Differential Interaction of the Staphylococcal Toxins Panton-Valentine Leukocidin and γ-Hemolysin CB with Human C5a Receptors

Ethics Statement

Human neutrophils were isolated after informed consent was obtained from all donors in accordance with the Declaration of Helsinki. For this, approval was obtained from the medical ethics committee of the UMC Utrecht, The Netherlands. Experiments involving animals were reviewed and approved by the animal ethics committees of Lyon, France (CECCAPP, protocol number ENS_2012_033), or Utrecht, The Netherlands (protocol number DEC.2012.II.09.136).

Recombinant protein production and purification

S. aureus strains used to amplify coding sequences of toxins and bacterial proteins include strain ATCC 49775 for LukS-PV and LukF-PV (30), strain Cagnant for HlgC and HlgB (30), strain Newman for CHIPS (31), and strain MW2 for FLIPr-Like (32). Coding sequences were cloned and expressed as described elsewhere (30-32). For the bicomponent pore-forming toxins, single protein coding sequences omitting the predicted signal peptide were cloned and transformed into E. coli M15 or BL21 pLys. Polyhistidine-tagged proteins were isolated from supernatant of E. coli cell lysates on Ni-nitrilotriacetic acid columns (Qiagen).

Cell Isolation

Human neutrophils, obtained from healthy volunteers, macaque (Macaca fascicularis), and rabbit (New Zealand White) neutrophils were isolated by Ficoll/Histopaque centrifugation (31). Neutrophils from cows (Holstein Frisian) were isolated using Percoll (1.09176 g/L) centrifugation. Mouse (C57BL/6) leukocytes were obtained from bone marrow and immune cells were collected as described (28). Unless specified otherwise, all in vitro experiments with cells were performed using RPMI (Invitrogen) supplemented with 0.05% human serum albumin (Sanquin), with cell concentrations adjusted to 5 × 10⁶ cell/mL.

Cell lines and transfections

HEK293T cells (a human embryonic kidney cell line) were used for overexpression of wild type and chimeric receptors. The genes encoding the C5a receptors of all mammalian species investigated reside on a single exon. Amplifications were performed on QUICK-Clone cDNA of human bone marrow or mouse liver (BD Biosciences Clontech), macaque, rabbit, and cow peripheral blood using PfuTurbo DNA polymerase (Stratagene). N-terminus and extracellular loop exchange chimeric receptors were constructed using overlap extension PCR as described elsewhere (33). The receptor sequences were amplified and fused together in multiple PCR steps. The 5’- and 3’- terminal primers contained respectively EcoRI and NotI restriction sites, respectively, allowing for specific ligation into the multiple cloning sites of the pcDNA3.1 vector (Invitrogen) (28), or the bicistronic expression plasmid pIRESPuro3 (Clontech) (24). To confirm surface expression of the receptors on transiently transfected cells during binding studies of LukS-PV, an N-terminal FLAG-tag (DYKDDDDK) was placed after the first methionine. An additional methionine was placed directly after the FLAG-tag to keep the N-terminal sequence intact. Expression vectors, primer designs, restriction sites, accession numbers, and primer combinations used in this study are listed in Supplementary Table I (wild type full length receptors), Supplementary Table II (primer designs used for chimeric receptors) and Supplementary Table III (primer combinations used for C5aR1 or chimeric receptors). Cloning of rabbit C5aR2 resulted in an amino acid sequence slightly divergent from the annotated sequence. All wild type amino acid sequences used in this study are aligned in Fig. 2. HEK293T cells stably transfected with a plasmid encoding human C5aR2 were donated by Peter N. Monk (Sheffield University Medical School, Sheffield, UK) (34).

Cells were either transiently or stably transfected as described elsewhere (24, 28). Briefly, cells were transfected with 4 μg DNA and 5 μL Lipofectamin 2000 (Life Technologies), according to the manufacturer's protocol. For transient transfections, cells were harvested after 24 hours with 0.05% Trypsin/0.53 mM EDTA. For stable transfections, selection pressure was applied using 1 μg/mL puromycin in DMEM with 10% fetal calf serum and cells were subcloned after several weeks.

