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Pyrazinamide Inhibits Trans-Translation in Mycobacterium tuberculosis

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Science  16 Sep 2011:
Vol. 333, Issue 6049, pp. 1630-1632
DOI: 10.1126/science.1208813

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.

Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug used in combination with isoniazid (INH), ethambutol, and rifampin for the treatment of drug-susceptible TB and frequently for multidrug-resistant TB (1). PZA shortens TB treatment from the 9 to 12 months required before its introduction to the current standard of 6 months, often referred to as short course chemotherapy (2). Despite PZA’s powerful in vivo sterilizing activity, it has no apparent activity for actively growing TB bacilli under normal culture conditions at neutral pH (3). It is instead preferentially active against nonreplicating persister bacteria with low metabolism at acid pH in vitro (4) or in vivo during active inflammation (5). Although several new drug candidates are currently in clinical development (6), they all have to be used together with PZA for optimal efficacy in the mouse model of TB infection (7, 8).

Despite its important role in shortening TB therapy, the mechanism of action of PZA is poorly understood (9). Structurally, PZA is an analog of nicotinamide, which, like INH (10), is a prodrug, requiring conversion into its active form pyrazinoic acid (POA) by the bacterial pyrazinamidase (PZase) (11). Mutation in the pncA gene encoding the PZase (11) is the major mechanism for PZA resistance in M. tuberculosis (1113). PZA is believed to enter M. tuberculosis by passive diffusion, where it is converted to POA by the PZase. POA is an acid with a pKa (where Ka is the acid dissociation constant) of 2.9 and is therefore trapped within the cell as the carboxylate anion, where it is excreted by a weak efflux pump and passive diffusion (14). Small amounts of protonated POA capable of diffusion across the membrane have been proposed to cause collapse of the proton gradient, reducing membrane potential and affecting membrane transport (15). The observations that energy inhibitors such as N,N′-dicyclohexylcarbodiimide (DCCD) (an F1F0 adenosine triphosphatase inhibitor) (15), as well as the drug candidate TMC207, synergize with PZA in vitro (7, 16) provide some support for this model. However, the molecular target of PZA is unknown. Although fatty acid synthase-I has been proposed as a target of PZA on the basis of studies with an analog (5-Cl-PZA) (17), a subsequent study negated this thesis (18).

Binding studies using a POA derivative, 5-hydroxyl-2-pyrazinecarboxylic acid, and ethanolamine as control extracted several proteins from M. tuberculosis cell lysates that bound to POA (Fig. 1). In contrast, no proteins bound to the control column, indicating that the proteins bound specifically to POA. Mass spectrometry analysis and subsequent database searches identified a major POA binding protein as RpsA (table S1), the largest 30S ribosomal protein S1 (Rv1630), along with three other proteins (Rv2731, Rv2783c, and Rv3169) from M. tuberculosis. Two of these proteins (Rv2731 and Rv3169) are conserved hypothetical proteins of unknown function, whereas Rv2783c is a bifunctional enzyme (polyribonucleotide nucleotidyltransferase and guanosine pentaphosphate synthetase), a component of the RNA degradosome involved in mRNA degradation. Because of the known biochemical functions of RpsA, we focused our attention first on understanding the implications of POA binding to this protein.

Fig. 1

M. tuberculosis whole-cell lysates were loaded onto the POA-linked and control columns, and the proteins that bound to POA (A) and the control column (B) were analyzed by SDS–polyacrylamide gel electrophoresis. Lane M, protein ladder; lane 1, whole-cell lysate; 2, flow-through fraction; 3, wash fraction; and 4, elution fraction. The bands indicated by arrowheads are Rv2783c, RpsA, Rv2731, and Rv3169, respectively.

Overexpression of the wild-type RpsA in M. tuberculosis caused a fivefold increase in the minimum inhibitory concentration (MIC) of PZA (MIC = 500 μg ml−1) compared with that of the vector control and the parental M. tuberculosis strain (MIC = 100 μg ml−1) at pH = 5.5. The susceptibility of the RpsA overexpressing M. tuberculosis strain to other drugs, including INH, rifampin, streptomycin, kanamycin, and norfloxacin, was not affected.

