Altered M. tuberculosis Mannosylation and the Macrophage

结核病继续在全世界人口中造成巨大的发病率和死亡率。关键在于建立 分枝结核杆菌（M.tb）感染的主要途径是进入巨噬细胞并在巨噬细胞中存活。结核分枝杆菌细胞被膜被大量甘露糖基化， 丰富的脂聚糖脂阿拉伯甘露聚糖（ManLAM）、脂甘露聚糖（LM）和磷脂酰肌醇甘露糖苷（PIM）。这些 脂聚糖与巨噬细胞和树突细胞（DC）上表达的C型凝集素结合。甘露糖受体（MR）是一种C型 介导人巨噬细胞对结核分枝杆菌强毒株的吞噬作用的凝集素。DC-SIGN在DC上大量表达 并且也在交替活化的巨噬细胞如肺泡巨噬细胞上表达。ManLAM是MR的主要配体 DC签名我们假设结核分枝杆菌表面甘露糖基化的性质对结核分枝杆菌的抗结核能力有重大影响。 细菌与C型凝集素（MR和DC-SIGN）相互作用，从而调节巨噬细胞功能和宿主 应答胞质核苷酸甘露糖载体GPD-甘露糖是细胞内直接或间接的必需供体。 LAM、LM和PIM以及细胞壁甘露糖蛋白的生物合成。这项建议的重点是核心酶， 需要在分枝杆菌中产生GPD-甘露糖。编码这些酶的基因将在M中过表达或被敲除。 然后将在人巨噬细胞和巨噬细胞中研究结核分枝杆菌和表面甘露糖基化改变的突变细菌。 TB发病机制的小鼠气溶胶模型。一个这样的基因是结核分枝杆菌manB，我们发现它编码一个功能性的 磷酸甘露变位酶在M.磺胺甲恶唑导致LAM、LM和PIM产生显著增加 以及与巨噬细胞结合的细菌增加13倍。除了靶向基因的方法， 表面甘露糖基化改变的克隆的结核分枝杆菌文库。具体目标：目标1：克隆、过表达和敲除a 目的2：确定结核分枝杆菌突变体是否在GDP-甘露糖生物合成中发生改变， 目标3：确定参与甘露糖生物合成的基因如何影响结核分枝杆菌与MR的相互作用， DC-SIGN、人巨噬细胞中细菌的生物学和小鼠中结核分枝杆菌的致病性，目的4：筛选结核分枝杆菌转座子 图书馆和M。使用沙门氏菌甘露糖结合（1型）皮利和表面活性剂过表达结核分枝杆菌基因的耻垢病文库 蛋白D以鉴定表面甘露糖基化改变的突变体和克隆。 该提案的总体目标是联合收割机结合分子、生物化学和细胞生物学技术，以确定关键酶， 结核分枝杆菌中的GDP-甘露糖生物合成，并通过改变其表达水平，确定它们对1）结核分枝杆菌细胞壁的影响 甘露糖基化和2）人巨噬细胞和小鼠中细菌行为的生物学。从这些信息中获得的信息 这些研究将提高我们对结核病发病机制的认识，并有可能确定新的治疗靶点， 已经证明代谢对于分枝杆菌的存活是必需的。

Tuberculosis continues to cause tremendous morbidity and mortality throughout the world's population. Critical in establishment of a M. tuberculosis (M.tb) infection are entry and survival in the macrophage. The M.tb cell envelope is heavily mannosylated with the abundant lipoglycans lipoarabinomannan (ManLAM), lipomannan (LM), and phosphatidyl inositol mannosides (PIMs). These lipoglycans bind to C-type lectins expressed on macrophages and dendritic cells (DC). The mannose receptor (MR) is a C-type lectin that mediates phagocytosis of virulent strains of M.tb by human macrophages. DC-SIGN is expressed abundantly on DCs and is also expressed on alternatively activated macrophages such as alveolar macrophages. ManLAM is a major ligand for the MR and DC-SIGN. We hypothesize that the nature of surface mannosylation of M.tb has a major impact on the ability of the bacterium to interact with C-type lectins (the MR and DC-SIGN) and thereby modulate macrophage function and host responses. The cytosolic nucleotide mannose carrier GPD-mannose is an essential donor either directly or indirectly in the biosynthesis of LAM, LM, and PIMs as well as cell wall mannoproteins. This proposal focuses on the core enzymes that are required to produce GPD-mannose in mycobacteria. Genes encoding these enzymes will be over-expressed or knocked out inM. smegmatis and M.tb and mutant bacteria with altered surface mannosylation will then be studied in human macrophages and in the mouse aerosol model of TB pathogenesis. One such gene is M.tb manB which we have found encodes a functional phosphomannomutase. Overexpression of manB in M. smegmatis leads to a significant increase in LAM, LM, and PIMs produced as well as a 13-fold increase in bacterial association with macrophages. In addition to the targeted gene approach, we will screen M.tb libraries for clones that are altered in surface mannosylation. Specific aims: Aim 1: Clone, over-express and knock out a defined set of M.tb genes involved in GDP-mannose biosynthesis, Aim 2: Determine whether mycobacteria mutants are altered in surface mannosylation, Aim 3: Determine how genes involved in mannose biosynthesis impact M.tb interactions with the MR and DC-SIGN, the biology of bacteria in human macrophages, and M.tb pathogenesis in the mouse, Aim 4: Screen a M.tb transposon library and a M. smegmatis library over-expressing M.tb genes using Salmonella mannose-binding (type 1)pili and surfactant protein D to identify mutants and clones altered in surface mannosylation. The overall goal of this proposal is to combine molecular, biochemical, and cell biology techniques to identify key enzymes for GDP-mannose biosynthesis in M.tb and, by altering then- level of expression, determine their impact on 1)M.tb cell wall mannosylation and 2) the biology of bacterial behavior in human macrophages and the mouse. Information gained from these studies should enhance our knowledge of TB pathogenesis and also potentially identify new therapeutic targets since mannose metabolism has been shown to be essential for the survival of mycobacteria.