The Staphylococcal Toxins γ-Hemolysin AB and CB Differentially Target Phagocytes by Employing Specific Chemokine Receptors

γ-Hemolysin AB and CB target chemokine receptors

To evaluate host target cell tropism of γ-Hemolysin, we tested toxin-mediated cell permeability in freshly isolated human leukocytes using a membrane-impermeant fluorescent dye. Figure 1a confirms that HlgAB and HlgCB target human neutrophils and monocytes²⁹. This pattern of cell specificity is similar to that of PVL¹⁸. In contrast to HlgCB, HlgAB shows limited cytotoxicity towards lymphocytes as well³⁰. The extended cellular tropism of HlgAB compared to HlgCB implies the involvement of different host target cell receptors.

The molecular basis of cell selectivity of both HlgAB and HlgCB is not understood. Because PVL and HlgCB were predicted to share proteinaceous receptors²⁹, we hypothesized that γ-Hemolysin targets chemokine receptors as well. As no specific predictions were available for HlgAB, we screened human embryonic kidney (HEK) cells transfected with a collection of human chemokine receptors for cytotoxic susceptibility to HlgAB and HlgCB. Untransfected cells were resistant to pore formation induced by both HlgAB and HlgCB (Fig. 1a, b).

Mirroring PVL specificity, HlgCB induced pore formation in cells transfected with the C5a receptors, C5aR or C5L2 (Fig. 1b). In addition, HlgCB induced pore formation in cells transfected with the complement receptor C3aR, though only at high toxin concentrations (Supplementary Fig. 1a). Assessment of half-maximal effective lytic concentrations (EC₅₀) identified C5aR and C5L2 as the most efficient targets for HlgCB cytotoxicity, with ± 100 fold lower EC₅₀ concentrations compared to C3aR (Supplementary Fig. 1b). HlgAB efficiently induced pore formation in cells transfected with the chemokine receptors CXCR1, CXCR2 and CCR2 (Fig. 1b). Furthermore, high concentrations of HlgAB induced pore formation in cells overexpressing the chemokine receptor CXCR4 (Supplementary Fig. 1a). Calculation of EC₅₀ concentrations identified CXCR1, CXCR2 and CCR2 as the receptors most efficiently employed by HlgAB, with at least 30 fold lower EC₅₀ concentrations compared to CXCR4 (Supplementary Fig. 1b). Importantly, the respective panel of HlgAB and HlgCB receptors did not overlap (Fig. 1b and Supplementary Fig. 1a), indicating that HlgA and HlgC control receptor specificity.

To obtain the affinity of the toxin-receptor interactions, Surface Plasmon Resonance (SPR) was performed using proteoliposomes containing C5aR, CXCR1, CXCR2 or CCR2. HlgC bound C5aR with a K_D of 5.64 (± 1.53) nM. HlgA bound CXCR1, CXCR2, and CCR2 with a K_D of 5.69 (± 1.94) nM, 27.20 (± 5.54 nM), and 3.51 (± 0.29) nM, respectively. Consistent with the cellular assays, no detectable binding of HlgC was observed on the HlgA receptors and vice versa (Table 1). The restricted receptor spectrum emphasizes HlgAB and HlgCB as two different and specific toxins.

In an attempt to better understand leukocyte tropism of γ-Hemolysin, we evaluated expression levels of the receptors most efficiently employed by HlgAB and HlgCB on the surface of freshly isolated human leukocyte populations. The receptors targeted by HlgCB are present on neutrophils and monocytes, but absent on lymphocytes (Fig. 1c). These observations are in line with cell specificity of HlgCB. The receptors employed by HlgAB are abundantly present on neutrophils and monocytes (Fig. 1c). CCR2 is exclusively expressed on monocytes. Low-level expression of CXCR1 and CXCR2 on lymphocytes likely accounts for the limited cytotoxicity of HlgAB towards these cells.

In summary, these data suggest that cellular tropism of γ-Hemolysin is determined by the specific interaction of HlgA and HlgC with their respective protein receptors. The abundant expression of the efficient receptors on phagocytes allows the toxins to discriminate phagocytic cells from other cells.