Most PZA-resistant M. tuberculosis strains have mutations in pncA that prevent conversion of PZA to POA (1113); however, a few PZA-resistant strains have been reported that do not have pncA mutations (12, 13). We previously identified a low-level PZA-resistant M. tuberculosis clinical isolate DHM444 (MIC = 200 to 300 μg ml−1 PZA compared with 100 μg ml−1 in the sensitive control strain H37Rv) that lacked pncA mutations (12), suggesting that its resistance may be caused by alterations in RpsA. Sequencing the rpsA gene from this strain revealed that it contained a 3–base pair GCC deletion at the nucleotide position 1314, resulting in loss of an alanine at amino acid 438 (ΔA438) in the C terminus of RpsA (Fig. 2), a region that is not considered to be strictly required for protein synthesis in vivo (19). Sequencing of an additional five PZA-resistant M. tuberculosis clinical isolates without pncA mutations identified two additional strains bearing RpsA mutations of T5→S5 (T5S) and D123A (20) in one strain and V262M in another strain.

Fig. 2

RpsA alignment and 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 RpsA sequence. The expanded region shows the variability in amino acid sequence (20) in the C terminus of RpsA among mycobacterial species. The red arrowhead at the position 438 amino acid residue indicates the deletion of alanine in the C-terminal region of the mutant RpsA. (B) ITC titration of POA binding to RpsA. ITC binding studies indicate that POA bound to the M. tuberculosis H37Rv RpsA [wild type (WT)] (line VI), but not DHM444 RpsA (Mutant) (line IV) and only weakly with the M. smegmatis RpsA (M. smeg) (line II). PZA did not bind to wild-type RpsA (line V) or mutant RpsA (line III). The top graph shows raw data, and the y axis indicates the heat released per second during RpsA and POA or PZA binding. The bottom graph shows integrated heat in each injection of POA together with a fit, and the y axis is expressed by heat release per mole in each injection. The association constants were obtained from fits of POA binding with M. tuberculosis RpsA (WT).

To determine whether the mutant RpsA from the PZA-resistant strain DHM444 has any defect in POA binding, we overexpressed and purified the mutant RpsA (RpsAΔA438), the wild-type M. tuberculosis RpsA, and the M. smegmatis RpsA and measured their ability to bind to POA by using isothermal titration calorimetry (ITC). The wild-type M. tuberculosis RpsA was found to specifically bind to POA (Fig. 2B, VI) with a Ka = 7.53 × 106 ± 2.21 × 106 M−1, enthalpy ΔH = –410.9 ± 8.693 kcal·mol−1, and entropy ΔS = 27.6 cal·mol−1·K−1 (Fig. 2B, bottom graph) but did not bind the prodrug PZA (Fig. 2B, V). However, the mutant RpsAΔA438 from the PZA-resistant strain DHM444 failed to bind either POA or PZA (Fig. 2B, IV and III), and the RpsA from naturally PZA-resistant M. smegmatis bound to POA only weakly (Fig. 2B, II). The mutant RpsAΔA438 did not bind to POA when subjected to the affinity column described above. Because M. tuberculosis DHM444 mutant RpsAΔA438 and M. smegmatis RpsA showed little or no binding to POA (Fig. 2B), it may be inferred that POA binds to the C terminus of the wild-type M. tuberculosis RpsA (Fig. 2A). From the protein sequence alignment of RpsA of different mycobacterial species, the C-terminal region, where the mutation occurs in the PZA-resistant M. tuberculosis strain DHM444, is also the region that varies most between PZA-sensitive and -resistant mycobacterial species (Fig. 2A), indicating that changes in this region may alter PZA susceptibility.

RpsA is essential for translation, binding directly both to the ribosome and upstream sequences of mRNA (21). In addition to its function in translation, the C terminus of the RpsA is also involved in trans-translation by specifically binding to transfer-messenger RNA (tmRNA) (2224). A Streptomyces homolog of one of the other proteins identified by the POA affinity column, Rv2783c, has been identified by affinity chromatography on a tmRNA column and has also been implicated in trans-translation (25). Trans-translation is a process involved in rescuing ribosomes that have stalled while in the process of decoding mRNA and involves displacement of the stalled message by tmRNA followed by translation of a short tag encoded by tmRNA targeting the stalled protein for degradation. Trans-translation has been associated with stress survival, virulence, and recovery from nutrient starvation (26). We therefore evaluated whether the mutant RpsA has any deficiency in tmRNA binding compared with the wild-type M. tuberculosis RpsA and whether POA affects the interaction between RpsA and tmRNA. Specific binding of RpsA to tmRNA was assessed by changes in gel mobility in the presence of excess tmRNA. The wild-type M. tuberculosis RpsA and the mutant RpsAΔA438 both bound to the tmRNA in the absence of POA (Fig. 3A, B), although the mutant appeared to bind more weakly overall (Fig. 3B). However, when POA was added to the system, it inhibited the wild-type M. tuberculosis RpsA from binding to tmRNA at its MIC concentration of 50 μg ml−1 (Fig. 3A) but did not inhibit binding of the mutant RpsAΔA438 (Fig. 3B). The prodrug PZA and the control drug INH had no effect on the binding of wild-type or mutant RpsA to the tmRNA (Fig. 3B).

