, Schmudde M et al. Clonal distribution of superantigen genes in

clinical Staphylococcus aureus isolates. Journal of clinical microbiology. 45(8), 2669–2680

(2007).

206 Blomfeldt A, Eskesen AN, Aamot HV, Leegaard TM, Bjornholt JV. Population-based

epidemiology of Staphylococcus aureus bloodstream infection: clonal complex 30 genotype is

associated with mortality. European journal of clinical microbiology & infectious diseases :

official publication of the European Society of Clinical Microbiology (2016).

207 Stentzel S, Sundaramoorthy N, Michalik S et al. Specific serum IgG at diagnosis of

Staphylococcus aureus bloodstream invasion is correlated with disease progression. Journal

of proteomics. 128, 1–7 (2015).

208 Dunbar SA, Hoffmeyer MR. Microsphere-Based Multiplex Immunoassays. In: The

Immunoassay Handbook, Elsevier, 157–174 (2013).

209 Reverberi R, Reverberi L. Factors affecting the antigen-antibody reaction. Blood

transfusion. 5(4), 227–240 (2007).

210 Mann HB, Whitney DR. On a Test of Whether one of Two Random Variables is

Stochastically Larger than the Other. Ann. Math. Statist., 50–60 (1947).

211 Holtfreter S, Grumann D, Schmudde M et al. Clonal Distribution of Superantigen Genes

in Clinical Staphylococcus aureus Isolates.

212 Yu NY, Wagner JR, Laird MR et al. PSORTb 3.0: improved protein subcellular localization

prediction with refined localization subcategories and predictive capabilities for all

prokaryotes. Bioinformatics (Oxford, England). 26(13), 1608–1615 (2010).

213 Corriere MD, Decker CF. MRSA: an evolving pathogen. Disease-a-month : DM. 54(12),

751–755 (2008).

214 Laal S, Samanich KM, Sonnenberg MG, Zolla-Pazner S, Phadtare JM, Belisle JT. Human

humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-

molecular-mass antigens. Clinical and diagnostic laboratory immunology. 4(1), 49–56 (1997).

215 Davies DH, Liang X, Hernandez JE et al. Profiling the humoral immune response to

infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen

discovery. Proceedings of the National Academy of Sciences of the United States of America.

102(3), 547–552 (2005).

Page 143: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

127

216 Kunnath-Velayudhan S, Salamon H, Wang H et al. Dynamic antibody responses to the

Mycobacterium tuberculosis proteome. Proceedings of the National Academy of Sciences of

the United States of America. 107(33), 14703–14708 (2010).

217 Hansenová Maňásková S, van Belkum A, Endtz HP, Bikker FJ, Veerman EC, van Wamel

WJ. Comparison of non-magnetic and magnetic beads in bead-based assays. Journal of

Immunological Methods.

218 Holtfreter S, Nguyen TTH, Wertheim H et al. Human immune proteome in experimental

colonization with Staphylococcus aureus. Clinical and vaccine immunology : CVI. 16(11),

1607–1614 (2009).

219 Kolata J, Bode LGM, Holtfreter S et al. Distinctive patterns in the human antibody

response to Staphylococcus aureus bacteremia in carriers and non-carriers. Proteomics.

11(19), 3914–3927 (2011).

220 Kloppot P, Selle M, Kohler C et al. Microarray-based identification of human antibodies

against Staphylococcus aureus antigens. Proteomics. Clinical applications. 9(11-12), 1003–

1011 (2015).

221 Alegre M, Chen L, David MZ et al. Impact of Staphylococcus aureus USA300 Colonization

and Skin Infections on Systemic Immune Responses in Humans. Journal of immunology

(Baltimore, Md. : 1950) (2016).

222 Mulcahy ME, Geoghegan JA, Monk IR et al. Nasal colonisation by Staphylococcus aureus

depends upon clumping factor B binding to the squamous epithelial cell envelope protein

loricrin. PLoS pathogens. 8(12), e1003092 (2012).

223 Dryla A, Prustomersky S, Gelbmann D et al. Comparison of Antibody Repertoires against

Staphylococcus aureus in Healthy Individuals and in Acutely Infected Patients. Clinical and

diagnostic laboratory immunology. 12(3), 387–398 (2005).