HlgAB and HlgCB lyse cells receptor-specifically

S. aureus isolates vary in both genetic contents and expression levels of leukocidins. Strain USA300 clone SF8300 is a minimally passed representative PVL-positive community-associated methicillin-resistant S. aureus (MRSA) isolate from the United States³¹. S. aureus Newman is a PVL-negative, methicillin-sensitive strain frequently used for in vivo studies³². We tested the interaction of native HlgAB and HlgCB with specific receptors using bacterial culture supernatants. CXCR2-mediated cytotoxicity of HlgAB was confirmed using supernatant of S. aureus USA300 SF8300 and its isogenic hlgACB mutant (hlgACB knockout) strain³³. Induction of pore formation in U937 promyelocytic cells stably transfected with CXCR2 was lost using supernatant of the strain lacking HlgACB. Pore forming capacity of culture supernatant was restored by complementation with a plasmid encoding HlgAB, but not with a plasmid encoding HlgCB (Fig. 2a). Due to production of PVL, HlgCB cytotoxicity towards C5aR-transfected cells could not be investigated using a USA300 strain¹⁸. C5aR-mediated cytotoxicity of HlgCB was therefore validated using supernatants of strain Newman and its isogenic hlgACB mutant (hlgACB knockout) strain²². Culture filtrates of Newman wild type but not of the strain lacking HlgACB were lytic to C5aR-transfected U937 cells. Cytotoxicity was restored by complementation with a plasmid encoding HlgCB, but not HlgAB (Fig. 2b).

These data confirm the results obtained with recombinant toxins, and demonstrate that HlgAB and HlgCB produced by S. aureus specifically target their respective host receptors.

HlgAB and HlgCB are major secreted S. aureus leukocidins

HlgCB shares receptors with PVL, while HlgAB partly shares receptors with LukED^18,25,26. This finding suggests redundancy of the secreted toxins. To evaluate the relative importance of the different toxin-receptor pairs, we composed a panel of representative European clinical S. aureus strains isolated from blood cultures. As most of European S. aureus strains are PVL-negative¹⁶, we included the PVL-positive major MRSA-clone currently causing infections in the United States (USA300 LAC) as a control³⁴. All the selected European clinical isolates were methicillin susceptible, and belonged to a variety of clonal complexes (Table 2).

We first determined which genes encoding secreted leukocidins are present in the different clinical isolates. Confirming expectations^7,15,16, all clinical strains contained the genes encoding hlgA and hlgC, while none contained the lukS-PV gene. Following clonal lineage³⁵, half of the isolates encoded the lukE gene (Table 2). Next, we evaluated expression of the leukocidin S-component mRNAs by qRT-PCR. For PVL, culture broth dependent differential expression of the toxin has been described^17,36,37. Strain USA300 LAC confirmed this broth dependent expression of lukS-PV (Supplementary Fig. 2 a, b). Quantities of lukE transcripts were broth dependent as well. In contrast, all clinical strains consistently expressed high levels of hlgA and hlgC mRNA in the two different culture broths tested (Table 2 and Supplementary Fig. 2a, b).

To investigate the relative contribution to pore formation of the respective secreted leukocidins, we subsequently tested supernatants of the different bacterial cultures on human neutrophils and HEK cells stably transfected with specific receptors. Untransfected HEK cells showed background pore formation of ± 15% on average (Supplementary Fig. 2b – d, f, g). Culture supernatants of all clinical isolates efficiently induced pore formation in HEK cells transfected with C5aR or CCR2 (Table 2 and Supplementary Fig. 2b, d), indicating that HlgCB and HlgAB are secreted in functional quantities. Although HEK cells transfected with CCR5 showed susceptibility to recombinant LukED as expected (Supplementary Fig. 2e)²⁵, pore formation by culture supernatants in CCR5-expressing HEK cells was inefficient and not linked to lukE expression (Table 2 and Supplementary Fig. 2 a, b, f). However, culture supernatants of the clinical isolates induced efficient pore formation in HEK cells transfected with CXCR1 (Supplementary Fig. 2c). As CCR5-expressing HEK cells remained largely unaffected by the culture supernatants, it is likely that HlgAB mainly drives cytotoxicity towards CXCR1-transfected cells. Similar to cells expressing CCR5, pore formation in HEK cells co-transfected with CD11b/CD18 (CR3) was inefficient (Supplementary Fig. 2g). All culture supernatants showed high cytotoxic activity towards human neutrophils, which abundantly express the γ-Hemolysin receptors C5aR, CXCR1, and CXCR2 (Fig. 1c, Table 2 and Supplementary Fig. 2h).

