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Pore-forming toxins are critical virulence factors for many bacterial pathogens and are central to Staphylococcus aureus-mediated killing of host cells. S. aureus encodes pore-forming bi-component leukotoxins that are toxic toward neutrophils, but also specifically target other immune cells. Despite decades since the first description of Staphylococcal leukocidal activity, the host factors responsible for the selectivity of leukotoxins toward different immune cells remain unknown. Here we identified the HIV co-receptor, CCR5, as a cellular determinant required for cytotoxic targeting of subsets of myeloid cells and T lymphocytes by the S. aureus leukotoxin ED (LukED). We further demonstrate that LukED-dependent cell killing is blocked by CCR5 receptor antagonists, including the HIV drug maraviroc. Remarkably, CCR5-deficient mice are largely resistant to lethal S. aureus infection, highlighting the importance of CCR5 targeting in S. aureus pathogenesis. Thus, depletion of CCR5+ leukocytes by LukED suggests a novel S. aureus immune evasion mechanism that can be therapeutically targeted.
Staphylococcus aureus is a bacterial pathogen that causes significant morbidity and mortality worldwide. The organism is responsible for a myriad of diseases, from skin and soft tissue infections, to more invasive diseases including necrotizing pneumonia and sepsis. S. aureus secretes a number of protein products that allow the organism to effectively subvert the host immune system. Such factors include super-antigens, antibody binding proteins, cytolytic peptides, and pore-forming cytotoxins 1.
Pore-forming toxins are secreted by a substantial number of pathogenic bacteria 2. The toxins are secreted as water-soluble monomers that recognize host cell membranes, oligomerize, and insert α-helical or β-barrel pores into the lipid bilayer 2. Pore-formation disrupts osmotic balance and membrane potential, ultmately leading to cell death 2. S. aureus strains that infect humans produce up to four different β-barrel, bi-component, pore-forming toxins (HlgACB, LukED, LukSF-PV/PVL, and LukAB/HG) that exhibit a unique tropism for host immune cells and contribute to the greater virulence of S. aureus 1,3,4. The precise repertoire of immune cells targeted by the pore-forming leukotoxins remains to be fully determined. Even now, more than a century since the first description of Staphylococcal leukocidal activity 5,6, our understanding of leukotoxin function in vivo is limited due to an absence of known host-derived specificity determinants.
To identify potential leukotoxin receptors, we purified recombinant LukED, LukAB, and LukSF-PV and assessed their ability to kill a set of human cell lines 4,7. Granulocyte-like human cells (PMN-HL60) were killed within 1 hour by LukAB and LukSF-PV, but not LukED (Fig. 1a). In contrast, LukED was cytotoxic to a human T cell line ectopically expressing CCR5 (HUT-R5); whereas another T cell line (Jurkat), which lacks detectable CCR5, was insensitive (Fig. 1a). This suggested that CCR5 was involved in LukED cytotoxicity towards HUT-R5 cells. Accordingly, when CCR5 levels were reduced in HUT-R5 cells using lentiviral CCR5 shRNA, the cells were protected from LukED-mediated killing (Fig. 1b and S1a–b).
Complementary to these findings, ectopic expression of CCR5 was sufficient to render Jurkat and H9 cells (Fig. S1c) susceptible to LukED cytotoxicity (Fig. 1c). As expected, based on the mode of action of the bi-component leukotoxins, CCR5-dependent LukED-mediated cytotoxicity required both LukE and LukD subunits (Fig. S2a–b). A human osteosarcoma cell line engineered to constitutively express CCR5 (GHOST.R5 cells) 8 was also sensitive to LukED, but not to LukAB or LukSF-PV (Fig. 1d). The sensitivity of GHOST cells to LukED was specific to CCR5 expression, as over-expression of additional T cell-specific chemokine receptors (CCR1, CCR2, CCR3, CXCR4, CCR8, and CXCR6) in these cells did not confer susceptibility to LukED (Fig. S2c).
