Abstract
ABSTRACTEnterohemorrhagic 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.
Highlights
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
We developed a genome-wide CRISPR/Cas9 screen to identify host factors that contribute to susceptibility to EHEC infection in the HT-29 colonic epithelial cell line using the Avana library of single-guide RNAs [23]
EHEC encodes two potent virulence factors that empower it to disrupt the colonic epithelium during infection: (i) its T3SS, which enables intimate attachment of bacteria as well as translocation of multiple effectors that disrupt epithelial cell processes, and (ii) Stx, a potent translation inhibitor that triggers multiple stress responses in cells within and outside the intestinal tract
Summary
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. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were targeted Mutations in these loci rescued cells from Stx-mediated cell death, and prevented cytotoxicity associated with the EHEC T3SS. 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. EHEC pathogenesis shares many features with that of enteropathogenic E. coli (EPEC), another extracellular pathogen that colonizes the intestine Successful colonization by both species is dependent upon a type III secretion system (T3SS) that enables tight adherence of bacteria to host epithelial cells by inducing characteristic actin cytoskeletal rearrangements and loss of microvillus structure (attaching and effacing [AE] lesions) [2]. Translocation of Stxs to tissues outside the intestinal tract is thought to underlie the development of HUS [3, 4]
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