CCR5 is a receptor for Staphylococcus aureus leukotoxin ED

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1 oi:1.138/nature11724 is a reeptor for Staphyloous aureus leukotoxin ED Franis Alonzo III 1, Lina Kozhaya 1 *, Stephen A. Rawlings 1 *, Tamara Reyes-Robles 1 *, Ashley L. DuMont 1, Davi G. Myszka 4, Nathaniel R. Lanau 1, Derya Unutmaz 1,2,3 & Vitor J. Torres 1 Pore-forming toxins are ritial virulene fators for many baterial pathogens an are entral to Staphyloous aureus-meiate killing of host ells. S. aureus enoes pore-forming bi-omponent leukotoxins that are toxi towars neutrophils, but also speifially target other immune ells. Despite eaes sine the first esription of staphylooal leukoial ativity, the host fators responsible for the seletivity of leukotoxins towars ifferent immune ells remain unknown. Here we ientify the human immunoefiieny virus (HIV) o-reeptor as a ellular eterminant require for ytotoxi targeting of subsets of myeloi ells an T lymphoytes by the S. aureus leukotoxin ED (). We further emonstrate that -epenent ell killing is bloke by reeptor antagonists, inluing the HIV rug maraviro. Remarkably, -efiient mie are largely resistant to lethal S. aureus infetion, highlighting the importane of targeting in S. aureus pathogenesis. Thus, epletion of 1 leukoytes by suggests a new immune evasion mehanism of S. aureus that an be therapeutially targete. S. aureus is a baterial pathogen that auses signifiant morbiity an mortality worlwie. The organism is responsible for a myria of iseases, from skin an soft-tissue infetions, to more invasive iseases inluing nerotizing pneumonia an sepsis. S. aureus seretes several protein prouts that allow the organism to subvert the host immune system. Suh fators inlue super-antigens, antiboy bining proteins, ytolyti pepties an pore-forming ytotoxins 1. Pore-forming toxins are serete by a substantial number of pathogeni bateria 2. The toxins are serete as water-soluble monomers that reognize host ell membranes, oligomerize, an insert a-helial or b-barrel pores into the lipi bilayer 2. Pore formation isrupts osmoti balane an membrane potential, ultimately leaing to ell eath 2. S. aureus strains that infet humans proue up to four ifferent b-barrel, bi-omponent, pore-forming toxins (HlgACB,, -PV/PVL an /HG) that exhibit a unique tropism for host immune ells an ontribute to the greater virulene of S. aureus 1,3,4. The preise repertoire of immune ells targete by the pore-forming leukotoxins remains to be fully etermine. Even now, more than a entury sine the first esription of staphylooal leukoial ativity,6, our unerstaning of leukotoxin funtion in vivo is limite beause of an absene of known host-erive speifiity eterminants. is require for ytotoxiity To ientify potential leukotoxin reeptors, we purifie reombinant, an -PV an assesse their ability to kill a set of human ell lines 4,7. Granuloyte-like human ells (PMN-HL6) were kille in 1 h by an -PV, but not (Fig. 1a). In ontrast, was ytotoxi to a human T-ell line etopially expressing (HUT-R); whereas another T-ell line (Jurkat), whih laks etetable, was insensitive (Fig. 1a). This suggeste that was involve in ytotoxiity towars HUT-R ells. Aoringly, when amounts were reue in HUT-R ells using lentiviral short hairpin RNA (shrna), the ells were protete from -meiate killing (Fig. 1b an Supplementary Fig. 1a, b). Complementary to these finings, etopi expression of was suffiient to rener Jurkat an H9 ells (Supplementary Fig. 1) suseptible to ytotoxiity (Fig. 1). As expete, on the basis of the moe of ation of the bi-omponent leukotoxins, - epenent -meiate ytotoxiity require both an LukD subunits (Supplementary Fig. 2a, b). A human osteosaroma ell line engineere to onstitutively express (GHOST.R ells) 8 was also sensitive to, but not to or -PV (Fig. 1). The sensitivity of GHOST ells to was speifi to expression, as overexpression of aitional T-ell-speifi hemokine reeptors (CCR1, CCR2, CCR3, CXCR4, CCR8 an CXCR6) in these ells i not onfer suseptibility to (Supplementary Fig. 2). antagonists blok ell killing is a o-reeptor require for HIV infetion 9 11 an has been targete with small moleule antagonists aime at restriting HIV entry into host ells 11. We foun that one suh linially approve reeptor antagonist, maraviro, potently bloke killing of 1 ells (Fig. 1e an Supplementary Fig. 3a) at onentrations similar to those require to blok HIV infetion (Supplementary Fig. 3b). Similar inhibitory effets were observe with the antagonists viriviro an TAK-779, as well as hemokines that are natural ligans of (Supplementary Fig. 3a, ) 12,13. We foun that maraviro resulte in omplete blokae of pore formation, an essential proess for ytotoxiity (Fig. 1f an Supplementary Fig. 3). We next investigate whether S. aureus was able to kill 1 ells in a -epenent manner. The expression of luked in S. aureus is inherently low uring in vitro growth 7. However, eletion of the transription fator Rot, a potent repressor, results in the enhane expression an proution of by S. aureus 7. Thus, to assess S. aureus ytotoxiity towars 1 ells, Jurkat or Jurkat-R ells were infete with S. aureus Drot (Sa 1 ) an S. aureus 1 Department of Mirobiology, New York University Shool of Meiine, New York, New York 116, USA. 2 Department of Pathology, New York University Shool of Meiine, New York, New York 116, USA. 3 Department of Meiine, New York University Shool of Meiine, New York, New York 116, USA. 4 Biosensor Tools LLC, Salt Lake City, Utah 8413, USA. *These authors ontribute equally to this work. 212 Mamillan Publishers Limite. All rights reserve MONTH 212 VOL NATURE 1

