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.
Highlights
These cells with a CRISPR library targeting 19,050 human genes and 1864 miRNAs and intoxicated with α HL to obtain survivors that contain a mutation in genes essential for α HL-mediated cell death
To identify host genes required for α HL toxicity, we used a genome-wide CRISPR library generated by the Zhang laboratory[10,12]
We intoxicated the cells with α HL or diphtheria toxin (DT), as control, for two weeks to allow outgrowth of resistant mutants
Summary
These cells with a CRISPR library targeting 19,050 human genes and 1864 miRNAs and intoxicated with α HL to obtain survivors that contain a mutation in genes essential for α HL-mediated cell death. Sequencing revealed the enrichment of genes that are involved in α HL toxicity including ADAM10. Single-cell cloning and complementation confirmed that all further analyzed hits from the screen are required for α HL-mediated cell death. Three genes regulate ADAM10 post-translationally and their deficiency reduces ADAM10 levels on the surface, thereby decreasing toxin binding. The importance of lipid rafts is underlined by the requirement of sphingomyelin synthase 1 (SGMS1) to allow binding of α HL to the host cell
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