224 Clarke SR, Brummell KJ, Horsburgh MJ et al. Identification of In Vivo–Expressed Antigens

of Staphylococcus aureus and Their Use in Vaccinations for Protection against Nasal Carriage.

Journal of Infectious Diseases. 193(8), 1098–1108 (2006).

225 Verkaik NJ, Lebon A, Vogel CP de et al. Induction of antibodies by Staphylococcus aureus

nasal colonization in young children. Clinical microbiology and infection : the official

publication of the European Society of Clinical Microbiology and Infectious Diseases. 16(8),

1312–1317 (2010).

226 Wertheim HFL, Walsh E, Choudhurry R et al. Key role for clumping factor B in

Staphylococcus aureus nasal colonization of humans. PLoS medicine. 5(1), e17 (2008).

227 Balaban N, Rasooly A. Staphylococcal enterotoxins. International journal of food

microbiology. 61(1), 1–10 (2000).

228 Holtfreter S, Roschack K, Eichler P et al. Staphylococcus aureus Carriers Neutralize

Superantigens by Antibodies Specific for Their Colonizing Strain: A Potential Explanation for

Their Improved Prognosis in Severe Sepsis. Journal of Infectious Diseases. 193(9), 1275–1278

(2006).

Page 144: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

128

229 van den Berg S, Vogel CP de, van Belkum A, Bakker-Woudenberg, Irma A. J. M. Mild

Staphylococcus aureus Skin Infection Improves the Course of Subsequent Endogenous

Bacteremia in Mice. PLoS ONE. 10(6), e0129150 (2015).

230 Ritz HL, Kirkland JJ, Bond GG, Warner EK, Petty GP. Association of high levels of serum

antibody to staphylococcal toxic shock antigen with nasal carriage of toxic shock antigen-

producing strains of Staphylococcus aureus. Infection and immunity. 43(3), 954–958 (1984).

231 Verkaik NJ, Vogel CP de, Boelens HA et al. Anti-staphylococcal humoral immune

response in persistent nasal carriers and noncarriers of Staphylococcus aureus. The Journal of

infectious diseases. 199(5), 625–632 (2009).

232 Stolz SJ, Davis JP, Vergeront JM et al. Development of serum antibody to toxic shock

toxin among individuals with toxic shock syndrome in Wisconsin. The Journal of infectious

diseases. 151(5), 883–889 (1985).

233 Blomfeldt A, Eskesen AN, Aamot HV, Leegaard TM, Bjornholt JV. Population-based

epidemiology of Staphylococcus aureus bloodstream infection: clonal complex 30 genotype is

associated with mortality. European journal of clinical microbiology & infectious diseases :

official publication of the European Society of Clinical Microbiology. 35(5), 803–813 (2016).

234 Feil EJ, Cooper JE, Grundmann H et al. How Clonal Is Staphylococcus aureus? Journal of

bacteriology. 185(11), 3307–3316 (2003).

235 Melles DC, Gorkink RFJ, Boelens HAM et al. Natural population dynamics and expansion

of pathogenic clones of Staphylococcus aureus. The Journal of clinical investigation. 114(12),

1732–1740 (2004).

236 Holtfreter S, Grumann D, Schmudde M et al. Clonal Distribution of Superantigen Genes

in Clinical Staphylococcus aureus Isolates.

237 Nienaber JJC, Sharma Kuinkel BK, Clarke-Pearson M et al. Methicillin-susceptible

Staphylococcus aureus endocarditis isolates are associated with clonal complex 30 genotype

and a distinct repertoire of enterotoxins and adhesins. The Journal of infectious diseases.

204(5), 704–713 (2011).

238 Xiong YQ, Fowler VG, Yeaman MR, Perdreau-Remington F, Kreiswirth BN, Bayer AS.

Phenotypic and genotypic characteristics of persistent methicillin-resistant Staphylococcus

aureus bacteremia in vitro and in an experimental endocarditis model. The Journal of

infectious diseases. 199(2), 201–208 (2009).

239 Fowler VG, JR, Nelson CL, McIntyre LM et al. Potential associations between

hematogenous complications and bacterial genotype in Staphylococcus aureus infection. The

Journal of infectious diseases. 196(5), 738–747 (2007).

