GENETIC SYSTEMS TO STUDY VIRULENCE IN BACTEROIDES

描述(改编自申请人的摘要)：长期 本提案的目标是了解以下因素 对脆弱类杆菌的毒力和致病性有贡献， 化脓性肠内最重要的厌氧微生物 男性的腹部和盆腔感染。调查人员最近 分离的转座子插入突变与几个 可能成为致病因子的功能；这些功能包括 对氧气的敏感度增加(空气耐受性丧失)；降低 在组织培养中与动物细胞结合(丧失特异性粘附性)； 降低摄取必需的血红素分子的能力，以获得酶和 辅酶形成。使用差异基因检测系统 表达，调查人员已经开始分离候选启动子 打开或打开到更高水平的基因片段 在感染和/或暴露后模型系统中的表达 脆弱芽孢杆菌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体 在组织培养单层系统中显示出生长缺陷 在大鼠肉芽肿小袋模型中。MoxR区域由 航空敏感型moxR同源突变体YT65.2.10和YT120.2.20。他们会 确定mox区的基因是否包含共同的转录 对这些基因进行分组并确定它们是如何被调控的。他们将使用一个 敏感的诱变试验以跟踪氧气暴露的早期影响。 他们预计在基因突变频率上会有显著的差异 MoxR同源突变株与亲本菌株比较。这个 Rubredosin区域特别有趣，因为这个蛋白质是 经常参与可能涉及氧的电子转移反应 以及它的一些有毒产品。他们将确定下游的基因 并产生特定的缺失突变 以测试它们体内生长潜力的变化。 脆弱芽孢杆菌结合缺陷突变体YT58.1.3将用于 描述与组织结合所需的基因和蛋白质 培养单层。将应用检测表面蛋白的方法 对这些突变体，试图鉴定受病毒影响的蛋白质(S) 插入。 他们将通过以下方式研究脆弱杆菌对铁和血红素的吸收过程 利用他们新发现的血红素渗透酶基因 实验室，在转座子中发现的新发现的AngR同系物 突变的MGD13.1，以及已知的hupA、B基因序列 与血红素结合有关。 用于检测环境激活基因表达的方法 在感染过程中对细菌细胞的影响可能揭示全球和基因特异性 与毒力有关的调控途径。调查人员将 继续应用他们已调整以用于 脆弱芽孢杆菌获得被激活的基因的更多候选基因 在体内或在暴露于氧气或过氧化氢之后。 为了鉴定在脓肿形成中有缺陷的转座子突变体， 脆弱芽孢杆菌感染的标志，他们会适应皮下 取自小鼠的脓肿模型用于大鼠。

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.