Holger Barth

Institut für Pharmakologie und Toxikologie

Universitätsklinikum Ulm

Cyclosporin A und FK506:

Neue pharmakologische Strategien gegen Bakterientoxine

Acknowledgements

Katharina Ernst, Leonie Schnell

Manuel Schuster, Peter Feigl, Carina Birkhofer, Claudia Kroll, Natalie Böhm, Nina Eberhardt, Michael Vollmer

Eva Kaiser, Gerd Haug, Simon Langer (former group members)

Collaborators in this project:

C. Schiene-Fischer & G. Fischer, Halle

K. Aktories, Freiburg

J. Buchner, München

R. J. Collier, Boston

M. R. Popoff, Paris

Financial support:

DFG Priority Program SPP 1150 (BA 2087/1)

Grant BA 2087 (2-1, 2-2)

SFB1149 (Project A4)

International Graduate School in Molecular Medicine Ulm (IGradU)

Medical Faculty Ulm (Bausteinförderung)

Bacterial toxins: An overview

BACTERIUM

Exotoxins

Proteins

secreted from living bacteria

Gram-positive & Gram-negative

EndotoxinsLPS (Lipopolysaccharide)

After lysis of Gram-negative bacteria

Released From cell wall

Pore-forming

ToxinsAB-Toxins

permeabilise

cell membranes

of eukaryotic cells

Act as enzymes

in cytosol

of eukaryotic cells

Act on immune cells:

mediator release,

fever,…

Botulinum-Toxin

(Clostridium botulinum)2,6 x 10-4 µg/kg

Batrachotoxin

Frog (Phyllobates bicolor)2 µg/kg

Taipoxin

(Snake, Australia)2 µg/kg

Kobra-Neurotoxin

(Naja naja siamensis)75 µg/kg

d-Tubocurarin (Curare) 200 µg/kg

Natriumcyanid 10.000 µg/kg

Bacterial AB-type toxins: The most toxic substances

(LD50,Mouse, i. v.)

proteins, secreted by bacteria

act independent from producing bacteria real intoxication!

enter mammalian cells (via B subunit)

act as enzymes in the cytosol (A subunit)

high substrate specificity, extremely potent

Bacterial AB-type toxins: Important virulence factors

receptor

substratemodified

substrate

cellular reaction

clinical symptoms:

diphtheria, cholera, pertussis, anthrax, enteric diseases, …

translocation

mechanism of toxin translocation widely unknown

scientific & medical interest: drug delivery, therapy

no A subunit in cytosol, no intoxication!

novel inhibitors to prevent A delivery into cytosolH. Barth, K. Aktories (2011) Eur. J. Cell Biol. 90

H. Barth H (2011) Naunyn-Schmied. Arch. Pharmacol. 383

Gefahr durch Klinik-Keime

Wenn keine Arznei mehr hilft:

Multiresistente Bakterien werden für kranke Menschen zu wachsender Gefahr

Experten-Schätzung: BRD 30.000 - 40.000 Todesfälle/Jahr

Südwestpresse Ulm, 20. 11. 2014

Bacterial AB-type exotoxins: Novel pharmacological inhibitors are required

Exotoxins are produced and released by bacteria but act independent from bacteria

antibiotics act against bacteria, not against toxins!

increasing resistance against antibiotics

Nucleus

EE

LE

TGN

Golgi

ER

EE

A-domain

B-domain

AB-toxin

(short-trip-toxin)Receptor

A-domain

B-domain

Substrate

Modified

Substrate

AB-toxin

(long-trip-toxin)

Cellular

Reactions

CytosolReceptor-mediated

endocytosis

AB-toxins exploit vesicular protein traffic pathways of host cells

to deliver their A-domain into the cytosol

Barth, H. & Stiles, B.G. (2008)

Curr. Med. Chem. 15

Diphtheria

toxinCholera

toxin

C. botulinum C2 toxin

C. perfringens iota toxin

C. difficile CDT

Binary actin ADP-ribosylating toxins from clostridia

H. Barth, K. Aktories (2011) Eur. J. Cell Biol. 90

Role of host cell factors in uptake of C2 toxin: Hsp90

HOP

Hsp

70

Hsp

90

Hsp

90Hsp

70

client

protein

HOPHOP

Hsp

90

Hsp

90Hsp

70Cyp 40

client

proteinclient

protein

ATP ADP

Geldanamycin /

Radicicolmodified from J. Buchner, München

Geldanamycin (GA), Radicicol (Rad) inhibit Hsp90 ATPase activity

prevent intoxication of cells with C2 toxin!