240 Gill SR, McIntyre LM, Nelson CL et al. Potential associations between severity of

infection and the presence of virulence-associated genes in clinical strains of Staphylococcus

aureus. PloS one. 6(4), e18673 (2011).

Page 145: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

129

241 Spaulding AR, Satterwhite EA, Lin Y et al. Comparison of Staphylococcus aureus strains

for ability to cause infective endocarditis and lethal sepsis in rabbits. Frontiers in cellular and

infection microbiology. 2, 18 (2012).

242 Wertheim HFL, Vos MC, Ott A et al. Risk and outcome of nosocomial Staphylococcus

aureus bacteraemia in nasal carriers versus non-carriers. Lancet (London, England).

364(9435), 703–705 (2004).

243 Delfani S, Mohabati Mobarez A, Imani Fooladi AA, Amani J, Emaneini M. Protection of

mice against Staphylococcus aureus infection by a recombinant protein ClfA-IsdB-Hlg as a

vaccine candidate. Medical microbiology and immunology. 205(1), 47–55 (2016).

244 Verkaik NJ, Boelens HA, Vogel CP de et al. Heterogeneity of the humoral immune

response following Staphylococcus aureus bacteremia. European journal of clinical

microbiology & infectious diseases : official publication of the European Society of Clinical

Microbiology. 29(5), 509–518 (2010).

245 Kolata J, Bode LGM, Holtfreter S et al. Distinctive patterns in the human antibody

response to Staphylococcus aureus bacteremia in carriers and non-carriers. Proteomics.

11(19), 3914–3927 (2011).

246 Dryla A, Prustomersky S, Gelbmann D et al. Comparison of Antibody Repertoires against

Staphylococcus aureus in Healthy Individuals and in Acutely Infected Patients. Clinical and

diagnostic laboratory immunology. 12(3), 387–398 (2005).

247 Jacobsson G, Colque-Navarro P, Gustafsson E, Andersson R, Mollby R. Antibody

responses in patients with invasive Staphylococcus aureus infections. European journal of

clinical microbiology & infectious diseases : official publication of the European Society of

Clinical Microbiology. 29(6), 715–725 (2010).

248 Holtfreter S, Nguyen TTH, Wertheim H et al. Human immune proteome in experimental

colonization with Staphylococcus aureus. Clinical and vaccine immunology : CVI. 16(11),

1607–1614 (2009).

249 Lindsay JA, Holden MTG. Staphylococcus aureus: superbug, super genome? Trends in

microbiology. 12(8), 378–385 (2004).

250 Smagur J, Guzik K, Bzowska M et al. Staphylococcal cysteine protease staphopain B

(SspB) induces rapid engulfment of human neutrophils and monocytes by macrophages.

Biological chemistry. 390(4), 361–371 (2009).

251 Smagur J, Guzik K, Magiera L et al. A new pathway of staphylococcal pathogenesis:

apoptosis-like death induced by Staphopain B in human neutrophils and monocytes. Journal

of innate immunity. 1(2), 98–108 (2009).

252 Imamura T, Tanase S, Szmyd G, Kozik A, Travis J, Potempa J. Induction of vascular

leakage through release of bradykinin and a novel kinin by cysteine proteinases from

Staphylococcus aureus. The Journal of experimental medicine. 201(10), 1669–1676 (2005).

Page 146: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

130

253 Ohbayashi T, Irie A, Murakami Y et al. Degradation of fibrinogen and collagen by

staphopains, cysteine proteases released from Staphylococcus aureus. Microbiology

(Reading, England). 157(Pt 3), 786–792 (2011).

254 White MJ, Boyd JM, Horswill AR, Nauseef WM. Phosphatidylinositol-Specific

Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood

and Neutrophils. Infection and immunity. 82(4), 1559–1571 (2014).

255 Katayama Y, Baba T, Sekine M, Fukuda M, Hiramatsu K. Beta-hemolysin promotes skin

colonization by Staphylococcus aureus. Journal of bacteriology. 195(6), 1194–1203 (2013).

256 Makris G, Wright JD, Ingham E, Holland KT. The hyaluronate lyase of Staphylococcus

aureus - a virulence factor? Microbiology (Reading, England). 150(Pt 6), 2005–2013 (2004).

