Staphylococcus aureus Pathogenesis

- Gene exchanges

- Gene regulation

- Gene products

- Gene product cooperation

Brussels 07

Host

Species

Coagulase

Clumping factor

Virulence

Human and other primates

S. aureus S. epidermidis S. capitis S. caprae S. saccharolyticus S. warneri S. pasteuri S. haemolyticus S. hominis S. lugdunensis S. auricularis S. saprophyticus S. cohnii S. xilosus S. simulans S. schleiferi

++ - - - + - - - - - - - - - - +

++ - - - - - - - - + - - - - - +

+++ +

(+) (+) - - - +

(+) +

(+) + - - - +

Carnivores S. intermedius S. felis

+ -

- -

++ ++

Some Staphylococcal Species from Mammals

Brussels 07

Baba T et al. Lancet 2002;359:1819

Whole genomes of S. aureus MW2, N315 and Mu50

MW2

N315

Mu50

Brussels 07

- GC content- Gene comparison- Codon usage- Genometrics

From Linguistics to Genometrics

theicaacisagreatconférenceparcequellegathermanygreatscientists

theicaacisagreatconférenceparcequellegathermanygreatscientists

http://www2.unil.ch/comparativegenometrics/

CA Roten and L. Guy

Brussels 07

Guy Lionel, Thesis 2007

Brussels 07

S. aureus Genometric Analysis

Baba T et al. Lancet 2002;359:1819

Methicillin resistance

Whole genomes of S. aureus MW2, N315 and Mu50

MW2

N315

Mu50

Provirus encoding

PVL toxin

tst1

set

Brussels 07

- DNA region (up to 200 kb) of foreign origin

- Contain pathogenic genes

- May have a different GC content

- Flanked by direct repats of IS

- Associated with tRNA genes

- Contain relics of integrase/excisase genesor of phage genes

Define Pathogenicity Islands

Brussels 07

Hacker J et al. Mol Microbiol 1997; 23:1089-97

- Transformation

- Conjugation

- Transduction (generalized)

How do they move ?

Brussels 07

attatt

int

The example of SaPI1

Moving Genes Around

Brussels 07

attatt

int

xis

Moving Genes Around

The example of SaPI1

Brussels 07

attatt

int

xis

Moving Genes Around

The example of SaPI1

Brussels 07

Moving Genes Around

The example of SaPI1

Brussels 07

attatt

int

Moving Genes Around

The example of SaPI1

Brussels 07

Novick, Schlievert, Ruzin. Microbes and Infect 2001; 3:585

Moving SaPI1 with Phage 80α

Brussels 07

Staphylococcus aureus Pathogenesis

- Gene exchanges

- Gene regulation

- Gene products

- Gene product cooperation

Brussels 07

Time

Growth

Transcriptionof exoproteins

Transcriptionof adhesins

agr-Regulation of Virulence DeterminantsDuring in vitro Growth of S. aureus (1)

Adapted from R.P. Novick ASM press, 2000

Brussels 07

AIP = auto-inducing peptide

A C D B

P2 P3

AgrB

AIP

AgrC

AgrA

δ

RNAIII

P

Novick et al. Curr. Op. Microbiol. 1999;2:40-45

Virulence Gene Regulation by Peptides in S. aureus (1)

Brussels 07

A C D B

P2 P3

AgrB

AIP

AIP = auto-inducing peptide

AgrC

AgrA

δ

RNAIII

AgrAP

Activation of Tx

Virulence Gene Regulation by Peptides in S. aureus (2)

Novick et al. Curr. Op. Microbiol. 1999;2:40-45

Translationof exoproteins

Brussels 07

Regulation of S. aureus super-antigens

Action of regulatory genes

Gene Product Activity/function agr Sae-RS

rot sarA sarS sarT tst

Superantigens

sea Enterotoxin A Food poisoning, TSS 0 0

seb Enterotoxin B Food poisoning, TSS + b -sec Enterotoxin C Food poisoning, TSS +sed Enterotoxin D Food poisoning, TSS +eta Exfoliatin A Scalded skin syndrome +etb Exfoliatin B Scalded skin syndrome +tst Toxic shock toxin-1 Toxic shock syndrome + - b -

0, no effect of gene on expression; +, upregulated; -, downregulated. b. Controversial.

Brussels 07

Host:- Lack of anti-TSST antibodies

Environment:- High protein concentration

- Neutral pH

- High pCO2

- High pO2

Conditions for Developing Toxic Shock Syndrome

Brussels 07

AlteredEcology !

Staphylococcus aureus Pathogenesis

- Gene exchanges

- Gene regulation

- Gene products

- Gene product cooperation

Brussels 07

Gene Protein Function Implication in disease

spa Protein A Binds Fc fragment Sepsis, osteoarthritis

clfA Clumping factor A Binding to fibrinogen Endocarditis

clfB Clumping factor B Binding to fibrinogen -

cna Collagen binding protein Binding to collagen Osteomyelitis

fnA Fn-binding protein A Binding to fibronectin Endocarditis, persistence

fnB Fn-binding protein B Binding to fibronectin -

sdrC Serine-aspartate repeat prot Binding to fibrinogen -

sdrD Serine-aspartate repeat prot Binding to fibrinogen ? -

sdrE Serine-aspartate repeat prot Binding to fibrinogen ? -

pls Plasmin-sensitive protein Binding to nasal mucosa Nasal colonization?

fmtB Methicillin resistance Putative cell-wall building Expression of methi-R

sasA S. aureus surface protein A Undetermined -

sasB S. aureus surface protein B Undetermined -

sasC S. aureus surface protein C Undetermined -

sasE S. aureus surface protein E Undetermined -

sasF S. aureus surface protein F Undetermined -

sasG S. aureus surface protein G Binding to nasal mucosa Invasive disease

sasH S. aureus surface protein H Undetermined Invasive disease

sasI S. aureus surface protein I Undetermined -

sasJ S. aureus surface protein J Undetermined -

sasK S. aureus surface protein K Undetermined -

Understanding Pathogenic FactorsWild Type Coa- Coa-/ClfA- Coa-/ClfA-/Fnb-

Coagulation

Vegetations

PlateletFactors

Tissuefactor

??

