GENETIC SYSTEMS TO STUDY VIRULENCE BACTEROIDES FRAGILES

本提案的长期目标是了解 有助于B的毒力和致病性。最脆弱的 腹腔及盆腔化脓性疾病重要厌氧菌 由于这需要遗传和生理分析， 我们的方法是开发新的基因， 用于脆弱B.fragilis以鉴定潜在毒力的系统 决定因素我们关注的是B fragilis的空气耐受性， 重要的因素在有机体的能力，以生存在氧合组织， 和对B很重要的神经氨酸酶。fragilis粘附 宿主细胞和从环境中获取必需的营养物质。由于许多 细胞被膜蛋白在细菌致病性中发挥作用，我们将使用 一种修饰的TnphoA转座子，用于在B中鉴定新的毒力因子。 fragilis表面或输出的蛋白质。突变体， 将测试潜在的毒力因子在两种环境中生长的能力， B的模型系统。脆弱性感染。 第一个目的是继续分析可能在这方面发挥作用的因素， B的毒力。脆弱的; B。脆壁神经氨酸酶我们将创建B。 仅在神经氨酸酶结构上不同的fragilis同基因突变体 基因并在模型系统中测试它们的性质。我们会研究 定位B中神经氨酸酶的序列和因子。fragilis和E. 杆菌我们将确定神经氨酸酶的表达是如何控制在B。 脆弱的B.空气耐受性可能是B.脆弱群岛 拟杆菌属的主要病原体。为了验证这一假设 我们将分离过氧化氢酶缺陷突变体，并确定它们是否 对空气敏感。如果是这样，我们将研究过氧化氢酶缺乏的B。脆弱拟 感染模型系统中的菌株。如果这些菌株减少了 在模型系统中生存的能力，然后我们将确定过氧化氢酶 结构基因，研究过氧化氢酶在B中的表达调控。 脆弱的 第二个目的是使用TnphoA诱变来鉴定编码以下的基因： 脆弱B.fragilis输出蛋白质，并测试突变体在其蛋白质中的改变。 在两个模型系统中生长的能力：a.我们将修改TnphoA， 含有在B中可选择的标记。脆弱的B.我们将诱变一个 B。fragilis染色体文库。大肠杆菌，并将诱变的 质粒到B。fragilis选择染色体中的phoA+融合。 第三个目的是测试组织中潜在毒力因子的突变体 B的培养和大鼠眼袋模型系统。fragilis感染：a.组织 培养模型将测试空气耐受性和定殖因素。B.的 大鼠肉芽肿袋是一个真正的体内系统。除了测试 在组织培养系统中的重要因素，它还测试了 生物体对宿主防御机制的全面反应 包括活跃的免疫系统

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.