257 Buhren BA, Schrumpf H, Hoff N, Bolke E, Hilton S, Gerber PA. Hyaluronidase: from

clinical applications to molecular and cellular mechanisms. European journal of medical

research. 21, 5 (2016).

258 Ibberson CB, Jones CL, Singh S et al. Staphylococcus aureus hyaluronidase is a CodY-

regulated virulence factor. Infection and immunity. 82(10), 4253–4264 (2014).

259 den Reijer PM, Lemmens-den Toom N, Kant S et al. Characterization of the Humoral

Immune Response during Staphylococcus aureus Bacteremia and Global Gene Expression by

Staphylococcus aureus in Human Blood. PLoS ONE. 8(1), e53391 (2013).

260 Bardoel BW, Vos R, Bouman T et al. Evasion of Toll-like receptor 2 activation by

staphylococcal superantigen-like protein 3. Journal of molecular medicine (Berlin, Germany).

90(10), 1109–1120 (2012).

261 Chung MC, Wines BD, Baker H, Langley RJ, Baker EN, Fraser JD. The crystal structure of

staphylococcal superantigen-like protein 11 in complex with sialyl Lewis X reveals the

mechanism for cell binding and immune inhibition. Molecular microbiology. 66(6), 1342–

1355 (2007).

262 Bestebroer J, Poppelier, Miriam J J G, Ulfman LH et al. Staphylococcal superantigen-like

5 binds PSGL-1 and inhibits P-selectin-mediated neutrophil rolling. Blood. 109(7), 2936–2943

(2007).

263 Koymans KJ, Bisschop A, Vughs MM, van Kessel, Kok P M, de Haas, Carla J C, van Strijp,

Jos A G. Staphylococcal Superantigen-Like Protein 1 and 5 (SSL1 & SSL5) Limit Neutrophil

Chemotaxis and Migration through MMP-Inhibition. International journal of molecular

sciences. 17(7) (2016).

264 Itoh S, Hamada E, Kamoshida G et al. Staphylococcal superantigen-like protein 10

(SSL10) binds to human immunoglobulin G (IgG) and inhibits complement activation via the

classical pathway. Molecular immunology. 47(4), 932–938 (2010).

265 Jongerius I, Kohl J, Pandey MK et al. Staphylococcal complement evasion by various

convertase-blocking molecules. The Journal of experimental medicine. 204(10), 2461–2471

(2007).

Page 147: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

131

266 Burlak C, Hammer CH, Robinson M et al. Global analysis of community-associated

methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro

and during infection. Cellular microbiology. 9(5), 1172–1190 (2007).

267 Sieprawska-Lupa M, Mydel P, Krawczyk K et al. Degradation of human antimicrobial

peptide LL-37 by Staphylococcus aureus-derived proteinases. Antimicrobial agents and

chemotherapy. 48(12), 4673–4679 (2004).

268 Laarman AJ, Ruyken M, Malone CL, van Strijp, Jos A G, Horswill AR, Rooijakkers SHM.

Staphylococcus aureus metalloprotease aureolysin cleaves complement C3 to mediate

immune evasion. Journal of immunology (Baltimore, Md. : 1950). 186(11), 6445–6453 (2011).

269 Holtfreter S, Roschack K, Eichler P et al. Staphylococcus aureus Carriers Neutralize

Superantigens by Antibodies Specific for Their Colonizing Strain: A Potential Explanation for

Their Improved Prognosis in Severe Sepsis. Journal of Infectious Diseases. 193(9), 1275–1278

(2006).

270 Krakauer T. Immune response to staphylococcal superantigens. Immunologic research.

20(2), 163–173 (1999).

271 LeClaire RD, Hunt RE, Bavari S. Protection against Bacterial Superantigen Staphylococcal

Enterotoxin B by Passive Vaccination. Infection and immunity. 70(5), 2278–2281 (2002).

272 Notermans S, van Leeuwen WJ, Dufrenne J, Tips PD. Serum antibodies to enterotoxins

produced by Staphylococcus aureus with special reference to enterotoxin F and toxic shock

syndrome. Journal of clinical microbiology. 18(5), 1055–1060 (1983).

