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. 2016 Apr 12:6:24242.
doi: 10.1038/srep24242.

Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity

Sebastian Virreira Winter  1 , Arturo Zychlinsky  1 , Bart W Bardoel  1
Affiliations

Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity

Sebastian Virreira Winter et al. Sci Rep. .

Abstract

Staphylococcus aureus causes a wide variety of infections and antibiotic resistant strains are a major problem in hospitals. One of the best studied virulence factors of S. aureus is the pore-forming toxin alpha hemolysin (αHL) whose mechanism of action is incompletely understood. We performed a genome-wide loss-of-function screen using CRISPR/Cas9 technology to identify host targets required for αHL susceptibility in human myeloid cells. We found gRNAs for ten genes enriched after intoxication with αHL and focused on the top five hits. Besides a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), the host receptor for αHL, we identified three proteins, Sys1 golgi trafficking protein (SYS1), ADP-ribosylation factor 1 (ARFRP1), and tetraspanin-14 (TSPAN14) which regulate the presentation of ADAM10 on the plasma membrane post-translationally. Interestingly, we also showed that cells lacking sphingomyelin synthase 1 (SGMS1) resist αHL intoxication, but have only a slightly reduced ADAM10 surface expression. SGMS1 regulates lipid raft formation, suggesting that αHL requires these membrane microdomains for attachment and cytotoxicity.

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Figures

Figure 1
Figure 1. Genome-wide CRISPR screen identifies novel αHL host targets.
(a) U937 cells were incubated with various concentrations of αHL overnight and the percentage of viable cells was analyzed by flow cytometry. Data represent mean values of three independent experiments + SD. (b,c) Individual enrichment (log2) of gRNAs is shown for the most enriched genes following intoxication with DT (b) or αHL (c). Ranking is based on the median enrichment (log2) among all six gRNAs present in the library. (d) Median enrichment of six gRNAs for the 20 most enriched genes in the αHL screen is shown .
Figure 2
Figure 2. Individual targeting of the αHL hits results in αHL-resistant cells.
(a–e) U937 clones derived from single cells by limiting dilution for each of the two gRNAs targeting (a) ADAM10, (b) SYS1, (c) ARFRP1, (d) TSPAN14 or (e) SGMS1 were incubated with 1 μg/ml of αHL overnight. Percentage of viable cells was determined by flow cytometry and gating for DRAQ7-negative and forward side scatter characteristics as control U937 cells. As a control, a random scrambled and a gRNA targeting an unrelated control gene (neutrophil elastase, NE) were included. (f) For each target gene, a CRISPR-targeted clone was complemented with a copy of the gene, modified via silent mutations to avoid editing by Cas9. Transduction with an empty vector served as a control. Cells were stimulated with 0.3 μg/ml αHL and after 40 hours the percentage of viable cells was measured by flow cytometry. All graphs (a–f) represent mean + SD of three independent experiments. (f) Statistical testing was performed using t test and p values are indicated.
Figure 3
Figure 3. αHL binding is impaired in toxin-resistant CRISPR-targeted cells.
(a) U937 cells were incubated with 0–1 μg/ml fluorescently labeled αHL for 30 minutes at 37 °C. For each gene we tested one αHL-resistant clone. Binding was analyzed by gating for DRAQ7-negative cells using flow cytometry and expressed relative to 0 μg/ml αHL. Graphs indicate mean + SD of three independent experiments and statistical testing was performed using unpaired one-way ANOVA with correction for multiple comparisons (Dunnett). (b) U937 clones were challenged with 2 μg/ml αHL-Cys-His-Alexa647 for 5 minutes, fixed and stained with DAPI. Images were obtained by confocal microscopy. Nuclei (blue) and cell-bound αHL-Cys-His-Alexa647 (magenta) are shown. These are representative confocal z-planes of two independent experiments.
Figure 4
Figure 4. Surface ADAM10 regulates susceptibility to αHL.
(a) CRISPR-targeted U937 cells were analyzed for gene expression of ADAM10, quantified by qPCR and expressed as relative value compared to the RPL32 gene. (b) U937 clones targeted with the gRNAs to the indicated genes were incubated with an ADAM10 antibody, followed by a PE-labeled secondary goat-anti-mouse antibody. Geometric mean of ADAM10-PE staining was analyzed by flow cytometry. Values represent the relative ADAM10 surface expression compared to control cells (WT U937 or cells transduced with scrambled gRNA). (c) U937 clones were intoxicated with 2 μg/ml αHL-Cys-His-Alexa647 (magenta) for the indicated times, fixed, permeabilized and ADAM10 was visualized by staining with an anti-ADAM10 antibody (green). Nuclei were stained with DAPI (blue). Shown are representative confocal z-planes of two independent experiments. (d) U937 CRISPR clones were stained with anti-ADAM15 and analyzed by flow cytometry. Data are expressed as fold expression relative to scrambled control. (e) U937 CRISPR clones were incubated with either 0 or 3 μg/ml αHL overnight at 37 °C. Subsequently, surface expression of ADAM10 was analyzed by flow cytometry. Dots represent individual experiments of αHL resistant CRISPR clones. Data are plotted as relative ADAM10 surface expression compared to WT U937 or scrambled at 0 versus 3 μg/ml αHL. (a,b,d) Shown are mean + SD of three independent experiments and statistical testing was performed using unpaired one-way ANOVA with correction for multiple comparisons (Dunnett). (c) representative images of two independent experiments.
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