The expression of receptors was confirmed using 10 μg/mL monoclonal antibody (PE-conjugated mouse anti-human C5aR1 clone S5/1, 1:50, Biolegend; FITC-conjugated rat anti-mouse C5aR1 clone 20/70, 1:50, Biolegend; PE-conjugated mouse anti-human C5aR2 clone 1D9-M12, 1:50, Biolegend) or 240 nM FITC-conjugated C5a (Bachem) where possible. Specific susceptibility towards PVL- and/or HlgCB-toxicity of cells stably transfected with plasmids encoding wild type C5a receptors from different mammalian species was interpreted as a confirmation of receptor expression. In the case of resistance of cells towards both PVL and HlgCB-toxicity, stable expression of wild type C5a receptors was confirmed (Supplementary Fig. 1A, B). Due to inability to bind monoclonal antibodies or C5a, expression of chimera Mouse C5aR1 Human ECL1+2+3 and chimera Mouse C5aR2 Human ECL1+3 on stably transfected cells could not be verified. The expression of the remaining chimeric receptors was verified similarly (Supplementary Fig. 1 C – P).

Cell Permeability Assays

Cells were exposed to recombinant toxins in a volume of 50 μL for 30 min at 37 °C. Human, macaque, rabbit, and cow neutrophils, and cell lines were subsequently analyzed by flow cytometry and gated for forward and side scatters. At least 10,000 cells per sample were analyzed. For graphical presentation, pore formation was defined as the percentage of cells positive for intracellular staining by 4’,6-diamidino-2-phenylindole (DAPI).

After treatment with Fcγ-receptor block, mouse leukocytes were stained with monoclonal antibodies directed towards CD11b (clone M1/70, 1:400, BD), Ly6G (clone 1A8, 1:100, BD), and Ly6C (clone AL-21, 1:200, BD). After exposure to toxins, mouse cells were incubated on ice with the fixable viability dye eFluor 780 (1:1,000, eBiosciences). Cell viability of mouse neutrophils was analyzed by flow cytometry in the presence of Flow-Count Fluorospheres (BD) and defined as eFluor 780 negative cells relative to buffer-treated cells.

For C5aR1-competition experiments, human neutrophils were preincubated for 10 min at room temperature with CHIPS (710 nM), mouse anti-human C5aR1 monoclonal antibody (clone S5/1, AbD SeroTec, 67 nM), NDT 9513727 (Tocris, R&D, 10 μM), W-54011 (Calbiochem, Merck Millipore, 10 μM), PMX 205 (Tocris, R&D, 10 μM), or AS-65122 (AnaSpec, 10 μM). Concentrations fof C5aR1-antagonists used are based on efficacy titration for PVL and HlgCB.

Analysis of pore formation in transiently transfected HEK293T cells was corrected for the fraction of receptor-positive cells. Transiently transfected or stably transfected cells were reciprocally compared in functional experiments.

Equimolar concentrations of toxin protein components were used.

Binding assays

For binding experiments with LukS-PV, cells transiently transfected with plasmids encoding receptors with an N-terminal FLAG tag (28) were pre-labeled with a mouse anti-FLAG monoclonal antibody (clone M2, 1:500, Sigma), followed by PE-labeled goat-anti-mouse antibody (1:80, Dako). For experiments with HlgC, stably transfected cells were directly used for subsequent binding. After incubation with the protein on ice for 30 min in a volume of 50 μL, cells were washed and incubated with a FITC-conjugated mouse anti-his monoclonal antibody (1:80, LifeSpan Biosciences). Binding was detected using flow cytometry, and at least 10,000 cells were analyzed. Analysis of binding to transiently transfected cells was limited to receptor-positive cells. Binding of the S-components was expressed as the percentage of mean fluorescence in relation to saturated binding of the S-component to human C5aR1 at 31 nM.

Quantification of leukocyte receptor expression levels

Freshly isolated macaque and human neutrophils were incubated in a total volume of 50 μL at 5 × 10⁶ cell/mL on ice with 10 μg/mL mouse anti-human chemokine receptor monoclonal antibodies for C5aR1 (clone S5/1, AbD SeroTec) or C5aR2 (clone 1D9-M12, BioLegend), followed by PE-conjugated goat-anti mouse antibody (1:80, Dako). Samples were analyzed using flow cytometry. At least 10,000 cells were analyzed, and cells were gated for forward and side scatters. Antibody binding was quantified by calibration to defined antibody binding capacity units using QIFIKIT (Dako) and corrected for isotype controls.

Graphical and statistical analyses

Flow cytometric data were analyzed with FlowJo (Tree Star Software). Statistical analyses were performed using Prism (GraphPad Software).