???

??

? ???

Brussels 07

Transfer or Staphylococcal Pathogenicgenes into less Virulent Bacteria

S. aureus donor(Coa+/ClfA+/Fnb+)

Lactococcusrecipient (ClfA-)

1: Transfer of S.aureus clfA geneinto viridans group streptococcus

2: Select clumping-positiverecombinants

Lactococcusrecombinant (ClfA+)

Que et al. I&I 2000; 68:3516-22Brussels 07

Adherence of L. lactis to PMMA coverslips coated with fibronectin

8325-4

pIL253

FnbpA (+) 8325-4

Adh

eren

t CFU

(x10

3 /cov

ersl

ip)

20

40

60

80

100

0

Soluble concentration of Fn (µg/ml)

0.0 0.25 0.50 0.75

Que et al, I&I 2001; 69:6296-302 Brussels 07

ExperimentalEndocarditis

Brussels 07

% In

fect

ed V

eget

atio

ns

100

20

40

60

80

0

L. lactis parent ClfA+ FnBPA+

Inoculum = 104 105 106 107 104 105 106 107 104 105 106 107

Infectivity of L. lactis Recombinants Binding to Fibrinogen or Fibronectin

Que et al. Infect. Immun. 2001Brussels 07

Log

CFU

/g V

eget

atio

ns

10

2

4

6

8

0

L. lactis parent ClfA+ FnBPA+

Time after infection (h) = 2 12 48 72 2 12 48 72 2 12 48 72

Dynamic of L. lactis Endocarditis

Brussels 07

Que et al. J. Exp. Med . 2005

ExperimentalEndocarditis

Brussels 07

Neighboring Aorta of Valves Infected with ClfA-positive Lactococci /Gram Stain

Que YA, J. Exp. Med 2005;201:1627Brussels 07

Que YA, J. Exp. Med 2005;201:1627 Brussels 07

Membrane Peptidoglycan

Binding Domains of FnBPA

Binding toFibrinogen Fibonectin

ClfA +++ -

FnBPA ++ +++

and Piroth et al. 2007, submittedQue YA, J. Exp. Med 2005;201:1627

M-LPXTG W D1-4 Du C B A L

Fibronect. Fibronect. Fibronect. Fibrinogen Leader

Brussels 07

Membrane Peptidoglycan

Binding Domains of FnBPA

M-LPXTG W L

M-LPXTG W D1-4 Du C L

M-LPXTG W A L

M-LPXTG W D1-4 Du C B A L

Fibronect. Fibronect. Fibronect. Fibrinogen Leader

Brussels 07and Piroth et al. 2007, submittedQue YA, J. Exp. Med 2005;201:1627

Binding Domains of FnBPA

Surrogaterecombinant Binding Infection Internalization Persistence

Fg +++ --- ---

Fn --- +++ ---

cis Fn/Fg +++ +++ +++

trans Fn/Fg +++ +++ +++

Brussels 07and Piroth et al. 2007, submittedQue YA, J. Exp. Med 2005;201:1627

- S. aureus needs both fibrinogen- and fibronectin-binding domains to be fully infective

- Fibronectin-binding protein represents an exampleof intra-molecular domain cooperation

- Fibronectin- and fibrinogen-binding represent anexample of inter-molecular domain cooperation

Brussels 07

E. Widmer et al. I&I , submitted Brussels 07

Que YA, J. Exp. Med 2005;201:1627 Brussels 07

Que YA, J. Exp. Med 2005;201:1627 Brussels 07

Que YA, J. Exp. Med 2005;201:1627

LPXTG-proteins Toxins

S. aureus 21 > 30

S. epidermidis > 10 No

S. haemolyticus 6 No

S. saprophyticus 1 No

But all have prophages and genomic islands,including the SCC staphylococcal K7 chromosome

Brussels 07

Staphylooccus aureus

a paradigm of host-parasite evolution

N. WidmerM. Giddey J. EntenzaS. PiuJ. Vouillamoz

YA QueL PirothCA. RotenL. GuyF. Collyn

Subcutaneous Infection of Mice with 108 CFU of AIP IS. aureus supplemented or not with AIP II

Lesi

on s

ize

in m

m2

1000

200

400

600

800

0

Day 1 Day 5n = 4 4 3 4 4 4 3 4

Strain 6390B, agr+Strain 6911 , agr-

6390B + 10 μg AIP II6390B + 5 μg AIP II

Mayville et al. PNAS 1999; 96:1218-1223

Brussels 07

- No, not sensu stricto

- All Staphylococcus spp. carry an agr

- agr is also required for regulationof house-keeping genes

Is agr a Virulence Factor ?

Brussels 07