CCR5 is a co-receptor required for HIV infection 9–11 and has been targeted with small molecule antagonists aimed at restricting HIV entry into host cells 11. We found that one such clinically approved receptor antagonist, maraviroc, potently blocked LukED killing of CCR5+ cells (Fig. 1e and S3a) at concentrations similar to those required to block HIV infection (Fig. S3b). Similar inhibitory effects were observed with the CCR5 antagonists Vicriviroc and TAK-779, as well as chemokines that are natural ligands of CCR5 (Fig. S3a and S3c) 12,13. We found that maraviroc resulted in complete blockade of LukED pore-formation, an essential process for cytotoxicity (Fig. 1f and S3d).
We next investigated whether S. aureus was able to kill CCR5+ cells in a LukED-dependent manner. The expression level of lukED in S. aureus is inherently low during in vitro growth 7. However, deletion of the transcription factor Rot, a potent repressor, results in the enhanced expression and production of LukED by S. aureus 7. Thus, to assess S. aureus cytotoxicity towards CCR5+ cells, Jurkat or Jurkat-R5 cells were infected with S. aureus Δrot (Sa LukED+) and S. aureus ΔrotΔlukED (Sa LukED−) mutants. Jurkat-R5 cells were killed by S. aureus in a LukED-dependent manner, while Jurkats lacking CCR5 were resistant to killing (Fig. 1g). Additionally, Jurkat-R5 killing by S. aureus was completely blocked by maraviroc (Fig. 1h).
To more precisely characterize the LukED-CCR5 interaction on target cells we first determined whether monoclonal antibodies specific towards extracellular regions of CCR5 14 were sufficient to block toxin activity (Fig. 2a). Antibodies against extracellular loop 2 (ECL-2), but not the N-terminus of the receptor or CXCR4, significantly blocked toxin killing (Fig 2a) and prevented association of functional GFP-labeled toxin (Fig. S4) with the cell surface of sorted primary human CD4+CCR5+ T cells (Fig 2b). Furthermore, toxin association with the cell surface of CCR5+ cells was also reduced in the presence of CCL5, and was completely blocked upon addition of maraviroc, similar to CD4+CCR5− T cells (Fig. 2c). To determine if LukED interacts with CCR5, pull-down assays were conducted with purified toxin and solubilized CCR5. We found that CCR5 interacted with LukE but not LukD (Fig 2d). This interaction was significantly reduced in the presence of maraviroc, natural ligands of CCR5, as well as monoclonal antibody 45531 directed against ECL-2, but not 3A9 directed against the N-terminus of CCR5 (Fig. 2e and f). Additionally, incubation of LukE (75-fold molar excess) with CCR5+ cells largely blunted native ligand-induced CCR5 signaling as measured via calcium mobilization (Fig. S5). Importantly, LukE itself does not appear to induce CCR5 signaling (Fig S6a–b). Surface plasmon resonance (SPR) studies with immobilized native CCR5 15 and purified LukE or LukD subunits confirmed the pull-down studies and determined that LukE, but not LukD, binds to CCR5 in a time-dependent and saturable manner with an apparent KD of 39.6+/−0.4nM (Fig. 2g and S7a–b). This interaction was specific as evidenced by an inability of LukE to bind native CXCR4 (Fig. 2g).
We next sought to determine the subsets of primary human lymphoid and myeloid cells targeted by LukED. Treatment of blood lymphocytes with LukED resulted in specific depletion of CCR5+ T cells, the majority of which were effector memory T lymphocytes (TEM) (Fig. 3a and S8). As with cell lines, the CCR5-dependent killing of primary cells was completely blocked by maraviroc (Fig. 3a and S8). A portion of individuals of Northern European heritage harbors a 32 base-pair deletion in the CCR5 gene (Δ32 CCR5) resulting in a truncated protein that cannot be surface localized, thus rendering the CD4+ T cells refractory to HIV infection 11,16,17. Similarly, primary T cells expanded from a Δ32 CCR5 donor were also resistant to LukED cytotoxicity (Fig. 3b). In keeping with the notion that CCR5 is required for HIV-1 entry into CD4+ T cells 9–11, selective depletion of CCR5+ T cells by LukED suppressed HIV-1 spread (Fig. S9).
