Pathogenesis

of complicated Staphylococcus aureus infections

Bhanu Sinha

Institute of Hygiene and Microbiology Julius Maximilians University Würzburg

S. aureus infections

•

No specific site

-

Localized:

furuncle, bullous impetigo, infected lesions (atopic dermatitis), surgical wounds, abscess formation, foreign body infection,

pneumonia, ...High potential for systemic spread

-

Hematogenous:

soft tissue, thrombophlebitis, septic arthritis, osteomyelitis, endocarditis,

sepsis, ...

-

Intoxication:

food intoxication, toxic shock syndrome, scalded skin syndrome, ...

S. aureus host interaction

ImmunomodulationChemotaxisOpsonisationPhagocytosisComplementAngiogenesisHost cell viability…

PersistenceMetabolic variation (SCV)Altered habitat (i.c.)Biofilm…

AdherenceAdhesinsWTAStress resistance

ComplementAntimicrobial peptidesLow pHOxidative conditions…

Antibiotic resistanceMRSA (β-lactam)

other classes

Redundancy, pleiotropy (variable prevalence/expression)

PlasticityGeneticRegulatory

High colonisation rate: 20-30% permanently, 30-50% transiently

Complicated S. aureus infectionsEndocarditis (Chronic) osteomyelitis

(www.thachers.org/ orthopedics.htm;D.P. Lew, Lancet 2004)(escuela.med.puc.cl/.../ patologia067-073.html%7F)

Complicated infection

•

Deep seated Associated with line-sepsis, osteomyelitis, septic arthritis, deep organ abscesses, infective endocarditis

•

Persistent Repeated positive bloodculture (>3 d therapy)

•

Recurrent Repeated isolation of same organism from blood culture after documented negative blood culture and/or clinical improvement

F.-Y. Chang, et al., Medicine, 2003. 82: 333 -

339

Pathogenesis of infective endocarditis

3.) Replication of micro-organismsGrowth of vegetations

5.) Survival of host defence(and therapy)

1.) Transmission(here: blood)

4.) Dissemination2.) Adherence tohost structures

(platelets, matrix, cells)+/-

invasion

of host cells

Prerequisite: often endothelial damgeApposition of platelets and matrix proteins

S. aureus endothelium interaction

S. aureus adhesins

2.) Anchorless (secreted/soluble;

back binding):e.g. Emp, Eap,

Ebh/GSSP

3.) Transmembraneous:EbpS, Ebh/GSSP?

1.) Cell wall-anchored(sortase motif

LPXTG ):e.g. FnBPA/B,

Cna, ClfA/B, Protein A, etc.

4.) Wall teichoic acids (WTA), Lipoteichoic acids (LTA)(negatively charged)-> WTA: adherence (nasal epithelia),

nasal colonization; flow interaction (HUVEC), virulence (IE)

C. Weidenmaier et al., Nat. Med. 2004; 10:243-245C. Weidenmaier et al., Infect. Dis. 2005, 191:1771-1777

Redundancy, pleiotropy (variable prevalence/expression)

Two fnb genes are more likely associated with invasive infections

Different regulation of fnbA and fnbBD. Li et al., Antimicrob. Agents Chemother. 2005, 49:916-924

⇒ Important for different steps during infection?

Endocarditis, osteomyelitis, foreign body infection

Invasive infections: 119

82 %

18 %

Nasal colonisation: 44

63 %

37 %

Fibronectin binding correlates with invasive infectionsR.A. Proctor et al., J. Lab. Clin. Med. 1984; 104:455-469

A. Johansson et al., Clin. Orthop. 2001; 382:241-246

163 clinicalisolates (PCR)

S.J. Peacock et al.,Infect. Immun. 2002,

70:4987-4996

1 fnb

fnbA+ fnbB

FnBP-dependent endothelial adherence in vivo

S. Kerdudou et al., Thromb. Haemost. 2006; 96: 183-189

Intravital microscopy (dorsal skinfold, postcapillary/collecting

venules)

Bact

eria

/mm

2

untreated + TNF-α

Cowan 1FnBPA-

Direct modulation of fibrinolysis by live staphylococci?

+

-

Pro-

thro

mbo

tic

activity

+

-

Pro-

fibr

inolyt

ic a

ctivity

Based on: B. Haslinger-Löffler et al., Thromb. Haemost. 2007; 98:813-822

24 h

HMC stimulated with conditioned media (MNC treated with ...)

