Regulation of Virulence Factors by Proteins of the Flagellar System in Enteric Bacteria

Non-Technical Summary: In order to have effective therapeutic interventions to treat microbial infectious diseases, it is essential to understand the survival strategies employed by pathogenic bacteria that enable them to persist in a colonized host. The environment in the host differs considerably from that in the free-living phase. Therefore, ultimate microbial survival is dependent upon accurate sensing of environmental signals and appropriate transcriptional responses to these environmental signals. The PI's long-term goal is to understand the regulation of virulence factors in enteric bacteria. The objective of this hatch project is the determination of the effect of flagella proteins, such as Aer and FliA upon virulence of E. coli and Y. enterocolitica. We will perform physiological experiments under laboratory conditions and develop an animal model for each pathogen. We will perform mechanistic studies on Aer and FliA. This study is expected to provide new insight into microbial gene regulation in response to the host environment, an important aspect in understanding microbial infectious disease. <P> Approach: Objective #1: 1.1.1 Determine the role of Aer in the transition from aerobic to anaerobic growth that occurs as bacteria grown in batch culture deplete the oxygen from the medium. 1.1.2 Determine the effect of Aer upon gene regulation in mouse mucus media. 1.2.1 Determine the competitive phenotype of the aer mutant under laboratory conditions. 1.2.2 Determine the role of Aer in the colonization process. 1.3.1 Determine the specificity of Aer mediated gene regulation. 1.3.2 Determine the domain of Aer that is required for gene regulation. Objective # 2: Occurrence of Y. enterocolitica in avian food products and other foods in the State of North Dakota. Y. enterocolitica has been found in food products around the world, predominantly in beef, pork, and chicken (2-6). However, occurrence in the State of North Dakota has not routinely been tested. We will determine the occurrence of Y. enterocolitica in a variety of different foods, starting out with avian food products, such as eggs. Other foods will include meat of beef and pork, and milk products. An enrichment protocol will be used as described (29). This protocol uses three enrichment procedures to insure that any serotype of pathogenic Y. enterocolitica present in the product will be recovered. Selection will be performed on two selective agars. Colonies of the correct morphology will be picked and subjected to a duplex PCR test (30), amplifying the 16S rDNA gene and ail gene. With this procedure we should be able to detect the majority of pathogenic Y. enterocolitica serotypes and answer the question whether Y. enterocolitica is a food hazard in the State of North Dakota. Objective # 3: 3.1 Investigate the inverse regulation of flagellar and plasmid-encoded virulence genes by FliA. 3.2 Determine the effect of FliA upon the pathogenic character of Y. enterocolitica in an avian embryo model.