273 Józefczyk Z. Specific Human Antibodies to Enterotoxins A, B, and C₁ of Staphylococcus:

Their Increased Synthesis in Staphylococcal Infection. The Journal of infectious diseases.

130(1), 1–7 (1974).

274 Jozefczyk Z, Robbins RN, Spitz JM, Bergdoll MS. Antibodies to staphylococcal enterotoxin

in laboratory personnel. Journal of clinical microbiology. 11(4), 438–439 (1980).

275 Holtfreter S, Bauer K, Thomas D et al. egc-Encoded superantigens from Staphylococcus

aureus are neutralized by human sera much less efficiently than are classical staphylococcal

enterotoxins or toxic shock syndrome toxin. Infection and immunity. 72(7), 4061–4071

(2004).

276 Kaul R, McGeer A, Norrby-Teglund A et al. Intravenous immunoglobulin therapy for

streptococcal toxic shock syndrome--a comparative observational study. The Canadian

Streptococcal Study Group. Clinical infectious diseases : an official publication of the

Infectious Diseases Society of America. 28(4), 800–807 (1999).

277 Darenberg J, Ihendyane N, Sjolin J et al. Intravenous immunoglobulin G therapy in

streptococcal toxic shock syndrome: a European randomized, double-blind, placebo-

controlled trial. Clinical infectious diseases : an official publication of the Infectious Diseases

Society of America. 37(3), 333–340 (2003).

278 George EA, Muir TW. Molecular mechanisms of agr quorum sensing in virulent

staphylococci. Chembiochem : a European journal of chemical biology. 8(8), 847–855 (2007).

Page 148: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

132

279 Kleerebezem M, Quadri LE, Kuipers OP, Vos WM de. Quorum sensing by peptide

pheromones and two-component signal-transduction systems in Gram-positive bacteria.

Molecular microbiology. 24(5), 895–904 (1997).

280 Montgomery CP, Boyle-Vavra S, Daum RS. Importance of the Global Regulators Agr and

SaeRS in the Pathogenesis of CA-MRSA USA300 Infection. PLoS ONE. 5(12), e15177 (2010).

281 Abdelnour A, Arvidson S, Bremell T, Rydén C, Tarkowski A. The accessory gene regulator

(agr) controls Staphylococcus aureus virulence in a murine arthritis model. Infection and

immunity. 61(9), 3879–3885 (1993).

282 Gillaspy AF, Hickmon SG, Skinner RA, Thomas JR, Nelson CL, Smeltzer MS. Role of the

accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis. Infection and

immunity. 63(9), 3373–3380 (1995).

283 Cheung AL, Eberhardt KJ, Chung E et al. Diminished virulence of a sar-/agr- mutant of

Staphylococcus aureus in the rabbit model of endocarditis. The Journal of clinical

investigation. 94(5), 1815–1822 (1994).

284 Wright JS, Jin R, Novick RP. Transient interference with staphylococcal quorum sensing

blocks abscess formation. Proceedings of the National Academy of Sciences of the United

States of America. 102(5), 1691–1696 (2005).

285 Cheung GYC, Wang R, Khan BA, Sturdevant DE, Otto M. Role of the accessory gene

regulator agr in community-associated methicillin-resistant Staphylococcus aureus

pathogenesis. Infection and immunity. 79(5), 1927–1935 (2011).

286 Le KY, Otto M. Quorum-sensing regulation in staphylococci—an overview. Frontiers in

microbiology. 6, 1174 (2015).

287 Shopsin B, Drlica-Wagner A, Mathema B, Adhikari RP, Kreiswirth BN, Novick R.

Prevalence of agr Dysfunction among Colonizing Staphylococcus aureus Strains. Journal of

Infectious Diseases. 198(8), 1171–1174 (2008).

288 Malachowa N, Whitney AR, Kobayashi SD et al. Global changes in Staphylococcus aureus

gene expression in human blood. PloS one. 6(4), e18617 (2011).

289 Hall PR, Elmore BO, Spang CH et al. Nox2 modification of LDL is essential for optimal

apolipoprotein B-mediated control of agr type III Staphylococcus aureus quorum-sensing.

PLoS pathogens. 9(2), e1003166 (2013).