Altogether, near-universal prevalence and consistent toxin expression discriminate γ-Hemolysin from other staphylococcal leukocidins, and identify HlgAB and HlgCB as highly significant secreted S. aureus toxins efficiently targeting phagocytic cells.

HlgAB and HlgCB induce pro-inflammatory responses

In addition to inducing pore formation, staphylococcal leukocidins have pro-inflammatory properties^33,38,39.

Sublytic concentrations of PVL prime neutrophil responses in a C5aR-dependent manner¹⁸. We thus tested if immune activating properties of HlgAB and HlgCB were receptor-mediated. HlgCB and HlgAB at low concentrations increased the N-formyl-methionine-leucine-phenylalanine (fMLP)-induced oxidative burst by neutrophils in a similar way as Tumor necrosis factor α (TNF-α), C5a, and Interleukin-8 (IL-8, CXCL8). Priming by HlgCB was fully antagonized after preincubation of cells with the specific C5aR-antagonist CHIPS_31–121 (Fig. 3a and Supplementary Fig. 3a)⁴⁰. This finding demonstrates that the C5aR drives priming of neutrophils by sublytic concentrations of HlgCB. To our knowledge, no molecules are available that antagonize both human CXCR1 and CXCR2. Therefore, we tested the CXCR2-specific proteolytic enzyme Staphopain-A for interference with HlgAB⁴¹. As expected based on the presence of CXCR1, preincubation of cells with Staphopain-A alone did not affect priming by HlgAB (Fig. 3b and Supplementary Fig. 3b).

In human macrophages, HlgCB activates inflammasome signaling³³. CHIPS, a specific C5aR antagonist⁴², antagonized IL-1β release induced by HlgCB (Fig. 3c). This finding implies that the C5aR mediates the release of pro-inflammatory cytokine IL-1β in response to HlgCB.

Taken together, these findings indicate that the C5aR mediates the pro-inflammatory properties of HlgCB. CXCR1 and CXCR2 likely mediate priming of neutrophils by HlgAB.

HlgA and HlgC are immunomodulatory molecules

Following activation with their natural ligands, signal transducing chemokine receptors provoke transient rises of intracellular calcium. Low concentrations of the single protein components HlgA and HlgC are not activating or cytotoxic⁴³. Since both proteins bind chemokine receptors, we tested if HlgA and HlgC act as functional antagonists of the respective receptors by detection of intracellular calcium mobilization.

As HlgC binds the signal transducing C5aR, we tested if HlgC acts as a functional inhibitor of this receptor. Just as LukS-PV, HlgC antagonized activation induced by C5a on neutrophils (Fig. 3d)¹⁸. Because C5L2 is an atypical chemokine receptor and not coupled to a G-protein⁴⁴, functional inhibition of this receptor by HlgC could not be tested. HlgA interacts with CXCR1, CXCR2, and CCR2. HlgA antagonized activation of neutrophils induced by IL-8 (a ligand of both CXCR1 and CXCR2) (Fig. 3e)⁴⁵. In addition, HlgA inhibited activation of monocytes induced by the CCR2-specific ligand MCP-1 (CCL2) (Fig. 3f).

These data suggest that the binding proteins of γ-Hemolysin as single components have receptor-specific immunomodulatory properties.

HlgAB and HlgCB feature receptor-selectivity in mice

S. aureus interacts with the host immune system in a highly human specific manner⁵. In order to explore murine susceptibility to γ-Hemolysin, we tested cytotoxicity of HlgAB and HlgCB towards murine bone marrow-derived leukocytes.

As observed for PVL, murine neutrophils and monocytes were resistant to HlgCB induced pore formation (Fig. 4a). In line with this result, HEK cells transfected with the murine C5aR or C5L2 were resistant to HlgCB (Fig. 4b).

