The human pathogen Yersinia enterocolitica causes gastroenteritis by penetrating the mucosal barrier and colonizing Peyer's patches and mesenteric lymph nodes. Ail promotes intracellular entry and resistance of Y. enterocolitica to human serum in vitro. Since environmental regulation of virulence factors is common, a reporter gene fusion to ail will be created, recombined back into the Yersinia chromosome, and tested under varying environmental conditions. Then transposon insertion mutants with altered regulation will be screened using the enzymatic activity of the reporter as a marker. The putative regulatory genes will be cloned, sequenced, and characterized. The effect of the transposon insertion on the regulation of other known virulence factors will also be examined. Both a reporter fusion and the regulatory mutant will be examined in the mouse model. The reporter fusion will be used to identify the tissues in which Ail is produced perhaps giving a clue to the function of Ail in vivo. The regulatory mutant will be assayed for virulence by LD50' colonization of Peyer's patches, and tissue spread. Alteration of virulence as compared to the parental strain will imply that the regulation of other virulence factors is affected since ail null mutations are not reduced for virulence by these three criteria. Therefore, the long term goal of the project is to characterized regulation of ail and the genes involved. A potential future goal not covered in the proposal will be the identification of other virulence genes which are also regulated by the same mechanism.
