Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA) is a first-line tuberculosis drug that plays a unique role in shortening the duration of tuberculosis chemotherapy. PZA is hydrolyzed intracellularly to pyrazinoic acid (POA) by pyrazinamidase (PZase, encoded by pncA), an enzyme frequently lost in PZA-resistant strains, but the target of POA in Mycobacterium tuberculosis has remained elusive. Here, we identify a previously unknown target of POA as the ribosomal protein S1 (RpsA), a vital protein involved in protein translation and the ribosome-sparing process of trans-translation. Three PZA-resistant clinical isolates without pncA mutation harbored RpsA mutations. RpsA overexpression conferred increased PZA resistance, and we confirmed that POA bound to RpsA (but not a clinically identified ΔAla mutant) and subsequently inhibited trans-translation rather than canonical translation. Trans-translation is essential for freeing scarce ribosomes in nonreplicating organisms, and its inhibition may explain the ability of PZA to eradicate persisting organisms.

Fig. 1

Structures of POA derivative (5-hydroxyl-2-pyrazinecarboxylic acid) (A) and the control compound ethanolamine (B) coupled to Sepharose 6B column for the identification of POA binding proteins from M. tuberculosis.

Fig. 2

RpsA alignment and isothermal titration calorimetry (ITC) titration of RpsA and POA. (A) Alignment of RpsA from M. tuberculosis H37Rv, M. tuberculosis PZA-resistant strain DHM444 and M. smegmatis. R1 to R4 represent the four homologous RNA-binding domains in RpsA. Colored vertical lines in gray boxes indicate sequence variations in the highly conserved RpsA sequences compared with the wild type M. tuberculosis sequence. The expanded region shows the variability in amino acid sequence in the C-terminus of RpsA among mycobacterial species. The red arrow at position 438 amino acid residue indicates the deletion of alanine in the C-terminal region of the mutant RpsA. ITC binding studies indicate POA bound to the M. tuberculosis H37Rv RpsA (WT) (B, inset VI), but not DHM444 RpsA (Mutant)(Inset, IV), and only weakly with the M. smegmatis RpsA (M. smeg) (Inset II). PZA did not bind to wild type RpsA (Inset V) or mutant RpsA (Inset III). The lower panel of the Fig. 2B shows the typical molar ratio saturation plot of POA with wild type Mtb RpsA.

Fig. 3

(A) Concentration-dependent inhibition of tmRNA binding to wild type M. tuberculosis RpsA by POA (Lanes 2–7). tmRNA from M. tuberculosis was used as RNA alone control (Lane 1). The wild type RpsA interaction with tmRNA was not affected by PZA (200 μg/ml) (Lane 8) or INH (1 μg/ml) (Lane 9). (B) tmRNA had impaired binding to the mutant RpsA (Lane 2), and POA at different concentrations did not inhibit the interaction of the DHM444 mutant RpsA with tmRNA (Lanes 3–7); The mutant RpsA interaction with tmRNA was not affected by PZA (200 μg/ml) (Lane 8) or INH (1 μg/ml) (Lane 9). (C) POA at 100, 50, and 25 μg/ml inhibited trans-translation of the DHFR product in a concentration-dependent manner in the in vitro system that contained ribosomes from M. tuberculosis, tmRNA and recombinant SmpB from M. tuberculosis, template pDHFR-8×AGG rare codons that are required for trans-translation (Lanes 1–5). Arrowheads indicate the trans-translation product DHFR was still present with low concentration of POA at 12.5 μg/ml (Lane 4) or in the absence of POA (Lane 5). POA at different concentrations did not inhibit canonical translation in in vitro translation system using ribosomes from M. tuberculosis, template pDHFR with stop codon (D, Lanes 6–10), nor the trans-translation of DHFR using ribosome from M. smegmatis (E, Lanes 1–4), or using ribosome from E. coli (F, Lanes 1–4) in the trans-translation system that contained tmRNA and recombinant SmpB from M. tuberculosis, template pDHFR-8×AGG rare codons.

Fig. 4

A new model for the mode of action of PZA. PZA is converted to the active form POA by M. tuberculosis PZase intracellularly and inhibits targets including RpsA. Upon stress, translating ribosomes are stalled and incomplete polypeptides may be toxic to the cell. The bacterial cell resolves this problem by adding tmRNA to the stalled mRNA (28, 36). tmRNA binds to SmpB and EF-Tu, activating the complex for ribosome interaction. The alanyl-tmRNA/SmpB/EF-Tu complex recognizes stalled ribosomes at the 3′ end of an mRNA without stop codon or with rare codons. Translation resumes using tmRNA as a message, resulting in addition of the tmRNA-encoded peptide tag to the C-terminus of the stalled polypeptide. The tagged protein and mRNA are then degraded by proteases and RNases, leading to rescue of stalled ribosomes. POA binding to RpsA interferes with the interaction of RpsA with tmRNA required for trans-translation. POA blockade of the trans-translation pathway leads to a defect in stalled ribosome rescue and depletion of available ribosomes and perhaps increased accumulation of toxic or deleterious proteins, ultimately affecting persister survival under stress conditions.