GENETIC SYSTEMS TO STUDY VIRULENCE IN B FRAGILIS

描述（改编自申请人摘要）：长期目标 这一建议的目的是了解有助于 脆弱类杆菌的毒力和致病性，最重要的 厌氧菌在化脓性腹腔内和盆腔感染中的作用 人类 因为这需要对基因和生理进行分析， 毒力因子，主要研究者的方法是 开发新的遗传系统用于B。fragilis以识别 潜在的毒力决定因素 研究者关注的是B的耐氧性。fragilis作为一个 这是生物体在含氧组织中生存能力的重要因素。 他将分析一组B。fragilis高表达的蛋白质 只有在氧气存在的情况下。 他将研究控制合成 这些氧反应蛋白（ORP），并试图确定全球和 基因特异性调节剂。 他还将研究关闭的过程 大多数B。fragilis蛋白质通常在缺氧期间产生 当生长的细胞受到氧气的挑战时。 他将继续他的 B超氧化物歧化酶的研究fragilis并确定其作用 在B。fragilis在体外和体内的存活。 因为研究者已经确定了B的重要角色。脆弱拟 神经氨酸酶（nanH）在B. fragilis在两个体内生长 测试系统，他将集中精力定义控制区域的 nanH结构基因，并了解其表达是如何调节的。 他将使用一个已在E. 大肠杆菌鉴定输出蛋白，以研究表面蛋白 B。脆弱的 由于许多细胞被膜蛋白在细菌中起作用， 致病性，他将使用TnCef来确定 B。fragilis表面或输出的蛋白质。 突变体， 将测试潜在的毒力因子在两种环境中生长的能力， B的模型系统。fragilis感染，大鼠肉芽肿袋，CHO或 Monika P15动物细胞培养。 他将寻找B。fragilis基因，只有在 细菌在体内生长。 这些基因很可能在 在建立和维持B中的作用。脆弱菌感染 进一步定义B中的初始步骤。大鼠脆性感染 袋，他将进行组织学分析的感染袋， 感染后早期。 他将确定 感染B。fragilis细胞在建立厌氧 这是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.