HlgAB and LukED both target human CXCR1 and CXCR2 (Fig. 1b)²⁶. However, murine neutrophils were resistant to HlgAB-induced pore formation (Fig. 4a). In mice, CXCR2 but not CXCR1 is expressed on neutrophils and monocytes⁴⁶. Although HEK cells transfected with murine CXCR1 were susceptible to HlgAB-induced cytotoxicity, cells transfected with murine CXCR2 remained unaffected by HlgAB (Fig. 4b). The incompatibility of HlgAB with murine CXCR2 explains resistance of murine neutrophils to the toxin. In contrast, LukED is compatible with murine CXCR2 (Supplementary Fig. 4a), accounting for susceptibility of murine neutrophils towards LukED (Fig. 4a).

Monocytes of wild type and ccr5^−/− mice were susceptible to HlgAB induced pore formation, whereas monocytes of ccr2^−/− mice were HlgAB-resistant (Fig. 4a and 4c). In agreement with this, HEK cells transfected with the murine CCR2 showed clear sensitivity to HlgAB (Fig. 4b). Murine bone marrow cells from ccr2^−/− mice did not release IL-1β when exposed to HlgAB, in constrast to cells from wild type mice (Supplementary Fig. 4b). These results provide further evidence for a specific interaction of HlgAB with CCR2.

Collectively, our data show that γ-Hemolysin features distinct receptor-mediated specificity in mice and human. As a consequence, murine neutrophils are resistant to both HlgAB and HlgCB. Importantly, the results identify HlgAB and CCR2 as potential subjects for investigation in murine models of staphylococcal infection.

HlgAB targeting of CCR2 enhances S. aureus pathogenesis

Although the genes encoding γ-Hemolysin are present in nearly all human S. aureus isolates, its contribution to staphylococcal pathophysiology is not fully understood. Since we discovered that HlgAB targets CCR2, we investigated the contribution of this interaction to S. aureus pathogenesis in vivo in a murine peritonitis model.

Inflammatory macrophages in the peritoneal cavity of mice, elicited by heat-inactivated S. aureus, express CCR2 (Fig. 5a)⁴⁷.

To investigate γ-Hemolysin mediated targeting of CCR2, we monitored the presence of CCR2⁺ inflammatory macrophages in the peritoneal cavity of mice infected with S. aureus USA300 clone SF8300 or its isogenic hlgACB mutant (hlgACB knockout) strain^33,48. Mice infected with wild type S. aureus showed a significant decrease in the proportion of CCR2⁺ inflammatory macrophages compared to mice infected with the hlgACB mutant strain (Fig. 5b). Notably, in the same mice, the fraction of CCR5⁺ macrophages was equal between wild type and hlgACB mutant infected mice. In agreement with resistance of murine neutrophils to γ-Hemolysin, mice infected with wild type and hlgACB mutant had similar proportions of neutrophils (Fig. 5b). Using another USA300 clone, LAC³⁴, and its isogenic hlgA mutant strain (hlgA::bursa), the observed modulation of the CCR2⁺ inflammatory macrophage population was shown to be HlgA specific (Supplementary Fig. 5a). These data demonstrate that during infection, HlgAB is active and specifically targets CCR2⁺ inflammatory macrophages in the peritoneal cavity.

Staphylococcal peritonitis is used as a model to study the spread of bacteria into the bloodstream⁴⁹. We observed that the number of viable bacteria in the peripheral blood of mice is dependent on the presence of both HlgAB and CCR2. Twenty four hours after intraperitoneal infection, wild type mice infected with S. aureus USA300 clone SF8300 had ± 100 fold higher counts of viable bacteria in the peripheral blood compared to wild type mice infected with the hlgACB mutant strain (Fig. 5c). Using USA300 clone LAC and its isogenic hlgA mutant, the bacteremia was confirmed to be driven specifically by HlgA (Supplementary Fig. 5b). Cellular recruitment in response to sterile inflammatory stimuli is impaired in ccr2^−/− mice⁵⁰. In contrast, infection with S. aureus induced similar influx of macrophages into the peritoneal cavity of wild type and ccr2^−/− mice (Supplementary Fig. 5c). Compared to wild type mice, infection of ccr2^−/− mice with S. aureus USA300 clone SF8300 resulted in ± 100 fold lower numbers of bacteria in the peripheral blood (Fig. 5c). The number of bacterial counts in the peripheral blood of ccr2^−/− mice was independent of γ-Hemolysin. Moreover, no statistically significant differences were observed in the bacterial counts of the peritoneal cavity after twenty four hours in all infected mice (Fig. 5c and Supplementary Fig. 5b), representing comparable levels of bacteria as the seeding source. These results demonstrate that HlgAB contributes to S. aureus bacteremia in a CCR2-dependent manner. In addition, the interaction of HlgAB with CCR2⁺ cells highlights the involvement of inflammatory macrophages during S. aureus infection.