PVL and HlgCB feature distinct species specificity

Some leukocidins exert cytotoxic activity in a species-specific manner (21, 24, 26, 28). For PVL, species-specific toxicity is well-described (21). Although PVL and HlgCB have shared receptors, species-specific susceptibility of primary cells towards HlgCB is not completely understood. To compare species specificity of HlgCB to PVL, we tested toxin-mediated cell permeability in freshly isolated neutrophils from different mammalian species using a membrane-impermeant fluorescent dye. Cells from mouse and rabbit were investigated, as both mammals are frequently used in preclinical experimental models of S. aureus infections (21). In addition, macaque neutrophils were investigated since this primate is closely related to humans. Finally, cow neutrophils were included, as this species is a natural host to S. aureus (35). As expected, PVL was only active towards human and rabbit neutrophils but not towards neutrophils from macaque, cow, and mouse (Fig. 1A). However, HlgCB was active towards neutrophils from all species tested, except for mice (Fig. 1A). These data identify that the rabbit is the only compatible species for establishing the contribution towards staphylococcal pathophysiology of both PVL and HlgCB in vivo. Furthermore, the divergent susceptibility of neutrophils from different mammalian species towards PVL and HlgCB suggests a differential interaction of both toxins with their receptors.

PVL and HlgCB differentially interact with C5aR1 and C5aR2 in a species-dependent manner

To further investigate the molecular interaction of PVL and HlgCB with neutrophils from different mammalian species, we screened human embryonic kidney (HEK) cells overexpressing the respective orthologues of human C5aR1 and C5aR2 for their susceptibility to these two toxins.

In line with susceptibility of neutrophils, only cells expressing human or rabbit C5aR1 were susceptible towards PVL. As illustrated in Figure 1B, cells expressing macaque, cow, or mouse C5aR1 were fully resistant towards pore formation induced by PVL. In accordance with the results obtained with primary cells, cells expressing different mammalian C5a receptors, with the exception of mouse C5aR1, were susceptible towards HlgCB-induced toxicity (Fig. 1B). PVL was cytotoxic towards cells expressing human or rabbit C5aR2, while mouse and cow C5aR2 were incompatible with PVL (Fig. 1C). In contrast to the resistance of neutrophils, HEK cells expressing macaque C5aR2 were susceptible to pore formation induced by PVL (Fig. 1C). Just as in humans (28), expression of C5aR2 on macaque neutrophils is very low compared to C5aR1 (Supplementary Fig. 1Q). Similarly to C5aR1, screening of C5aR2 with HlgCB showed broad species compatibility, again with the exception of mouse C5aR2 (Fig. 1C). Cells transfected with plasmids encoding PVL- and HlgCB-incompatible receptors expressed equal levels of receptors compared to cells transfected with plasmids encoding PVL- or HlgCB-compatible receptors (Supplementary Fig. 1A, B).

Jointly, these analyses demonstrate that the mouse is a poor model to investigate both PVL and HlgCB as a virulence factor due to a molecular incompatibility of the toxins with its respective receptors. Furthermore, these data show that although PVL and HlgCB target the complement receptors C5aR1 and C5aR2, both toxins do so in a species-dependent differential manner.

Different domains of C5aR1 define human specificity of PVL and HlgCB

Although C5aR1 and C5aR2 are relatively conserved among different mammalian species, amino acid sequence identities vary from 60% to 93% when compared to the human receptors (Fig. 2A, B). In an attempt to define the regions of the receptors responsible for the observed species-specific toxin-receptor interaction, we tested cells expressing chimeric C5a receptors for susceptibility towards PVL and HlgCB. Since most amino acid sequence divergence between the C5a receptors of the different mammalian species is clustered in the N-termini and ECLs (Fig. 2C, D), we focused on the extracellular domains of the respective receptors.

While macaque C5aR1 is most closely related to human C5aR1, this receptor is incompatible with PVL (Fig. 1B, 2C). Therefore, we used macaque and human C5aR1 as chimeric templates to investigate the importance of the different extracellular receptor domains for interaction with PVL on receptor-expressing HEK cells. Receptor expression was similar for all constructs tested (Supplementary Fig. 1C – L). For PVL, exchange of the N-terminus of human C5aR1 with macaque or mouse N-terminal C5aR1 did not result in loss of susceptibility (Fig. 3A). Exchange of the individual human ECL2 or ECL3 with those of macaque resulted in loss of susceptibility to PVL, however replacement of ECL1 showed no effect (Fig. 3B). Since the amino acid sequence of human and macaque ECL1 is nearly identical (Fig. 2C), we additionally tested a chimeric human C5aR1 with mouse ECL1. Cells expressing this receptor were resistant to pore formation induced by PVL, just as cells expressing a human C5aR1 chimera with mouse ECL2 or ECL3 (Fig. 3C). Interestingly, although replacement of human ECL2 with macaque ECL2 is sufficient to prevent PVL-toxicity, the sequences of ECL2 differ in only few amino acids (Fig. 2C). Thus, we introduced single amino acid mutations in the ECL2 of human C5aR1 following the macaque sequence. Cells expressing a P183S mutant human C5aR1 were resistant to pore formation induced by PVL (Fig. 3D), highlighting the critical role of C5aR1-ECL2 in human-specific cytotoxicity of PVL. In addition, mutation of the human amino acid cassette sequence SHDK to the respective macaque sequence NNDT resulted in loss of susceptibility towards PVL. Introduction of the individual human ECL2 or ECL3 in macaque C5aR1 did not restore susceptibility of cells towards PVL (Fig. 3E). Cells expressing a macaque C5aR1 chimera with both human ECL2 and ECL3 showed a partially restored susceptibility towards PVL, just as cells expressing a mouse C5aR1 chimera with human ECL1, 2, and 3 (Fig. 3F). This observation suggests that, in addition to the extracellular domains, other parts of the receptor are involved in the interaction with PVL as well.