Haug et al. (2003) J.Biol.Chem. 278

C2 toxin + Rad C2 toxin control C2 toxin + GA

Vero cells, 1 h GA/Rad, 3 h C2 toxin

(50 ng/ml C2I+100 ng/ml C2IIa) GA: 10 µM; Rad: 1 µM

FKBP 51/52

120 min 240 min

0

5

10

15

20

25

30

35

40

45

50

rou

nd

ed

ce

lls

( %)

GA – + – +

1. Bafilomycin A1 (Baf) +/- Geldanamycin (GA)

2. C2 toxin binding (C2I + C2II, 4°C)

3. acidic pulse, pH 4.5, 5 min +/- GA

4. further incubation at pH 7.5 + Baf

5. photos: counting of rounded cells

Haug et al. (2003) J.Biol.Chem. 278

** ***

pH 4.5

(37°C)

bafilomycin

H+

H+

Barth et al. (2000).J.Biol.Chem. 275

pH 7.5

(4°C)

C2I

C2IIa

Membrane translocation assay

Role of Hsp90 in membrane translocation of C2 toxin

GA, Rad block pH-driven

membrane translocation of C2I:

Hsp90 crucial for translocation!

In GA-treated cells:

C2I trapped in early endosomes (IF microscopy).

Co-IP of Hsp90 with C2I from cell lysate:

• HeLa cells, C2IIa (600 ng/ml) + GST-C2I (300 ng/ml)

• lysis, pull-down of GST-C2I

• Western blotting of co-precipitated Hsp90

Dot blot analysis:

• Hsp90 spotted onto membrane

• overlay +/- C2I

• Western blotting of bound C2I

95

72

WB: anti-Hsp90

GST-C2I con

+ C2IIa

Interaction between Hsp90 and C2I

Hsp90

con

C2I PBS

[µg]1 0.5 0.25 1 0.5 0.25

Kaiser et al. (2012). Cell. Microbiol. 14

Hsp90 binds to C2I in intact cells.

Hsp90 directly binds to C2I in vitro.

iota Rad + iota Rad + CDT CDT

CDTa

Hsp90

iota a PBS

[µg]

overlay

1 0.5 0.25 1 0.5 0.25 1 0.5 0.25

Dot blot analysis:

Hsp90 binds to the

enzyme components

in vitro (and in cells).

Role of Hsp90 in cellular uptake of iota toxin and CDT

Inhibition of Hsp90 by GA or Rad

delays intoxication of Vero cells with iota toxin and CDT.

prevents pH-triggered membrane translocation of enzyme components.

Hsp90 interacts with and facilitates membrane translocation of the

ADPRT-subunits of binary clostridial toxins.

Kaiser et al. (2012). Cell. Microbiol. 140

Kaiser et al. (2011). Infect. Immun. 79

active C2I

H+

H+

H+

H+

Cyclophilins

FK506-binding proteins

Parvulins

PPIases

Are PPIases involved in membrane translocation of C2I?

• Cyclosporin A (CsA) Cyclophilins

• FK506 (= Tacrolimus) FK506-binding proteins

Fischer et al. (1984), Biomed Biochim Acta. 43

PPIases = Peptidyl prolyl cis/trans isomerases (Rotamases)

• folding helper enzymes, act in concert with Hsp90

• catalyze cis/trans isomerization of peptide-proline-bonds in proteins

• accelerate refolding of proteins after membrane translocation in cells?

Role of PPIases in cellular uptake of C2 toxin

??Hsp90

C2 toxin

CsA CsA + C2

con

10 µM CsA/ FK506; 50/100 ng/mL;C2I/C2IIa, 2 h

Effect of pharmacological PPIase inhibitors on intoxication of HeLa cells with C2 toxin.

ADP-rib. actin

actin

C2 toxin

CsA + - +-

Cyclosporine A (CsA) and FK506 protect HeLa cells from intoxication with C2 toxin:

Cyclophilin(s) and FKBP(s) involved in mode of action of C2 toxin.

con

FK506

C2 toxin

FK506 + C2

C2 toxin

ADP-rib. actin

FK506 - + - +

Role of PPIases in cellular uptake of C2 toxin

Kaiser et al. (2009) Cell. Microbiol. 11 Kaiser et al. (2012) Cell. Microbiol. 14

***

** ** **

** ** **

***

**

******

***

******

**

2.5 3.0 3.5 4.0 4.5

0

20

40

60

80

100 C2 toxin

CsA + C2 toxin

FK506 + C2 toxin

CsA + FK506 + C2 toxin

time [h]

rou

nd

ed

ce

lls [%

]

CsA and FK506 protect mammalian cells from intoxication with C2 toxin

Kaiser et al. (2012) Cell. Microbiol. 14

Effect of CsA and FK506 on intoxication of HeLa cells

CsA and FK506 delay intoxication with C2 toxin.

CsA + FK506 act synergistic / additive.