0

0,5

1

1,5

contr

olTN

Falph

aS.

epi. 1

9S.

epi. 2

0044

S. ep

i. 0-4

7Cow

an

8325

-4Woo

d

6850

ST 23

9

HMC

t-PA

/PA

I-1

rati

o

48 h

HMC infected with live staphylococci

WTA required for adherence and virulence (IE) in vivo

C. Weidenmaier et al., Infect. Dis. 2005, 191:1771-1777

S. aureus strain SA113 and ΔtagO (WTA-deficient)

Virulence (rabbits, traumatic IE)48 h post challenge (5 x 105 CFU)

CFU

Vegetations

Kidney/SpleenWT

~ 109

~ 107

ΔtagO

~ 106

~ 103

HUVECFlow conditions

S. aureus : a facultative intracellular microorganism

S. aureus invades host cells in vitro and in vivo

S. Clement et al., J. Infect. Dis. 2005; 192:1023-1028

Nasal epithelium; IF

Green: α-S. aureus (PG)Red: α-Cytoceratin (polyclonal)

Blue: Nuclei (TOTO-III)

B. Sinha et al., Cell. Microbiol. 1999; 1:101-117

293 cells; TEM

+ Tannic acidCowan I, fixed

MSSA

B. Sinha, et al., Cell. Microbiol. 1999, 1:101-117M. Grundmeier, et al., Infect. Immun. 2004; 72:7155 -7163

Clinical S. aureus isolates are invasive for host cells

Regulationdefects

293 cells

FnBPdefects

Clinicalisolates(n > 150)

Internalizedbacteria

[% of Cowan(AFU)]

Mechanism

of S. aureus cellular invasion

Staphylococcus aureus

Host cellmembrane Integrin (α5

)β1

Fn binding:> D1-D4 and Du

involved

T. Fowler et al., Eur. J. Cell Biol. 2000 79:672-679

R. Massey et al., Cell. Microbiol.2001 3:839-851

[U. Schwarz-Linek et al., Nature 2003, 423:177-181]

FnBPs

Fn

Pls

MRSAPls expression anti-invasive Mechanism?K. Juuti/B. Sinha et al., J. Infect. Dis. 2004,

189:1574-1584 C. Werbick, et al. J. Infect. Dis. 2007, 195:1678-1685

Eap: partially compensatory for

loss of FnBPs (Newman)A. Haggar

et al., Infect. Immun. 2003; 71:2310-2317

Mechanism?

Eap

Role of WTA:Adherence (HUVEC: flow; nasal

keratinocytes)=> Host cell binding partner?

Required for invasion?

C. Weidenmaier et al., Nat. Med. 2004; 10:243-245C. Weidenmaier et al., . Infect. Dis. 2005, 191:1771-1777

Adapted and updated from B. Sinha et al., Thromb. Haemost. 2005; 94:266-277

F-Actin re-arrangement;Signaling: Tyr-P, Src kinase;

T. Fowler et al. Cell. Microbiol. 2003; 5:417-426; K. Dziewanowska et al. . Infect Immun 1999; 67:4673-4678

F. Agerer et al., J. Biol. Chem. 2003; 278:42524-42531

Cortactin;

F. Agerer et al., J. Cell Sci. 2005; 118:2189-2200

Tensin, nWASP, Rab5

A. Schröder

et al., Mol. Biol. Cell 2006; 17:5198–5210

Endocarditis: FnBP-dependent pathogenesis and invasion in vivo

Y.A. Que et al., J. Exp. Med. 2005; 201:1627-1635

Traumatic endocarditis (rats); immuno-histochemistry (24 h p. i.) Heterologous expression of S. aureus adhesins

αClfA F(ab)’2

αFnBPA F(ab)’2

αvWF

αvWF

Invasion of HUVEC in vitro

No

Yes

Disease

Limited

Progression

Dircect (extracellular) leukocyte cytotoxicity of S. aureus

S. aureus toxins

4.) Pyrogenic toxin-super- antigens (PTSAg):

-

Enterotoxins(SEA, SEB, SEC-SEH,…)

-

Toxic shock syndrome toxin-1(TSST-1)

-

Other enterotoxins (no SAg?)

2.) Hemolysins/cytotoxins: (structurally not related!)

