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

Author:

Pacheco Alline R.12,Lazarus Jacob E.123,Sit Brandon12,Schmieder Stefanie456,Lencer Wayne I.456,Blondel Carlos J.12,Doench John G.7,Davis Brigid M.12,Waldor Matthew K.1278

Affiliation:

1. Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, USA

2. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

3. Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA

4. Division of Gastroenterology, Boston Children’s Hospital, Boston, Massachusetts, USA

5. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA

6. Department of Pediatrics, Harvard Digestive Diseases Center, Boston, Massachusetts, USA

7. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

8. Howard Hughes Medical Institute, Boston, Massachusetts, USA

Abstract

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.

Funder

National Sciences and Engineering Research Council of Canada

HHS | National Institutes of Health

Howard Hughes Medical Institute

Publisher

American Society for Microbiology

Subject

Virology,Microbiology

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