Biosynthesis and transbilayer flipping of mycobacterial PIM glycolipids

描述（由申请人提供）：分枝杆菌属，例如M.结核和M.麻风病是人类主要疾病的病因。在这个R21“高风险/高影响”的应用程序中，我们提出了一个探索性的计划，以确定磷脂酰肌醇甘露糖苷（PIM）的生物合成的早期步骤。PIM是在分枝杆菌的发病机制中起关键作用的甘露糖脂家族，因为它们是关键细胞表面糖缀合物脂甘露聚糖（LM）和脂阿拉伯甘露聚糖（LAM）的组装中的生物合成前体。PIM生物合成发生在细胞质膜中，并且有相当多的证据表明它是通过将甘露糖残基顺序添加到PI上而发生的。我们假设PIM生物合成的前三个甘露糖基转移反应发生在膜的细胞质表面上，并使用GDP-甘露糖作为甘露糖基供体;这些反应由甘露糖基转移酶（ManTs）PimA、PimB和我们称为PimC* 的未定义的酶催化。我们进一步提出，随后的甘露糖基化反应使用聚戊烯醇-磷酸-甘露糖（PPM）作为甘露糖基供体，并发生在膜的周质面上。催化这些周质定向反应中的第一个的酶是未知的;我们将其称为PimD*。为了适应PIM生物合成中的拓扑分裂，我们提出由PimC* 产生的三甘露糖基化糖脂（AcPIM 3）必须翻转穿过膜以进一步甘露糖基化。由于非催化的磷脂翻转是一种罕见的事件，我们提出，AcPIM 3翻转需要一个脂质转运蛋白或翻转酶（在本文中称为Flp*），一种蛋白质，仍有待确定。因此，围绕PIM组装路径中的拓扑分裂的三个步骤的关系仍有待定义。在这个“主要证据”项目中，我们提出了两个具体目标，以确定这些步骤所需的蛋白质组分。我们的研究将利用实验模型耻垢分枝杆菌结合多方面的方法，包括生物信息学，突变分析，和生化重建的运输活动。我们的目标是：1）鉴定PimC* 和PimD*，即催化紧接在PIM中间体的跨双层易位之前和之后发生的甘露糖基转移反应的ManT。2)开发重现Flp* 催化的PIM翻转的重构系统，并使用该系统鉴定负责翻转AcPIM 3穿过膜的翻转酶Flp*。因此，该提案的统一主题包括填补PIM生物发生中的“缺失步骤”，建立我们假设的途径，所有这些都与识别独特的细胞外靶标有关。公共卫生相关性：在此，提出了两个探索性的具体目标，以解决分枝杆菌核心糖脂组装中的重要缺失缺口。

DESCRIPTION (provided by applicant): Mycobacteria species, exemplified by M. tuberculosis and M. leprae are the cause of major diseases in humans. In this R21 `high risk/high impact' application we propose an exploratory program to define early steps in the biosynthesis of phosphatidylinositol mannosides (PIMs). PIMs are a family of mannolipids that plays a critical role in the pathogenesis of mycobacteria, because they are biosynthetic precursors in the assembly of the key cell surface glycoconjugates lipomannan (LM) and lipoarabinomannan (LAM). PIM biosynthesis occurs in the cytoplasmic membrane and there is considerable evidence to suggest that it occurs by the sequential addition of mannose residues to PI. We hypothesize that the first three mannosyltransfer reactions of PIM biosynthesis occur on the cytoplasmic face of the membrane and use GDP-mannose as the mannosyl donor; these reactions are catalyzed by the mannosyltransferases (ManTs) PimA, PimB and an undefined enzyme that we term PimC*. We further propose that subsequent mannosylation reactions use polyprenol-phosphate-mannose (PPM) as the mannosyl donor and occur on the membrane's periplasmic face. The enzyme that catalyzes the first of these periplasmically oriented reactions is unknown; we refer to it as PimD*. To accommodate the topological split in PIM biosynthesis, we propose that the trimannosylated glycolipid (AcPIM3) produced by PimC* must flip across the membrane in order to be further mannosylated. Since non-catalyzed phospholipid flipping is a rare event, we propose that AcPIM3 flipping requires a lipid translocator or flippase (referred to herein as Flp*), a protein that remains to be identified. The nexus of three steps surrounding the topological split in the PIM assembly pathway thus remains to be defined. In this "proof of principal" project evolving from close interactions between the three principal investigators over the last year, we propose two specific aims to identify the protein components required for these steps. Our studies will exploit the experimental model Mycobacterium smegmatis in conjunction with a multi-faceted approach including bioinformatics, mutational analysis, and biochemical reconstitution of transport activity. Our aims are: 1) To identify PimC* and PimD*, the ManTs that catalyze the mannosyltransfer reactions that occur immediately prior to and after the transbilayer translocation of a PIM intermediate. 2) To develop a reconstitution system that recapitulates Flp*-catalyzed PIM flip- flop and to use this system to identify Flp*, the flippase responsible for flipping AcPIM3 across the membrane. Thus the unifying theme of this proposal encompasses filling in `missing steps' in the biogenesis of PIMs, establish OUR hypothesized pathway, all in relation to identifying unique, extracellular targets. PUBLIC HEALTH RELEVANCE: Herein, two exploratory specific aims are proposed to address the vital missing gaps in the assembly of core glycolipids in Mycobacterium.