Identification of Differentiality Expressed Genes in C. Jejuni Virulence

<ol> <li>Identification of differentially expressed genes by gene fusion technology. The goal of this Specific Aim is to identify differentially expressed C. jejuni genes using a gfp transcriptional fusion vector.
<li>Molecular cloning and characterization of C. jejuni genes. The goals of this Specific Aim are to clone each gene in its entirety, ensure that the cloned gene is from C. jejuni, generate a null mutant, determine the cellular location of the gene product, and perform complementation analysis of the C. jejuni mutant.
<li>Phenotypic analysis of the C. jejuni mutants. The goal of this Specific Aim is to determine the role of the products, which are encoded by the genes that are differentially expressed in C. jejuni pathogenesis.</ol>
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Gene function will be determined and a detailed restriction map constructed to facilitate mutagenesis work. Completion of these goals will better define the virulence mechanisms used by C. jejuni to cause disease. Knock-outs of genes responsible for intra-macrophage survival may lead to the development of live, attenuated vaccines to prevent campylobacteriosis.
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Campylobacter jejuni is the number one cause of bacterial gastroenteritis in the U.S. with an estimated cost of treatment and loss of productivity exceeding $1 billion, annually. Clearly, understanding the mechanisms by which this major pathogen causes disease is needed to develop an effective vaccine to prevent campylobacteriosis. Our work with the piglet model has shown that C. jejuni invades intestinal epithelial cells in vivo, spreads to deeper tissue in the intestinal lamina propria, and resides in the deeper tissue as well as in lamina propria macrophages. Reporter systems emitting differential fluorescence have been used successfully to identify genes active under host- cell invasion and survival.
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For this proposal, our goal is to identify, clone and characterize genes of C. jejuni which are required for intra-macrophage survival. The proposed research will use GFP as a reporter to identify genes preferentially expressed when C. jejuni invades host intestinal tissue and lamina propria macrophages. Gene function will be determined and a detailed restriction map constructed to facilitate mutagenesis work. Completion of these goals will better define the virulence mechanisms used by C. jejuni to cause disease. Knock-outs of genes responsible for intra-macrophage survival may lead to the development of live, attenuated vaccines to prevent campylobacteriosis.