290 Peterson MM, Mack JL, Hall PR et al. Apolipoprotein B Is an innate barrier against

invasive Staphylococcus aureus infection. Cell host & microbe. 4(6), 555–566 (2008).

291 Edwards AM, Bowden MG, Brown EL, Laabei M, Massey RC. Staphylococcus aureus

Extracellular Adherence Protein Triggers TNFα Release, Promoting Attachment to Endothelial

Cells via Protein A. PLoS ONE. 7(8), e43046 (2012).

292 Edwards AM, Potts JR, Josefsson E, Massey RC. Staphylococcus aureus host cell invasion

and virulence in sepsis is facilitated by the multiple repeats within FnBPA. PLoS pathogens.

6(6), e1000964 (2010).

Page 149: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

133

293 Palmqvist N, Foster T, Fitzgerald JR, Josefsson E, Tarkowski A. Fibronectin-binding

proteins and fibrinogen-binding clumping factors play distinct roles in staphylococcal arthritis

and systemic inflammation. The Journal of infectious diseases. 191(5), 791–798 (2005).

294 Sinha B, Francois P, Que YA et al. Heterologously expressed Staphylococcus aureus

fibronectin-binding proteins are sufficient for invasion of host cells. Infection and immunity.

68(12), 6871–6878 (2000).

295 Sinha B, Francois PP, Nusse O et al. Fibronectin-binding protein acts as Staphylococcus

aureus invasin via fibronectin bridging to integrin alpha5beta1. Cellular microbiology. 1(2),

101–117 (1999).

296 Meenan NAG, Visai L, Valtulina V et al. The tandem beta-zipper model defines high

affinity fibronectin-binding repeats within Staphylococcus aureus FnBPA. The Journal of

biological chemistry. 282(35), 25893–25902 (2007).

297 Lindsay JA, Moore CE, Day NP et al. Microarrays reveal that each of the ten dominant

lineages of Staphylococcus aureus has a unique combination of surface-associated and

regulatory genes. Journal of bacteriology. 188(2), 669–676 (2006).

298 Malachowa N, DeLeo FR. Staphylococcus aureus survival in human blood. Virulence.

2(6), 567–569 (2011).

299 Adhikari RP, Ajao AO, Aman MJ et al. Lower antibody levels to Staphylococcus aureus

exotoxins are associated with sepsis in hospitalized adults with invasive S. aureus infections.

The Journal of infectious diseases. 206(6), 915–923 (2012).

300 White MJ, Boyd JM, Horswill AR, Nauseef WM. Phosphatidylinositol-specific

phospholipase C contributes to survival of Staphylococcus aureus USA300 in human blood

and neutrophils. Infection and immunity. 82(4), 1559–1571 (2014).

301 Oesterreich B, Lorenz B, Schmitter T et al. Characterization of the biological anti-

staphylococcal functionality of hUK-66 IgG1, a humanized monoclonal antibody as

substantial component for an immunotherapeutic approach. Human vaccines &

immunotherapeutics. 10(4), 926–937 (2014).

302 van den Berg S, Bonarius HPJ, van Kessel, Kok P M et al. A human monoclonal antibody

targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus

aureus bacteremia. International journal of medical microbiology : IJMM. 305(1), 55–64

(2015).

303 Holtfreter S, Nguyen TTH, Wertheim H et al. Human immune proteome in experimental

colonization with Staphylococcus aureus. Clinical and vaccine immunology : CVI. 16(11),

1607–1614 (2009).

304 Wertheim HFL, Vos MC, Ott A et al. Risk and outcome of nosocomial Staphylococcus

aureus bacteraemia in nasal carriers versus non-carriers. Lancet (London, England).

364(9435), 703–705 (2004).

Page 150: Exploring humoral responses during Staphylococcus aureus · Sak – Staphylokinase SA4Ag – Staphylococcus aureus 4-Antigen SAg – Super antigen SasG – Surface protein G S. aureus

134

305 Verkaik NJ, Vogel CP de, Boelens HA et al. Anti-staphylococcal humoral immune

response in persistent nasal carriers and noncarriers of Staphylococcus aureus. The Journal of

infectious diseases. 199(5), 625–632 (2009).

