Grant: Discovery Grants Program - Individual

Enterohemorrhagic E.coli (EHEC) is a serious food and water-borne pathogen and a leading cause of hemorrhagic colitis and hemolytic uremic syndrome. While major advances have been made in the field of EHEC pathogenesis, we still do not fully understand the impact of environmental stress on EHEC virulence. Our research has revealed that exposure to environmental stress elicits a set of specific, highly regulated responses designed not only to protect EHEC from the stress but also to modulate virulence potential and antimicrobial susceptibility. We have examined the impact of exposure to stresses including acid, bile salts and short chain fatty acids (SCFA), agents encountered both inside and outside the host and have found striking evidence that these stresses, rather than compromising EHEC survival, enhance its fitness and ability to cause infection. Our long term goal for this research proposal is to develop a systematic understanding of how EHEC responds to these commonly encountered environmental stresses with specific emphasis on pathogen survival and virulence potential. *Our first objective is to define the molecular mechanisms underlying EHEC's response to the selected stresses. Specifically, we plan to determine how bile and bile salts enhance EHEC's ability to resist human defensins, molecules produced by the intestines against bacterial pathogens. Our latest data indicate that EHEC may be using exposure to bile/bile salts to arm itself against defensins. Our aim is to define the molecular basis of this resistance. Secondly, we have evidence that the pathogen modulates its motility in response to SCFAs that it encounters during washing of meat carcasses in the food processing industry and during transit through the intestines. Motility provides the pathogen with a profound advantage during intestinal transit, helping it to avoid detrimental locales and to find favorable niches. We plan to examine how SCFAs encountered in different regions of the GI tract and in the external environment, modulate the pathogen's motility and how this modulation is regulated. *Our second objective is to investigate ways of enhancing the natural killing action of gastric acid on EHEC using selected antimicrobials. We know from our recent research how EHEC responds to acid, in particular by increasing mechanisms to repair its damaged DNA. We plan to employ targeted antimicrobials that prevent the pathogen from repairing its DNA after acid stress and to compare their action in both in vitro and in vivo experiments. Using EHEC and an EHEC-related pathogen that infects mice, we will compare the ability of the antimicrobials to compromise survival of both pathogens after exposure to acid on the bench top and in the stomach of mice. By doing so, we should be able to exploit the natural antimicrobial action of gastric acid stress in a way that targets the killing of newly ingested EHEC without damaging the beneficial commensal flora. This strategy could be used to develop an antimicrobial agent for application to potentially contaminated foods prior to ingestion. Since EHEC survives in acidic foods, this could also compromise its survival there.*This research proposal will permit us to build on our significant progress with an in-depth investigation of the effect of commonly encountered environmental agents on specific critical virulence properties and pathways, identifying the molecular basis and regulation of pathways. Results of this research will enhance our understanding of the biological pathways engaged during the response of EHEC and related enteric pathogens to selected environmental stresses and may provide new molecular targets that can be exploited during food processing to modulate the virulence potential of these pathogens.