Genetic Systems to Study Virulence in Bacteroides

描述（由申请人提供）：本研究将侧重于使专性厌氧菌脆弱拟杆菌成为成功病原体的因素，尽管它是正常结肠微生物群的组成部分。这些包括在感染的早期阶段，它耐受有氧环境的能力（空气耐受性）；存在将血红素导入细胞的系统，用于中心代谢和防御活性氧的血红素依赖途径；脆弱芽孢杆菌从宿主成分中去除唾液酸残基的能力，以及从复杂的低聚糖和糖蛋白中获取体内生长所需营养的精湛技艺。具体目标包括：1，继续研究允许脆弱芽孢杆菌承受长时间氧挑战（空气耐受性）的因素：我们提出脆弱芽孢杆菌周质中的活性可作为抵抗活性氧（ROS）形成的初始防线，保护敏感靶标免受ROS挑战并逆转ROS损伤。此外，我们已经确定了特定的功能（超氧化物歧化酶，SOD）和一个广泛的基因簇（Bat操纵子）是空气耐受性所必需的。我们将验证Bat操纵子在细胞膜中形成多蛋白复合物的假设，该复合物在将还原电位从细胞质输出到外周质中起重要作用。2. 铁和血红素的获取对脆弱芽孢杆菌的体外和体内生长都很重要。我们将研究脆弱芽孢杆菌通过血红素渗透酶系统摄取血红素的过程，我们已经描述了血红素渗透酶的基因和功能。我们还将继续研究血红素依赖酶和含Fe-S簇的酶在中枢代谢的双重途径中，以确定它们在空气耐受性和氧气挑战时提供能量的作用。3. 目的：探讨侵染宿主获得生长底物的操纵子的组成、功能和调控。我们将重点关注含有神经氨酸酶（nanH1）基因的操纵子和其他几种糖水解酶，这些酶能够将许多人类细胞表面的复杂Lewis抗原转化为单个单糖。我们将继续分析NANA利用的操纵子、NanLET操纵子，并确定三个NanR抑制启动子中与NanR结合的位点。我们将确定神经氨酸酶是否是一种毒力因子，因为它为生长提供NANA，还是因为它的活性改变了宿主细胞的表面，或者两者兼而有之。

DESCRIPTION (provided by applicant): This study will focus on factors that allow the obligate anaerobe Bacteroides fragilis, although a component of the normal colonic microbiota, to be a successful pathogen. These include its ability to withstand an aerobic environment (aero-tolerance) during early stages of infection; the presence of systems to import heme into the cell for the heme-dependent pathways of central metabolism and defense against reactive oxygen species; the ability of B. fragilis to remove sialic acid residues from host components, and its virtuosity in obtaining nutrients for growth in vivo from complex oligosaccharides and glycoproteins. Specific aims include: 1, to continue to study factors that allow B. fragilis to withstand prolonged oxygen challenge (aerotolerance): We propose that activities in the B. fragilis periplasm serve as the initial line of defense to combat the formation of reactive oxygen species (ROS), protect sensitive targets from ROS challenges and to reverse ROS damage. In addition we have identified specific functions (superoxide dismutase, SOD), and an extensive gene cluster (the Bat operon) that are required for aerotolerance. We will test the hypothesis that the Bat operon forms a multi-protein complex in the cell membrane that plays an important role in exporting reducing potential from the cytoplasm to the periplasm. 2. Acquisition of iron and heme is important for B. fragilis growth in vitro and in vivo. We will study the process of heme uptake in B. fragilis by the heme permease systems whose genes and functions we have described We will also continue to study the heme-dependent, and Fe-S cluster-containing enzymes in the dual pathways of central metabolism to establish their roles in aerotolerance and in providing energy during oxygen challenge. 3. to investigate the composition, functions and control of operons for the acquisition of growth substrates from the infected host. We will focus on the operon containing the neuraminidase (nanH1) gene and several other glycohydrolases capable of converting the complex Lewis antigen found on the surface of many human cells to individual monosaccharides. We will continue to analyze the operon for NANA utilization, the NanLET operon and to define the sites in the three NanR repressed promoters for NanR binding. We will determine if neuraminidase is a virulence factor because it supplies NANA for growth, or because its activity alters the surface of host cells, or both.