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. IMPORTANCE 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. 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.

[1]  I. Smith,et al.  Phospholipases C are involved in the virulence of Mycobacterium tuberculosis , 2002, Molecular microbiology.

[2]  M. Braunstein,et al.  The ins and outs of Mycobacterium tuberculosis protein export. , 2012, Tuberculosis.

[3]  K. Tanneeru,et al.  Human resistin, a proinflammatory cytokine, shows chaperone-like activity , 2013, Proceedings of the National Academy of Sciences.

[4]  P. D. de Boer,et al.  The Escherichia coli amidase AmiC is a periplasmic septal ring component exported via the twin‐arginine transport pathway , 2003, Molecular microbiology.

[5]  W. Wickner,et al.  Functional reconstitution of bacterial Tat translocation in vitro , 2001, The EMBO journal.

[6]  K. Cline,et al.  A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase , 2002, The Journal of cell biology.

[7]  M. Brennan,et al.  A rational vaccine pipeline for tuberculosis. , 2012, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[8]  E. Brown,et al.  A Mycobacterial Operon Essential for Virulence In Vivo and Invasion and Intracellular Persistence in Macrophages , 2006, Infection and Immunity.

[9]  Vinay Dahiya,et al.  Chaperones GroEL/GroES Accelerate the Refolding of a Multidomain Protein through Modulating On-pathway Intermediates , 2013, The Journal of Biological Chemistry.

[10]  G. Riccardi,et al.  Determination of a 15437 bp nucleotide sequence around the inhA gene of Mycobacterium avium and similarity analysis of the products of putative ORFs. , 1998, Microbiology.

[11]  S. Hasnain,et al.  Mechanistic Insights into a Novel Exporter-Importer System of Mycobacterium tuberculosis Unravel Its Role in Trafficking of Iron , 2008, PloS one.

[12]  W. Houry,et al.  Structure of RavA MoxR AAA+ protein reveals the design principles of a molecular cage modulating the inducible lysine decarboxylase activity , 2010, Proceedings of the National Academy of Sciences.

[13]  B. Berks.  A common export pathway for proteins binding complex redox cofactors? , 1996, Molecular microbiology.

[14]  R. Garrett,et al.  Chaperone Role for Proteins p618 and p892 in the Extracellular Tail Development of Acidianus Two-Tailed Virus , 2011, Journal of Virology.

[15]  S. Hasnain,et al.  Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenases show strong B cell response and distinguish vaccinated controls from TB patients. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  C. Santini,et al.  Translocation of Jellyfish Green Fluorescent Protein via the Tat System of Escherichia coli and Change of Its Periplasmic Localization in Response to Osmotic Up-shock* , 2001, The Journal of Biological Chemistry.

[17]  J. D. Reid,et al.  Current understanding of the function of magnesium chelatase. , 2001, Biochemical Society transactions.

[18]  Detlef D. Leipe,et al.  Evolutionary history and higher order classification of AAA+ ATPases. , 2004, Journal of structural biology.

[19]  O. Meyer,et al.  The CoxD Protein of Oligotropha carboxidovorans Is a Predicted AAA+ ATPase Chaperone Involved in the Biogenesis of the CO Dehydrogenase [CuSMoO2] Cluster , 2009, Journal of Biological Chemistry.

[20]  V. Mizrahi,et al.  Global expression profiling of strains harbouring null mutations reveals that the five rpf-like genes of Mycobacterium tuberculosis show functional redundancy. , 2004, Tuberculosis.

[21]  S. Cole,et al.  Inactivation of Rv2525c, a Substrate of the Twin Arginine Translocation (Tat) System of Mycobacterium tuberculosis, Increases β-Lactam Susceptibility and Virulence , 2006, Journal of bacteriology.

[22]  A. Tyagi,et al.  Gene cooption in mycobacteria and search for virulence attributes: comparative proteomic analyses of Mycobacterium tuberculosis, Mycobacterium indicus pranii and other mycobacteria. , 2014, International journal of medical microbiology : IJMM.

[23]  F. Berven,et al.  Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv , 2007, Proteomics.

[24]  S. Fortune,et al.  A partner for the resuscitation‐promoting factors of Mycobacterium tuberculosis , 2007, Molecular microbiology.

[25]  V. Nandicoori,et al.  Mycobacteria modulate host epigenetic machinery by Rv1988 methylation of a non-tail arginine of histone H3 , 2015, Nature Communications.

[26]  Seyed E. Hasnain,et al.  Comparative genomic and proteomic analyses of PE/PPE multigene family of Mycobacterium tuberculosis H37Rv and H37Ra reveal novel and interesting differences with implications in virulence , 2012, Nucleic acids research.

[27]  T. Brüser,et al.  Conservation and Variation between Rhodobacter capsulatus and Escherichia coli Tat Systems , 2006, Journal of bacteriology.

[28]  R. Daniel,et al.  Export of active green fluorescent protein to the periplasm by the twin‐arginine translocase (Tat) pathway in Escherichia coli , 2001, Molecular microbiology.

[29]  E. Rubin,et al.  A Mycobacterial Enzyme Essential for Cell Division Synergizes with Resuscitation-Promoting Factor , 2008, PLoS pathogens.

[30]  A. Emili,et al.  The MoxR ATPase RavA and Its Cofactor ViaA Interact with the NADH:Ubiquinone Oxidoreductase I in Escherichia coli , 2014, PloS one.

[31]  Sangwei Lu,et al.  Recombinant Mycobacterium tuberculosis protein associated with mammalian cell entry , 2001, Cellular microbiology.

[32]  B. Aldridge,et al.  Protein Complexes and Proteolytic Activation of the Cell Wall Hydrolase RipA Regulate Septal Resolution in Mycobacteria , 2013, PLoS pathogens.

[33]  Long-Fei Wu,et al.  Involvement of the twin‐arginine translocation system in protein secretion via the type II pathway , 2001, The EMBO journal.

[34]  Matthias Müller,et al.  Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli. , 2003, Molecular cell.