Abstract

Escherichia coli (EHEC) 0157:H7 is a pathogenic bacterial species that is most commonly linked to severe diarrhea, but is also the leading cause of the potentially fatal hemolytic-uremic syndrome (HUS). In order to establish infection in the colon, EHEC must endure different stresses encountered in the gastrointestinal (GI) tract, such as acid stress in the stomach, bile salt stress in the small intestine, and short-chain fatty acid (SCFA) stress in the colon. These bacteria are likely able to use GI stresses as indicators of their location, impacting gene expression of adhesion, motility, and virulence factors. The E. coli Common Pilus (ECP) has been shown to be an important factor for EHEC adhesion to epithelial cells, which is increased after either acid or SCFA stress. It has also been demonstrated via microarray that genes of this operon are upregulated after acid stress. The aim of this study is to determine how expression of the main subunit of this structure, EcpA, is regulated upon exposure of EHEC 0157:H7 to acid or SCFA-stress. Both transcriptional and translational regulation are hypothesized to be involved. Isogenic mutants have been constructed that lacked key regulators suspected to be important for each system. Two approaches are used to determine if the predicted regulatory systems are playing a role in response to stress: observing EcpA protein expression analysis through Western blotting with anti-EcpA antibodies, and examining differences in ecp operon promoter activity in regulatory mutants. In this study Western blots reconfirmed H-NS does not modulate ecpA expression in direct response to acute acid stress. This suggests an alternate regulatory response in EHEC 0157:H7 to acute acid stress resulting in the upregulation of ecpA expression previously observed with microarray analysis.

Highlights

  • 1.1 General IntroductionEscherichia coli (E. coli) is a species of rod shaped, gram-negative bacteria that most frequently colonize the guts of the host organism[7,45]

  • Processing of cattle for meat production allows for bacteria, such as EHEC, on the exterior surface of the cattle to come into contact with the interior tissues humans consume as meat; with many large-scale meat-processing plants currently in operation, there are ample opportunities for these accidentally contaminated products to make it to market[11,45]

  • In a DNA microarray analysis of gene expression in O157:H7 it was shown that many genes related to virulence and adhesion were upregulated after acid stress[31]

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Summary

Introduction

1.1 General IntroductionEscherichia coli (E. coli) is a species of rod shaped, gram-negative bacteria that most frequently colonize the guts of the host organism[7,45]. The enterohaemorrhagic (EHEC) pathotype is one of the E. coli subsets known to be pathogenic to humans[45]. EHEC O157:H7 was first identified in 1982 after being implicated as the cause of several outbreaks of food poisoning in humans, some of them fatal[7,11,13,45]. Those food poisoning incidents were traced back to the consumption of beef products that had not been cooked thoroughly, leaving the bacteria viable[7,11]. EHEC O157:H7 outbreaks have been traced back to dairy products, as well as contaminated produce[7,11,45]. The largest outbreak of EHEC O157:H7 infection, with 7966 confirmed cases, occurred in 1996 in Japan and was traced back to contaminated radish sprouts[7,45]. Studies have found that this type of contamination of produce can be linked back to exposure to feces of EHEC colonized cattle,

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