2 RESEARCH ARTICLE a PMN-HL6 HUT-R 1 Toxin onentration (μg ml 1 ) e g HUT-R + (1 μg ml 1 ).1.1 H9-R Jurkat-R H9 HSA Jurkat HSA Jurkat 1 1, Maraviro onentration (ng ml 1 ) Uninfete Jurkat Sa luked + Sa luked Uninfete 1. Jurkat-R Sa luked + Sa luked b 1 DrotDlukED (Sa 2 ) mutants. Jurkat-R ells were kille by S. aureus in a -epenent manner, whereas Jurkats laking were resistant to killing (Fig. 1g). Aitionally, Jurkat-R killing by S. aureus was ompletely bloke by maraviro (Fig. 1h). interats iretly with To haraterize more preisely the interation on target ells, we first etermine whether monolonal antiboies speifi towars extraellular regions of (ref. 14) were suffiient to blok toxin ativity (Fig. 2a). Antiboies against extraellular loop 2 (ECL-2), but not the amino (N) terminus of the reeptor or CXCR4, signifiantly bloke toxin killing (Fig. 2a) an prevente assoiation of funtional green fluoresent protein (GFP)-labelle toxin (Supplementary Fig. 4) with the ell surfae of sorte primary human CD4 1 1 T ells (Fig. 2b). Furthermore, toxin assoiation with the ell surfae of 1 ells was also reue in the presene of Toxin onentration (μg ml 1 ) 1 GHOST GHOST 8 R f EtBr (RFU/1 4 ) h HUT-R + sh(ctrl) HUT-R + shr 1 No MVC + MVC (1 μg ml 1 ) 4 6 Time (min) Jurkat-R MVC: + + Uninfete Sa luked + Figure 1 requires for ell killing. a, Viability of ells expose to ifferent leukotoxins (1 mgml 21 ). b, Viability of HUT-R ells transue with ontrol (Ctrl) or shrnas., Viability of Jurkat an H9 ells transue with (-R) or mouse CD24 (-HSA) followe by treatment with., Viability of GHOST ells overexpressing an treate with iniate leukotoxins. e, Viability of HUT-R pre-inubate with maraviro an treate with. f, Pore formation, as measure by ethiium bromie uptake, on Jurkat-R with or without maraviro (MVC; 1 ng ml 21 ) followe by inubation with. g, h, Viability of Jurkat or Jurkat-R ells infete with S. aureus (g), in the presene or absene of MVC (h). Means 6 s.. (n 3) are shown. a 1 GFP positive (%) f Anti-HA No toxin Anti-His g Response (RU)1 No toxin CXCR A9 CXCR4 <6% GFP positive (%) <8% E + CCL E + CCL4 E + CCL3 E CXCR Response (RU)1 3A b GFP GFP 8 8 Anti-HA Time (s) CCL MVC Anti-His 1 2 MVC MVC+ E + 3A9 2 3 E + CXCR4 CCL, an was ompletely bloke upon aition of maraviro, similar to CD4 1 2 T ells (Fig. 2). To etermine whether interats with, pull-own assays were onute with purifie toxin an solubilize. We foun that interate with but not LukD (Fig. 2). This interation was signifiantly reue in the presene of maraviro, natural ligans of, as well as monolonal antiboy 431 irete against ECL-2, but not 3A9 irete against the N terminus of (Fig. 2e, f). Aitionally, inubation of (7-fol molar exess) with 1 ells largely blunte native ligan-inue signalling as measure by alium mobilization (Supplementary Fig. ). itself oes not seem to inue signalling (Supplementary Fig. 6a, b). Surfae plasmon resonane stuies with immobilize native (ref. ) an purifie or LukD subunits onfirme the pull-own stuies an etermine that, but not LukD, bins to in a e Anti-HA Anti-His N 3A Input No Time (s) C Multiomain toxin LukD LukD Figure 2 iretly interats with. a, Viability of ells treate with anti- monolonal antiboies (3 mgml 21 ) followe by exposure to (1 mgml 21 ). b, Membrane assoiation of GFP (1 mgml 21 )to the surfae of primary CD4 1 1 T ells with or without the iniate monolonal antiboies (2 mgml 21 ) as etermine by fluoresene-ativate ell sorting (FACS)., Membrane assoiation of GFP (1 mgml 21 )on the surfae of primary CD4 1 1 T ells with or without maraviro (MVC) (1 ng ml 21 ), CCL ( mgml 21 )oroncd4 1 2 T ells. f, Interation between His-, LukD, or an HA- (), with or without MVC ( mgml 21 )(e), CCL, CCL4, CCL3 (1 mgml 21 )(f) an monolonal antiboies 431, 3A9, CXCR4 (3 mgml 21 )(f). In f, E stans for the toxin subunit. Immunoblots are representative of at least two inepenent experiments. g, Interation of with an CXCR4 by surfae plasmon resonane. Representative sensorgrams (g) of two experiments performe in upliate are shown. Where relevant, means 6 s.. (n 3) are shown. 2 NATURE VOL MONTH Mamillan Publishers Limite. All rights reserve

