Biogenesis of lipoarabinomannan in mycobacteria

摘要：磷脂酰肌醇甘露聚糖（PIM）及其多糖基化对应物，脂甘露聚糖（LM）和脂阿拉伯糖甘露聚糖（LAM）是在所有分枝杆菌种类的包膜中发现的复合糖脂和脂聚糖。它们在分枝杆菌生理学中发挥着各种重要的作用，尽管定义尚不明确，是结核病和麻风病过程中重要的免疫调节分子，也是促进分枝杆菌进入吞噬细胞和非吞噬细胞的关键配体。尽管在过去的20年里，在阐明这些分子的结构方面取得了很大的进展，但关于它们的生物合成、组装和运输到细胞表面的途径的知识仍然不完整。对这些途径的阐明，除了提供有关结核分枝杆菌生物化学的基本知识外，预计还将导致发现必要的酶和转运体，这些酶和转运体可能代表新的抗结核药物的有趣靶点。积累这些分子的生物合成前体的重组分枝杆菌菌株的可用性将促进结构-功能关系的研究，而在PIM， LM和LAM合成的各个方面缺乏的明确的结核分枝杆菌突变体将允许精确评估这些分子对体内结核病免疫发病机制的贡献。采用生物信息学、遗传学和生物化学的综合方法，我们将继续研究与PIM、LM和LAM的伸长和组装有关的酶的鉴定和功能表征，并进一步扩展这项工作，以表征负责生物合成中间体和最终产物跨细胞包膜不同层易位的转运蛋白。缩写：AM，阿拉伯甘露聚糖；AcylT酰基转移酶;Araf arabinofuranosyl;AraT arabinosyltransferase;CZE：毛细管区带电泳；医生,脱氧胆酸盐;ESI，电喷雾电离；GT,糖基转移酶;LAM lipoarabinomannan;LM lipomannan;MALDI-TOF，矩阵辅助激光解吸/电离飞行时间Manp mannopyranosyl;ManT mannosyltransferase;甘露糖化磷脂酰肌醇；质谱法；myo-Ins,肌醇;ORF：开放阅读框；调查员,phosphatidyl-myo-inositol;PIM，磷脂酰肌醇甘露糖；TFA，三氟乙酸；薄层色谱法。术语：PIM用于描述全球磷脂酰肌醇甘露糖苷家族，该家族含有1至4种脂肪酸（与甘油、肌醇和/或甘露糖相连）和1至6种甘露糖残基。在AcXPIMY中，x为甘露糖或肌醇残基上酯化成可用羟基的酰基数，y为甘露糖残基数；例如，Ac1PIM1对应于磷脂酰肌醇单甘露糖苷PIM1，携带两个酰基连接到甘油（二酰基甘油取代基），一个酰基酯化到甘露糖残基。

DESCRIPTION (provided by applicant): Biogenesis of lipoarabinomannan in mycobacteria Summary Phosphatidylinositol mannosides (PIM) and their multiglycosylated counterparts, lipomannan (LM) and lipoarabinomannan (LAM) are complex glycolipids and lipoglycans found in the envelopes of all mycobacterial species. They play various essential although poorly defined roles in mycobacterial physiology and are important immunomodulatory molecules in the course of tuberculosis and leprosy as well as key ligands promoting the entry of mycobacteria into phagocytic and non-phagocytic cells. Although much progress has been made over the last 20 years in elucidating the structures of these molecules, knowledge of the pathways leading to their biosynthesis, assembling and transport to the cell surface is still incomplete. The elucidation of these pathways, in addition to providing fundamental knowledge about the biochemistry of Mycobacterium tuberculosis, is expected to lead to the discovery of essential enzymes and transporters that could represent interesting targets for novel anti-TB drugs. The availability of recombinant mycobacterial strains accumulating biosynthetic precursors of these molecules would facilitate structure-function relationship studies and that of defined M. tuberculosis mutants deficient in various aspects of PIM, LM and LAM synthesis would allow a precise assessment of the contribution of these molecules to the immunopathogenesis of tuberculosis in vivo. Following an integrated approach in the form of bioinformatics, genetics and biochemistry, we propose to pursue our work on the identification and functional characterization of the enzymes involved in the elongation and assembling of PIM, LM and LAM and to further extend this work to the characterization of the transporters responsible for the translocation of biosynthetic intermediates and end products across the different layers of the cell envelope. Abbreviations: AM, arabinomannan; AcylT, acyltransferase; Araf, arabinofuranosyl; AraT, arabinosyltransferase; CZE, capillary zone electrophoresis; DOC, deoxycholate; ESI, electrospray ionization; GT, glycosyltransferase; LAM, lipoarabinomannan; LM, lipomannan; MALDI-TOF, Matrix-Assisted Laser desorption/ionization time of flight; Manp, mannopyranosyl; ManT, mannosyltransferase; MPI, mannosylated phosphatidylinositol; MS, mass spectrometry; myo-Ins, myo-inositol; ORF, open reading frame; investigator, phosphatidyl-myo-inositol; PIM, phosphatidyl-myo-inositol mannosides; TFA, trifluoroacetic acid; TLC, thin-layer chromatography. Nomenclature: PIM is used to describe the global family of phosphatidylinositol mannosides that carries one to four fatty acids (attached to the glycerol, inositol and/or mannose) and one to six mannose residues. In AcXPIMY, x refers to the number of acyl groups esterified to available hydroxyls on the mannose or myo-inositol residues, y refers to the number of mannose residues; e.g. Ac1PIM1 corresponds to the phosphatidylinositol mono-mannoside PIM1 carrying two acyl groups attached to the glycerol (the diacylglycerol substituent) and one acyl group esterified to the mannose residue.