GENETIC SYSTEMS TO STUDY VIRULENCE BACTEROIDES FRAGILES

研究脆性拟杆菌毒力的遗传系统

• 批准号: 3128855
• 负责人: MICHAEL H MALAMY
• 金额: $ 21.71万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-01-01 至 1993-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3128855
• 关键词: Bacillus; Bacteroides; Escherichia coli; antibiotics; bacterial cytopathogenic effect; bacterial genetics; biological models; catalase; chromosomes; exo alpha sialidase; gene expression; genetic library; genetic manipulation; genetic markers; genetic strain; laboratory rat; molecular cloning; mutant; protein signal sequence; site directed mutagenesis; structural genes; tissue /cell culture; transcription factor; transport proteins; transposon /insertion element; virulence

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.

该提议的长期目标是了解 有助于B. fragilis的毒力和致病性，最多 化脓性腹内和骨盆中重要的厌氧病原体 人类感染。由于这需要遗传和生理分析 在毒力因素中，我们的方法是发展新的遗传 为了识别潜在的毒力，用于b.fragilis的系统 决定因素。我们将重点放在B Fragilis的空气耐受性上 生物体在含氧组织中生存的能力的重要因素， 而神经氨酸酶对于脆弱的芽孢杆菌粘附可能很重要 宿主细胞并从环境中得出必需的营养。自从很多 细胞包膜蛋白在细菌致病性中起作用，我们将使用 修饰的tnphoa转座子，以鉴定B中的新毒力因子。 脆弱的表面或导出的蛋白质。变化的突变体 潜在的毒力因子将测试其在两个中生长的能力 脆弱芽孢杆菌感染的模型系统。 第一个目的是继续分析可能在 B. fragilis的毒力： B.脆弱的神经氨酸酶。我们将创建B。 仅在神经氨酸酶结构中有所不同的脆弱的等源性突变体 基因并在模型系统中测试其性质。我们将研究 将神经氨酸酶定位在脆弱芽孢杆菌和E.中的序列和因子。 大肠杆菌。我们将确定如何在B中控制神经氨酸酶的表达。 Fragilis。 b。空气耐受性可能是B. fragilis是的重要原因 细菌基团的前主导病原体。检验这一假设 我们将隔离过氧化氢酶缺乏突变体，并确定它们是否是 空气敏感。如果是这样，我们将研究过氧化氢酶缺乏脆弱的B. fragilis 感染模型系统中的菌株。如果这些菌株减少了 然后，我们将在模型系统中生存，然后确定过氧化氢酶 结构基因并研究B中过氧化氢酶表达的调节。 Fragilis。 第二个目的是使用tnphoa诱变来识别编码的基因 B. fragilis导出蛋白质并测试突变体的改变 在两个模型系统中生长的能力：我们将将tnphoa修改为 包含在B. fragilis中选择的标记。 b。我们将诱变 大肠杆菌中的B.脆弱的染色体文库并转移诱变 质粒到脆弱的B. b.b。在染色体中选择phoa+融合。 第三个目的是测试组织潜在毒力因子中的突变体 B. fragilis感染的培养和大鼠袋模型系统：组织 培养模型将测试空气耐受性和定植因子。 b。这 大鼠肉芽瘤袋是一个真正的体内系统。除了测试 在组织培养系统中重要的因素，它还测试了 有机体对一系列宿主防御机制的反应 包括主动免疫系统。