Pyrazinoic Acid Inhibits the Bifunctional Enzyme (Rv2783) in Mycobacterium tuberculosis by Competing with tmRNA

Abstract

Pyrazinamide (PZA) is a key drug for tuberculosis treatment. The active form of PZA, pyrazinoic acid (POA), appears to inhibit multiple targets in M. tuberculosis. Recently, the bifunctional enzyme Rv2783 was reported as a new target of POA. However, the mechanism by which POA inhibits Rv2783 is not yet clear. Here, we report how a new A2104C substitution in Rv2783c, identified in PZA-resistant clinical isolates, conferred resistance to PZA in M. tuberculosis. Expression of the mutant allele recapitulated the PZA resistance. All catalytic activities of Rv2783, but not the mutant, were inhibited by POA. Additionally, POA competed with transfer-messenger RNA (tmRNA) for binding to Rv2783, other than the mutant. These results provide new insight into the molecular mechanism of the antitubercular activity of PZA.

Keywords: PNPase; Rv2783c gene; mycobacterium tuberculosis; pyrazinamide resistance; tmRNA.

Figure 1

Rv2783c mRNA levels in the expression strains. Wild-type Rv2783c and Rv2783c A2104C mutants were expressed in the M. tuberculosis H37Ra strain, using the plasmid pOLYG. The fold increase is expressed as mRNA levels after normalizing to rrs mRNA values and then comparing to the parental M. tuberculosis control. Abbreviations are as follows: “E” = pOLYG, “WT” = pOLYG+Rv2783c, “Mutant” = pOLYG+Rv2783c_A2104C.

Figure 2

Effect of pyrazinoic acid (POA) on the RNA degradative ability of Rv2783. Rv2783 plays a major role in the degradation of RNA from the 3′- to 5′-ends in M. tuberculosis. (a) Coupled ADP-Glo™ assay protocol (adapted from Promega Corporation). The ADP-Glo™ Kinase Assay is a luminescent ADP detection assay that can measure the kinase activity of polyribonucleotide nucleotidyltransferase (PNPase) by quantifying the amount of ADP produced during a kinase reaction; (b) PNPase ability of Rv2783 is Rv2783 concentration-dependent; (c) PNPase ability of Rv2783_K702Q is also protein concentration-dependent; (d) inhibition of POA on the PNPase activity of WT Rv2783 is POA concentration-dependent; (e) no significant effect of POA on the PNPase activity of Rv2783_K702Q. RLU = relative light units.

Figure 3

Rv2783 domain analysis and alignment with RpsA domains. (a) Rv2783 domain analysis shows the S1_PNPase domain (in the red circle) at its C-terminus, originally identified in ribosomal protein S1 (Rv1630) for RNA binding; (b) alignment of Rv2783 with four Rv1630 domains. Rv1630-1 to Rv1630-4 represent the four homologous RNA-binding domains (R1–R4) in RpsA. Both the Rv2783 S1_domain and the Rv1630-4 domain carry the KXXXFXXF motif (in the left red rectangle), which has been reported to comprise the main residues for POA and transfer-messenger RNA (tmRNA) binding. The mutant site of K702Q in Rv2783 is also located in the S1_domain (in the right red rectangle).

Figure 4

Effect of POA on the tmRNA binding ability of Rv2783. (a) Concentration-dependent inhibition of tmRNA binding to WT M. tuberculosis (Mtb) Rv2783 by POA (Lanes 2–7). tmRNA from Mtb was used as an RNA alone control (Lane 1); (b) no effect of tmRNA binding to Mtb Rv2783_K702Q by POA (Lanes 8–11). tmRNA had impaired binding to Rv2783_K702Q (Lane 8), and POA at different concentrations did not inhibit the interaction between Rv2783_K702Q and tmRNA (Lanes 9–11).