Kaur Devinder, Obregón-Henao Andrés, Pham Ha, Chatterjee Delphi, Brennan Patrick J, Jackson Mary
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17973-7. doi: 10.1073/pnas.0807761105. Epub 2008 Nov 12.
Biosynthesis of phosphatidylinositol (PI)-containing lipoarabinomannan (LAM) and lipomannan (LM) of Mycobacterium spp. follows a conserved pathway involving multiple membrane-associated, substrate-specific mannosyltransferases (ManTs) responsible for the sequential addition of alpha-mannopyranosyl (Manp) units donated by decaprenyl-P-Manp on the periplasmic side of the plasma membrane. Because of their receptor-binding and immunomodulatory properties, the alpha(1-->2)-linked di- and tri-Manp motifs that functionalize the nonreducing arabinan termini of LAM (ManLAM) in Mycobacterium tuberculosis are of crucial importance. We now show that the M. tuberculosis ManT, Rv2181, is required for the addition of these alpha(1-->2)-linked Manp residues but also at other locations of the LAM molecule. Structural analyses of the LM and LAM variants produced by a M. tuberculosis Rv2181 knockout mutant revealed the presence of but a single Manp residue on the nonreducing arabinan termini of LAM and also a complete absence of alpha(1-->2)-linked Man branching on the mannan backbones of LM and LAM. A recombinant strain was constructed in ManLAM-deficient Mycobacterium smegmatis that coexpressed Rv2181 and Rv1635c-the ManT responsible for the addition of the first Manp capping residue of ManLAM. Analysis revealed LAM termini fully capped with di- and tri-Manp motifs in addition to alpha(1-->2)Man branching on the mannan backbones of LM and LAM, confirming the involvement of the alpha(1-->2)ManT Rv2181 in the dual role of Man capping and mannan-core branching, and in the process generated a rapidly growing, ManLAM-containing strain, a tool for the study of the role of ManLAM in the pathogenesis of tuberculosis.
分枝杆菌属含磷脂酰肌醇（PI）的脂阿拉伯甘露聚糖（LAM）和脂甘露聚糖（LM）的生物合成遵循一条保守途径，该途径涉及多种与膜相关的、底物特异性甘露糖基转移酶（ManTs），这些酶负责在质膜周质侧依次添加由癸异戊二烯基-P-甘露糖（Manp）提供的α-甘露吡喃糖基（Manp）单元。由于其受体结合和免疫调节特性，结核分枝杆菌中使LAM（ManLAM）的非还原阿拉伯聚糖末端功能化的α（1→2）连接的二甘露糖基和三甘露糖基基序至关重要。我们现在表明，结核分枝杆菌的ManT，Rv2181，是添加这些α（1→2）连接的Manp残基所必需的，但在LAM分子的其他位置也是必需的。对结核分枝杆菌Rv2181基因敲除突变体产生的LM和LAM变体的结构分析表明，LAM的非还原阿拉伯聚糖末端仅存在一个Manp残基，并且在LM和LAM的甘露聚糖主链上也完全不存在α（1→2）连接的Man分支。在缺乏ManLAM的耻垢分枝杆菌中构建了一个重组菌株，该菌株共表达Rv2181和Rv1635c——负责添加ManLAM的第一个Manp封端残基的ManT。分析显示，除了LM和LAM的甘露聚糖主链上的α（1→2）Man分支外，LAM末端还完全被二甘露糖基和三甘露糖基基序封端，证实了α（1→2）ManT Rv2181参与了Man封端和甘露聚糖核心分支的双重作用，并在此过程中产生了一种快速生长的、含ManLAM的菌株，这是一种研究ManLAM在结核病发病机制中作用的工具。