Memory T cells can be classified into functional subsets based on differential chemokine receptor profiles and cytokine production. Among T cell subsets, CCR6+CCR5+ subset produces more IL-17 and IFNγ than CCR6+CCR5 T cells 18. Consistent with this association, depletion of CCR5+CD4+ T cells with LukED greatly reduced the proportion of IFNγ- and IL-17-producing cells compared to purified CD4+ T cell controls (Fig. 3c, Day 0). Incubation with the γc-cytokines IL-7 and IL-15 significantly enhances the proportion of IL-17+ and IL-17+/IFNγ+ by CCR6+ memory T cells 19. We found that when human CD4+ T cells were first treated with LukED, followed by 7-days of culture with IL-7 and IL-15, there was a substantial reduction in the induction of IFNγ and IL-17/IL-22-secreting CCR6+ T cells (Fig. 3c); a finding that correlates well with depletion of the CCR6+CCR5+ memory progenitor subset (Fig. S10). In addition to Th1 and Th17 effector cells, LukED also killed macrophages and dendritic cells in a CCR5-dependent manner (Fig. 3d).
Next we examined the contribution of CCR5 to S. aureus pathogenesis and determined the influence of LukED on the targeted killing of CCR5+ cells in vivo. We found that murine CCR5 (mCCR5) renders transfected 293T cells fully susceptible to the toxin (Fig. S11a–b). Additionally, primary murine macrophages treated with high concentrations of maraviroc were partially protected from toxin-mediated killing, confirming that LukED is directly targeting mCCR5 (Fig. S11c). Because maraviroc is potent toward human CCR5 but not mCCR5 (Fig 1e and S11)20 we chose to study WT and CCR5-deficient mice with the hypothesis that the latter would be resistant to LukED cytotoxicty. S. aureus-elicited lymphocytes and macrophages from WT mice were highly susceptible to purified LukED, while lymphocytes and macrophages isolated from CCR5−/− mice were markedly resistant (Fig. 4a–b). To further validate that S. aureus kills CCR5+ leukocytes in vivo, we implemented a peritonitis model where WT and CCR5−/− mice were infected with S. aureus. CCR5 surface expression was not required for the initial influx of immune cells to the infection site, as the cells recovered and their profiles were identical among all mice (Fig. S12). However, lymphocytes and macrophages elicited in vivo in WT mice were more susceptible to S. aureus killing compared to those from the CCR5−/− mice (Fig. 4c–d). LukED is associated with S. aureus pathogenesis in a murine model of systemic infection7. Using this model, CCR5−/− mice infected with wildtype S. aureus exhibited significantly reduced bacterial burden in the kidneys compared to those of infected WT mice (Fig. 4e), a phenotype similar to that observed for mice infected with a S. aureus ΔlukED mutant7. After 96 hours, infected CCR5−/− mice also exhibited significantly reduced serum pro-inflammatory cytokines and chemokines and displayed a commensurate reduction in innate immune cells in the kidney compared to WT mice (Fig. 4f–g), signs consistent with infection resolution. Additionally, when WT mice were challenged systemically with WT or a ΔlukED mutant we observed LukED-dependent killing of CCR5+ macrophages within infected kidneys, consistent with our hypothesis that LukED is capable of targeting CCR5+ leukocytes during infection (Fig. 4h). In support of the importance of CCR5 targeting in vivo, the mortality associated with S. aureus bloodstream infection was reduced for CCR5-deficient mice, a phenotype similar to that of mice challenged with strains of S. aureus lacking lukED (Fig. 4i).
To our knowledge, CCR5 is the first described cellular receptor that is necessary and sufficient for the killing of mammalian cells by a Staphylococcal bi-component leukotoxin. Thus, in addition to HIV, Toxoplasma gondii and poxviruses (vaccinia and myxoma)9,21–24, S. aureus can also exploit CCR5 to target immune cells. Interestingly, the Δ32 allele of CCR5 is thought to have been acquired through selective pressure imparted by a deadly pathogen25,26. Yersinia pestis or variola virus were postulated as potential driving forces behind this selection, but these hypotheses have either been discounted or remain uncertain in favor of an older selection event incited by an immune cell-targeting pathogen24,27. Our findings put forth the possibility that resistance to S. aureus leukotoxins may have influenced the selection of the Δ32 allele.
