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During passage through the gastrointestinal tract, enterohaemorrhagic Escherichia coli (EHEC) encounters numerous stresses, each producing unique antimicrobial conditions. Beyond surviving these stresses, EHEC may also use them as cues about the local microenvironment to modulate its virulence. Of particular interest is how exposure to changing concentrations of short-chain fatty acids (SCFAs) associated with passage through the small and large intestines affects EHEC virulence, as well as flagella expression and motility specifically. In this study, we investigate the impact of exposure to SCFA mixes simulating concentrations and compositions within the small and large intestines on EHEC flagella expression and function. Using a combination of DNA microarray, quantitative real-time PCR, immunoblot analysis, flow cytometry and motility assays, we show that there is a marked, significant upregulation of flagellar genes, the flagellar protein, FliC, and motility when EHEC is exposed to SCFA mixes representative of the small intestine. By contrast, when EHEC is exposed to SCFA mixes representative of the large intestine, there is a significant downregulation of flagellar genes, FliC and motility. Our results demonstrate that EHEC modulates flagella expression and motility in response to SCFAs, with differential responses associated with SCFA mixes typical of the small and large intestines. This research contributes to our understanding of how EHEC senses and responds to host environmental signals and the mechanisms it uses to successfully infect the human host. Significantly, it also suggests that EHEC is using this key gastrointestinal chemical signpost to cue changes in flagella expression and motility in different locations within the host intestinal tract.

Citrobacter rodentium is a Gram-negative, murine-specific enteric pathogen that infects epithelial cells in the colon. It is closely related to the clinically relevant human pathogen, enterohemorrhagic Escherichia coli (EHEC), a leading cause of haemorrhagic colitis and haemolytic uremic syndrome. We have previously reported that a novel antimicrobial peptide, wrwycr, compromises bacterial DNA repair and significantly reduces the survival of acid-stressed EHEC, suggesting an antimicrobial strategy for targeting the survival of ingested EHEC. This study examines the impact of peptide pretreatment on survival of the closely related murine pathogen, C. rodentium, before and after acid stress, using both in vitro and in vivo investigations. Peptide pretreatment of C. rodentium significantly and dramatically increases acid-stress-induced killing in a peptide-dose-dependent and time-dependent manner. Reduction in survival rates after brief pretreatment with peptide (25-65 µM) followed by 1 h at pH 3.5 ranges from 6 to 8 log fold relative to untreated C. rodentium, with no detectable bacteria after 65 µM peptide-acid treatment. Using a C57BL/6 mouse model of infection, peptide pretreatment of C. rodentium with wrwycr prior to orogastric gavage eliminates evidence of infection based on C. rodentium colonization levels, faecal scores, colonic histology, faecal microbiome and visual observation of overall animal health. These findings provide compelling evidence for the role of the peptide wrwycr as a potential strategy to control the growth and colonization of enteric pathogens.

Enterohaemorrhagic Escherichia coli (EHEC) survives exposure to acute acid stress during gastric passage and progresses to colonize the large intestine. We previously reported that acid stress significantly increases host adhesion of EHEC O157 : H7 and is associated with a coincident upregulation of the expression of a putative adhesin gene, yadK. Further gene expression analysis now confirms that yadK is minimally transcribed under unstressed conditions and is significantly upregulated under acid stress. Immunoblotting with an anti-YadK polyclonal antiserum demonstrates that YadK protein is also upregulated after acid stress. Disruption of yadK results in loss of the acid-induced adhesion increase seen for wild-type EHEC to human epithelial cells in vitro and complementation in trans fully restores the acid-induced adhesion phenotype to the wild-type level. Significantly, no difference is observed in adhesion of the unstressed yadK mutant relative to wild-type, indicating that YadK does not play a role in adhesion of unstressed EHEC. Anti-YadK antiserum inhibits the acid-induced adhesion enhancement of EHEC but has no effect on adhesion of unstressed EHEC. There is no significant difference in the viability of either the unstressed or the acid-stressed yadK mutant relative to the similarly treated wild-type, suggesting that yadK is not involved in acid tolerance. These results provide persuasive evidence that YadK plays a significant role in the adhesion of acid-stressed EHEC to epithelial cells, and support a role for acid stress as a factor which may regulate bacteria-host attachment and lead to increased EHEC colonization and virulence.