Mouse C5aR1 is the most divergent receptor from the panel of mammalian C5a receptors investigated, and it is the only HlgCB-incompatible C5aR1 (Fig. 1B, 2C). Thus, we used human and mouse C5aR1 as chimeric templates to study the interaction between HlgCB and C5aR1. Similar to PVL, exchange of the N-terminus of human C5aR1 with mouse N-terminal C5aR1 did not result in loss of susceptibility of cells towards HlgCB (Fig. 4A). In contrast to PVL, introduction of mouse ECL2 in human C5aR1 did not affect susceptibility of cells towards HlgCB (Fig. 4B). Introduction of mouse ECL1 or ECL3 in human C5aR1 resulted in partial and complete loss of cell susceptibility towards HlgCB-induced pore formation, respectively (Fig. 4B). ECL3 of mouse C5aR1 is highly divergent from the human amino acid sequence (Fig. 2C). Introduction of single amino acid mutations in the ECL3 of human C5aR1 following the mouse sequence did not completely abolish susceptibility of cells towards HlgCB (Fig. 4C), indicating that the interaction between HlgCB and human C5aR1 involves a combination of multiple amino acid residues in ECL3. As expected based on the studies with loss of function chimeric receptors, introduction of human ECL1 in mouse C5aR1 was insufficient to result in susceptibility of cells towards HlgCB (Fig. 4D). However, introduction of human ECL3 in mouse C5aR1 resulted in a substantial restoration of susceptibility towards HlgCB (Fig. 4D). Introduction of both human ECL1 and ECL3 did not further enhance susceptibility of cells towards HlgCB. These data indicate that specificity of HlgCB for human C5aR1 is mostly defined by ECL3 and ECL1.

Together, these findings demonstrate that PVL and HlgCB differentially interact with C5aR1 in a complex manner. Results with chimeric receptors suggest that both PVL and HlgCB interact with multiple extracellular domains of C5aR1. While human C5aR1-specificity of PVL is critically driven by ECL2, human C5aR1-specificity of HlgCB is mostly determined by ECL3 and ECL1. Incomplete functional restoration of macaque and mouse C5aR1 chimeric receptors towards PVL and HlgCB indicate that, in addition to the extracellular domains, other domains of the receptor are likely involved in the interaction with both toxins as well.

PVL and HlgCB differentially interact with human C5aR2

In addition to C5aR1, we tested cells expressing chimeric C5aR2 receptors for susceptibility towards PVL and HlgCB. Mouse C5aR2 is the most divergent receptor from the mammalian C5aR2 receptors tested, and the only PVL- and HlgCB-incompatible C5aR2 (Fig. 1C). The N-terminus of mouse C5aR2 is most divergent compared to the amino acid sequence of other mammalian species (Fig. 2D). Thus, we used human and mouse C5aR2 as chimeric templates to study the interaction of PVL and HlgCB with receptor-expressing HEK cells. Receptor expression was similar for all constructs tested (Supplementary Fig. 1L – P).

Exchange of the N-terminus of human C5aR2 with the N-terminus of mouse C5aR2 resulted in a complete and partial loss of susceptibility for PVL and HlgCB, respectively (Fig. 5A, B). While introduction of mouse ECL1 and ECL3 into human C5aR2 caused a complete loss of susceptibility of cells towards PVL (Fig. 5C), none of the mouse ECLs substantially affected susceptibility towards HlgCB-toxicity (Fig. 5D). Introduction of mouse ECL2 into human C5aR2 only moderately affected susceptibility of cells towards both PVL (Fig. 5C). Introduction of individual human ECLs, or all three human ECLs together, into mouse C5aR2 was insufficient to restore susceptibility towards PVL and HlgCB (Fig. 5E, F). Additional introduction of the human N-terminus did not result in PVL- and HlgCB-susceptibility either (Fig. 5E, F).