Ethics Statement

Human leukocytes were isolated after informed consent was obtained from all subjects in accordance with the Declaration of Helsinki. Approval was obtained from the medical ethics committee of the UMC Utrecht, The Netherlands. All experiments involving animals were reviewed and approved by the animal ethics committees of Lyon, France (CECCAPP, protocol number ENS2012_033).

Recombinant Protein Production and Purification

S. aureus strains used to amplify coding sequences of toxins are shown in Supplementary Table 1. HlgA, HlgB, HlgC, LukE, LukD, LukS-PV and LukF-PV were cloned and expressed as described elsewhere³³. Briefly, the coding sequences omitting the regions predicted to encode the signal peptide were cloned and transformed into E. coli M15 or BL21 pLys. Protein expression was induced by adding isopropyl-b-d-thiogalactopyranoside during exponential growth of transformed E. coli. Polyhistidine-tagged proteins were isolated from supernatants of cell lysates on Ni-nitrilotriacetic acid columns (Qiagen). Contaminating LPS was eliminated using a Polymixin-B column (Thermo Scientific).

Bacterial Strains and Culture

S. aureus strains used for this study are listed in Table 2 and Supplementary Table 2. Complementation of ΔhlgACB mutants and generation of an erythromycin sensitive USA300 LAC ΔhlgA strain is described in Supplementary Methods. Clinical S. aureus strains, isolated from blood cultures with a diverse infection focus and originating from patients admitted to the intensive care unit of the University Medical Center Utrecht, The Netherlands, were randomly chosen from a database created for tertiary hospital routine diagnostics. Standard microbiological diagnostic techniques were applied for microbial identification and antibiotic resistance profiling. All strains were cultured in casein hydrolysate and yeast extract (CCY)^36,37 or brain heart infusion (BHI), and overnight supernatants filter sterilized. For in vivo experiments, mid-exponential subcultures were washed extensively in PBS.

Typing of clinical S. aureus isolates

All clinical S. aureus isolates used in this study were typed by multi-locus sequence typing (MLST)⁵⁷. Using the eBURST algorithm, isolates were assigned to a clonal complex⁵⁸. Leukocidin S-component PCRs were performed on genomic DNA, which was isolated as previously described⁴². Primers were designed to be highly gene specific (Supplementary Table 3).

S. aureus RNA Extraction and cDNA Synthesis

S. aureus RNA was isolated from samples using an RNeasy Plus Mini Kit (Qiagen) following instructions of the manufacturer. Bacteria were pre-cultured overnight and subsequently diluted in fresh medium for culture to early stationary phase (6,5 hrs; OD₆₆₀ ± 1,5). Bacteria were spun for 30 s at 13.000 rpm. Pellets were briefly kept in liquid nitrogen to stop bacterial metabolism, and resuspended in buffer RLT containing β-mercaptoethanol (Sigma). Resuspended bacteria were added to silica beads (Merlin), and disrupted using a mini-Beadbeater (BioSpec Products) for 1 min at 3.500 rpm. After cooling of the samples for 1 min on ice, an additional two cycles of disruption were applied. Contaminating chromosomal DNA was removed by consecutive treatment of samples with RNAse Free DNAse (Qiagen) and a RapidOut DNA Removal Kit (Thermo Scientific). RNA yield was measured using a NanoDrop ND1000 (NanoDrop Technologies), and quality was checked on agarose gel. Contamination with genomic DNA was excluded by a PCR specific for genomic DNA. Standardized quantities of total RNA were used immediately for synthesis of cDNA with a Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific).