306 Kolata J, Bode LGM, Holtfreter S et al. Distinctive patterns in the human antibody

response to Staphylococcus aureus bacteremia in carriers and non-carriers. Proteomics.

11(19), 3914–3927 (2011).

307 van der Kooi-Pol, Magdalena M, Vogel CP de, Westerhout-Pluister GN et al. High anti-

staphylococcal antibody titers in patients with epidermolysis bullosa relate to long-term

colonization with alternating types of Staphylococcus aureus. The Journal of investigative

dermatology. 133(3), 847–850 (2013).

308 Fritz SA, Tiemann KM, Hogan PG et al. A serologic correlate of protective immunity

against community-onset Staphylococcus aureus infection. Clinical infectious diseases : an

official publication of the Infectious Diseases Society of America. 56(11), 1554–1561 (2013).

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Summary

Staphylococcus aureus (S. aureus) is the leading cause of serious diseases in human both

from hospital and community associated infections. Some clinical manifestations of S.

aureus infections are infective endocarditis (IE), osteoarticular infections, skin and soft

tissue, pleuropulmonary, and device-related infections. In Germany, S. aureus is the

second most common cause of hospital-acquired (HA) infections. About 16.7% of these

nosocomial infections are caused by HA-MRSA clinical isolates. It has been a huge

threat for the clinicians/scientists to control the emergence of such infections caused by S.

aureus. S. aureus exhibits increasing virulence and resistance to various antibiotics,

complicating prevention and treatment of infections. Eventually, active and passive

vaccines might be the alternative strategy to deal with S. aureus related diseases. An

effective S. aureus vaccine would provide great potential security and many societal

benefits. However, so far vaccine trials have failed often due to limited number of

available antigen candidates (monovalent/single antigen) in the clinical trials. Efforts to

develop not only S. aureus vaccine but also prognosis or diagnosis tools are challenging

tasks. That was the motivation point for the current thesis to identify potential antigen

candidates for the aid of vaccine development using immunoproteomics approaches.

From the earlier studies, passive immunisation with CP5, CP8, PNAG, ClfA, SdrG,

alpha-hemolysin and active immunisation with IsdB, SEB, ClfA, CP5, CP8 were

examined during preclinical trials and found to be the best examples for potential vaccine

candidates. The antibody responses against S. aureus infections are heterogenous, still it

is possible to identify the antibody signatures to a number of corresponding S. aureus

antigens, whose abundance and presence could correlate to the disease state and may

predict treatment outcome. To support this hypothesis, goals were set to develop and

validate serological assay by indirect detection using suspension array technology (SAT).

During the study, an antigen library of 140 recombinant S. aureus antigens was

generated. Further serological assay were developed and validated to monitor the insights

of antibody mediated humoral responses during S. aureus infection from various episodes

of S. aureus infection. As an outcome, potential immunogenic antigen candidates were

identified which may be used as candidates in active/passive vaccination and to stratify

the patient. In total, three studies were carried out using serum and plasma samples from

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S. aureus nasal colonised healthy individuals (carriers and non-carriers) and bacteraemia

patients (control, complicated and uncomplicated sepsis). Bead-based assays were

performed and subsequent statistical analyses were done to identify immunogenic

antigens that might discriminate between the different clinical status and outcome.

Screening of healthy individuals (study-1) have shown significantly higher IgG responses

against 14 antigens in S. aureus nasal carriers compared to non-carriers. Furthermore, the

clonal complex 30 group of healthy carriers has shown significantly higher IgG responses

against toxic shock syndrome toxin-1 (Tsst1) in comparison to non-clonal complex 30

healthy carriers. Study-2 have shown extensively higher IgG responses against 67

antigens in control samples compared to sepsis patients. 50% of the antigens eliciting

different immune responses belonged to the extracellular components of S. aureus. The

IgG responses against MSCRAMM proteins such as FnbA, FnbB, Efb-1 have been

shown to be significantly higher in complicated sepsis. Study-3 have shown notably

higher IgG responses against 8 antigens (Plc, SspB, IsaA, SEM, GlpQ, HlgC,

SACOL0444, SACOL0985) at baseline in uncomplicated sepsis patients compared to

patients subsequently developing complicated sepsis.

