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

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

Resultado de la investigación: Article

8 Citas (Scopus)

Resumen

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.

Idioma originalEnglish
Número de artículoe01003-18
PublicaciónmBio
Volumen9
N.º3
DOI
EstadoPublished - 1 may 2018

Huella dactilar

Clustered Regularly Interspaced Short Palindromic Repeats
Shiga Toxin
Enterohemorrhagic Escherichia coli
Sphingolipids
Escherichia coli Infections
Infection
Virulence
Shiga Toxins
Mutation
Intestines
Guide RNA
Epithelial Cells
Genome
Virulence Factors
Cell Death
Anti-Bacterial Agents
Lipids

ASJC Scopus subject areas

  • Microbiology
  • Virology

Citar esto

Pacheco, A. R., Lazarus, J. E., Sit, B., Schmieder, S., Lencer, W. I., Blondel, C. J., ... Waldor, M. K. (2018). CRISPR screen reveals that EHEC’s T3SS and Shiga Toxin rely on shared host factors for infection. mBio, 9(3), [e01003-18]. https://doi.org/10.1128/mBio.01003-18
Pacheco, Alline R. ; Lazarus, Jacob E. ; Sit, Brandon ; Schmieder, Stefanie ; Lencer, Wayne I. ; Blondel, Carlos J. ; Doench, John G. ; Davis, Brigid M. ; Waldor, Matthew K. / CRISPR screen reveals that EHEC’s T3SS and Shiga Toxin rely on shared host factors for infection. En: mBio. 2018 ; Vol. 9, N.º 3.
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title = "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 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.",
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Pacheco, AR, Lazarus, JE, Sit, B, Schmieder, S, Lencer, WI, Blondel, CJ, Doench, JG, Davis, BM & Waldor, MK 2018, 'CRISPR screen reveals that EHEC’s T3SS and Shiga Toxin rely on shared host factors for infection', mBio, vol. 9, n.º 3, e01003-18. https://doi.org/10.1128/mBio.01003-18

CRISPR screen reveals that EHEC’s T3SS and Shiga Toxin rely on shared host factors for infection. / Pacheco, Alline R.; Lazarus, Jacob E.; Sit, Brandon; Schmieder, Stefanie; Lencer, Wayne I.; Blondel, Carlos J.; Doench, John G.; Davis, Brigid M.; Waldor, Matthew K.

En: mBio, Vol. 9, N.º 3, e01003-18, 01.05.2018.

Resultado de la investigación: Article

TY - JOUR

T1 - CRISPR screen reveals that EHEC’s T3SS and Shiga Toxin rely on shared host factors for infection

AU - Pacheco, Alline R.

AU - Lazarus, Jacob E.

AU - Sit, Brandon

AU - Schmieder, Stefanie

AU - Lencer, Wayne I.

AU - Blondel, Carlos J.

AU - Doench, John G.

AU - Davis, Brigid M.

AU - Waldor, Matthew K.

PY - 2018/5/1

Y1 - 2018/5/1

N2 - 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.

AB - 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.

KW - CRISPR screen

KW - EHEC

KW - EPEC

KW - Host susceptibility

KW - LAPTM4A

KW - Shiga toxin

KW - Sphingolipid synthesis

KW - T3SS

KW - TM9SF2

UR - http://www.scopus.com/inward/record.url?scp=85048743651&partnerID=8YFLogxK

U2 - 10.1128/mBio.01003-18

DO - 10.1128/mBio.01003-18

M3 - Article

C2 - 29921669

AN - SCOPUS:85048743651

VL - 9

JO - mBio

JF - mBio

SN - 2161-2129

IS - 3

M1 - e01003-18

ER -