The finding that LukED selectively kills CCR5+ T cells, macrophages, and dendritic cells extends the repertoire of immune cells targeted by this leukotoxin and supports a role for these leukocytes in the resolution of S. aureus infection. The lukED gene is believed to be present in a large number of clinically relevant strains (>70%) including clones responsible for the majority of infections in the United States and Germany, though it is absent in a subset of strains causing hospital acquired infection (e.g. EMRSA15/16) in Great Britain28–30. The majority of isolates lacking lukED appear to be of clonal complex 30 (USA200/EMRSA16), which are known to produce low levels of cytotoxins31. Conceivably, the pathogenesis of these strains is influenced by the weakened immune status of hospitalized patients rather than toxic molecules. In contrast, we envisage that virulent clinical strains producing large amounts of LukED (e.g. clonal complex 8)7 use the toxin to eliminate antigen presenting cells as well as S. aureus-specific CCR5+ Th1/Th17 cells, which are induced by the bacterium32 and are protective against infection33,34. In support of this hypothesis, we demonstrate that LukED kills CCR5+ cells in vivo during systemic infection and that mice lacking CCR5 are protected from the mortality associated with acute S. aureus disease. Current systemic murine infection models are insufficient to reliably evaluate CCR5hi T cell susceptibility to LukED (data not shown). However, our in vitro data and in vivo studies with CCR5+ macrophages strongly support the notion that subsets of CCR5hi T cells are also targeted in vivo.
Interestingly, LukED-mediated toxicity toward neutrophils and monocytes is not blocked by maraviroc (data not shown), suggesting LukED targets these cells via alternate and non-redundant mechanisms. This point also implies a role for CCR5+ myeloid cells and T cells in resolving acute infection, one that extends beyond the initial control of infection imparted by neutrophils. Importantly, the finding that LukED toxicity toward CCR5+ cells is potently neutralized by a clinically approved CCR5 antagonist (maraviroc) suggests that these types of drugs could provide much-needed therapeutic alternatives in the treatment of S. aureus infections.
Mammalian cells were maintained at 37°C with 5% CO2 in RPMI supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and Penicillin (100 U/ml) and Streptomycin (0.1mg/ml) (P/S; Mediatech) unless stated otherwise. Lentivirus-based over-expression and knockdown of human CCR5, was conducted according to previously described transduction methods19. Virus stocks were produced by calcium phosphate-mediated DNA transfection as described35. CCR5 over-expressing and shRNA-encoding viruses, including non-coding shRNA or HSA (mCD24)-over-expressing controls, were used at a multiplicity of infection (MOI) of 1–3. HIV-R5 virus used for infection of primary T cells was used at an MOI of 0.3.
Blood was obtained from de-identified, consenting healthy adult donors as Buffy coats (New York Blood Center) and from Δ32/Δ32 CCR5 donors. PBMCs were isolated from blood using a Ficoll-Paque PLUS (GE Amersham) gradient. Resting CD4+ and CD8+ human T cells were purified as previously described19. Briefly, CD4+ and CD8+ T cells were isolated from purified PBMC using Dynal CD4+ or CD8+ Isolation Kits (Life Technologies, Grand Island, NY) and were >99% pure. To purify naïve, central memory and effector memory subsets, isolated CD4+ and CD8+ cells were stained with CCR7 and CD45RO antibodies, and CD45RO−CCR7+ (TN), CD45RO+CCR7+ (TCM), CCR7− (TEM) subsets were sorted using a flow cytometer (FACSAria; BD Biosciences). In some experiments, total CD45RO+ (TM) cells were sorted into CCR5+ and CCR5−subsets. Sorted subsets were >98% pure. Primary human CD4+ T cells for HIV-R5 infections were activated using anti-CD3/CD28 coated beads (Dynabeads, Invitrogen) and maintained in RPMI + Penicillin and Streptomycin + 10% FBS supplemented with 200 U/mL IL-2 and 2mM L-glutamine (Mediatech). In some experiments, CD4+ T cells were cultured in 20 ng/ml IL-7 plus IL-15 (R&D systems) for 7 days. All experiments with primary PBMC from wildtype CCR5 donors were performed with cells from at least 3 independent donors. Experiments using Δ32 CCR5 PBMC were performed with cells from 2 donors.
