Bhuwan Manish, Arora Naresh, Sharma Ashish, Khubaib Mohd, Pandey Saurabh, Chaudhuri Tapan Kumar, Hasnain Seyed Ehtesham, Ehtesham Nasreen Zafar
Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, New Delhi, India.
Kusuma School of Biological Sciences, Indian Institute of Technology, New Delhi, India.
mBio. 2016 Mar 1;7(2):e02259. doi: 10.1128/mBio.02259-15.
Mycobacterium tuberculosis is a leading cause of death worldwide. The M. tuberculosis TAT (twin-arginine translocation) protein secretion system is present at the cytoplasmic membrane of mycobacteria and is known to transport folded proteins. The TAT secretion system is reported to be essential for many important bacterial processes that include cell wall biosynthesis. The M. tuberculosis secretion and invasion protein RipA has endopeptidase activity and interacts with one of the resuscitation antigens (RpfB) that are expressed during pathogen reactivation. MoxR1, a member of the ATPase family that is associated with various cellular activities, was predicted to interact with RipA based on in silico analyses. A bimolecular fluorescence complementation (BiFC) assay confirmed the interaction of these two proteins in HEK293T cells. The overexpression of RipA in Mycobacterium smegmatis and copurification with MoxR1 further validated their interaction in vivo. Recombinant MoxR1 protein, expressed in Escherichia coli, displays ATP-enhanced chaperone activity. Secretion of recombinant RipA (rRipA) protein into the E. coli culture filtrate was not observed in the absence of RipA-MoxR interaction. Inhibition of this export system in M. tuberculosis, including the key players, will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing β-lactam antibiotics, opening up new candidates for drug repurposing.
The virulence mechanism of mycobacteria is very complex. Broadly, the virulence factors can be classified as secretion factors, cell surface components, enzymes involved in cellular metabolism, and transcriptional regulators. The mycobacteria have evolved several mechanisms to secrete its proteins. Here, we have identified one of the virulence proteins of Mycobacterium tuberculosis, RipA, possessing peptidoglycan hydrolase activities secreted by the TAT secretion pathway. We also identified MoxR1 as a protein-protein interaction partner of RipA and demonstrated chaperone activity of this protein. We show that MoxR1-mediated folding is critical for the secretion of RipA within the TAT system. Inhibition of this export system in M. tuberculosis will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing β-lactam antibiotics, opening up new candidates for drug repurposing.
结核分枝杆菌是全球主要的死亡原因。结核分枝杆菌双精氨酸转运（TAT）蛋白分泌系统存在于分枝杆菌的细胞质膜上，已知可转运折叠蛋白。据报道，TAT分泌系统对于包括细胞壁生物合成在内的许多重要细菌过程至关重要。结核分枝杆菌分泌与侵袭蛋白RipA具有内肽酶活性，并与病原体重新激活期间表达的复苏抗原之一（RpfB）相互作用。基于计算机分析预测，与各种细胞活动相关的ATP酶家族成员MoxR1与RipA相互作用。双分子荧光互补（BiFC）分析证实了这两种蛋白在HEK293T细胞中的相互作用。RipA在耻垢分枝杆菌中的过表达以及与MoxR1的共纯化进一步验证了它们在体内的相互作用。在大肠杆菌中表达的重组MoxR1蛋白表现出ATP增强的伴侣活性。在不存在RipA-MoxR相互作用的情况下，未观察到重组RipA（rRipA）蛋白分泌到大肠杆菌培养滤液中。抑制结核分枝杆菌中的这种输出系统，包括关键成分，将阻止肽聚糖水解酶的定位，并导致对现有β-内酰胺抗生素敏感，从而为药物重新利用开辟新的候选药物。
分枝杆菌的毒力机制非常复杂。广义上，毒力因子可分为分泌因子、细胞表面成分、参与细胞代谢的酶和转录调节因子。分枝杆菌进化出了几种分泌其蛋白质的机制。在这里，我们鉴定出结核分枝杆菌的一种毒力蛋白RipA，它具有通过TAT分泌途径分泌的肽聚糖水解酶活性。我们还鉴定出MoxR1是RipA的蛋白质-蛋白质相互作用伙伴，并证明了该蛋白的伴侣活性。我们表明，MoxR1介导的折叠对于RipA在TAT系统中的分泌至关重要。抑制结核分枝杆菌中的这种输出系统将阻止肽聚糖水解酶的定位，并导致对现有β-内酰胺抗生素敏感，从而为药物重新利用开辟新的候选药物。
Zotero 插件
