Virulence Determinants in Helicobacter hepaticus

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DESCRIPTION (provided by applicant): The presence of H. hepaticus in the liver of mice is correlated with the development of hepatitis and liver carcinomas, and these symptoms can be studied by inoculating mice with the bacterium. The specific aim of the proposed work is to begin to identify some of the bacterial factors that may be virulence components in H. hepaticus via a targeted mutagenesis approach. A complete genome sequence for the type strain is expected to be available sometime in 2003, and some suspected virulence factors (superoxide dismutase, alkyl hydroperoxide reductase, and hydrogenase) have been identified by enzyme assays on H. hepaticus extracts (in the Principal Investigator's lab). The above three enzymes have been shown to affect virulence in the related bacterium Helicobacter pylori (H. pylori), by the same approach (targeted mutagenesis) proposed here. The sequences for the three genes (sodB, ahpC, and hydA) are being made available to the Principal Investigator prior to the full genome sequence release so that targeted mutants can be generated in these three potential virulence factors. However, a mutant H. hepaticus has never been recovered in any gene. Therefore, an initial pilot study is needed to ascertain the feasibility of this approach, using kanamycin-resistance cassette alMic exchange mutagenesis (the method that is currently used for H. pylori). Once mutants are made they will be compared to the parent strain for colonization and infections of mice, and the resulting liver tissue will be assessed for disease by a board certified veterinary pathologist. If specific mutants can be created, then virulence determinants can begin to be systematically studied.