Monocyte derived macrophages and DCs from healthy donors were generated from CD14+ cells as previously described35. Monocytes (CD14+) cells were isolated from PBMC using anti-CD14 antibody coated bead based sorting using AutoMACS (Miltenyi Biotec) and were typically >99% pure. Monocyte-derived macrophages were generated from CD14+ cells by supplementing the culture medium with human GMCSF (50 ng/ml)36. Monocyte-derived dendritic cells (DCs) were generated from CD14+ cells by supplementing the culture medium with human GMCSF (50 ng/ml) + IL-4 (40 ng/ml)37. Cells were cultured for 5 days in the differentiation condition, folowed by addition of LukED as already described.
Maraviroc and TAK-779 were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. Vicriviroc was purchased from Selleck Chemicals. Recombinant human Rantes (CCL-5) and Macrophage inflammatory protein-1β (MIP-1β, CCL-4) were obtained from R&D Systems. Macrophage inflammatory protein 1α (MIP-1α CCL-3) was obtained from Biolegend. Maraviroc was used at 100 ng/ml unless otherwise indicated.
Cells were stained as previously described35. For intracellular staining, CD4+ T cell cultures were stimulated for 5 hours at 37°C with PMA, Ionomycin and Golgistop (BD Biosciences). Stimulated cells were washed with PBS and stained with Fixable Viability Dye to gate on live cells. Cells were then fixed and permeabilized by a commercially available intracellular staining kit (eBioscience) as per the manufacturer's protocol. All FACS data were acquired on an LSRII flow cytometer (BD Biosciences) using FACSDiva software. Data analyses were done using Flowjo software (Treestar Inc.).
CD3-Percp Cy5.5 (clone UCHT1), CD4-Alexa700 (clone OKT4), CD8-Pacific Blue (clone RPA-T8), CXCR3-Percp Cy5.5 (clone G025H7), IL-17-Alexa488 (clone BL168), IFNγ-Alexa700 (clone 4S.B3) (Biolegend), CD45RO-PeCy7 (clone UCHL1), CCR6-biotin (clone 11A9), CCR4-PE (clone 1G1), CCR5-PE (clone 2D7) or CCR5-APC-Cy7 (clone 2D7), Streptavidin-APC, HSA-PE (clone M1/69) (BD Biosciences), and CCR7-FITC (clone 150503) (R&D systems), IL-22-PerCP-eFluor710 (clone 22URTI) (eBioscience).
CD3e-APC (clone 145-2C11), CD11b-PeCy7 (clone M1/70), CD11b-FITC (clone M1/70) Ly6G-FITC (clone 1A8), Ly6G-PE (clone 1A8), CCR5-biotin (C34-3448), CD16/CD32 Fc Block (clone 2.4G2)(BD Biosciences), F4/80-APC (clone BM8), F4/80-PeCy7 (clone BM8) Streptavidin-PerCP.Cy5.5, and B220-A700 (clone RA3-6B2) (Biolegend). Fixable viability dyes eFluor-450 and eFluor-780 were obtained from eBioscience.
CCR5 clones 45533, 45529, 45531, 45517, 45523, 45549, 3A9, 45502, and 4551914 (R&D systems). The control CXCR4 antibody used in these studies was clone 44716 (R&D systems).
Jurkat, H9, Hut-R5, and GHOST cell lines, primary human PBMCs and their sorted subsets, as well as primary murine peritoneal elicited cells were incubated with LukE, LukD, or LukED as previously described7. In all experiments cells were seeded into a 96-well plate (1×105–2×105 cells per well), treated for 1 hour at 37°C and evaluated for morphological changes and ethidium bromide (EtBr) uptake via microscopy, or viability using CellTiter (Promega), CytotoxOne (Promega), cell scatter via FACS and staining with commercial viability dyes (eBioscience). CellTiter, CytotoxOne, and EtBr measurements were made using an EnVision 2103 Plate Reader (Perkin-Elmer). Intoxications were done in the presence of specific inhibitors (maraviroc, TAK779, and vicriviroc), chemokines (CCL3, CCL5, CCL5), or monoclonal CCR5 and CXCR4 antibodies where indicated in the text.
