The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo
The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo
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DOI：
10.1128/jb.01346-13
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发表时间：
2014-10-01
影响因子：
3.2
通讯作者：
Manganelli, Riccardo
中科院分区：
文献类型：
--
作者：
Boldrin, Francesca;Ventura, Marcello;Manganelli, Riccardo
结核分枝杆菌的细胞包膜含有特殊结构的聚糖和脂质，在结核的生物学和发病机制中起着重要作用。因此，细胞壁的化学结构和生物合成已被深入研究，以确定新的药物靶点。在这里，我们验证了磷脂酰肌醇甘露糖基转移酶PimA在体外和体内对结核分枝杆菌的功能至关重要。PimA通过将甘露糖基残基从GDP-Man转移到质膜细胞质侧的磷脂酰肌醇，启动磷脂酰肌醇甘露糖苷的生物合成。为了证明pimA在结核分枝杆菌中的本质，我们利用ter - pip关闭系统构建了一个pimA条件突变体，并发现pimA表达下调可在批量培养中产生杀菌作用。与PimA催化的生化反应一致，这种表型与分枝杆菌细胞包膜的基本结构成分磷脂酰肌醇二甘醇苷水平显著降低有关。此外，在巨噬细胞感染和两种不同的感染小鼠模型中，PimA对生存能力的要求被清楚地证明，在沉默该基因后，观察到存活数量急剧下降。值得注意的是，在感染的急性和慢性阶段，PimA的消耗导致小鼠肺部完全清除。总之，实验数据强调了磷脂酰肌醇甘露糖苷生物合成途径对结核分枝杆菌的重要性，并证实PimA是未来药物发现计划的新靶点。
The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.