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CRISPR screen reveals that EHEC’s T3SS and Shiga Toxin rely on shared host factors for infection

dc.contributor.authorPacheco A.R.
dc.contributor.authorLazarus J.E.
dc.contributor.authorSit B.
dc.contributor.authorSchmieder S.
dc.contributor.authorLencer W.I.
dc.contributor.authorBlondel C.J.
dc.contributor.authorDoench J.G.
dc.contributor.authorDavis B.M.
dc.contributor.authorWaldor M.K.
dc.date.accessioned2020-09-02T22:25:16Z
dc.date.available2020-09-02T22:25:16Z
dc.date.issued2018
dc.identifier10.1128/mBio.01003-18
dc.identifier.citation9, 3, -
dc.identifier.issn21612129
dc.identifier.urihttps://hdl.handle.net/20.500.12728/5722
dc.descriptionEnterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors—a type III secretion system (T3SS) and Shiga toxins (Stxs)—that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with Cas9) loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors—a type III secretion system (T3SS) and Shiga toxins (Stxs)—that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3) and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogen’s key virulence factors. © 2018 Pacheco et al.
dc.language.isoen
dc.publisherAmerican Society for Microbiology
dc.subjectCRISPR screen
dc.subjectEHEC
dc.subjectEPEC
dc.subjectHost susceptibility
dc.subjectLAPTM4A
dc.subjectShiga toxin
dc.subjectSphingolipid synthesis
dc.subjectT3SS
dc.subjectTM9SF2
dc.subjectglobotriaosylceramide
dc.subjectShiga toxin
dc.subjectsphingolipid
dc.subjectceramide trihexoside
dc.subjectglobotriaosylceramide
dc.subjectShiga toxin
dc.subjectsphingolipid
dc.subjectvirulence factor
dc.subjectArticle
dc.subjectbacterial colonization
dc.subjectbacterial growth
dc.subjectbacterial strain
dc.subjectbacterial virulence
dc.subjectcontrolled study
dc.subjectCRISPR-CAS9 system
dc.subjectcytotoxicity
dc.subjectenterohemorrhagic Escherichia coli
dc.subjecthost pathogen interaction
dc.subjectintestine epithelium cell
dc.subjectlimit of quantitation
dc.subjectnonhuman
dc.subjectpriority journal
dc.subjectbiosynthesis
dc.subjectcell line
dc.subjectcell survival
dc.subjectclustered regularly interspaced short palindromic repeat
dc.subjectenterohemorrhagic Escherichia coli
dc.subjectepithelium cell
dc.subjectEscherichia coli infection
dc.subjectgene locus
dc.subjectgene targeting
dc.subjectgenetics
dc.subjectgenome-wide association study
dc.subjecthost pathogen interaction
dc.subjecthuman
dc.subjectmetabolism
dc.subjectmicrobiology
dc.subjectmutation
dc.subjectpathogenicity
dc.subjectphysiology
dc.subjecttype III secretion system
dc.subjectBiosynthetic Pathways
dc.subjectCell Line
dc.subjectCell Survival
dc.subjectClustered Regularly Interspaced Short Palindromic Repeats
dc.subjectEnterohemorrhagic Escherichia coli
dc.subjectEpithelial Cells
dc.subjectEscherichia coli Infections
dc.subjectGene Targeting
dc.subjectGenetic Loci
dc.subjectGenome-Wide Association Study
dc.subjectHost-Pathogen Interactions
dc.subjectHumans
dc.subjectMutation
dc.subjectShiga Toxin
dc.subjectSphingolipids
dc.subjectTrihexosylceramides
dc.subjectType III Secretion Systems
dc.subjectVirulence Factors
dc.titleCRISPR screen reveals that EHEC’s T3SS and Shiga Toxin rely on shared host factors for infection
dc.typeArticle


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