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The type 6 secretion system immunity protein RhsFI has been repurposed for small RNA regulation in pathogenic E. coli

ABSTRACT: RNA-binding proteins (RBPs) play key roles in regulating virulence gene expression in pathogenic, enterohaemorrhagic E. coli (EHEC). Under virulence-inducing conditions total RNA associated protein purification (TRAPP) identified 443 RNA-associated proteins. These included 35 encoded within pathogenicity islands, with RNA-binding confirmed in vitro for a sub-set that included RhsFI. In vivo RNA targets for RhsFI included regulatory small RNAs (sRNAs) encoded in 3’ UTRs. The rhsF-rhsFI locus encodes effector and immunity proteins for the type 6 secretion system (T6SS). T6SS expression is repressed in EHEC, but internal initiation generates a transcript encoding a truncated toxin (RhsFint) and downstream RhsFI. Modelling predicted that RhsFint can dimerise with RhsFI. Deletion of rhsFint rhsFI alters the expression of multiple operons in which embedded sRNAs are direct RhsFI targets. Loss of RhsFint:RhsFI dramatically reduced adhesion to bovine cells in vitro, altered cell morphology and increased acid resistance, phenotypes that impact host colonisation.

ORGANISM(S): Escherichia coli O157:H7 str. Sakai

PROVIDER: GSE291334 | GEO | 2025/08/28

REPOSITORIES: GEO

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Project description:EHEC is a food-borne pathogen that colonizes human GI tract and leads to infection. To understand the process of colonization and to decipher if any factors secreted by intestinal epithelial cells help EHEC during the infection process, we studied expression of EHEC virulence gene expression when exposed to intestinal epithelial cell conditioned medium. In our study, we observed that exposure to epithelial cell conditioned medium for 1 h and 3 h increases expression of 32 out of 41 EHEC LEE virulence genes. In addition, expression of the shiga toxin 1 (Stx1) gene is up-regulated at 1 h of exposure. Also, 17 genes encoded by prophage 933W, including those for Stx2, are also upregulated at both time-points. The increase in 933W prophage expression is mirrored by a 2.7-fold increase in intracellular Stx2 phage titers. Consistent with the increase in virulence gene expression, we observed a 5-fold increase in EHEC attachment to epithelial cells when exposed to conditioned medium, suggesting that EHEC utilizes host cell molecules to increase virulence and infectivity. The molecule(s) responsible for increased EHEC virulence is heat-sensitive as heating the conditioned medium to 95oC abolishes the increase in attachment to epithelial cells. A similar decrease was observed when the conditioned medium was treated with proteinase-K to degrade the proteins. The secreted molecule(s) was found to be larger than 3 kDa and strongly suggests that the HCT-8 secreted molecule that increases EHEC virulence and colonization is a protein-based molecule. Affymetrix E. coli Genome 2.0 Arrays were used to determine the changes in EHEC virulence gene expression on exposure to intestinal epithelial cell-secreted factors (through growth in conditioned medium). Overnight cultures of EHEC were diluted in LB medium to a turbidity of 0.1 at 600 nm. The cells were allowed to grow to a turbidity of 1.0 at 600 nm at 37Â°C and the EHEC cells were then resuspended in either fresh or conditioned medium. The cultures were then allowed to grow for 1 h or 3 h before cell pellets were collected by centrifugation and stored at -80Â°C. Total RNA was isolated from the cell pellets (173) and RNA quality was assessed using gel electrophoresis. Escherichia coli Genome 2.0 arrays (Affymetrix, Santa Clara, CA, USA) containing 10,208 probe sets for all 20,366 genes present in four strains of E. coli, including EHEC, were used to profile changes in gene expression using RNA samples for each treatment.

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The type 6 secretion system immunity protein RhsFI has been repurposed for small RNA regulation in pathogenic E. coli

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