3 RESEARCH time-epenent an saturable manner, with an apparent issoiation onstant (K ) of nm (Fig. 2g an Supplementary Fig. 7a, b). This interation was speifi, as eviene by an inability of to bin native CXCR4 (Fig. 2g). kills 1 myeloi ells an T ells We next sought to etermine the subsets of primary human lymphoi an myeloi ells targete by. Treatment of bloo lymphoytes with resulte in speifi epletion of 1 T ells, most of whih were effetor memory T lymphoytes (Fig. 3a an Supplementary Fig. 8). As with ell lines, the -epenent killing of primary ells was ompletely bloke by maraviro (Fig. 3a an Supplementary Fig. 8). A proportion of iniviuals of Northern European heritage harbour a 32 base-pair eletion in the gene (D32 ), resulting in a trunate protein that annot be surfae loalize, thus renering the CD4 1 T ells refratory to HIV infetion 11,16,17. Similarly, primary T ells expane from a D32 onor were also resistant to ytotoxiity (Fig. 3b). In keeping with the notion that is require for HIV-1 entry into CD4 1 T ells 9 11, seletive epletion of 1 T ells by suppresse HIV-1 sprea (Supplementary Fig. 9). Memory T ells an be lassifie into funtional subsets on the basis of ifferential hemokine reeptor profiles an ytokine proution. Among T-ell subsets, the CCR6 1 1 subset proues more interleukin (IL)-17 an interferon (IFN)- than CCR6 1 2 T ells 18. Consistent with this assoiation, epletion of 1 CD4 1 T ells with greatly reue the proportion of IFN-- an IL-17-prouing ells ompare with purifie CD4 1 T-ell ontrols (Fig. 3, ay ). Inubation with the -ytokines IL-7 an IL- a CD3 b WT Δ Meia + MVC Meia + MVC Marophages Meia + MVC Denriti ells Meia + MVC CCR6 + CCR Day Control IL IL-22 Day 7 (IL-7 + IL-) Control IL-17 IL Figure 3 kills 1 human memory T ells, marophages an enriti ells. a, Total 1 primary human T ells (CD3 1 / 1 ) inubate with meia, (2. mgml 21 ) or maraviro (MVC; 1 ng ml 21 ) followe by treatment. b, Suseptibility of T ells isolate from a D32- or WT- onor. Cell viability an expression evaluate by flow ytometry as in a., Cytokine proution of CD4 1 T ells with or without treatment ( mgml 21 ) that were stimulate on ay with PMA an ionomyin (P 1 I; top panel) or ulture in meia supplemente with IL-7/IL- (2 ng ml 21 ) for 7 ays followe by stimulation with P1I (bottom panel)., Viability of monoyte-erive marophages an enriti ells inubate with (3. mgml 21 with or without MVC). For FACS plots (a ), a representative from one of three inepenent onors is shown. Bar graphs, mean 6 s.. of results from three inepenent onors. signifiantly enhanes the proportion of IL-17 1 an IL-17 1 /IFN- 1 by CCR6 1 memory T ells 19. We foun that when human CD4 1 T ells were first treate with, followe by 7 ays of ulture with IL-7 an IL-, there was a substantial reution in the inution of IFN- an IL-17/IL-22-sereting CCR6 1 T ells (Fig. 3). This fining orrelates well with epletion of the CCR6 1 1 memory progenitor subset (Supplementary Fig. 1). In aition to Th1 an Th17 effetor ells, also kille marophages an enriti ells in a - epenent manner (Fig. 3). targets 1 ells in vivo Next we examine the ontribution of to S. aureus pathogenesis an etermine the influene of on the targete killing of 1 ells in vivo. We foun that murine (m) reners transfete 293T ells fully suseptible to the toxin (Supplementary Fig. 11a, b). Aitionally, primary murine marophages treate with high onentrations of maraviro were partly protete from toxinmeiate killing, onfirming that is iretly targeting m (Supplementary Fig. 11). Beause maraviro is potent towars human but not m (Fig. 1e an Supplementary Fig. 11) 2, we hose to stuy wil-type (WT) an -efiient mie with the hypothesis that the latter woul be resistant to ytotoxiity. S. aureuseliite lymphoytes an marophages from WT mie were highly suseptible to purifie, whereas lymphoytes an marophages isolate from 2/2 mie were markely resistant (Fig. 4a, b). To valiate further that S. aureus kills 1 leukoytes in vivo, we implemente a peritonitis moel in whih WT an 2/2 mie were infete with S. aureus. surfae expression was not require for the initial influx of immune ells to the infetion site, as the ells reovere an their profiles were iential among all mie (Supplementary Fig. 12). However, lymphoytes an marophages eliite in vivo in WT mie were more suseptible to S. aureus killing than those from the 2/2 mie (Fig. 4, ). is assoiate with S. aureus pathogenesis in a murine moel of systemi infetion 7. Using this moel, 2/2 mie infete with WT S. aureus exhibite signifiantly reue baterial buren in the kineys than those of infete WT mie (Fig. 4e), a phenotype similar to that observe for mie infete with a S. aureus DlukED mutant 7. After 96 h, infete 2/2 mie also exhibite signifiantly reue serum pro-inflammatory ytokines an hemokines an showe a ommensurate reution in innate immune ells in the kiney ompare with WT mie (Fig. 4f, g), signs onsistent with infetion resolution. Aitionally, when WT mie were hallenge systemially with WT or a DlukED mutant, we observe -epenent killing of 1 marophages in infete kineys, onsistent with our hypothesis that is apable of targeting 1 leukoytes uring infetion (Fig. 4h). In support of the importane of targeting in vivo, the mortality assoiate with S. aureus bloostream infetion was reue for -efiient mie, a phenotype similar to that of mie hallenge with strains of S. aureus laking luked (Fig. 4i). Disussion To our knowlege, is the first esribe ellular reeptor that is neessary an suffiient for the killing of mammalian ells by a staphylooal bi-omponent leukotoxin. Thus, in aition to HIV, Toxoplasma gonii an poxviruses (vainia an myxoma) 9,21 24, S. aureus an also exploit to target immune ells. Interestingly, the D32 allele of is thought to have been aquire through seletive pressure imparte by a ealy pathogen 2,26. Yersinia pestis or variola virus were postulate as potential riving fores behin this seletion, but these hypotheses have either been isounte or remain unertain in favour of an oler seletion event inite by an immuneell-targeting pathogen 24,27. Our finings put forth the possibility that resistane to S. aureus leukotoxins may have influene the seletion of the D32 allele. 212 Mamillan Publishers Limite. All rights reserve MONTH 212 VOL NATURE 3