-

α-Toxin (pore-forming)-

β-Toxin/Sphingomyelinase C

-

δ-Toxin/δ-Lysin

Redundancy, pleiotropy (variable prevalence/expression)

1.) Exfoliative toxins:(Proteases: Desmoglein)

- ETA- ETB- ETC- ETD

-> Staphylococcal scalded skin syndrome (SSSS)

3.) Hemolysins/cytotoxins : (Two component toxins,

related, pore-forming)-

γ-Toxins

-

Panton-Valentin Leukocidin (PVL)

Mononuclear leukocytes

Neutrophil granulocytes

S. aureus α-toxin•

Pore-forming toxin (homo-heptameric), 33 kDa monomer (homologous to LukF-PVL)

•

Highly selective small pores at low (< 6 ng/ml) concentrations (K+-permissive, not Ca2+)

•

Host cell membrane receptor ?•

Highest sensitivity: rabbit RBC;

(other RBC ~ x 10-2, human RBC ~ x 10-3)

A. Valeva, et al. J. Biol. Chem. 2001

Multistep assembly

Mitochondria are “targeted“Intact Jurkat T cells (8 h) Isolated mitochondria (30 min)

H. Bantel/B. Sinha, et al., J. Cell. Biol. 2001;, 155:637-647

Mitochondrial transmembrane potential (ΔΨm

): TMRE uptake by FACS (Fl-2H)

Jurkat: WTJurkat Bcl-2 +++

(4 h)10 ng/ml 100 ng/ml

Caspase activation byS. aureus α-toxin in mononuclear leukocytes

Casp-8 Pro-Casp-9Apaf1

Bcl-2

Apaf1dATP

DEATH

C LLSubstrates

Casp-3EFFECTORCASPASE

INITIATOR CASPASE Casp-9

Mitochondrium

α-Toxin

JurkatT cellsandMNC

?

Cytochrome c

MNConly

EndogenousTNF

Bid?

tBid?

?

?FADD

TNF-R1

Pro-Casp-8

Similar data in PMN:

PVLA.L. Genestier, et al., 2005; 115:3117-3127 Based on H. Bantel/B. Sinha, et al., J. Cell. Biol. 2001; 155:637-647

B. Haslinger, et al., Cell. Microbiol. 2003; 5:729-741

Caspase inhibition prevents α-toxin- induced cell death in Jurkat T cells

Trypan blue exclusion (light microscopy)

B. Haslinger, et al., Cell. Microbiol. 2003; 5:729-741

Invasiveness required for induction of apoptosis

Correlation of S. aureus properties

Based on B. Haslinger-Löffler et al., Cell. Microbiol. 2005; 7:1087-1097

-

Invasion and hemolysis

required-

Invasion not

sufficient-

Hemolysis

not

sufficient

HUVEC

Supernatants (6850), α-toxin (100 µg/ml):

No effect

in HUVEC

(Jurkat: 3-10 ng/ml sufficient)

B. Haslinger-Löffler, et al. (unpublished data)

Summary- S. aureus : facultative intracellular pathogen-

Endothelial adherence WTA-

and FnBP-dependent

(in vivo flow conditions)- Proinflammatory

stimulation of HMC (and EC?) indirect

-

Cellular invasiveness crucial for pathogenesis of complicated S. aureus infections (IE) in vivo

-

Cytotoxicity

for leukocytes by pore-forming toxins (α-toxin, PVL); apparently different mechanism in EC

-

Leucocyte

cell death occurs via mitochondrial pathway

=> Both aspects may contribute to pathogenesis of complicated infections

Acknowledgements•

Institut of Hygiene and Microbiology, Würzburg

Karina Lamprecht, Nadine LeitschuhAnke HellrungJohanna Priller, Matthias RaspeDaniel SchäferThiên-Trí

LâmDeepak Chikkaballi

Matthias Frosch

and co-workers

•

Theodor-Boveri-Institut Biozentrum, Würzburg

Georg Krohne

and co-workers

•

Institute of Medical Microbiology, Münster

Katrin Strangfeld, Michaela BrückCornelia WerbickMatthias GrundmeierBettina Haslinger-LöfflerMuzaffar

HussainGeorg Peters and co-workers

•

Division of Infectious Diseases, GenevaSabine Couzinet

(formerly

Bern)Daniel Lew and co-workers

•

Department for Pathology, GenevaKarl-Heinz Krause and co-workers

•

Institut of Bacteriology and Hygiene, Homburg

Mathias Herrmann and co-workers

•

University of DüsseldorfHeike Bantel

(now

Hannover)Klaus Schulze-Osthoff

and co-workers

•

University of HelsinkiKatri

Juuti-Savolainen, Pentti

Kuusela

•

University of LausanneYok-Ai

Que, Philippe Moreillon

•

Funding (cumulative)DFG (Deutsche Forschungsgemeinschaft),

IZKF and IMF Münster, Else-Kröner-

Fresenius-Stiftung, DKFZ-Scholarship