Quantitative Real-Time PCR

cDNA from the equivalent of 10 ng mRNA was used to perform qPCR for hlgA, hlgC, lukE, and lukS-PV using SYBR Green Master Mix (Qiagen) in a 7300 Real Time PCR System (Applied Biosystems) according to manufacturer’s instructions. cDNA from the equivalent of 1 ng mRNA was used to amplify 16S as an endogenous control. Primers used to detect specific transcripts are listed in Supplementary Table 4. These primers are based on toxin sequences from strains Newman and USA300 (lukS-PV). Triplicate reactions for each primer set containing approximately 100 copies of S. aureus USA300 clone LAC genomic DNA were included to calibrate transcript abundance of each toxin. C_T values were collected over 40 cycles of PCR, with primer annealing at 60°C, and data collection during the 30 second extension step at 72°C.

Analysis was performed using 7300 Real Time PCR System Software. Relative transcript abundance was calculated by the comparative C_T (ΔΔC_T) method, using 16S as the endogenous control and USA300 clone LAC genomic DNA as the calibrator sample. Samples with an average C_T value greater than 32 were considered background and were excluded from analysis. Toxin expression is indicated as the log₁₀ of the fold change in relative transcript abundance of each toxin compared to abundance from amplification from USA300 clone LAC genomic DNA.

Cell Isolations

Human leukocytes, obtained from healthy volunteers, were isolated by Ficoll/Histopaque centrifugation⁴². Bone marrow of mice was harvested and immune cells collected as described¹⁸. Mice receiving intraperitoneal injections with heat-inactivated S. aureus USA300 SF8300 ΔhlgACB (1×10⁸ CFU, at 0 hr and 24 hr) were sacrificed after 48 hr. Immune cells were collected by peritoneal lavage with PBS.

All in vitro experiments with cells were performed with RPMI (Invitrogen) supplemented with 0.05% human serum albumin (Sanquin) unless specified otherwise, with cell concentrations adjusted to 5×10⁶ cell mL⁻¹.

Quantification of Human Leukocyte Receptor Expression Levels

Freshly isolated human leukocytes were incubated in a total volume of 50 µL at 5×10⁶ cell mL⁻¹ on ice with 10 µg mL⁻¹ mouse anti-human chemokine receptor monoclonal antibodies for C5aR (clone S5/1, AbD SeroTec), C5L2 (clone 1D9-M12, Bio Legend), CXCR1 (clone 42705, R&D), CXCR2 (clone 48311, R&D), and CCR2 (clone 48607, R&D), followed by FITC-conjugated goat-anti-mouse antibody (1:50, Dako). Antibody binding was quantified by calibration to defined antibody binding capacity units using QIFIKIT (Dako) and corrected for isotype controls.

Cell Lines and Transfections

U937 cells (a promyelocytic cell line) stably transfected with C5aR, CXCR2 or an empty expression vector were obtained from Eric R. Prossnitz (University of New Mexico, Albuquerque, NM, USA)⁵⁹.

Human C5aR, C5L2, C3aR, CXCR1, CXCR2, CCR1, CCR2 and CCR5, and murine C5aR, C5L2, CXCR1, CXCR2, and CCR2 were cloned into a pcDNA3.1 vector (Invitrogen) as described previously¹⁸. All genes reside on a single exon. Amplification reactions were performed on QUICK-Clone cDNA of human bone marrow or mouse liver (BD Biosciences Clontech) using PfuTurbo DNA polymerase (Stratagene). Plasmids encoding human C5L2 and CXCR2 were purchased from UMR cDNA Resource Center. Plasmids encoding CXCR3, CXCR4, and CXCR6 were purchased from Thermo Fisher. Primer designs, restriction sites and accession numbers used in this study are displayed in Supplementary Table 5. HEK293T cells (a human embryonic kidney cell line) were transiently transfected according to the manufacturer’s protocols with 4 µg DNA and 5 µl Lipofectamine 2000 (Life Technologies)¹⁸. After 24 hr, transfected cells were harvested with 0.05% Trypsin / 0.53 mM EDTA. Maximal pore formation of HEK293T cells transiently transfected with plasmids encoding chemokine receptors was defined by the percentage of receptor expressing cells as independently determined by flow cytometry.