4 RESEARCH ARTICLE a Lymphoytes b Marophages e Kiney f GCSF RANTES GM-CSF IL-6 MCP * * 3 * 1,2 12 * , ml NS ng 2 * Lymphoytes Marophages CFU ml 1 3 * Figure 4 1 ell killing is important for S. aureus pathogenesis. a, b, Viability of primary murine peritoneal-eliite immune ells from R 1/1 (n 3) or R 2/2 (n 3) mie after inubation with (1 mgml 21 ).,, In vivo viability of reruite immune ells from R 1/1 (n 1) or R 2/2 (n 1) mie hallenge with live S. aureus Drot. e, Baterial olony-forming units (CFU) reovere from the kineys of R 1/1 (n 8) or R 2/2 (n 9) mie infete for 96 h with WT S. aureus. f, Serum ytokine an hemokine amounts from animals in e. g, Quantifiation of neutrophils an marophages g Neutrophils (%) i Survival (%) Marophages (%) Time (h).1.1 * * h R / + WT Sa R +/+ + Sa ΔlukED R +/+ + WT Sa Marophages WT ΔlukED WT ΔlukED reovere from infete kineys 96 h after infetion. h, In vivo viability of reruite marophages from R 1/1 mie hallenge with S. aureus WT (n 1) or DlukED (n 1). i, Survival of R 1/1 mie infete with WT S. aureus (n 1) or a DlukED mutant (n 1) an R 2/2 infete with WT S. aureus (n 2). FACS plots show a representative from one of 1 infete animals. *P,.; P #.1; *P #.1 by one-way analysis of variane (a, b), Stuent s t-test ( h) an Mantel Cox test (i). Bar graphs, mean 6 s.. The fining that seletively kills 1 T ells, marophages an enriti ells extens the repertoire of immune ells targete by this leukotoxin an supports a role for these leukoytes in the resolution of S. aureus infetion. The luked gene is believe to be present in many linially relevant strains (.7%) inluing lones responsible for most infetions in the USA an Germany, although it is absent in a subset of strains ausing hospital-aquire infetion (for example EMRSA/16) in the UK Most isolates laking luked seem to be of lonal omplex 3 (USA2/EMRSA16), whih is known to proue low amounts of ytotoxins 31. Coneivably, the pathogenesis of these strains is influene by the weakene immune status of hospitalize patients rather than toxi moleules. In ontrast, we preit that virulent linial strains prouing large amounts of (for example, lonal omplex 8) 7 use the toxin to eliminate antigen-presenting ells as well as S. aureus-speifi 1 Th1/ Th17 ells, whih are inue by the baterium 32 an are protetive against infetion 33,34. In support of this hypothesis, we emonstrate that kills 1 ells in vivo uring systemi infetion an that mie laking are protete from the mortality assoiate with aute S. aureus isease. Current systemi murine infetion moels are insuffiient to evaluate reliably hi T-ell suseptibility to (ata not shown). However, our in vitro ata an in vivo stuies with 1 marophages strongly support the notion that subsets of hi T ells are also targete in vivo. Interestingly, -meiate toxiity towars neutrophils an monoytes is not bloke by maraviro (ata not shown), suggesting targets these ells through alternative an non-reunant mehanisms. This point also implies a role for 1 myeloi ells an T ells in resolving aute infetion, one that extens beyon the initial ontrol of infetion imparte by neutrophils. The fining that toxiity towars 1 ells is potently neutralize by a linially approve antagonist (maraviro) suggests that these types of rug oul provie muh-neee therapeuti alternatives in the treatment of S. aureus infetions. METHODS SUMMARY Cell lines an primary human ells were maintaine in RPMI plus 1% fetal bovine serum with peniillin an streptomyin, unless supplemente as otherwise iniate, an were inubate with, LukD or as previously esribe 7. All bloo samples were obtaine from anonymous healthy onors as buffy oats (New York Bloo Center). The New York Bloo Center obtaine written informe onsent from all partiipants. Animal experiments were performe in aorane with the Institutional Animal Care an Use Committee at New York University Shool of Meiine. -overexpressing ell lines an shrna knokowns were generate by lentiviral-base tranution as previously esribe 3. Isolation of human peripheral bloo mononulear ells (PBMC) an their sorte subsets was performe as previously esribe 19. Full Methos an any assoiate referenes are available in the online version of the paper. Reeive 28 Marh; aepte 26 Otober 212. Publishe online 12 Deember Foster, T. J. Immune evasion by staphylooi. Nature Rev. Mirobiol. 3, (2). 2. Bishofberger, M., Iaovahe, I. & Gisou van er Goot, F. Pathogeni pore-forming proteins: funtion an host response. Cell Host Mirobe 12, (212). 3. Menestrina, G. et al. Ion hannels an baterial infetion: the ase of b-barrel poreforming protein toxins of Staphyloous aureus. FEBS Lett. 2, 4 6 (23). 4. Dumont, A. L. et al. Charaterization of a new ytotoxin that ontributes to Staphyloous aureus pathogenesis. Mol. Mirobiol. 79, (211). 4 NATURE VOL MONTH Mamillan Publishers Limite. All rights reserve

