mechanismsofaconof staphylococcaltoxins(partii) bd

Mechanisms of ac-on of staphylococcal toxins (Part II)

Pore-‐Forming Toxins

Phenol-‐Soluble Modulins (PSMs)

Centre Interna)onal de Recherche en Infec)ologie (CIRI, Lyon, France) Thomas Henry ([email protected])

ESCMID Online Lecture Library

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Staphylococcus aureus and its arsenal of ly-c factors

PFTs: e.g. α-‐hemolysin

Panton-‐Valen)ne Leukocidin (PVL)


β-‐hemolysin (sphingomyelinase)

Vandenesch F et al. 2012, FrontiersGaldiero S et al, Science 2004Wang R et al, Nat Med 2007


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Phenol Soluble Modulins (PSMs): defini-on Short pep)des: ≈20 AA for PSM-‐α and ≈40AA for PSM-‐βα-‐type: PSMα1-‐4, δ-‐toxin (encoded in RNAIII) , PSM-‐mec (encoded in a methicillin resistant cassePe) β-‐type: PSMβ1, β2

α-helical structure Amphipa)c: one face of the helix is hydrophilic one face is hydrophobic

Wang R. et al. Nat Med 2007 Mehlin C. et al. JEM 1999


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Phenol Soluble Modulins (PSMs): defini-on

Short pep)des: ≈20 AA for PSM-‐α and ≈40AA for PSM-‐βα-helical structure Amphipa)c: one face of the helix is hydrophilic one face is hydrophobic

Surfactant-‐like proper)es

Peschel A and OPo M. Nat Rev. Microbiol. 2014

PSM-‐α3 helical wheel projec)on


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Phenol Soluble Modulins (PSMs): ly-c pep-des wo receptors

Laabei M et al. BBA 2014

Synthe)c vesicles


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Phenol Soluble Modulins (PSMs): ly-c pep-des wo receptors

Laabei M et al. BBA 2014

Synthe)c vesicles T cells

Wang R et al. Nat Med 07


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Phenol Soluble Modulins (PSMs): ac-ng from within the phagosomes?

Surewaard BGB et al. PlosPath 2012

Liproteins inac)vate PSMs


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Phenol Soluble Modulins (PSMs): ac-ng from within the phagosomes?

Surewaard BGB et al. PlosPath 2012

Liproteins inac)vate PSMs

Surewaard BGB et al. CellMicro 2013 promoter PSMα::GFP

Neutrophil killing assay

Post-‐phagocytosis ESCMID Online Lectu

re Library

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Phenol Soluble Modulins (PSMs): Mode of secre-on and therapeu-c target

Secre-on by an ABC transporter

Red: ATPases Purple: Membrane Prot

ChaPerjee SS et al. Nat. Med 13


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Phenol Soluble Modulins (PSMs): Potent pro-‐inflammatory signals (FpR2 ac-va-on)

Kretschmer D et al. CHM 10


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Phenol Soluble Modulins (PSMs): required for virulence

Wang R et al. Nat Med 07


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Peschel A. and OPo M. , Nat Rev Microbiol. 2013

Phenol Soluble Modulins (PSMs): Virulence factors but not only!


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Staphylococcus aureus and its arsenal of ly-c factors: PFTs







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Staphylococcus aureus: 6 PFTs

homoheptamer: α-‐hemolysin

γ-‐hemolysin (HlgBC)γ-‐hemolysin (HlgAB)


LukED

Vandenesch F et al. 2012, Frontiers in cellular and Infection Microbiology

LukAB/GH

Bi-‐components PFTs: hetero-‐octamer


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Staphylococcus aureus: 6 PFTs of various prevalence

homoheptamer: α-‐hemolysin ≈100%

γ-‐hemolysin (HlgBC) ≈100%γ-‐hemolysin (HlgAB) ≈100%

Panton-‐Valen)ne Leukocidin (PVL) 3%≈100% depending of the countries

LukED≈80%

Vandenesch F et al. 2012, Frontiers in cellular and Infection MicrobiologyMcCarthy AJ et al. 2013, Infection, Genetics and Evolution

LukAB/GH≈100%

Bi-‐components PFTs: hetero-‐octamer


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PVL: a bi-‐component leukocidin targe-ng specifically phagocytes

•  Hetero-‐octamer β-‐barrel pore forming toxin.

