Host-Receptor Post-Translational Modifications Refine Staphylococcal Leukocidin Cytotoxicity

Abstract

Staphylococcal bi-component pore-forming toxins, also known as leukocidins, target and lyse human phagocytes in a receptor-dependent manner. S-components of the leukocidins Panton-Valentine leukocidin (PVL), γ-haemolysin AB (HlgAB) and CB (HlgCB), and leukocidin ED (LukED) specifically employ receptors that belong to the class of G-protein coupled receptors (GPCRs). Although these receptors share a common structural architecture, little is known about the conserved characteristics of the interaction between leukocidins and GPCRs. In this study, we investigated host cellular pathways contributing to susceptibility towards S. aureus leukocidin cytotoxicity. We performed a genome-wide CRISPR/Cas9 library screen for toxin-resistance in U937 cells sensitized to leukocidins by ectopic expression of different GPCRs. Our screen identifies post-translational modification (PTM) pathways involved in the sulfation and sialylation of the leukocidin-receptors. Subsequent validation experiments show differences in the impact of PTM moieties on leukocidin toxicity, highlighting an additional layer of refinement and divergence in the staphylococcal host-pathogen interface. Leukocidin receptors may serve as targets for anti-staphylococcal interventions and understanding toxin-receptor interactions will facilitate the development of innovative therapeutics. Variations in the genes encoding PTM pathways could provide insight into observed differences in susceptibility of humans to infections with S. aureus.

Keywords: G-protein coupled receptors; Staphylococcus aureus; bi-component pore-forming toxins; leukocidins; post-translational modifications; receptors.

Figure 1

Genome-wide CRISPR/Cas9 library screen reveals post-translational modification pathways involved in PVL and HlgCB toxicity. Cellular factors involved in PVL- and HlgCB-mediated cytotoxicity as identified by the introduction of a genome-wide sgRNA library in U937-C5aR1-SpCas9 cells coupled to deep sequencing. Visualized are the most significantly enriched genes after PVL and HlgCB challenge as calculated by the MaGeCK ‘positive enrichment score’. See Tables S1 and S2 for the full list of genes. Grey: genes encoding known host cellular factors; blue: genes belonging to the tyrosine sulfation pathway; red: genes involved in the sialylation pathway.

Figure 2

Sulfation of C5aR1 facilitates PVL and HlgCB cytotoxicity. (a) Anti-C5aR1 (clone S5/1) and anti-sulfotyrosine antibodies were used to assess the overall expression of C5aR1 and total sulfotyrosine in U937-C5aR1-SpCas9 cell lines transduced with sgRNA for PAPSS1 (C5aR1⁺ PAPSS1⁻), TPST2 (C5aR1⁺ TPST2⁻), SLC35B2 (C5aR1⁺ SLC35b2⁻), non-targeting control sgRNA (NTC, C5aR1⁺), and U937-SpCas9 (WT, C5aR1⁻) cells. Antibody binding was determined by a fluorescent secondary antibody and the fluorescence measured and analyzed by flow cytometry. Dashed line: expression in U937-SpCas9 (WT, C5aR1⁻) cells; dotted line: C5aR1 expression in NTC (C5aR1⁺) U937 cells. Histograms depict representative examples of two independently repeated experiments. (b) Validation of the sulfation-pathway hits after genome-wide CRISPR/Cas9 screen for PVL and HlgCB resistance in U937-C5aR1-SpCas9 cells. As a readout for cell permeability, internalization of DAPI was tested at 30 min post-toxin treatment on a monochromator-based microplate reader and expressed in relation to U937-C5aR1-SpCas9 cells transduced with an NTC sgRNA. Mean and s.d. are shown, with n = 3. Statistical significance was calculated using ANOVA analysis of variance with Bonferroni posttest correction for multiple comparison. Statistical significance is displayed as ** for p < 0.01 and **** for p < 0.0001. (c) Expression of sulfated C5aR1 in, and binding of polyhistidine-tagged LukS-PV and HlgC to, U937-C5aR1-SpCas9 cell lines transduced with sgRNA for PAPSS1 (C5aR1⁺ PAPSS1⁻), TPST2 (C5aR1⁺ TPST2⁻), SLC35B2 (C5aR1⁺ SLC35b2⁻), non-targeting control sgRNA (NTC, C5aR1⁺), and U937-SpCas9 (WT, C5aR1⁻) cells. Anti-C5aR1 (clone 347214) antibodies were used to assess the expression of sulfated C5aR1. Antibody binding was determined by a fluorescent secondary antibody. Binding of LukS-PV and HlgC was detected with anti-His-FITC antibodies. Fluorescence was measured and analyzed by flow cytometry. Dashed line: expression on and binding to U937-SpCas9 (WT, C5aR1⁻) cells; dotted line: expression on and binding to NTC (C5aR1⁺) U937 cells. Histograms depict representative examples of two independently repeated experiments.