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Genes Immun. 6, (2). 26. Luotte, G. Frequenies of 32 basepaireletion ofthe (D32) allele ofthe HIV- 1 o-reeptor gene in Cauasians: a omparative analysis. Infet. Genet. Evol. 1, 21 2 (22). 27. Herik, P. W. & Verrelli, B. C. Groun truth for seletion on -D32. Trens Genet. 22, (26). 28. Moore, P. C. & Linsay, J. A. Moleular haraterisation of the ominant UK methiillin-resistant Staphyloous aureus strains, EMRSA- an EMRSA-16. J. Me. Mirobiol. 1, (22). 29. Vanenesh, F. et al. Community-aquire methiillin-resistant Staphyloous aureus arrying Panton-Valentine leukoiin genes: worlwie emergene. Emerg. Infet. Dis. 9, (23). 3. von Eiff, C., Frierih, A. W., Peters, G. & Beker, K. Prevalene of genes enoing for members of the staphylooal leukotoxin family among linial isolates of Staphyloous aureus. Diagn. Mirobiol. Infet. Dis. 49, (24). 31. DeLeo,F.R.et al. Moleular ifferentiation of histori phage-type 8/81 an ontemporary epiemi Staphyloous aureus. Pro Natl Aa Si USA 18, (211). 32. Zielinski, C. E. et al. Pathogen-inue human T H 17 ells proue IFN- or IL-1 an are regulate by IL-1b. Nature 484, (212). 33. Cho, J. S. et al. IL-17 is essential for host efense against utaneous Staphyloous aureus infetion in mie. J. Clin. Invest. 12, (21). 34. Lin, L. et al. Th1-Th17 ells meiate protetive aaptive immunity against Staphyloous aureus an Cania albians infetion in mie. PLoS Pathog., e173 (29). 3. Oswal-Rihter, K. et al. Ientifiation of a -expressing T ell subset that is resistant to R-tropi HIV infetion. PLoS Pathog. 3, e8 (27). Supplementary Information is available in the online version of the paper. Aknowlegements We thank members of the Torres laboratory, D. R. Littman, M. Lu, an A. Darwin for reaing this manusript. We also thank V. KewalRamani for proviing reagents, an S. Polsky for assistane with purifiation of PBMCs. This researh was supporte by New York University Shool of Meiine Development Funs, an Amerian Heart Assoiation Sientist Development Grant (9SDG2636) to V.J.T. an National Institutes of Health (NIH) grants R6-AI9186-1A1 to V.J.T., NIH training grant T32-AI718 to F.A., A.L.D. an S.A.R., NIH R42-MH A1 to D.G.M., an NIH R21-AI87973 an R1-AI633 grants to D.U. Author Contributions F.A. an V.J.T. ientifie as the reeptor. F.A., A.L.D. an T.R.-R. purifie the toxins. S.A.R. generate the shrna knokownan over-expressing ells. F.A., S.A.R. an A.L.D. performe the ytotoxiity assays of ell lines. L.K. purifie an sorte primary ells. D.U. esignetheexperiments for the effet of on human ells. L.K. performe the experiments with primary human ells an S.A.R. performe the HIV infetion experiments. F.A. an T.R.-R. onute the biohemial an ell bining stuies with an GFP fusion proteins. F.A. an T.R.-R. onute the animal stuies. D.M. performe the surfae plasmon resonane experiments. N.R.L. provie DNA plasmis an the D32 primary ells. V.J.T. an D.U. oorinate an irete the projet. All authors isusse the ata an ommente on the manusript. F.A., D.U. an V.J.T. interprete the ata an wrote the manusript. Author Information Reprints an permissions information is available at The authors elare no ompeting finanial interests. Reaers are welome to omment on the online version of the paper. Corresponene an requests for materials shoul be aresse to V.J.T. (vitor.torres@nyum.org) or D.U. (erya.unutmaz@nyum.org). 212 Mamillan Publishers Limite. All rights reserve MONTH 212 VOL NATURE

6 RESEARCH ARTICLE METHODS Cell ulture onitions an viruses. Mammalian ells were maintaine at 37 uc with % CO 2 in RPMI supplemente with 1% fetal bovine serum (FBS; Atlanta Biologials) an peniillin (1 U ml 21 ) an streptomyin (.1 mg ml 21 ) (Meiateh) unless state otherwise. Lentivirus-base overexpression an knokown of human were onute aoring to previously esribe transution methos 19. Virus stoks were proue by DNA transfetion meiate by alium phosphate as esribe 3. overexpressing an shrna-enoing viruses, inluing non-oing shrna or HSA (mcd24)-overexpressing ontrols, were use at a multipliity of infetion of 1 3. HIV-R virus use for infetion of primary T ells was use at a multipliity of infetion of.3. Isolation of human PBMC, T-ell purifiation an ativation. Bloo was obtaine from e-ientifie, onsenting healthy ault onors as Buffy oats (New York Bloo Center) an from D32/D32 onors. Human peripheral bloo mononulear ells (PBMCs) were isolate from bloo using a Fioll-Paque PLUS (GE Amersham) graient. Resting CD4 1 an CD8 1 human T ells were purifie as previously esribe 19. Briefly, CD4 1 an CD8 1 T ells were isolate from purifie PBMCs using Dynal CD4 1 or CD8 1 Isolation Kits (Life Tehnologies) an were more than 99% pure. To purify naive, entral memory an effetor memory subsets, isolate CD4 1 an CD8 1 ells were staine with CCR7 an CD4RO antiboies, an CD4RO 2 CCR7 1 (TN), CD4RO 1 CCR7 1 (TCM), CCR7 2 (effetor memory T lymphoyte) subsets were sorte using a flow ytometer (FACSAria; BD Biosienes). In some experiments, total CD4RO 1 (T M ) ells were sorte into 1 an 2 subsets. Sorte subsets were more than 98% pure. Primary human CD4 1 T ells for HIV-R infetions were ativate using anti-cd3/cd28 oate beas (Dynabeas, Invitrogen) an maintaine in RPMI 1 peniillin an streptomyin 1 1% FBS supplemente with 2 U ml 21 IL-2 an 2 mm L-glutamine (Meiateh). In some