CsA and FK506 inhibit intoxication of other cell lines and cell types.

CsA (and FK506) inhibit uptake of C2I into the cytosol of target cells.

Biotin-C2I

Hsp90 (cytosolic marker)

Rab5 (EE marker)

cytosolic

fraction

C2 toxin+ - +-

extracted

cells

Cytosolic fractions obtained by digitonin extraction

C2 toxin

CsA - - +-

Baf A1 - + --

+- + +

0

10

20

30

40

50

bio

tin

-C2I

(in

ten

sit

y a

rbit

rary

un

its) ***

NS

NS

No EE in digitonin-extract!Kaiser et al. (2009) Cell. Microbiol. 11

Why do CsA and FK506 inhibit intoxication of cells with C2 toxin?

No effect on enzyme activity of C2I. Effect on uptake of C2I into the cytosol?

C2IIa + C2I

C2IDigitonin

C2I

C2I

C2 toxin C2 toxin + FK506

H+ H+ Endosome

pH 5

37°C

Bafilomycin

pH 7.5

4°C

C2I

C2I

No effect on: binding of C2 toxin to cells, endocytosis, pore formation by C2IIa.

Effect on membrane translocation of C2I?

pH-dependent membrane translocation of C2I in intact cells

CsA and FK506 inhibit membrane translocation of C2I:

Cyp(s) and FKBP(s) involved in translocation.

Why do CsA and FK506 inhibit uptake of C2I into the cytosol?

0

20

40

60

80

into

xica

ted

cel

ls [

%]

- - +

- + +

FK506

(20 µM)

**

C2 toxin

CsA

(10 µM)

-- +

+- +

***

C2 toxin

0

10

20

30

40

50

into

xic

ate

d c

ells

[%

]

Kaiser et al. (2012) Cell. Microbiol. 14

Kaiser et al. (2009) Cell. Microbiol. 11

Which CyP(s) and FKBP(s) interact with C2 toxin in living cells?

Kaiser et al. (2012) Cell. Microbiol. 14

Co-IP of PPIases with C2I from lysates of C2 toxin-treated cells

• HeLa cells incubated with C2IIa (600 ng/ml) + biotin-C2I/GST-C2I (300 ng/ml)

• lysis, pull-down of C2I-bound proteins (streptavidin- or glutathion-sepharose)

• identification of co-precitipted proteins: mass-spectrometry (MALDI-TOF), Western blotting

IP: glutathion-sepharose

55

43

55

43

IP: streptavidin-sepharose

95

72

17

GST-C2I con

+ C2IIa

17

Biot-C2I con

+ C2IIa

95

72WB: -Hsp90

WB: -FKBP 51

WB: -CyPA

WB: -Hsp90

WB: -FKBP 51

WB: -CyPA

In intact cells, C2I interacts with Hsp90, CypA, Cyp40 and FKBP 51.

Dot blot analysis:

• PPIases spotted onto membrane

• Overlay +/- C2I

• Western blot analysis of bound C2I

0.5 µg

Kaiser et al. (2012) Cell. Microbiol. 14

0.5 0.2 0.1 µg

FKBP 52

FKBP 51

FKBP 12

Cyp A

+ C2I - C2I

In vitro, C2I binds to CypA, Cyp40, FKBP 51/52, Hsp90.

Unfolded C2I binds stronger.

1 0.5 0.25 [µg] 1 0.5 0.25 1 0.5 0.25 1 0.5 0.25

FKBP51

con

overlay: C2I

guanidine

hydrochloride

pH 4,3 urea native

Which CyP(s) and FKBP(s) interact with C2I in vitro?

ADP-rib. actin

denat.

C2I

native

C2I con

Ernst et al. (2014). J. Mol. Biol., in press

Cyp A crucial for membrane translocation of C2I!

Experimental setup:

purified early endosomes (EE)

+ cytosol, + biotin-NAD+

+ CsA (10 µM) or α-CypA

CsA -- + -

cytosol + +

++ + +

α-CypA -- - +

0

10

20

30

40

AD

P-r

ib. acti

n

(in

ten

sit

y a

rbit

rary

un

its)

+

EE

-

***NS

NS

cytosol (95 °C) - - -+

Kaiser et al. (2009) Cell. Microbiol. 11

Assay modified from Tamayo et al. (2008) PNAS 105

α-CypA

Hsp90

H+

H+

H+

H+

CypA

CsA

Does anti-CypA inhibit translocation of C2I from endosomes in vitro?

Cytosol

Role of host cell factors in cellular uptake of C2 toxin: PPIases

Cyp40 co-precipitates with C2I in lysates from C2 toxin-treated cells

Simon Langer

Time-dependent interaction pattern

Cyp40 interacts with Biotin-C2I

Ernst et al. (2014) J. Mol. Biol

HeLa cells + Biotin-C2I + C2IIa

Lysis + Streptavidin beads Identification of co-precipitated proteins by WB

Is there a direct interaction between Cyp40 and C2I?