S. aureus (Newman) Δrot, and Δrot ΔlukED7 were subcultured for 5 hours in tryptic soy broth followed by washing in RPMI + 10% FBS and normalization to 1×109 CFU/ml in this same media. Normalized bacteria were then added to 2×105 Jurkat and Jurkat-R5 cells (multiplicity of infection 10:1) that had been prestained with α-CCR5-PE antibody (clone 2D7) and mixed at a ratio of 50/50. Staining of CCR5 with α-CCR5-PE antibody (clone 2D7) was previously determined to be stable for greater than six hours on the surface of Jurkat-R5 cells yet does not influence the killing of these cells by LukED (data not shown and Fig S13). Infected cells were incubated at 37 °C + 5% CO2 for four hours followed by the addition of lysostaphin to kill all bacteria. Samples were then analyzed on a BD LSRII flow cytometer. Depletion of CCR5+ vs CCR5− cells was evaluated and displayed graphically as percent dead cells relative to no toxin controls. For studies with maraviroc, the inhibitor was added to cells 30 minutes prior to the addition of bacteria as described above. Experiments were conducted a total of three times in triplicate.
To generate recombinant N-terminal His6x-GFP-tagged LukE and LukD, the mature protein coding sequences of LukE and LukD from S. aureus Newman genomic DNA were PCR-amplified using the following primers: lukE-F-SalI (5'-CCCC-GTCGAC-AATACTAATATTGAAAAT- 3'), lukD-F-SalI (5'-CCCCGTCGAC-GCTCAACATATCACA- 3'), lukE-R-NotI (5'-CCCC-GCGGCCGC-tta-ATTATGTCCTTTCACTTTAATTTCGTG-3 '), and lukD-R-NotI (5'-CCCCGCGGCCGC-tta-TACTCCAGGATTAGTTTCTTTAGAATC-3'). Amplified sequences were subcloned into pET-41b (Novagen) resulting in a fusion of His6x-GFP with the N-terminus of mature LukE or LukD. Recombinant plasmids were transformed into E.coli DH5α and transformants selected by kanamycin resistance. Positive clones were transformed into Escherichia coli LysY/LacQ (New England BioLabs) for protein expression and purification.
LukE, LukD, GFP-LukE, GFP-LukD, LukS, LukF, LukA, and LukB were purified from E. coli LysY/LaqQ as previously described4,7 followed by endotoxin removal with Detoxi-Gel Endotoxin Removal Gel (Thermo Scientific). The following alterations were made for purification of recombinant GFP-LukE and GFP-LukD: upon sonication of bacterial cell pellets, lysates were incubated with 1% Triton X-100 for 1 hour at room temperature. After incubation, lysates were centrifuged for 60 minutes at 12000 RPM and passed through a 0.22 μm filter prior to completing the purification protocol as described7.
Association of LukED with the surface of CCR5+ cells was measured as follows. A toxic dose of purified recombinant GFP-LukE or GFP-LukD with LukD or LukE respectively (final concentration 10 μg/ml) was incubated for 30 minutes on ice with sorted CD4+CCR5+ or CD4+CCR5− T cells (5×104 cells/well) from three independent donors. Cells were gated as GFP positive compared to baseline fluorescence of untreated cells. A total of 50,000 events were collected in all conditions tested. Due to the high level of background fluorescence of GFP toxins with the membranes of transduced cell lines, we were unable to use these cells for membrane association assays (data not shown). As an alternative we used primary CD4+ T cells for membrane association studies. To increase the abundance of CCR5 on these cells and foster reproducible measures of membrane association, CD4+CCR5+ cells were generated from CD4+ cells infected with a lentivirus encoding CCR5 and sorted by FACS as CCR5+ from the resulting CD4+ population after surface staining for CCR5 using 2D7 clone (PE). CD4+CCR5− cells were sorted from the same population as those cells with undetectable CCR5 surface expression. Importantly, CCR5 surface staining with 2D7 antibody does not influence toxin killing kinetics and therefore is unlikely to adversely influence membrane association, as the latter is required for the former (Fig. S13). Somewhat paradoxically, clone 2D7 also binds to ECL-2 of CCR5 similar to that of clone 45531, which blocks toxin activity. However, 2D7 and 45531 do bind to distinct portions of ECL-2 (the N-terminal portion and C-terminal portion, respecitvely) perhaps explaining this phenomenon38. Alternatively, our staining protocols may not sufficiently saturate all receptor sites, thereby allowing functional characterization of toxin in the presence of 2D7.
