Genetic System to Study Virulence in Bacteroides

描述（由申请方提供）：本研究将重点关注允许专性厌氧菌脆弱B.fragilis（尽管是正常结肠微生物群的组成部分）成为成功病原体的因素。这些包括其在感染的早期阶段耐受有氧环境（空气耐受性）的能力;存在将血红素输入到细胞中以用于中心代谢的血红素依赖性途径和对活性氧的防御的系统;脆弱B.fragilis从宿主组分中去除唾液酸残基的能力，以及其在从复杂寡糖和糖蛋白中获得用于体内生长的营养物方面的精湛技艺。具体目标包括：1、继续研究使脆弱B.fragilis耐受长时间氧挑战（耐氧性）的因素：我们提出脆弱B.fragilis周质中的活性作为对抗活性氧（ROS）形成的初始防线，保护敏感靶点免受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），和广泛的基因簇（蝙蝠操纵子）所需的耐氧性。我们将测试的假设，蝙蝠操纵子在出口还原电位从细胞质到周质中起着重要的作用。2.铁和血红素的获得对于脆弱B.fragilis在体外和体内的生长是重要的。我们将通过血红素通透酶系统研究脆弱B.fragilis对血红素的吸收过程，我们已经描述了血红素通透酶系统的基因和功能。 我们还将继续研究血红素依赖性和含Fe-S簇的酶在中枢代谢的双重途径中的作用，以确定它们在耐氧性和在氧挑战期间提供能量中的作用。 3.研究操纵子的组成、功能和控制，以从受感染的宿主获得生长底物。我们将集中在操纵子含有神经氨酸酶（nanH 1）基因和其他几个糖水解酶能够转换复杂的刘易斯抗原发现在许多人类细胞表面的单个单糖。 我们将继续定义NANA利用的操纵子，NanL 1操纵子。神经氨酸酶是一种毒力因子，是因为它为脆弱B.fragilis生长提供NANA，还是因为它的活性在感染过程中改变了宿主细胞的表面，还是两者兼而有之？ 4.我们将开发额外的工具来定义在9氧挑战期间大分子合成和DNA，蛋白质和膜稳定性的变化。

DESCRIPTION (provided by applicant): This study will focus on factors that allow the obligate anaerobe B.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 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 define the operon for NANA utilization, the NanL1 operon. Is neuraminidase a virulence factor because it supplies NANA for B.fragilis growth, or because its activity alters the surface of host cells during infection, or both? 4.We will develop additional tools to define changes in macromolecular synthesis and stability of DNA, proteins and membranes during 9 oxygen challenge.