For construction of HEK293T cells stably expressing human C5aR, CXCR1, CCR2, CCR5, or CR3 (CD11b/CD18), we cloned the complete coding regions of these receptors (Supplementary Table 6) into the bicistronic expression plasmid pIRESpuro3 (Clontech). CD18 was cloned into pIRESpuro3, whereas CD11b was cloned into pIREShyg3 (Clontech). For stable expression, the constructs were transfected into HEK293T cells using Lipofectamine 2000 (Life Technologies). One day after transfection, cells were put under selection pressure using 1 µg mL⁻¹ puromycin in DMEM with 10% FCS. A few weeks later, cells were subcloned and tested for stable receptor expression by flow cytometry.

Expression of human chemokine receptors on transfected cells was defined using 10 µg mL⁻¹ mouse anti-human chemokine receptor monoclonal antibodies for C5aR (clone S5/1, AbD SeroTec), C5L2 (clone 1D9-M12, Bio Legend), C3aR (clone 534625, R&D), CXCR1 (clone 42705, R&D), CXCR2 (clone 48311, R&D), CXCR3 (clone 49801, R&D), CXCR4 (clone 12G5, R&D), CXCR6 (clone MAB699, R&D), CCR1 (clone 53504, R&D), CCR2 (clone 48607, R&D), and CCR5 (clone 2D7, BD), followed by PE-labeled goat anti-mouse antibody (1:80, DAKO). For cells expressing CR3, cells were stained with an APC-labeled mouse monoclonal antibody directed towards CD18 (clone L130, 1:50, BD) and a FITC-labeled mouse monoclonal antibody detecting CD11b (clone ICRF44, 1:10, BD). Receptor expression levels were similar within each panel of transfected cells for all receptors (Supplementary Fig. 6a, b, c). As no monoclonal antibodies are available for all involved murine chemokine receptors, expression of murine receptors was defined by co-transfection with an IRES-GFP encoding pCDNA3 vector (Invitrogen)⁶⁰. GFP expression was similar for all murine receptors investigated, and fully correlated with susceptibility of cells transfected with toxin-compatible receptors (Supplementary Fig. 6d).

Cell Permeability Assays

Cells were exposed to recombinant proteins or overnight bacterial culture supernatant in a volume of 50–100 µL. Human PBMC were pre-stained for CD14 expression (clone M5E2, 1:10, BD). Cells were incubated with toxin for 30 min at 37°C. Human leukocytes and cell lines were subsequently analyzed by flow cytometry and pore formation was defined as intracellular staining by propidium iodide (PI) or 4',6-diamidino-2-phenylindole (DAPI). After treatment with Fcγ-receptor block, murine cell surfaces were stained with antibodies directed towards CD11b (clone M1/70, 1:400, BD), Ly6G (clone 1A8, 1:100, BD), Ly6C (clone AL-21, 1:200, BD) and F4/80 (clone BM8, 1:600, Bio Legend) antibodies. Following exposure to toxins, murine cells were incubated on ice with the fixable viability dye eFluor 780 (1:1000, eBiosciences) and cell viability was subsequently analyzed by flow cytometry in the presence of Flow-Count Fluorospheres (BD). Cell viability was defined as eFluor 780 negative cells in relation to buffer treated cells.

Analysis of pore formation in transiently transfected HEK293T cells was limited to receptor-positive cells. Half maximal effective lytic concentrations were calculated using nonlinear regression analyses.

As leukocidins are 2-component toxins, equimolar concentrations of polyhistidine-tagged proteins were used. To minimize bias due to production of the nonspecifically cytotoxic phenol soluble modulins (PSMs), experiments with culture supernatants were performed in the presence of 10% fetal calf serum^61,62.

Surface Plasmon Resonance Studies

Surface Plasmon Resonance of HlgA and HlgC and chemokine receptor proteoliposomes was performed using the Biacore T100 system and the series S L1 sensor chip using 10 mM Tris 1 mM MgCl₂ and 1 mM CaCl₂ as the running buffer. Wheat-germ proteoliposomes of CXCR1, CXCR2, CCR2 and C5aR (Abnova) at a final protein concentration of 20 µg mL⁻¹ were immobilized onto test flow cells with a flow rate of 10 µL min⁻¹ for 10 min with a minimum capture level of 600RU. Empty, mock wheat-germ proteoliposomes (Abnova) were immobilized onto the reference flow cell using the same conditions with a minimum capture level of 300RU being obtained. Single cycle kinetics was initially used to establish concentration ranges for HlgA and HlgC for each chemokine receptor liposome with concentrations ranging from 0.01 to 20 µg mL⁻¹ with 5 µg mL⁻¹ used as the maximum concentration used in multiple cycle kinetics. A flow rate of 30 µL min⁻¹ was used for analysis with a contact time of 1 min and a disassociation time of 10 min for each concentration used. To ensure the chemokine receptors captured on the L1 chip were biologically relevant MCP-1 (Peprotech) was tested against CCR2. Concentrations of between 16 ng mL⁻¹ and 500 ng mL⁻¹ were tested. The affinity of MCP-1 with the captured CCR2 liposome was found to be 0.84 nM ± 014 nM on three replicate experiments. This was within the reported values found in the literature indicating that the proteoliposomes were in their native conformation⁶³.

