GENETIC SYSTEMS TO STUDY VIRULENCE BACTEROIDES FRAGILES

这项建议的长期目标是了解以下因素 对脆弱芽孢杆菌的毒力和致病性的贡献最大的 腹盆腔内化脓性感染的重要厌氧菌 人类的感染。因为这需要遗传和生理分析 在毒力因子中，我们的方法是开发新的基因 脆弱芽孢杆菌中用于识别潜在毒力的系统 决定因素。我们正在关注脆弱芽孢杆菌的耐气性作为一种 生物体在氧气组织中存活的能力的重要因素， 和神经氨酸酶，这可能是脆弱芽孢杆菌附着的重要因素 寄主细胞，并从环境中获取必要的营养。因为有很多人 细胞膜蛋白在细菌致病中起作用，我们将使用 一种改良的转座子用于鉴定芽孢杆菌的新毒力因子 脆性表面蛋白或出口蛋白。基因突变的突变体 潜在的毒力因子将进行测试，以确定它们是否有能力在两个 脆弱芽孢杆菌感染的模型系统。 第一个目标是继续分析可能在 脆弱芽孢杆菌的毒力：脆弱芽孢杆菌神经氨酸酶。我们将创建B。 仅在神经氨酸酶结构上不同的脆性杆菌等基因突变体 并在模型系统中测试它们的性质。我们会研究 脆性芽孢杆菌和E. Coli.我们将确定神经氨酸酶在B细胞中的表达是如何控制的。 脆弱的。B.对空气的耐受性可能是脆弱杆菌 类杆菌群的主要病原体。为了检验这一假说 我们将分离出缺乏过氧化氢酶的突变体，并确定它们是否 对航空敏感。如果是这样的话，我们将研究缺乏过氧化氢酶的脆弱芽孢杆菌 感染模型系统中的菌株。如果这些菌株减少了 在模型系统中存活的能力，然后我们将确定过氧化氢酶 结构基因，研究过氧化氢酶的表达调控。 脆弱的。 第二个目标是使用TnPhoA突变来识别编码 脆弱芽孢杆菌输出蛋白质，并测试突变体在其 在两个模型系统中增长的能力：a.我们将修改TnPhoA以 包含可在B.Fragilis中选择的标记。B.我们将突变一个 脆性芽孢杆菌染色体文库在大肠杆菌中的表达及诱变转移 脆弱芽孢杆菌的质粒选择在染色体上进行PhoA+融合。 第三个目标是测试组织中潜在毒力因子的突变。 脆弱芽孢杆菌的培养及大鼠眼袋模型系统：a.组织 培养模型将测试空气耐受性和定植因素。B. 大鼠肉芽肿袋是一个真实的活体系统。除了测试之外 在组织培养系统中的重要因素，它还测试了 生物体对各种寄主防御机制的反应 包括主动免疫系统。

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.