In summary, the group of immunogenic antigens that have been identified in these studies

using immunoproteomics approach could be a starting point for the development of S.

aureus vaccines. Moreover, the suspension array technology approach facilitated the

identification of new S. aureus antigen candidates in addition to earlier reports. The

current results of this study support the hypothesis that it is possible to identify a

serological response to potential S. aureus antigens that correlate to progression of S.

aureus infections.

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Acknowledgements

This thesis was prepared at the department of functional genomics at the University of

Medicine, Greifswald, headed by Prof. Dr. Uwe Völker.

At first, I would like to express my deepest sense of gratitude to my supervisor Prof. Dr.

Uwe Völker for giving me this great opportunity to perform my doctoral study in the

most interesting field Host-Pathogen interactions using Immunoproteomics and for the

agreement to supervise my thesis as the first examiner.

I would like to thank immensely my advisor/guru Dr. Frank Schmidt for the supervision

since April 2012. You have been a tremendous mentor for me. I am grateful for his

concrete support, advice and critics during my work. He has also provided insightful

discussions about the research. He has given me the opportunities to learn many new

analytical techniques, softwares etc.

I am deeply grateful to Dr. Stephan Michalik for his cooperation during my Ph.D. in the

process of developing and establishing the serological assay using FLEXMAP 3D®,

developing the scripts for the data analysis.

I gratefully thank to Prof. Dr. Barbara M. Bröker, Dr. Sebastian Stentzel, Dr. Silva

Holtfreter, Dr. Julia Kolata and Dr. Hege Vangstein Aamot (Akershus

Universitetssykehus, Oslo) for their collaboration and their valuable scientific tips during

the Ph.D.

I would like to thank Dr. Vishnu Dhople, Dr. Manuela Gesell Salazar, and Ms. Annette

Murr for their assistance in the mass spectrometric measurements.

I sincerely thank to Dr. Maren Depke and Ms. Tanya Meyer for the proofreading of this

thesis.

I deeply acknowledge the support by our laboratory assistants, especially Mrs. Kirsten

Bartels, Mrs. Jette Anklam, Mrs. Ulrike Lissner, Mr. Marc Schaffer, Mrs. Anja Wiechert,

Mrs. Katrin Schoknecht, and Ms. Sophie Eisenlöffel.

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I also thank to my friends (too many to list here but you know who you are!) for

providing the support and friendship that I needed.

A special thanks to my family. Words are not enough to express how grateful I am to my

wife Nithya, my mother, and my father for all of the sacrifices made on my behalf. Your

prayer for me was what sustained me thus far.

I would like to express my thanks to my beloved daughter Skandana and son Skawin for

being such a good children’s always cheering me up.

I would like to express my thanks to my sisters and my brothers for their encouraging

support throughout my life.

And now; Last but not least, I would like to thank God for letting me through all the

difficulties. I have experienced your guidance day by day. You are the one who let me

finish my degree. I will keep on trusting you for my future. Thank you God.

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Appendix

The appendix of this thesis is saved on the enclosed CD. It contains the raw data

collected from the FLEXMAP 3D®

for all the 3 studies, stability study data, validation

data and supplementary documents. Furthermore the supplementary documents contains

the list of antigens used for the study-1 and 2 and it is labelled as supplementary 1 and the

supplementary 2 contains the list antigens used for the study-3. Furthermore, it contains

the list of samples with clinical status, meta data information, protein annotation, half

maximal calculations and other graphs we generated during the data analysis for all the 3

studies.

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Eidesstattliche Erklärung

Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig verfasst und keine

anderen als die angegebenen Hilfsmittel benutzt habe.

Die Dissertation ist bisher keiner anderen Fakultät, keiner anderen wissenschaftlichen

Einrichtung vorgelegt worden.

Ich erkläre, dass ich bisher kein Promotionsverfahren erfolglos beendet habe und dass

eine Aberkennung eines bereits erworbenen Doktorgrades nicht vorliegt.

Datum Unterschrift

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-Thirukkural (World ethics and moral written by a Tamil poet between 3rd and 1st centuries BCE)

Explanation: The stalks of water-flowers are proportionate to

the depth of water; so is men's greatness proportionate to their

minds.