Neutrophil Priming

Priming of human neutrophils has been described in detail recently¹⁸. Briefly, neutrophils were incubated for 5 min with 700 nM CHIPS_31–121 to block the C5aR, or for 15 min with 500 nM Staphopain-A to cleave the N-terminal CXCR2. Cells were primed with 10 ng mL⁻¹ TNFα, 1 nM C5a, 10 nM IL-8, or a range of sublytic concentrations of HlgCB or HlgAB for 30 min at 37°C. Subsequently, chemiluminescence was recorded in a luminometer. After 30 s of baseline recording, a final concentration of 1 µM fMLP was injected to stimulate the oxidative burst. For each sample, the area under the curve (AUC) after addition of fMLP was calculated. For comparison of experiments, AUC values were expressed relative to buffer-treated cells stimulated with fMLP.

Interleukin-1β Production

Derivation of primary human macrophages from monocytes is described elsewhere³³. To induce pro-IL-1β and prime the NLRP3-inflammasome, human macrophages were treated with heat-killed S. aureus at a multiplicity of 100:1 for 16 hr³³. Cells were washed twice to remove heat-killed S. aureus and exposed to toxin during 1 hr at 37°C. Murine bone marrow cells were pretreated with 250 ng mL⁻¹ lipopolysaccharide for 3 hr and subsequently intoxicated with 6 nM HlgAB or 20 µM Nigericin during 30 min at 37°C. Release of human and murine IL-1β into the supernatant was quantified by enzyme linked immunosorbent assays (DuoSet ELISA kits, R&D Systems)³³.

Calcium Mobilization Assays

Activation of human neutrophils and monocytes was performed by real-time detection of calcium mobilization using flow cytometry¹⁸. Human monocytes were labeled with an anti-CD14 monoclonal antibody (BD). Cells, labeled with Fluo-3AM, were pre-incubated with HlgA or HlgC for 1 min at room temperature. Subsequently, baseline fluorescence was recorded in a flow cytometer. After 10 s of baseline recording, C5a (Sigma), IL-8, or MCP-1 (PeproTech) was added and fluorescence was recorded for an additional 50 s. Relative increase in fluorescence was expressed in comparison to uninhibited cells.

Murine In Vivo Experiments

C57BL/6 wild type mice were obtained from Charles River Laboratories. ccr2^−/− and ccr5^−/− mice (Jackson Laboratories) were bred in house. 8 to 9 week old age and sex matched mice were infected intraperitoneally with 0.5 mL PBS containing 1×10⁸ CFU of bacteria. At 24 hr postinfection, peripheral blood was obtained and mice were sacrificed in order to recover peritoneal lavage fluid. Peripheral blood and peritoneal lavage fluid were plated to evaluate the bacterial burden. After incubation on ice with the fixable viability dye eFluor 780, peritoneal cells were treated with Fcγ-receptor block and stained with monoclonal antibodies detecting CD11b, Ly6G, Ly6C, F4/80 and CCR2 (clone 475301, 1:100, R&D) or CCR5 (clone HMCCR5, 1:100, Bio Legend). Cells were subsequently analyzed by flow cytometry and viable cells were defined as eFluor 780 negative cells. In the model of staphylococcal peritonitis, mortality of mice was equal for all groups (± 20%).

Graphical and Statistical Analyses

Flow cytometric analyses were performed with FlowJo (Tree Star Software). Statistical analyses were performed with Prism (GraphPad Software). Statistical significance was calculated using ANOVA and Student’s t-tests where appropriate.