title: Interaction of Mycobacterium tuberculosis Virulence Factor RipA with Chaperone MoxR1 Is Required for Transport through the TAT Secretion System.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, New Delhi, India.
authors.forename: Manish
authors.initials: M
authors.lastname: Bhuwan
authors.affiliation: Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, New Delhi, India.
authors.forename: Naresh
authors.initials: N
authors.lastname: Arora
authors.affiliation: Kusuma School of Biological Sciences, Indian Institute of Technology, New Delhi, India.
authors.forename: Ashish
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, New Delhi, India.
authors.forename: Mohd
authors.initials: M
authors.lastname: Khubaib
authors.affiliation: Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, New Delhi, India.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Pandey
authors.affiliation: Kusuma School of Biological Sciences, Indian Institute of Technology, New Delhi, India.
authors.forename: Tapan Kumar
authors.initials: TK
authors.lastname: Chaudhuri
authors.affiliation: Molecular Infection and Functional Biology Laboratory, Kusuma School of Biological Sciences, Indian Institute of Technology, New Delhi, India Dr. Reddy's Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, India seyedhasnain@gmail.com nzehtesham@gmail.com.
authors.forename: Seyed Ehtesham
authors.initials: SE
authors.lastname: Hasnain
authors.affiliation: Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, New Delhi, India seyedhasnain@gmail.com nzehtesham@gmail.com.
authors.forename: Nasreen Zafar
authors.initials: NZ
authors.lastname: Ehtesham
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000378:metabolism*; D025941:Protein Interaction Mapping; D000069294:Twin-Arginine-Translocation System / Q000378:metabolism*; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D000069294:Twin-Arginine-Translocation System; D037521:Virulence Factors