GENETIC SYSTEMS TO STUDY VIRULENCE IN BACTEROIDES

研究拟杆菌毒力的遗传系统

• 批准号: 2611175
• 负责人: MICHAEL H MALAMY
• 金额: $ 30.28万
• 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 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2611175
• 关键词: Bacteroides; anaerobiosis; bacterial genetics; bacterial proteins; gene expression; genetic promoter element; genetic regulation; heme; iron metabolism; laboratory rat; microorganism growth; mutant; operon; oxygen; reporter genes; rubredoxins; tissue /cell culture; transposon /insertion element; virulence

DESCRIPTION (Adapted from the applicant's abstract): The long term objectives of this proposal are to understand the factors that contribute to the virulence and pathogenicity of Bacteroides fragilis, the most important obligately anaerobic organism in suppurative intra- abdominal and pelvic infections in man. The investigators have recently isolated transposon insertion mutations with alterations in several functions which are candidates to be virulence factors; these include increased sensitivity to oxygen (loss of aero-tolerance); decreased binding to animal cells in tissue culture (loss of specific adherence); reduced ability to take up the essential heme molecule for enzyme and co-enzyme formation. Using a detection system for differential gene expression, the investigators have begun to isolate candidate promoter fragments for genes that are turned on, or turned on to a higher level of expression in the model systems of infection and/or after exposure of B. fragilis cells to oxygen or hydrogen peroxide. Their first objective is to analyze the new insertion mutants with demonstrated defects in growth in the tissue culture monolayer systems and in the rat granuloma pouch model. The moxR-region is defined by the aero-sensitive moxR-homolog mutants YT65.2.10 and YT120.2.20. They will determine if the genes in the mox-region comprise a common transcription group and establish how these genes are regulated. They will use a sensitive mutagenesis assay to follow early effects of oxygen exposure. They expect to see a significant difference in mutation frequencies in the moxR-homolog mutants compared with the parental strain. The rubredoxin region is particularly interesting since this protein is often involved in electron transfer reactions that may involve oxygen and some of its toxic products. They will determine the genes downstream of the rubredoxin structural gene and create specific deletion mutations in them to test for alterations in their in vivo growth potential. The B. fragilis binding deficient mutant, YT58.1.3 will be used to characterize genes and proteins required for binding to the tissue culture monolayer. Methods to detect surface proteins will be applied to these mutants to try and identify the protein(s) affected by the insertion. They will study the process of iron and heme uptake in B. fragilis by taking advantage of the heme permease gene newly identified in their laboratory, the newly identified angR-homolog discovered in transposon mutant MGD13.1, and the sequences of the hupA,B genes known to be involved in heme binding. Methods for detecting environmentally activated gene expression applied to bacterial cells during infection may reveal global and gene specific regulatory pathways involved in virulence. The investigators will continue to apply the cre/lox system that they have adapted for use in B. fragilis to obtain additional candidates for genes that are activated in vivo or after exposure to oxygen or hydrogen peroxide. To identify transposon mutants with defects in abscess formation, the hallmark of B. fragilis infections, they will adapt a sub-cutaneous abscess model from the mouse for use in the rat.

描述（改编自申请人的摘要）：长期 该提案的目标是了解 有助于细菌的毒力和致病性， 化脓性内最重要的厌氧生物 人的腹部和骨盆感染。 调查人员最近有 分离的转座子插入突变，有几个变化 是候选毒力因素的功能；这些包括 对氧气的敏感性提高（空气耐受性的丧失）；减少 在组织培养中与动物细胞的结合（特定依从性的丧失）； 降低了用于酶的基本血红素分子的能力和 共酶形成。 使用检测系统进行差异基因 表达，研究人员已经开始隔离候选者 打开或打开的基因片段 感染模型系统和/或暴露后的表达 脆弱的芽孢杆菌细胞的氧或过氧化氢。 他们的第一个目标是用 证明了组织培养单层系统的生长缺陷 并在大鼠肉芽瘤模型中。 Moxr-Region由 空气敏感的Moxr--词素突变体YT65.2.10和YT120.2.20。 他们会的 确定MOX区域中的基因是否包含共同转录 组并确定如何调节这些基因。 他们将使用 敏感的诱变测定法需要遵循氧气暴露的早期作用。 他们期望看到突变频率的显着差异 MOXR-总体突变体与亲本菌株相比。 这 Rubredoxin区域特别有趣，因为该蛋白是 通常参与可能涉及氧气的电子转移反应 以及一些有毒产品。他们将确定下游的基因 Rubredoxin结构基因并创建特定的缺失突变 在他们中测试其体内生长潜力的改变。 B. fragilis结合缺陷突变体，YT58.1.3将用于 表征与组织结合所需的基因和蛋白质 文化单层。 检测表面蛋白的方法将应用 这些突变体试图鉴定受到该突变的蛋白质 插入。 他们将通过B. fragilis中的铁和血红素摄取的过程 利用血红素渗透基因在其中新鉴定 实验室，在转座子中发现的新鉴定 突变MGD13.1和HUPA的序列，B基因已知为 参与血红素结合。 检测应用环境激活的基因表达的方法 感染期间细菌细胞可能显示全球和基因特异性 涉及毒力的调节途径。 调查人员会 继续应用他们已适应的CRE/LOX系统 B. fragilis获得激活基因的其他候选者 体内或暴露于氧或过氧化氢后。 为了鉴定脓肿形成缺陷的转座子突变体， 脆弱的芽孢杆菌感染的标志，它们将适应下肠 来自鼠标的脓肿模型用于大鼠。