GENETIC SYSTEMS TO STUDY VIRULENCE IN BACTEROIDES

描述（改编自申请人的摘要）：长期 本提案的目的是了解 有助于脆弱拟杆菌的毒力和致病性， 最重要的专性厌氧生物在化脓性内- 男性腹部和盆腔感染。研究人员最近 分离的转座子插入突变，在几个 作为毒力因子的候选者的功能;这些功能包括 对氧的敏感性增加（丧失空气耐受性）;降低 在组织培养中与动物细胞结合（丧失特异性粘附）; 降低酶吸收必需血红素分子的能力， 辅酶形成。 利用差异基因检测系统 表达，研究人员已经开始分离候选启动子 基因的片段被打开，或者被打开到更高的水平， 在感染和/或暴露后的模型系统中的表达 的B。fragilis细胞对氧气或过氧化氢的反应。 他们的第一个目标是分析新的插入突变， 证明了在组织培养单层系统中的生长缺陷 和大鼠肉芽肿袋模型。 moxR区域由以下定义： 空气敏感性moxR同源物突变体YT 65.2.10和YT 120.2.20。 他们将 确定MOX区域中的基因是否包含共同的转录 分组并确定这些基因是如何调节的。 他们将使用一个 敏感的诱变试验以跟踪氧暴露的早期效应。 他们希望看到基因突变频率的显著差异， moxR-同源物突变体与亲本菌株相比。 的 rubredoxin区域特别有趣，因为该蛋白质 通常涉及电子转移反应， 和一些有毒的产品。他们将决定下游的基因 Rubredoxin结构基因并产生特定的缺失突变 以测试其体内生长潜力的变化。 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 单层培养 将应用检测表面蛋白的方法 对这些突变体，以尝试并确定蛋白质（S）的影响， 插入。 他们将研究B吸收铁和血红素的过程。fragilis通过 利用在他们的研究中新发现的血红素通透酶基因， 实验室，新确定的angR同源物发现转座子 突变体MGD 13.1，以及已知的hupA、B基因的序列， 参与血红素结合。 用于检测环境激活的基因表达的方法， 在感染过程中对细菌细胞的作用可能揭示全局性和基因特异性 参与毒力的调节途径。 调查人员将 继续采用经调整后的综合招聘考试/本地招聘考试制度， B。fragilis获得额外的候选基因， 体内或暴露于氧气或过氧化氢后。 为了鉴定具有脓肿形成缺陷的转座子突变体， B的标志。fragilis感染，他们将适应皮下 用于大鼠的小鼠脓肿模型。

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.