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。在建立的关键 结核分枝杆菌(M.tb)感染进入巨噬细胞并存活。结核分枝杆菌的细胞膜上有大量的甘露糖 富含脂肪糖脂阿拉伯甘露糖(ManLAM)、脂甘露聚糖(LM)和磷脂酰肌醇甘露糖苷(PIMs)。这些 脂多糖与巨噬细胞和树突状细胞(DC)上表达的C型凝集素结合。甘露糖受体(MR)为C型 介导人巨噬细胞吞噬结核分枝杆菌强毒株的凝集素。DC-SIGN在DC上大量表达 也表达在交替激活的巨噬细胞上，如肺泡巨噬细胞。ManLAM是MR的主要配体 和DC-Sign。我们推测结核分枝杆菌表面甘露糖化的性质对结核分枝杆菌的能力有重要影响 细菌与C型凝集素(MR和DC-SIGN)相互作用，从而调节巨噬细胞的功能和宿主 回应。胞浆核苷酸甘露糖载体GPD-甘露糖是体内直接或间接的必需供体。 LAM、LM、PIM以及细胞壁甘露糖蛋白的生物合成。这项建议重点关注的核心酶是 在分枝杆菌中生产GPD-甘露糖所需的。编码这些酶的基因将在INM过度表达或被敲除。 污垢杆菌、结核分枝杆菌和表面甘露糖化改变的突变细菌随后将在人的巨噬细胞和 结核病发病机制的小鼠气溶胶模型。其中一个基因是结核分枝杆菌manB，我们已经发现它编码一种功能性的 磷酸甘露糖变位酶。耻垢分枝杆菌中manB的过表达导致LAM、LM和PIM的产生显著增加 细菌与巨噬细胞的结合增加了13倍。除了靶向基因方法外，我们还将筛选 在表面甘露糖化过程中改变的克隆的M.tb文库。具体目标：目标1：克隆、过度表达和敲除 参与GDP-甘露糖生物合成的一组确定的M.tb基因，目标2：确定分枝杆菌突变是否在 表面甘露糖化作用，目标3：确定参与甘露糖生物合成的基因如何影响M.tb与MR和 DC-SIGN，细菌在人巨噬细胞中的生物学，以及结核分枝杆菌在小鼠中的发病机制，目标4：筛选结核分枝杆菌转座子 利用沙门氏菌甘露糖结合(1型)菌毛和表面活性物质过表达结核分枝杆菌基因的文库和耻垢分枝杆菌文库 蛋白D用于鉴定在表面甘露糖化过程中改变的突变体和克隆。 这项提案的总体目标是结合分子、生化和细胞生物学技术来确定关键的酶 在结核分枝杆菌中GDP-甘露糖的生物合成，并通过改变当时的表达水平，确定它们对结核分枝杆菌细胞壁的影响 甘露糖化作用和2)细菌在人巨噬细胞和小鼠体内的生物学行为。从这些信息中获得的信息 研究应该增强我们对结核病发病机制的了解，并可能确定新的治疗靶点，因为甘露糖 新陈代谢已被证明对分枝杆菌的生存至关重要。

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.