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.

FIG 1

(A) STRING analysis reveals the top 10 interaction partners, both known and putative, of Mycobacterium tuberculosis H₃₇Rv RipA (inv1) protein. (B) The score for each interaction partner is assigned and given in the table. The highest score for RipA association was found to be 0.847 for inv2/RipB. The confidence score for association of RipA and MoxR1 corresponds to 0.514, and that for Mce2B was found to be 0.547.

FIG 2

RipA interacts with MoxR1 and Mce2B, as is evident from the bimolecular florescence complementation assay. (A) Plasmids expressing the RipA and MoxR1 proteins in transfected HEK293T cells are shown. Fluorescence image for BiFC interaction was observed by imaging fixed cells (upper left panel) using confocal microscopy. The bright-field image for the same view is shown in the upper second panel. The overlay of the fluorescence and bright-field images is shown in the upper right panel. The plasmid expressing the negative control resulted in no fluorescence (middle left panel). Strong fluorescence intensity was observed for the positive control (lower left panel), and a merged image for fluorescence and the bright-field positive control is shown in the lower right panel (60× magnifications). (B) Fluorescence was also detected for interaction of RipA with Mce2B. The HEK293T cells transfected with plasmids that express both RipA and Mce2B exhibit fluorescence (upper left panel). The bright-field image of same view is shown in the upper second panel. The overlay of the fluorescence and bright-field images is shown in the upper right panel. In the negative control, no detectable fluorescence could be seen (middle left panel). The bright-field and merged images for the same view are also shown (middle second and right panels, respectively). The highest fluorescence intensity was detected for the positive control (lower left panel).

FIG 3

RipA protein interacts with MoxR1 in M. smegmatis. The vector alone was used as a negative control (lane 1). FLAG-tagged RipA binds in the presence of either MoxR1 protein purified from E. coli (lane 2) or MoxR1 expressed in both E. coli as well as M. smegmatis (lane 3). Note the absence of signal for MoxR1 alone (lane 4). Lane M, molecular mass marker lane.

FIG 4

Functional domain analysis of M. tuberculosis MoxR1 protein. The InterProScan analysis result shows the presence of the nucleoside triphosphate hydrolase, AAA type, and ATPase chaperone domain in MoxR1 protein.

FIG 5

(A) MoxR1 prevents thermal aggregation of MalZ protein. Shown are results from the light-scattering assay of thermal aggregation. Absorbance at 500 nm (OD₅₀₀) was measured for MoxR1 protein at 47°C for 15 min. (B) MoxR1 prevents thermal aggregation of MalZ protein in the presence of 1 mM ATP and Mg²⁺, as a cofactor. The kinetics of prevention of thermal aggregation of MalZ by MoxR1 at a similar concentration was enhanced in the presence of ATP. GroEL was used as a control.

FIG 6

Restriction enzyme activity of NdeI enzyme is protected from thermal denaturation in the presence of MoxR1 protein. SmaI-linearized pcDNA 3.1(+) plasmid is present at 5.4 kb (lane 1). Digestion with NdeI restriction enzyme cuts pcDNA 3.1(+) to generate two bands of 3.8 kb and 1.6 kb, respectively (lane 2). These bands are seen in the case of native NdeI (lane 2) and denatured NdeI plus MoxR1 proteins (lane 4).

FIG 7

ANS fluorescence spectrum for MoxR1 protein shows the presence of hydrophobic residue at the protein surface. Shown are a comparison of the fluorescence spectra of MoxR1 and buffer at 40 µM ANS. The concentration of the MoxR1 protein was 5 µM. Each experiment was carried out in triplicate.

FIG 8

(A) In vivo folding of RipA protein in the presence of MoxR1 chaperone activity. The different lanes are as follows: Lane M, marker; lanes 1 and 2, RipA pellet (lane 1) and folded RipA (supernatant) (lane 2) in the absence of any chaperone. RipA proteins in the pellet (lane 3) and supernatant fraction (lane 4) in the presence of GST are also shown. Insoluble RipA and folded RipA (supernatant) in the presence of MoxR1 can be seen in lanes 5 and 6, respectively. (B) Change in the levels of folded RipA in the presence of GST and MoxR1 at 37°C in vivo. The dark gray bars represent the RipA concentration in the pellet, and light gray represents the RipA concentration in the supernatant fraction.

FIG 9

(A) The N-terminal region of RipA protein consists of TAT signal peptide. (B) Schematic of RipA N-terminal signal peptide consensus amino acid. The conserved twin arginine is underlined, and the conserved 3-amino-acid LepB cleavage site starts at the 37th amino acid. The mature protein starting at the amino acid aspartate (D) is shown as +1. (C) MoxR1 protein aids in secretion of mature RipA through the TAT secretion pathway. The different lanes show the protein marker (lane 1), recombinant C-terminal His₆-RipA used as a positive control (lane 2), E. coli BL21 (DE3) cells expressing RipA protein in the soluble fraction used as an intracellular control (lane 5), and culture filtrate protein from transformed E. coli cells expressing RipA alone (lane 6), as well as both RipA and GST alone (lane 7), probed for mature RipA protein. Similarly, the culture filtrate proteins from cotransformed E. coli cells expressing both His₆-RipA and GST-MoxR1 were probed for mature RipA peptide and can be seen as an ~47.0-kDa band (lane 9).

FIG 10

Proposed model of MoxR action. The nascent polypeptide of RipA protein after release from the ribosome interacts with the MoxR1 protein. MoxR1 protein acts as a chaperone that helps RipA fold properly to protect the signal peptide containing RR sequence from proteasomal degradation and target the protein-chaperone complex to the TAT secretion system. The binding of ATP to the AAA+ ATPase domain and divalent Mg cations as a cofactor to the metal ion-dependent adhesion site of MoxR1 enhances its chaperone activity. The processed and secreted RipA might then associate with the mammalian cell entry protein and gain entry into host cells to exhibit its virulence.