GENETIC SYSTEMS TO STUDY VIRULENCE IN B FRAGILIS

描述(摘自申请者的摘要)：长期目标 这一建议的关键是了解导致 最重要的脆弱类杆菌的毒力和致病性 化脓性腹腔炎和盆腔炎的厌氧菌检测 人类。因为这需要进行遗传和生理分析 毒力因素，首席调查员的方法是 开发新的遗传系统用于脆弱芽孢杆菌以鉴定 潜在的毒力决定因素。 研究人员将脆弱芽孢杆菌的耐气性作为一种 生物体在含氧组织中生存能力的重要因素。 他将分析一组高度表达的脆弱芽孢杆菌蛋白质 只有在氧气存在的情况下。他将研究合成的控制 这些氧反应蛋白(ORPs)，并试图识别全球和 基因特异性调节因子。他还将研究关闭的过程 大多数脆弱芽孢杆菌蛋白通常是在厌氧菌发生时产生的 当生长中的细胞受到氧气的挑战时。他将继续他的 脆弱芽孢杆菌超氧化物歧化酶的研究及其作用 在脆弱芽孢杆菌体内和体外存活。 由于调查员已经确定了脆弱芽孢杆菌的重要角色 神经氨酸酶(NanH)对脆弱芽孢杆菌体内生长能力的影响 测试系统，他将专注于定义 了解其表达是如何调控的。 他将使用已在E. 以鉴定出口蛋白，以研究其表面蛋白。 脆性假单胞菌。由于许多细胞被膜蛋白在细菌中发挥作用 致病性，他将使用TnCef来识别毒力决定因素 脆弱芽孢杆菌表面蛋白或出口蛋白。基因突变的突变体 潜在的毒力因子将进行测试，以确定它们是否有能力在两个 脆弱芽孢杆菌感染、大鼠肉芽肿囊和CHO或 培养中的Monika P15动物细胞。 他将寻找只有在细菌感染后才表达的脆弱杆菌基因。 细菌在体内生长。这些基因很可能发挥重要的作用 在建立和维持脆弱芽孢杆菌感染中的作用。 进一步明确脆弱芽孢杆菌感染大鼠的初始步骤 ，他将对感染的眼袋进行组织学分析 感染后早期。他将确定 在厌氧菌建立过程中感染脆弱芽孢杆菌细胞 细菌生长所需的环境。

DESCRIPTION (Adapted from applicant's abstract): The long term objective of this proposal is to understand the factors that contribute to the virulence and pathogenicity of Bacteroides fragilis, the most important anaerobic pathogen in suppurative intra-abdominal and pelvic infections in human beings. Since this requires a genetic and physiological analysis of the virulence factors, the principal investigator's approach has been to develop new genetic systems for use in B. fragilis in order to identify potential virulence determinants. The investigator is focusing on the aerotolerance of B. fragilis as an important factor in the organisms' ability to survive in oxygenated tissue. He will analyze a set of B. fragilis proteins that are highly expressed only in the presence of oxygen. He will study the control of the synthesis of these oxygen response proteins (ORPs) and attempt to identify global and gene specific regulators. He will also study the process of the shut-off of most B. fragilis proteins normally made during anaerobiosis that occurs when the growing cells are challenged with oxygen. He will continue his studies on the superoxide dismutase of B. fragilis and determine its role in B. fragilis survival in vitro and in vivo. Since the investigator has determined an important role for the B. fragilis neuraminidase (nanH)in the ability of B. fragilis to grow in two in vivo test systems, he will concentrate on defining the control region of the nanH structural gene and in understanding how its expression is regulated. He will use an adaptation of the TnphoA system that has been used in E. coli to identify exported proteins, in order to study surface proteins of B. fragilis. Since many cell envelope proteins play a role in bacterial pathogenicity, he will use TnCef to identify virulence determinants 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 rat granuloma pouch, and CHO or Monika P15 animal cells in culture. He will search for B. fragilis genes that are only expressed when the bacteria are growing in vivo. These genes are likely to play an important role in the establishment and maintenance of B. fragilis infections. To further define the initial steps in B. fragilis infection of the rat pouch, he will perform a histological analysis of the infected pouch at early times after infection. He will determine the location of the infecting B. fragilis cells during the establishment of the anaerobic environment that is required for bacterial growth.