Biosynthesis and transbilayer flipping of mycobacterial PIM glycolipids

描述（由申请人提供）：分枝杆菌种类，例如结核分枝杆菌和麻风分枝杆菌是导致人类主要疾病的原因。在这个R21“高风险/高影响”申请中，我们提出了一个探索性计划，以确定磷脂酰肌醇甘露聚糖（pim）生物合成的早期步骤。pim是一个甘露脂家族，在分枝杆菌的发病机制中起着关键作用，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）的生物合成前体。PIM的生物合成发生在细胞质膜中，有大量证据表明，它是通过向PI中连续添加甘露糖残基而发生的。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，并以gdp -甘露糖为甘露糖基供体；这些反应是由甘露糖基转移酶（ManTs）、PimA、PimB和一种我们称之为PimC*的未定义酶催化的。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，并发生在膜的质周表面。催化第一个质周导向反应的酶是未知的；我们称之为PimD*。为了适应PIM生物合成中的拓扑分裂，我们提出PimC*产生的三糖基化糖脂（AcPIM3）必须在膜上翻转才能进一步甘糖基化。由于非催化磷脂翻转是一种罕见的事件，我们提出AcPIM3翻转需要脂质转运体或翻转酶（此处称为Flp*），这是一种尚待鉴定的蛋白质。因此，围绕PIM组装路径中拓扑分裂的三个步骤的联系仍有待定义。在这个“主要证明”项目中，我们提出了两个具体的目标，以确定这些步骤所需的蛋白质成分。我们的研究将利用耻垢分枝杆菌的实验模型，结合多方面的方法，包括生物信息学、突变分析和运输活性的生化重建。我们的目标是：1)鉴定在PIM中间体跨层易位之前和之后催化甘露糖基转移反应的ManTs PimC*和PimD*。2)开发一个重组系统，再现Flp*催化的PIM触发器，并使用该系统识别Flp*，负责将AcPIM3翻转过膜的翻转酶。因此，该提案的统一主题包括填补PIMs生物发生中的“缺失步骤”，建立我们的假设途径，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：本文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重要的缺失缺口。

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.