Grant: Discovery Grants Program - Individual

This application is for funding to support the investigation of stress-induced virulence changes in enterohemorrhagic (EHEC) E.coli O157:H7. This enteric pathogen is a leading cause of bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. During ingestion, EHEC is exposed to several stresses including gastric acid as well as bile salts and short chain fatty acids in the intestine. It can also be exposed to stress during acid-washing of meat carcasses. We have found that acid and bile salt stress significantly enhance adhesion of EHEC and a closely related pathogen, enteropathogenic E.coli(EPEC),to human epithelial cells. We have also identified a bacterial protein and a host cell lipid that play a role in the enhanced adhesion of acid-stressed EPEC. The goal of this proposal is to identify genes and pathways encompassing stress-induced EHEC virulence changes. Using DNA microarrays, we will examine the mRNA expression profiles of EHEC before and after exposure to acid, bile salts, and short chain fatty acids to identify bacterial proteins responsible for the stress-induced virulence changes. Differential gene expression will be confirmed by quantitative real-time PCR and protein changes will be validated by 2D gel electrophoresis and electrospray tandem mass spectrometry. To confirm the role of stress-induced genes in EHEC virulence, we will construct mutants defective in the individual genes and test them for changes in virulence phenotype before and after stress exposure. We will also identify regulators of the virulence genes using transposon mutagenesis. Stress-induced changes in virulence of EHEC O157:H7 can have a profound impact on the development and progression of the disease and therefore provide exciting targets to modulate the virulence potential of this pathogen. This targeted approach recognizes the value of commensal bacteria by permitting selective attenuation of EHEC O157:H7 while maintaining the nonpathogenic, commensal bacteria that play a role in preventing infection. The research will also identify and characterize the infection risk associated with sublethal environmental stress including food processing practices.