Miles G et al.JBC. 2006


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15 years old

70 years old

The Panton Valen-ne Leukocidin: a leukocidin associated with severe infec-ons

•  Pro-‐phage encoded-‐toxin present in 3 to >90% of the clinical strains –  Associated with severe diseases (necro)zing pneumonia, furuncles)

–  Over-‐represented in CA-‐MRSA (present in clone USA300 highly prevalent in the USA) Panton PN et al. Lancet 1932 Lina G et al. CID 1999 Gillet Y et al., The Lancet 2002

Death of patients Severe S.aureus pneumonia


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The Panton Valen-ne Leukocidin: a human specific leukocidin

Loffler B et al. PLoS Path 2010


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The Panton Valen-ne Leukocidin: lesson from a rabbit model of infec-on

Diep B et al, PNAS 2010


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Current model to explain the role of PVL in inflamma-on

Diep B et al, PNAS 2010 Perret M et al, Cell Micro 2012

IL-‐1β

IL-‐1β


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The Panton Valen-ne Leukocidin: Iden-fica-on of the host receptor

X •  56 receptors, leukocytes •  An)body binding inhibi)on assay

LukS-‐PV interacts with Human C5a Receptor

Spaan et al. CHM 2012


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The Panton Valen-ne Leukocidin: LukS-‐PV Binds C5a Receptors

Human C5a receptor (CD88) n  G-‐protein coupled receptor

C5a

C5a

C5a


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PVL Targets C5a Receptors PI

PVL induced pore forma)on is mediated by C5aR


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C5aR expression level determines PVL Cell Specificity

C5aR is most abundantly expressed on neutrophils and monocytes but not on lymphocytes


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C5aR Determines PVL Species Specificity

C5aR mediated suscep)bility is in line with suscep)bility of primary cells

J Infect Dis (2009); PLoS Pathog (2010). Spaan A. et al, CHM 2013


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Leukocidin Myeloid Host Counterparts-‐Progressing beyond redondancy

HlgCB

LukAB

HlgAB

LukED PVL

C5aR

C5L2

CCR5

CXCR1

CXCR2

CCR2

CD11b

a n  All receptor are myeloid-‐specific n  They define both the cell and the species specifici)es n  Mostly extracellular but intracellular ac)on for LukAB n  Subly)c roles to be determined. Cell Host Microbe (2013); Nature (2013); Proc Natl Acad Sci USA (2013); Cell Host Microbe (2014); Nat Commun (2014).


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Therapies targe-ng PFTs-‐1: An-bio-c

Dumitrescu O. et al CMI 2008 Dumitrescu O. et al AAC 2011

Oxacillin

PB1

sarA + rot

LukS/F-‐PV induc)on


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Therapies targe-ng PFTs-‐2: Monoclonal an-bodies

i.n. Ab 24h BI

i.v. Ab 24h BI

i.v. Ab 2h PI

Rouha H et al. mAbs 2014


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DARC n  Duffy An)gen Receptor of Chemokines

n  7-‐Transmembrane receptor n  Related to CXCR1/2 n  Binds CXCL8

n  Erythrocytes n Duffy blood groups n P. vivax entry-‐receptor

Nat Genet (1995); Nat Immunol (2009).