Figure 3

The role of sialylation in HlgCB- and PVL-induced cytotoxicity. (a) Anti-C5aR1 and anti-CD15s antibodies were used to assess the expression of C5aR1 and CD15s on U937-C5aR1-SpCas9 cell lines transduced with sgRNA for CMAS (C5aR1⁺ CMAS⁻), SLC35A1 (C5aR1⁺ SLC35a1⁻), non-targeting control sgRNA (NTC, C5aR1⁺), and U937-SpCas9 (WT, C5aR1⁻) cells. Antibody binding was determined by a fluorescent secondary antibody and the fluorescence measured and analyzed by flow cytometry. Dashed line: expression in U937-SpCas9 (WT, C5aR1⁻) cells; dotted line: C5aR1 expression in NTC (C5aR1⁺) U937 cells. Histograms depict representative examples of two independently repeated experiments. (b) Validation of the sialylation-pathway hits after genome-wide CRISPR/Cas9 screen for PVL and HlgCB resistance in U937-C5aR1-SpCas9 cells. As a readout for cell permeability, internalization of DAPI was tested at 30 min post-toxin treatment on a monochromator-based microplate reader and expressed in relation to U937-C5aR1-SpCas9 cells transduced with an NTC sgRNA. Mean and s.d. are shown, with n = 3. Statistical significance was calculated using ANOVA analysis of variance with Bonferroni posttest correction for multiple comparison. Statistical significance is displayed as ** for p < 0.01 and NS for not significant. (c) Binding of polyhistidine-tagged LukS-PV and HlgC to U937-C5aR1-SpCas9 cell lines transduced with sgRNA for CMAS (C5aR1⁺ CMAS⁻), SLC35A1 (C5aR1⁺ SLC35a1⁻), non-targeting control sgRNA (NTC, C5aR1⁺) and wild type U937 (C5aR1⁻). Cells were subsequently incubated with anti-His-FITC antibodies and the fluorescence measured and analyzed by flow cytometry. Dashed line: binding to U937-SpCas9 (WT, C5aR1⁻) cells; dotted line: binding to NTC (C5aR1⁺) U937 cells. Histograms depict representative examples of two independently repeated experiments.

Figure 4

Sulfation and sialylation of C5aR1 refine susceptibility to PVL and HlgCB. Susceptibility of U937-C5aR1-SpCas9 cell lines transduced with sgRNA for (a) the sulfation pathway genes SLC35B2 (C5aR1⁺ SLC35b2⁻), PAPSS1 (C5aR1⁺ PAPSS1⁻), TPST2 (C5aR1⁺ TPST2⁻), and (b) the sialylation pathway genes SLC35A1 (C5aR1⁺ SLC35a1⁻), CMAS (C5aR1⁺ CMAS⁻), a non-targeting control sgRNA (NTC, C5aR1⁺), and U937-SpCas9 (WT, C5aR1⁻) cells to PVL and HlgCB. As a readout for cell permeability, internalization of DAPI was measured during 30 min post-toxin treatment on a monochromator-based microplate reader and expressed in relation to the area under the curve for NTC sgRNA transduced U937-C5aR1-SpCas9 cells at 80 nM PVL or 20 nM HlgCB.

Figure 5

Sialylation and sulfation of CXCR2 refine susceptibility to LukED and HlgAB. (a) Anti-CXCR2, anti-sulfotyrosine and anti-CD15s antibodies were used to assess the expression of CXCR2, total sulfotyrosine, and CD15s on U937-CXCR2-SpCas9 cell lines transduced with sgRNA for SLC35A1 (CXCR2⁺ SLC35a1⁻), SLC35B2 (CXCR2⁺ SLC35b2⁻), non-targeting control sgRNA (NTC, CXCR2⁺), and U937-SpCas9 (WT, CXCR2⁻) cells. Antibody binding was determined by a fluorescent secondary antibody and the fluorescence measured and analyzed by flow cytometry. Dashed line: expression in U937-SpCas9 (WT, CXCR2⁻) cells; dotted line: expression in NTC (CXCR2⁺) U937 cells. Histograms depict representative examples of two independently repeated experiments. (b) Susceptibility of U937-CXCR2-SpCas9 cell lines transduced with sgRNA for SLC35B2 (CXCR2⁺ SLC35b2⁻), SLC35A1 (CXCR2⁺ SLC35a1⁻), non-targeting control sgRNA (NTC, CXCR2⁺), and U937-SpCas9 (WT, CXCR2⁻) cells to HlgAB and LukED. As a readout for cell permeability, internalization of DAPI was measured during 30 min post-toxin treatment on a monochromator-based microplate reader and expressed in relation to the area under the curve for NTC sgRNA transduced U937-CXCR2-SpCas9 cells at 20 nM HlgAB or LukED. (c) Binding of Alexa Fluor 647 maleimide-based labeled HlgA and polyhistidine-tagged LukE to U937-CXCR2-SpCas9 cell lines transduced with sgRNA for SLC35B2 (CXCR2⁺ SLC35b2⁻), SLC35A1 (CXCR2⁺ SLC35a1⁻), non-targeting control sgRNA (NTC, CXCR2⁺), and U937-SpCas9 (WT, CXCR2⁻) cells. Cells were subsequently incubated with anti-histidine-FITC secondary antibodies and/or the fluorescence was directly measured and analyzed by flow cytometry. Dashed line: binding to U937-SpCas9 (WT, CXCR2⁻) cells; dotted line: binding to NTC (CXCR2⁺) U937 cells. Histograms depict representative examples of two independently repeated experiments.