These findings indicate that PVL and HlgCB interact with human C5aR2 in a different manner. As the introduction of all human extracellular domains in mouse C5aR2 did not result in susceptibility of cells towards both toxins, other parts of the receptor are clearly also involved in the interaction with the toxins.

S-component binding to the receptor does not necessarily allow subsequent pore formation

Recently, it was identified that staphylococcal leukocidins act via cell surface receptors (24-26, 28). Although expression of the specific receptor is a prerequisite for the bicomponent toxins to form pores in the cell membrane, the role of the receptors during the process of binding, oligomerization, and pore formation itself has not been fully elucidated. This led us to investigate, in addition to pore formation, the binding of the S-components of PVL (LukS-PV) and HlgCB (HlgC) to an exemplary selection of HEK cells overexpressing chimeric C5a receptors.

Figure 6A confirms that LukS-PV cannot bind to HEK cells expressing macaque C5aR1, in contrast to human C5aR1 (28). In line with the loss of pore-forming capacity of PVL (Fig. 6B), introduction of macaque ECL2 in human C5aR1 resulted in a complete loss of detectable binding of LukS-PV (Fig. 6A). Although a replacement in macaque C5aR1 with human ECL2 did not restore pore-forming ability of PVL (Fig. 6B), binding of LukS-PV to cells expressing this chimeric receptor was similar when compared to wild type human C5aR1 (Fig. 6A). Similarly, HlgC could not bind to HEK cells expressing mouse C5aR1 (Fig. 6C). Partial restoration of susceptibility towards HlgCB-cytotoxicity in cells expressing a mouse C5aR1 with human ECL3 (Fig. 6D) was associated with suboptimal binding of HlgC when compared to cells expressing wild type human C5aR1 (Fig. 6C). Although introduction of mouse ECL3 in human C5aR1 resulted in a nearly complete loss of susceptibility towards HlgCB-toxicity (Fig. 6D), HlgC binding was still observed (Fig. 6C).

These data show, as an example, that binding of the S-component to the specific receptor itself is not necessarily sufficient to allow subsequent pore formation.

Toxicity of PVL but not HlgCB is neutralized by specific C5aR1-antagonists

C5aR1 is considered the major host receptor for PVL, and C5aR1 competition interferes with PVL-toxicity on human neutrophils (28). Since PVL and HlgCB target the human C5a receptors in a specific but divergent manner, we investigated if toxicity of PVL and HlgCB towards human neutrophils can be equally neutralized by different C5aR1-antagonists in vitro.

As observed before, pretreatment of neutrophils with the chemotaxis inhibiting protein of S. aureus (CHIPS)(31), which binds human C5aR1, significantly shifted the half-maximal effective concentration (EC₅₀) of PVL (Fig. 7A, D). However, CHIPS could not neutralize toxicity of HlgCB (Fig 7A, D). The staphylococcal protein FLIPr-Like, targeting two unrelated receptors, did not confer protection to neutrophils for either of the toxins. Pretreatment of neutrophils with a monoclonal antibody, directed towards the N-terminus of human C5aR1 (clone S5/1), resulted in a minimal shift of the EC₅₀ for PVL but again not for HlgCB (Fig 7B, D). In the next step, we tested the potency of different commercially available C5aR1 antagonists to neutralize PVL and HlgCB toxicity. NDT 9513727 and W-54011 are small molecules with potent C5aR1 antagonistic properties (36, 37). PMX 205 and AS-65122 are C5amimetic peptides blocking the binding of C5a (38, 39). While pretreatment of human neutrophils with all compounds resulted in 5- to 10-fold increased EC₅₀-values for PVL, none of the compounds reduced susceptibility of cells towards HlgCB (Fig 7C, D). Remarkably and in contrast to the other C5aR1-antagonists tested, both small molecules NDT 9513727 and W-54011 significantly enhanced susceptibility to HlgCB and pretreatment of neutrophils with these molecules resulted in 5-fold decreased EC₅₀-values for HlgCB (Fig. 7C, D).

Together, these data show that although PVL and HlgCB both target the human C5a receptors, only toxicity of PVL can be antagonized in vitro by C5aR1 competition on human neutrophils. This observation provides further evidence that both toxins differentially interact with human C5aR1.