Post on 05-Nov-2021
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