Genetic Systems to Study Virulence in Bacteroides

描述(由申请人提供)：这项研究将集中在使专性厌氧菌脆弱类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一个组成部分。这些问题包括：在感染的早期阶段抵抗有氧环境的能力(空气耐受性)；存在将血红素导入细胞以进行依赖于血红素的中心代谢途径和防御活性氧物种的系统；脆弱芽孢杆菌从宿主成分中去除唾液酸残留物的能力；以及从复杂的寡糖和糖蛋白中获得体内生长所需的营养的技巧。具体目标包括：1、继续研究使脆弱芽孢杆菌耐受长期氧气挑战(有氧耐受)的因素：我们建议脆弱芽孢杆菌周质中的活动作为最初的防线，以对抗活性氧物种(ROS)的形成，保护敏感靶标免受ROS挑战，并逆转ROS损伤。此外，我们还确定了耐氧性所需的特定功能(超氧化物歧化酶，SOD)和广泛的基因簇(蝙蝠操纵子)。我们将检验这样的假设，即蝙蝠操纵子在细胞膜上形成一个多蛋白质复合体，在将还原电位从细胞质输出到周质中发挥重要作用。2.铁和血红素的获得对脆弱芽孢杆菌的体外和体内生长具有重要意义。我们将通过我们描述的血红素渗透酶系统来研究脆弱假单胞菌对血红素的吸收过程。我们还将继续研究中枢代谢双重途径中的血红素依赖酶和含铁S簇的酶，以确定它们在有氧耐受和氧挑战时提供能量的作用。3.研究从受感染宿主获取生长底物的操纵子的组成、功能和调控。我们将专注于含有神经氨酸酶(NanH1)基因和其他几种糖水解酶的操纵子，这些糖水解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。我们将继续分析利用NaNA的操纵子，NanLET操纵子，并确定三个NanR抑制的启动子中用于NanR结合的位点。我们将确定神经氨酸酶是一种毒力因子，是因为它为生长提供了NaA，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.