Amino acids critical for the binding of CXCL-8As shown above, quantitative flow cytometric analysisusing three different anti-Fy mAbs as well as immunopre-cipitation experiments indicated that sufficient cell surfaceexpression of DARC protein was achieved on all recombin-ant cells, enabling the testing of CXCL-8 binding. K562-transfected cells were incubated for 1 h with 125I-labelledCXCL-8 (0Æ5 nmol/l), and the specific cell-associated radio-activity was measured as described in Materials andmethods. Results were adjusted to take into account therelative surface expression of the different DARC mutants.As shown in Fig 4, 30 out of the 39 alanine substitutionshad no or moderate effect on chemokine-binding capacity ofmutant cells with a residual binding capacity above 40%that of wild-type DARC. Nine other mutants exhibited adrastic reduction in CXCL-8 binding capacity (Fig 4).Alanine substitution of F22–E23 and P50 located in theECD1, of D263, R267 and D283 located in the ECD4 as wellas mutation of C51, C129, C195 and C276 located in ECD1,ECD2, ECD3 and ECD4, respectively, abrogated CXCL-8binding (at least 85% reduction compared with wild type). It

is assumed that these four cysteine residues are involved indisulphide bridges as these mutations also impaired thebinding of anti-Fy3, which recognizes amino acids presentin three ECDs (Table I). Of note, mutation of cysteine C54had no effect on anti-Fy binding (Table I) or CXCL-8 binding(Fig 4). Q19 and L20 were also mutated because theybelong to the QLDFEDV epitope, recognized by the i3A mAb(Wasniowska et al, 2000a), another anti-Fy6 mAb, which,like 2C3, is an antagonist of CXCL-8 binding (Tournamilleet al, 1997). As shown in Fig 4, these mutations did notalter CXCL-8 binding. Finally, in preliminary experiments,we found that the binding of CC chemokine ligand 5 (CCL5;RANTES) was not affected by mutations that severelyaltered the binding of CXCL-8 (data not shown).

N-glycosylation status of DARC and ligand bindingto transfected cellsAsparagines at positions 16 and 27 were individuallymutated to disrupt the two potential N-glycosylation sites(N16-SS and N27-SS) of DARC. To analyse the consequenceof these mutations on the N-glycosylation status of DARC,

Fig 2. Schematic representation of DARC showing the amino acid residues that are critical for anti-Fy mAbs and CXCL-8 binding. Role ofmutated residues present in the extracellular domains (ECDs): black, critical for CXCL-8 binding; grey, not involved in the CXCL-8 binding site.Residues critical for Fya and Fy6 epitopes, recognized by the 655 and 2C3 mAbs, respectively, are depicted according to our presentmutagenesis analysis and differ slightly from those characterized previously by PEPSCAN analysis (Wasniowska et al, 2000b, 2002). Starshighlight the amino acid residues participating in the Fy3 epitope recognized by the CRC-512 anti-Fy3 mAb. Putative disulphide bridges (C51–C276 and C129–C195) are indicated by broken arrows.

1018 C. Tournamille et al

! 2003 Blackwell Publishing Ltd, British Journal of Haematology 122: 1014–1023

Duffy Fya Fyb Fyweak Fynull ESCMID Online Lecture Library

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n  DARC expression is required and sufficient for hemolysis by both HlgAB and LukED

DARC is the Erythroid Leukocidin Receptor

Fyb+/b+

Fya-/b

-

Fyb+w

eak /Fyb

+wea

k

Fyb+w

eak /Fyb

-

102

103

104 DARC

Rec

epto

r num

ber

B

D

C D

1 10 1000

20

40

60

80

100Fy a+/b+Fy a+/a+Fy a+/b+weak

Fy b+/b+Fy b+weak/Fy b+weak

Fy b+weak/Fy b-Fy a-/b-

Concentration HlgAB (nM)

% H

emol

ysis

Cell Host Microbe (2015).