Experiments assessing maraviroc, natural ligand, or antibody inhibition of LukED membrane association were conducted in a similar fashion. However, in these instances cells were first preincubated for 30 minutes with maraviroc (100 ng/ml), CCL5 (5 μg/ml), 3A9, 45531, or CXCR4 monoclonal antibodies (25 μ-g/ml) or buffer prior to addition of a lethal concentration of LukE-GFP+LukD to the cells (5–10 μg/ml). After treatment, cells were washed, resuspended in fixation buffer (FACS buffer + 2% paraformaldehyde) for 15 minutes at room temperature, washed again, resuspended in FACS buffer, and fluorescence of bound toxin was monitored by flow cytometry. Cells are displayed as % GFP positive.
Binding kinetics of LukE and LukD to CCR5 and CXCR4 by Surface Plasmon resonance were measured as previously described15,39–42. This approach hasc also been employed to detect ligand interactions with CXCR1 and CXCR243,44. A C9-tagged CCR5 was solubilized using 50 mM HEPES, pH 7.0, 150 mM NaCl, 0.1% DDM, 0.1% CHAPS, 0.02% CHS15. This solubilzation scheme is known to retain conformationally specific antibody binding to both CCR5 and CXCR415. ~700 RU of the CCR5 receptor was captured onto a 1D4 antibody-bound CM5 chip15,40,41. Cells expressing a C9-tagged CXCR4 receptor were also solubilized as a control surface in the same buffer41. C9-CXCR4 was captured to ~1200 RU. LukE or LukD was diluted to 1.7 μM in running buffer containing 50 mM Hepes, pH 7.0, 150mM NaCl, 0.02% CHS, 0.1% DDM, and 0.1% Chaps and tested for binding in a 3-fold dilution series at a flow rate of 50μl/min. Each concentration series was replicated two times as shown by the overlaid sensorgrams. All data were collected at 25 °C and conducted at least twice in duplicate.
293T cells were transfected with a vector containing HA-tagged CCR5 (Missouri S&T cDNA Resource Center-www.cdna.org), followed by solubilization (~2.0×107 cells per condition) in PBS + 1% Brij010 + Complete EDTA-free protease inhibitor cocktail (Roche). Solubilized CCR5 was then added to 25μl of nickel resin containing no toxin or bound LukE, LukD, or LukED. For the maraviroc, natural ligand, and antibody inhibition experiments the solubilized CCR5 was pre-incubated for 30 minutes at room temperature with 5 μg/ml of maraviroc, 10 μg/ml of each chemokine, or 35 μg/ml of each antibody followed by incubation with nickel resin containing LukE. After incubation with cell lysates, the resin/protein complexes were fixed with 2 mM DTSSP (Pierce) for 30 minutes, quenched with 20 mM Tris pH 8.0 for 15 minutes, washed 4 times in PBS + 1% Brij010, and boiled in 4× SDS boiling buffer. All samples were run on a 10% SDS-PAGE gel at 80V, followed by transfer to nitrocellulose at 1 Amp for 1 hour. Membranes were blocked in PBS + 0.01% Tween + 5% milk for 1 hour and incubated overnight with either α-HA antibody for CCR5 (Covance) or α-His antibody (Cell Sciences) for LukE and LukD. The following day secondary goat α-mouse-HRP antibody (Bio-Rad) was added to the membranes for 1 hour followed by the addition of SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) for detection.
Assessment of CCR5 activation by calcium mobilization in cell lines and primary cells was carried out using the commercial dye Fluo4-AM (Invitrogen). Cells were labeled for 30 minutes at room temperature with 3 μM Fluo4 in Hanks Balanced Salt Solution, followed by three washes in HBSS and incubation at 37° C for 30 minutes. Cells were analyzed on a flow cytometer over time and at 100 seconds, ligand (CCL3, CCL4, CCL5, 10ng/ml) or LukE (10–20 μg/ml) was added to the cells. Fluorescence was monitored thereafter by flow cytometry (500 events were collected per second) until the indicated completion of each experiment. For conditions in which inhibition of receptor activation was monitored cells were pre-incubated with either maraviroc (1 μg/ml) or LukE (10–20 μg/ml) during the 30 minute incubation at 37° C described above. Graphs show the mean fluorescence of all events collected in five second intervals.
