CRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection

ABSTRACT Enterohemorrhagic 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 regular...

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Autores principales:	Alline R. Pacheco, Jacob E. Lazarus, Brandon Sit, Stefanie Schmieder, Wayne I. Lencer, Carlos J. Blondel, John G. Doench, Brigid M. Davis, Matthew K. Waldor
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	CRISPR screen EHEC EPEC LAPTM4A Shiga toxin T3SS Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/340bfdb97ab94b00822ca588d8794534
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spelling	oai:doaj.org-article:340bfdb97ab94b00822ca588d87945342021-11-15T16:00:26ZCRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection10.1128/mBio.01003-182150-7511https://doaj.org/article/340bfdb97ab94b00822ca588d87945342018-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01003-18https://doaj.org/toc/2150-7511ABSTRACT Enterohemorrhagic 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.Alline R. PachecoJacob E. LazarusBrandon SitStefanie SchmiederWayne I. LencerCarlos J. BlondelJohn G. DoenchBrigid M. DavisMatthew K. WaldorAmerican Society for MicrobiologyarticleCRISPR screenEHECEPECLAPTM4AShiga toxinT3SSMicrobiologyQR1-502ENmBio, Vol 9, Iss 3 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR screen EHEC EPEC LAPTM4A Shiga toxin T3SS Microbiology QR1-502
spellingShingle	CRISPR screen EHEC EPEC LAPTM4A Shiga toxin T3SS Microbiology QR1-502 Alline R. Pacheco Jacob E. Lazarus Brandon Sit Stefanie Schmieder Wayne I. Lencer Carlos J. Blondel John G. Doench Brigid M. Davis Matthew K. Waldor CRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection
description	ABSTRACT Enterohemorrhagic 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.
format	article
author	Alline R. Pacheco Jacob E. Lazarus Brandon Sit Stefanie Schmieder Wayne I. Lencer Carlos J. Blondel John G. Doench Brigid M. Davis Matthew K. Waldor
author_facet	Alline R. Pacheco Jacob E. Lazarus Brandon Sit Stefanie Schmieder Wayne I. Lencer Carlos J. Blondel John G. Doench Brigid M. Davis Matthew K. Waldor
author_sort	Alline R. Pacheco
title	CRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection
title_short	CRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection
title_full	CRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection
title_fullStr	CRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection
title_full_unstemmed	CRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection
title_sort	crispr screen reveals that ehec’s t3ss and shiga toxin rely on shared host factors for infection
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/340bfdb97ab94b00822ca588d8794534
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CRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection

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