E F

1 10 100 10000

20

40

60

80

100Fy a+/b+Fy a+/b-Fy a-/b+

Fy a-/b-

Fy b+weak/Fy b+weak

Fy b+weak/Fy b-

Concentration LukED (nM)

% H

emol

ysis


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n  Hemolysis during bacterial growth is mediated by leukocidins and DARC

Hemolysis by S. aureus

Cell Host Microbe (2015).

DARC+

DARC+ DARC-

DARC-

USA300 LAC WT USA300 LAC WT

B

A

Figure S4 (Related to Figure 4). S. aureus lyses erythrocytes in a γ-hemolysin and DARC dependent manner.(A) Bacteria were grown in the presence of erythrocytes from donors with or without erythroid expression of DARC. Microscopic images of S. aureus strain USA300 clone LAC and its isogen-ic hlgA mutant strain (hlgA::bursa) grown overnight show depletion of erythrocytes in a DARC and hlgA dependent manner. Pictures show representative samples, with infectious dose set at 1x106 CFU per sample. (B) Bacteria were grown in the presence of erythrocytes from donors with or without erythroid expression of DARC. Hemolysis induced overnight during growth of S. aureus strain USA300 clone SF8300, strain Newman, strain V8, and their respective hlgACB mutant isogenic strains (inoculation set at 1x106 CFU per sample). Hemolysis is expressed as OD405 nm of the super-natant. Bars indicate SEM, with n = 2. (C) Immunoblot analyses of filtered culture supernatants of S. aureus strains Newman wild type (WT) and isogenic mutants Δhla, ΔhlgΔlukED and ΔisdBH against LukA, Hla, LukD, and HlgC antisera. Immunoblots are representative of two individual isolates per strain.

USA300 LAC ΔhlgAUSA300 LAC ΔhlgA

C

0.0

0.5

1.0

1.5

2.0

WThlgACB

DARC+

USA300-SF8300

DARC-

OD

405

nm

0.0

0.5

1.0

1.5

2.0

WThlgACB

DARC+

Newman

DARC-

0.0

0.5

1.0

1.5

2.0

WThlgACB

DARC+

V8

DARC-

WT ΔhlaΔhlg

ΔlukEDΔisdBHAnti-LukA

Anti-Hla

Anti-LukD

Anti-HlgC

*

*

for initial binding of LukS-PV (Spaan et al., 2013a). Possibly,sulfated N-terminal tyrosines define a conserved host interactionsite for the staphylococcal leukocidins. Otherwise, our datashow that HlgAB and LukED interact differentially with DARC.An N-terminal cysteine (C51) identified as involved in the interac-tion of DARCwith LukED is also involved in bindingCXCL8 (Tour-namille et al., 2003), supporting the notion that this chemokinedirectly blocks receptor binding by LukE.

The genes encoding HlgAB are present in over 99.5% of hu-man S. aureus isolates (Prevost et al., 1995). Strictly followingclonal lineage, approximately 80% of S. aureus strains carrythe genes encoding LukED (McCarthy and Lindsay, 2013). TheS. aureus strains investigated in this study all contain the genesencoding HlgAB and LukED, thus demonstrating that S. aureus-mediated hemolysis requires DARC and these leukocidins.

S. aureus is remarkably well adapted to the human host, thusmultiple virulence factors of this bacterium are not compatiblewith non-human species frequently used during in vivo studies.One such factor is the staphylococcal hemoglobin receptorIsdB, which exhibits low affinity for murine hemoglobin ascompared to human hemoglobin (Pishchany et al., 2010). Never-theless, our in vivo studies revealed a remarkable similarity in thephenotypes of isogenic mutants lacking either the hemoglobinreceptors or the hemolytic leukocidins, suggesting that thesetoxins contribute to nutrient acquisition during infection. How-ever, to unequivocally demonstrate that the attenuated pheno-type exhibited by theDhlgDlukED strain is due to impaired eryth-rocyte lysis, additional studies uncoupling the leukocidal andhemolytic activities of HlgAB and LukED are required.