Cyp40 directly interacts

with C2I, CDTa and Ia

Stronger binding of denatured

C2I, CDTa, Ia to Cyp40

Loss of enzyme activity does not

interfere with binding of C2I to Cyp40

Ernst et al. (2015) J. Mol. Biol.

CDT kindly provided by Dr. Carsten Schwan & Dr. Klaus Aktories, Freiburg

Iota toxin kindly provided by Dr. Michel R. Popoff, Pasteur Institute Paris

Quantitative analysis of the interaction between Cyp40 and C2I or Ia

KD = 101 nM

N = 0.42

C2I and Cyp40 Control = Cyp40 and bufferIa and Cyp40

KD = 1010 nM

N = 0.36

ITC measurements: In cooperation with Dr. Cordelia Schiene-Fischer, Halle (Saale) Ernst et al. (2015) J. Mol. Biol.

Steinbach et al. (2007) Nature Reviews Microbiology 5

CsA and FK506 are immunosuppressive drugs

Effect of suppressing the Cyp40 activity by using specifically designed inhibitors

The non-immunosuppressive CsA derivative VK112 inhibits the intoxication of cells

with C2, iota and CDT toxin.

Ernst et al. (2015) J. Mol. Biol.

Effect of suppressing the Cyp40 activity by using specifically designed inhibitors

The non-immunosuppressive CsA derivative VK112 inhibits the intoxication of cells with

C2, iota and CDT toxin.

1.75 2.250

20

40

60

80

100

*

*****

***

*

*****

*

***

ns

iota toxin

iota toxin + CsA (20 µM)

iota toxin + VK112 (25 µM)

iota toxin + VK112 (50 µM)

iota toxin + VK112 (100 µM)

time [h]

rounded c

ells [

%]

3 4 5

0

20

40

60

80

100 CDT

CDT + VK112 (200 µM)

**

**

**

time [h]

rounded c

ells [

%]1.75 2.25

0

20

40

60

80

100

*

*****

***

*

*****

*

***

ns

iota toxin

iota toxin + CsA (20 µM)

iota toxin + VK112 (25 µM)

iota toxin + VK112 (50 µM)

iota toxin + VK112 (100 µM)

time [h]

rounded c

ells [

%]

Ernst et al. (2015) J. Mol. Biol.

Effect of VK112 on membrane translocation

VK112 inhibits the pH-dependent translocation of C2I across the cytoplasmic membrane.

50 µm

VK112 ?

Ernst et al. (2015) J. Mol. Biol.

HeLa cells + PA63 / His-TccC3hvr (= ADP-RT domain of Photorhabdus luminscens toxin)

Lysis + TalonTM CellThru beads Identification of co-precipitated proteins by WB

Cyp40 interacts with the ADP-ribosyltransferase domain of PTC3

Cyp40 co-precipitates with isolated ADP-RT domain

Uptake of LF via PA63 independent of Cyps!

Investigation of theisolated ADP-RT

Blanke et al. (1996)Collier and Young (2003)

Lang et al. (2010)Beitzinger et al. (2012)

Uptake via B. anthracis toxin

Isolated ADP-RT= His-TccC3hvr

In close collaboration with Alexander E. Lang & Klaus Aktories, Freiburg

PA63 kindly provided by Dr. R. John Collier, Boston

CsA protects HeLa cells from intoxication with the isolated ADP-RT

Lang, Ernst, Lee et al. (2013) Cell. Microbiol.

3.50

20

40

60

80

***

**

*

roun

de

dce

lls(%

)

PA63 +

HisTccC3hvr

+ C

sA

Summary and Conclusions

CypsA and 40 and FKBP51/52 co-precipitate with C2I in lysates from toxin-treated cells interaction with ADP-RT

Cyps A and 40 and FKBP51/52 directly interact with C2I, CDTa and Ia in vitro

CsA and FK506 inhibit translocation of A subunit into cytosol protect cells from C2, CDT, iota toxins

Non-immunosuppressive CsA-derivative VK112 inhibits intoxication of cells

VK112 prevents membrane translocation of C2I into cytosol of target cells

Cyp A and Cyp 40 are novel interaction partners of ADP-RTs, but not of other toxins.

Common Cyp-dependent translocation of ADP-RTs: Chaperone complex?

Cyps as new drug targets to prevent uptake of ADP-RT toxins.

Medical relevance:

Many toxins that cause severe diseases are ADP-RTs (diphtheria, cholera, pertussis,…)

potential novel therapeutic strategies against toxin-associated diseases