experiments, CD4 1 T ells were ulture in 2 ng ml 21 IL-7 plus IL- (R&D Systems) for 7 ays. All experiments with primary PBMCs from WT onors were performe with ells from at least three inepenent onors. Experiments using D32 PBMCs were performe with ells from two onors. Generation of primary human monoyte-erive marophages, an enriti ells. Monoyte-erive marophages an enriti ells from healthy onors were generate from CD14 1 ells as previously esribe 3. Monoyte (CD14 1 ) ells were isolate from PBMCs using anti-cd14 antiboy-oate bea-base sorting using AutoMACS (Miltenyi Biote) an were typially more than 99% pure. Monoyte-erive marophages were generate from CD14 1 ells by supplementing the ulture meium with human granuloyte marophage olonystimulating fator ( ng ml 21 ) 36. Monoyte-erive enriti ells were generate from CD14 1 ells by supplementing the ulture meium with human granuloyte marophage olony-stimulating fator ( ng ml 21 ) 1 IL-4 (4 ng ml 21 ) 37. Cells were ulture for ays in the ifferentiation onition, followe by aition of as alreay esribe. ligans an inhibitors. Maraviro an TAK-779 were obtaine through the AIDS Researh an Referene Reagent Program, Division of AIDS, NIAID, NIH. Viriviro was purhase from Sellek Chemials. Reombinant human Rantes (CCL-) an marophage inflammatory protein-1b (MIP-1b, CCL-4) were obtaine from R&D Systems. Marophage inflammatory protein 1a (MIP- 1a, CCL-3) was obtaine from Biolegen. Maraviro was use at 1 ng ml 21 unless otherwise iniate. FACS analysis. Cells were staine as previously esribe 3. For intraellular staining, CD4 1 T-ell ultures were stimulate for h at 37 uc with PMA, ionomyin an Golgistop (BD Biosienes). Stimulate ells were washe with PBS an staine with Fixable Viability Dye to gate on live ells. Cells were then fixe an permeabilize by a ommerially available intraellular staining kit (ebiosiene) aoring to the manufaturer s protool. All FACS ata were aquire on an LSRII flow ytometer (BD Biosienes) using FACSDiva software. Data were analyse using Flowjo software (Treestar). Antiboies an yes. Antiboies use for surfae an intraellular staining of primary human ells inlue the following: CD3-Perp Cy. (lone UCHT1), CD4-Alexa7 (lone OKT4), CD8-Paifi Blue (lone RPA-T8), CXCR3-Perp Cy. (lone G2H7), IL-17-Alexa488 (lone BL168), IFN--Alexa7 (lone 4S.B3) (Biolegen), CD4RO-PeCy7 (lone UCHL1), CCR6-biotin (lone 11A9), CCR4-PE (lone 1G1), -PE (lone 2D7) or -APC-Cy7 (lone 2D7), streptaviin-apc, HSA-PE (lone M1/69) (BD Biosienes), an CCR7-FITC (lone 3) (R&D systems), IL-22- PerCP-eFluor71 (lone 22URTI) (ebiosiene). Antiboies use for surfae staining of primary murine ells inlue the following: CD3e-APC (lone 14-2C11), CD11b-PeCy7 (lone M1/7), CD11b- FITC (lone M1/7) Ly6G-FITC (lone 1A8), Ly6G-PE (lone 1A8), - biotin (C ), CD16/CD32 F Blok (lone 2.4G2) (BD Biosienes), F4/ 8-APC (lone BM8), F4/8-PeCy7 (lone BM8) streptaviin-percp.cy., an B22-A7 (lone RA3-6B2) (Biolegen). Fixable viability yes efluor-4 an efluor-78 were obtaine from ebiosiene. Antiboies use for - interation mapping inlue the following: lones 433, 429, 431, 417, 423, 449, 3A9, 42 an 419 (ref. 14) (R&D systems). The ontrol CXCR4 antiboy use in these stuies was lone (R&D systems). Leukotoxin treatments. Jurkat, H9, Hut-R an GHOST ell lines, primary human PBMCs an their sorte subsets, as well as primary murine peritonealeliite ells, were inubate with, LukD or as previously esribe 7. In all experiments ells were seee into a 96-well plate (1 3 1 to ells per well), treate for 1 h at 37 uc an evaluate for morphologial hanges an ethiium bromie (EtBr) uptake by mirosopy, or viability using CellTiter (Promega), CytotoxOne (Promega), ell satter by FACS an staining with ommerial viability yes (ebiosiene). CellTiter, CytotoxOne an EtBr measurements were mae using an EnVision 213 Plate Reaer (Perkin-Elmer). Intoxiations were one in the presene of speifi inhibitors (maraviro, TAK-779 an viriviro), hemokines (CCL3, CCL4, CCL) or monolonal an CXCR4 antiboies where iniate in the text. S. aureus in vitro infetion experiments. S. aureus (Newman) Drot, an Drot DlukED 7, were subulture for h in trypti soy broth followe by washing in RPMI plus 1% FBS an normalization to CFU per millilitre in this same meia. Normalize bateria were then ae to Jurkat an Jurkat-R ells (multipliity of infetion 1:1) that ha been pre-staine with a--pe antiboy (lone 2D7) an mixe at a ratio of :. Staining of with a- -PE antiboy (lone 2D7) was previously etermine to be stable for longer than 6 h on the surfae of Jurkat-R ells yet i not influene the killing of these ells by (ata not shown an Supplementary Fig. 13). Infete ells were inubate at 37 uc 1 % CO 2 for 4 h followe by the aition of lysostaphin to kill all bateria. Samples were then analyse on a BD LSRII flow ytometer. Depletion of 1 ompare with 2 ells was evaluate an shown graphially as the perentage of ea ells relative to ontrols with no toxin. For stuies with maraviro, the inhibitor was ae to ells 3 min before the aition of bateria as esribe above. Experiments were onute three times in tripliate. Generation of GFP fusion proteins. To generate reombinant N-terminal His 6 GFP-tagge an LukD, the mature protein oing sequenes of an LukD from