The current epidemic of CA-MRSA in the United States andelsewhere disproportionally affects individuals of African

descent with severe and invasive infections (Fridkin et al.,2005). Socio-economic factors and other underlying diseaseslikely contribute to this predisposition, precluding epidemiolog-ical assessment of the contribution of erythroid DARCexpression to S. aureus infection. However, the resistance ofDARC negative erythrocytes to the parasites P. vivax andP. knowlesi, together with our findings, further support thenotion that this gene could undergo positive selection inresponse to different diseases caused by important humanpathogens.

EXPERIMENTAL PROCEDURES

Ethics StatementDARC blood samples were provided by the Centre National de Reference sur

les Groupes Sanguins (CNRGS, Paris). Additional blood samples of consent-

ing, healthy volunteers were obtained in accordance with the Declaration of

Helsinki. Approval was obtained from the medical ethics committee of the

UMC Utrecht, The Netherlands. Blood was also obtained from de-identified,

consenting donors from the New York Blood Center.

All experiments involving animals were reviewed and approved by the Insti-

tutional Animal Care andUseCommittee of New York University and were per-

formed according to NIH guidelines, the Animal Welfare Act, and US Federal

law.

Hemolysis Assays with Recombinant ToxinsErythrocytes were washed thrice in 0.9% saline, adjusted to 5 3 107 cells/ml,

and then intoxicated at a final of 2.5 3 107 cells/ml per reaction with purified

recombinant toxins for 30 min at 37!C + 5% CO2 in a final volume of 160 ml.

Equimolar concentrations of 6xHis-tagged proteins were used. Samples

were centrifuged for 10 min at 1,780 3 g, 4!C, and 100 ml of cell-free lysates

were used to measure absorbance (OD405 nm). Hemolysis is expressed as

the OD405 nm of cell-free lysates using an EnVision Plate Reader. The hemo-

lysis experiments with recombinant proteins were performed using buffer

A B

C D

Figure 4. S. aureus Lyses Erythrocytes in aHlgAB-, LukED-, and DARC-DependentManner to Release Iron and Promote Growth(A) S. aureusUSA300 LAC grown in the presence of

erythrocytes from donors with or without erythroid

expression of DARC and hemolysis measured.

Curves depict a representative sample.

(B) Hemolysis induced during overnight growth of

S. aureus strain USA300 LAC and its hlgA mutant

(hlgA::bursa) strain (infectious dose set at 1 3 106

CFU per sample). n = 3 ± SEM.

(C) Growth after 20 hr of S. aureus strains Newman

WT or isogenic DisdBH as a result of erythrocyte

lysis by LukED and HlgAB in iron-restricted me-

dium. n = 9 ± SEM. Statistical significance is dis-

played as ns (not significant), *p < 0.05, **p < 0.01,

and ****p < 0.0001 using one-way ANOVA with

Tukey’s post hoc test correction for multiple com-

parisons. Bacterial growth was measured at

OD600 nm.

(D) Swiss-Webster female mice (n = 10 mice per

group) infected systemicallywithS. aureusNewman

isogenicstrains:WT,DhlgDlukED,Dhla, andDisdBH

("1 3 107 colony forming units, CFU). 96 hr post

infection, mice were sacrificed and bacterial burden

in the liver determined. Lines represent median log

CFU. Statistical significance is displayed as ns (not

significant), *p < 0.05, **p < 0.01, and ****p < 0.0001

using one-way ANOVA with Tukey’s post hoc test

correction for multiple comparisons.

See also Figure S4.

CHOM 1319

6 Cell Host & Microbe 18, 1–8, September 9, 2015 ª2015 Elsevier Inc.

Please cite this article in press as: Spaan et al., Staphylococcus aureus Targets the Duffy Antigen Receptor for Chemokines (DARC) to Lyse Eryth-rocytes, Cell Host & Microbe (2015), http://dx.doi.org/10.1016/j.chom.2015.08.001


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