in vitro assessment of peritoneal elicited immune cell killing by LukED was conducted as follows. Female age-matched (4–6 weeks) C57Bl6 WT or CCR5−/− mice (Taconic) were injected with 1×107 CFU heat killed S. aureus Newman ΔlukED intraperitoneally. 24 hours later mice were injected with an additional 1×107 CFU of the same strain. After another 24 hours, mice were sacrificed and peritoneal elicited immune cells were lavaged with 7ml of PBS followed by lysis of red blood cells in ACK lysing buffer and resuspension in RPMI + 10% FBS. LukED was then added to cells as described above and incubated for 1 hour at 37° C + 5% CO2. After incubation cells were washed in PBS and stained with viability dye followed by surface staining for B220, CD11b, F480, Ly6G, CD3, and CCR5. The percent dead cells shown are an average of cells isolated and intoxicated from three independent mice. Means and standard deviation are shown.
For experiments designed to measure S. aureus killing of CCR5+ cells in vivo, female age-matched (4–6 weeks) C57Bl6 WT or CCR5−/− mice (Taconic) were injected on day 1 with 1×107 heat-killed S. aureus to promote the recruitment of CCR5+ macrophages and lymphocytes to the peritoneum. On day 2 mice were challenged with live S. aureus Δrot followed by the isolation of peritoneal immune cells 16–20 hours later. Isolated cells were processed for FACS as described above and the viability of lymphocytes and macrophages was evaluated. The percent dead lymphocytes were averaged from 10 WT and 10 CCR5−/− animals and representative FACS plots are shown.
For murine systemic infections, female age-matched (4–6 weeks) C57Bl6 WT or CCR5−/− mice (Taconic) were infected with WT S. aureus Newman as previously described 7. After 96 hours serum was collected and kidneys removed, homogenized, processed for FACS, and plated as previously described 7. All survival curves were conducted as previously described using WT S. aureus Newman and an isogenic ΔlukED mutant 7. For flow cytometry of immune cells from WT or ΔlukED infected kidneys, organs were removed after 96 hours and mechanically homogenized. Immune cells in homogenized tissues were enriched by performing a 40/80 Percoll (GE Healthcare) density gradient centrifugation. Cells were subsequently processed for surface and viability staining thereafter (see above).
We thank members of the Torres laboratory, Drs. Dan R. Littman and Min Lu for critically reading this manuscript. We also thank Dr. Vineet KewalRamani for providing reagents, and Sylvie Polsky for assistance with purification of PBMCs. This research was supported by New York University School of Medicine Development Funds and American Heart Association Scientist Development Grant (09SDG2060036) to V.J.T and National Institutes of Health (NIH) grants R56-AI091856-01A1 to V.J.T., NIH training grant T32 AI007180 to F.A., A.L.D and S.A.R., NIH R42MH084372-02A1 to D.G.M., and NIH R21AI087973 and R01AI065303 grants to D.U.
AUTHOR CONTRIBUTIONS: F.A. and V.J.T identified CCR5 as the LukED receptor. F.A., A.L.D., and T.R.R. purified the toxins. S.A.R. generated the CCR5 shRNA knockdown and CCR5 over-expressing cells. F.A., S.A.R., and A.L.D performed the cytotoxicity assays of cell lines. L.K. purified and sorted primary cells. D.U. designed the experiments for the effect of LukED on human cells. L.K. performed the experiments with primary human cells and S.A.R performed the HIV infection experiments. F.A. and T.R.R. conducted the biochemical and cell binding studies with LukED and GFP fusion proteins. F.A. and T.R.R conducted the animal studies. D.M performed the SPR experiments. N.R.L. provided cDNA plasmids and the Δ32 CCR5 primary cells. V.J.T. and D.U. coordinated and directed the project. All authors discussed the data and commented on the manuscript. F.A., D.U., and V.J.T interpreted the data and wrote the manuscript.
COMPETING INTEREST STATEMENT: The authors declare that they have no competing financial interest.