S. aureus Newman genomi DNA were PCR-amplifie using the following primers: luke-f-sali (9-CCCC-GTCGAC-AATACTAA TATTGAAAAT-39), lukd-f-sali (9-CCCC-GTCGAC-GCTCAACATATCA CA-39), luke-r-noti (9-CCCC-GCGGCCGC-tta-ATTATGTCCTTTCACTT TAATTTCGTG-39) an lukd-r-noti (9-CCCC-GCGGCCGC-tta-TACTCC AGGATTAGTTTCTTTAGAATC-39). Amplifie sequenes were sublone into pet-41b (Novagen), resulting in a fusion of His 6 GFP with the N terminus of mature or LukD. Reombinant plasmis were transforme into Esherihia oli DHa an transformants selete by kanamyin resistane. Positive lones were transforme into E. oli LysY/LaQ (New Englan BioLabs) for protein expression an purifiation. Leukotoxin purifiation., LukD, GFP, GFP LukD, LukS, LukF, LukA an LukB were purifie from E. oli LysY/LaqQ as previously esribe 4,7 followe by enotoxin removal with Detoxi-Gel Enotoxin Removal Gel (Thermo Sientifi). The following alterations were mae for purifiation of reombinant GFP an GFP LukD. Upon soniation of baterial ell pellets, lysates were inubate with 1% Triton X-1 for 1 h at room temperature. After inubation, lysates were entrifuge for 6 min at 12,3g an passe through a.22 mm filter before ompleting the purifiation protool as esribe 7. membrane assoiation stuies. Assoiation of with the surfae of 1 ells was measure as follows. A toxi ose of purifie reombinant GFP or GFP LukD with LukD or, respetively, (final onentration 1 mgml 21 ) was inubate for 3 min on ie with sorte CD4 1 1 or CD4 1 2 T ells ( ells per well) from three inepenent onors. Cells were gate as GFP positive ompare with baseline fluoresene of untreate ells. A total of, events were ollete in all onitions teste. Owing to the high amount of bakgroun fluoresene of GFP toxins with the membranes of transue ell lines, we were unable to use these ells for membrane assoiation assays (ata not shown). As an alternative, we use primary CD4 1 T ells for membrane assoiation stuies. To inrease the abunane of on these ells an foster reprouible measures of membrane assoiation, CD4 1 1 ells were generate from CD4 1 ells infete with a lentivirus enoing an sorte by FACS as 1 from the resulting CD4 1 population after surfae staining for using 2D7 lone (PE). CD4 1 2 ells were sorte from the same population as those ells with unetetable surfae expression. surfae staining with 2D7 antiboy oes not influene toxin killing kinetis an therefore is unlikely to aversely influene membrane assoiation, as the latter is require for the former (Supplementary Fig. 13). 212 Mamillan Publishers Limite. All rights reserve

7 RESEARCH Paraoxially, lone 2D7 also bins to ECL-2 of similar to that of lone 431, whih bloks toxin ativity. However, 2D7 an 431 o bin to istint portions of ECL-2 (the N-terminal portion an arboxy (C)-terminal portion, respetively) perhaps explaining this phenomenon 38. Alternatively, our staining protools may not have suffiiently saturate all reeptor sites, thereby allowing funtional haraterization of toxin in the presene of 2D7. Experiments assessing maraviro, natural ligan or antiboy inhibition of membrane assoiation were onute in a similar fashion. However, in these instanes ells were first pre-inubate for 3 min with maraviro (1 ng ml 21 ), CCL ( mgml 21 ), 3A9, 431 or CXCR4 monolonal antiboies (2 mgml 21 ) or buffer before aition of a lethal onentration of GFP 1 LukD to the ells ( 1 mgml 21 ). After treatment, ells were washe, re-suspene in fixation buffer (FACS buffer 1 2% paraformalehye) for min at room temperature, washe again, re-suspene in FACS buffer, an the fluoresene of boun toxin was monitore by flow ytometry. Cells are shown as the perentage that were GFP positive. Surfae plasmon resonane analysis of an LukD bining to solubilize an CXCR4. Bining kinetis of an LukD to an CXCR4 by surfae plasmon resonane were measure as previously esribe, This approah has also been use to etet ligan interations with CXCR1 an CXCR2 (refs 43, 44). A C9-tagge was solubilize using mm HEPES, ph 7., mm NaCl,.1% DDM,.1% CHAPS,.2% CHS. This solubilization sheme is known to retain onformationally speifi antiboy bining to both an CXCR4 (ref. ). Approximately 7 relative units (RU) of the reeptor was apture onto a 1D4 antiboy-boun CM hip,4,41. Cells expressing a C9-tagge CXCR4 reeptor were also solubilize as a ontrol surfae in the same buffer 41. C9-CXCR4 was apture to approximately 1,2 RU. or LukD was ilute to 1.7 mm in running buffer ontaining mm HEPES, ph 7., mm NaCl,.2% CHS,.1% DDM an.1% Chaps an teste for bining in a threefol ilution series at a flow rate of ml min 21. Eah onentration series was repliate twie as shown by the overlai sensorgrams. All ata were ollete at 2 uc an onute at least twie in upliate. Biohemial stuies to etet interations between an. 293T ells were transfete with a vetor ontaining HA-tagge (Missouri S&T DNA Resoure Center; followe by solubilization (approximately ells per onition) in PBS 1 1% Brij1 1 Complete EDTAfree protease inhibitor oktail (Rohe). Solubilize was then ae to 2 ml of nikel resin ontaining no toxin or boun, LukD or. For the maraviro, natural ligan an antiboy inhibition experiments, the solubilize was pre-inubate for 3 min at room temperature with mgml 21 of maraviro, 1 mgml 21 of eah hemokine or 3 mgml 21 of