GENETIC SYSTEMS TO STUDY VIRULENCE BACTEROIDES FRAGILES

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The long term objective of this proposal is to understand the factors that contribute to the virulence and pathogenicity of B. fragilis, the most important anaerobic pathogen in suppurative intra-abdominal and pelvic infections in man. Since this requires a genetic and physiological analysis ofthe virulence factors, our approach has been to develop new genetic systems for use in B.fragilis in order to identify potential virulence determinants. We are focussing on the aero-tolerance of B fragilis as an important factor in the organisms' ability to survivein oxygenated tissue, and the neuraminidase which may be important for B. fragilis to adhere to host cells and derive essential nutrients from the environment. Since many cell envelope proteins play a role in bacterial pathogenicity, we will use a modified TnphoA transposon to identify new virulence factors among B. fragilis surface or exported proteins. Mutants with alterations in potential virulence factors will be tested for their ability to grow in two model systems of B. fragilis infection. The first aim is to continue to analyze factors that may play a role in the virulence of B. fragilis: a. B. fragilis neuraminidase. We will create B. fragilis isogenic mutants that differ only in the neuraminidase structural gene and test their properties in the model systems. We will study the sequences and factors that localize neuraminidase in B. fragilis and E. coli. We will determine how neuraminidase expression is controlled in B. fragilis. b. Aero-tolerance may be an important reason why B. fragilis is the pre-dominant pathogen of the Bacteroides group. To test this hypothesis we will isolate catalase deficient mutants and determine if they are aero-sensitive. If this is so, we will study catalase deficient B. fragilis strains in the model systems of infection. If these strains have reduced ability to survive in the model systems, we will then identify the catalase structural gene and study the regulation of catalase expression in B. fragilis. The second aim is to use TnphoA mutagenesis to identify genes that code for B.fragilis exported proteins and test the mutants for alterations in their ability to grow in the two model systems: a. We will modify TnphoA to contain a marker that is selectable in B. fragilis. b. We will mutagenize a B. fragilis chromosomal library in E. coli and transfer the mutagenized plasmids to B. fragilis selecting for phoA+ fusions in-the chromosome. The third aim is to test mutants in potential virulence factors in a tissue culture and Rat Pouch model system of B. fragilis infection: a. The tissue culture model will test for aero-tolerance and colonization factors. b. The Rat Granuloma Pouch is a true in vivo system. In addition to testing factors important in the tissue culture system, it also tests for the response of the organism to the full array of host defense mechanisms including an active immune system.