eah antiboy followe by inubation with nikel resin ontaining. After inubation with ell lysates, the resin/protein omplexes were fixe with 2 mm DTSSP (Piere) for 3 min, quenhe with 2 mm Tris ph 8. for min, washe four times in PBS 1 1% Brij1 an boile in 43 SDS boiling buffer. All samples were run on a 1% SDS PAGE gel at 8 V, followe by transfer to nitroellulose at 1 A for 1 h. Membranes were bloke in PBS 1.1% Tween 1 % milk for 1 h an inubate overnight with either a-ha antiboy for (Covane) or a-his antiboy (Cell Sienes) for an LukD. The following ay, seonary goat a-mouse-hrp antiboy (Bio-Ra) was ae to the membranes for 1 h followe by the aition of SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Sientifi) for etetion. Measurement of ativation by alium mobilization. ativation by alium mobilization in ell lines an primary ells was assesse using the ommerial ye Fluo4-AM (Invitrogen). Cells were labelle for 3 min at room temperature with 3 mm Fluo4 in Hanks balane salt solution, followe by three washes in Hanks balane salt solution an inubation at 37 uc for 3 min. Cells were analyse on a flow ytometer over time an, at 1 s, ligan (CCL3, CCL4, CCL, 1 ng ml 21 ) or (1 2 mgml 21 ) was ae to the ells. Fluoresene was monitore thereafter by flow ytometry ( events were ollete per seon) until the iniate ompletion of eah experiment. For onitions in whih inhibition of reeptor ativation was monitore, ells were pre-inubate with either maraviro (1 mgml 21 ) or (1 2 mgml 21 ) uring the 3 min inubation at 37 uc esribe above. Graphs show the mean fluoresene of all events ollete in s intervals. Murine in vitro an in vivo experiments. In vitro assessment of peritonealeliite immune ell killing by was onute as follows. Female agemathe (4 6 weeks) C7BL/6 WT or 2/2 mie (Taoni) were injete with CFU of heat-kille S. aureus Newman DlukED intraperitoneally. Twenty-four hours later, mie were injete with an aitional CFU of the same strain. After another 24 h, mie were kille an peritoneal-eliite immune ells were lavage with 7 ml of PBS followe by lysis of re bloo ells in ACK lysing buffer an re-suspension in RPMI 1 1% FBS. was then ae to ells as esribe above an inubate for 1 h at 37 uc with % CO 2. After inubation, ells were washe in PBS an staine with viability ye followe by surfae staining for B22, CD11b, F48, Ly6G, CD3 an. The perentages of ea ells shown are an average of ells isolate an intoxiate from three inepenent mie. Means an stanar eviations are shown. For experiments esigne to measure S. aureus killing of 1 ells in vivo, female age-mathe (4 6 weeks) C7BL/6 WT or 2/2 mie (Taoni) were injete on ay 1 with heat-kille S. aureus to promote the reruitment of 1 marophages an lymphoytes to the peritoneum. On ay 2, mie were hallenge with live S. aureus Drot followe by the isolation of peritoneal immune ells 16 2 h later. Isolate ells were proesse for FACS as esribe above an the viability of lymphoytes an marophages was evaluate. The perentages of ea lymphoytes were average from 1 WT an 1 2/2 animals; representative FACS plots are shown. For murine systemi infetions, female age-mathe (4 6 weeks) C7BL/6 WT or 2/2 mie (Taoni) were infete with WT S. aureus Newman as previously esribe 7. After 96 h, serum was ollete an kineys remove, homogenize, proesse for FACS an plate as previously esribe 7. All survival urves were onute as previously esribe using WT S. aureus Newman an an isogeni DlukED mutant 7. For flow ytometry of immune ells from WT or DlukED infete kineys, organs were remove after 96 h an mehanially homogenize. Immune ells in homogenize tissues were enrihe by performing a 4/8 Peroll (GE Healthare) ensity graient entrifugation. Cells were subsequently proesse for surfae an viability staining thereafter (see above). 36. Gramberg, T., Sunseri, N. & Lanau, N. R. Eviene for anativation omain at the amino terminus of simian immunoefiieny virus Vpx. J. Virol. 84, (21). 37. Manel, N. et al. A rypti sensor for HIV-1 ativates antiviral innate immunity in enriti ells. Nature 467, (21). 38. Berro, R. et al. Multiple onformations on the ell surfae are use ifferentially by human immunoefiieny viruses resistant or sensitive to inhibitors. J. Virol. 8, (211). 39. Stenlun, P., Babok, G. J., Soroski, J. & Myszka, D. G. Capture an reonstitution of G protein-ouple reeptors on a biosensor surfae. Anal. Biohem. 316, (23). 4. Navratilova, I., Soroski, J. & Myszka, D. G. Solubilization, stabilization, an purifiation of hemokine reeptors using biosensor tehnology. Anal. Biohem. 339, (2). 41. Navratilova, I., Dioszegi, M. & Myszka, D. G. Analyzing ligan an small moleule bining ativity of solubilize GPCRs using biosensor tehnology. Anal. Biohem. 3, (26). 42. Navratilova, I., Panera, M., Wyatt, R. T. & Myszka, D. G. A biosensor-base approah towar purifiation an rystallization of G protein-ouple reeptors. Anal. Biohem. 33, (26). 43. Cauri, F. et al. HIV-1 matrix protein p17 promotes angiogenesis via hemokine reeptors CXCR1 an CXCR2. Pro. Natl Aa. Si. USA 19, (212). 44. Giagulli, C. et al. HIV-1 matrix protein p17 bins to the IL-8 reeptor CXCR1 an shows IL-8-like hemokine ativity on monoytes through Rho/ROCK ativation. Bloo 119, (212). 212 Mamillan Publishers Limite. All rights reserve