Fig. 3

(A) Concentration-dependent inhibition of tmRNA binding to WT M. tuberculosis RpsA by POA (lanes 2 to 7). tmRNA from M. tuberculosis was used as the RNA-alone control (lane 1). The WT RpsA interaction with tmRNA was not affected by PZA (200 μg ml−1) (lane 8) or INH (1 μg ml−1) (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 to 7). The mutant RpsA interaction with tmRNA was not affected by PZA (200 μg ml−1) (lane 8) or INH (1 μg ml−1) (lane 9). (C) POA at 100, 50, and 25 μg ml−1 inhibited trans-translation of the DHFR product in a concentration-dependent manner in the in vitro system that contained ribosomes, tmRNA, and recombinant SmpB from M. tuberculosis and template pDHFR-8×AGG rare codons that are required for trans-translation (lanes 1 to 3). Arrowheads indicate the trans-translation product DHFR was still present with a low concentration of POA at 12.5 μg ml−1 (lane 4) and in the absence of POA (lane 5). POA at different concentrations did not inhibit canonical translation in the in vitro translation system using ribosomes from M. tuberculosis and template pDHFR with a stop codon (lanes 6 to 10). NA, an analog of POA as a control, did not inhibit trans-translation in the above system (lane 11). POA failed to inhibit the trans-translation of DHFR using ribosomes from M. smegmatis (D) or ribosomes from E. coli (E) in the trans-translation system that contained tmRNA and recombinant SmpB from M. tuberculosis and template pDHFR-8×AGG rare codons. (F) Concentration-dependent inhibition of trans-translation by POA with M. tuberculosis ribosomes and a DHFR template with a rare codon cluster. This bar graph is from a densitometry scan of the samples in the same type of experiment as in (C), lanes 1 to 5, performed independently five times (P < 0.03, n = 5). Error bars indicate SEM.

In Escherichia coli, RpsA is known to bind to the 3′ terminus of the mRNA-like portion of the tmRNA (22), and, because POA bound to M. tuberculosis RpsA (Fig. 2B), we tested whether POA inhibited the translation or the trans-translation function of RpsA. POA had no effect on conventional protein synthesis (Fig. 3D). We then tested the effects of POA on trans-translation by using the target gene coding for dihydrofolate reductase (DHFR) in an in vitro cell-free translation system containing ribosomes from M. tuberculosis, M. smegmatis, or E. coli. In the presence of a rare codon cluster in the target DHFR gene, ribosomes stall and translation is blocked (27). When recombinant M. tuberculosis SmpB and in vitro transcribed M. tuberculosis tmRNA were added, a higher molecular weight protein with the tmRNA-derived peptide tag was observed from message carrying the rare codon cluster (Fig. 3C, lane 5). POA inhibited the trans-translation of DHFR with the rare codon cluster at concentrations greater than 25 μg ml−1 (Fig. 3C, lanes 1 to 3). However, POA did not affect the translation of the wild-type DHFR gene even at 100 μg ml−1 with M. tuberculosis ribosomes (Fig. 3D), nor did it inhibit the trans-translation of the template bearing the rare codon cluster with either M. smegmatis or E. coli ribosomes (Fig. 3, E and F). These observations support that POA does not inhibit M. tuberculosis SmpB or tmRNA function but instead directly binds to RpsA to cause the inhibition of trans-translation. Under the same conditions, nicotinic acid (NA) (50 μg ml−1), an analog of POA as a control compound, did not inhibit trans-translation (Fig. 3C, lane 11).

The M. tuberculosis RpsA protein consists of four imperfect repeats of the S1-like domain thought to function directly in binding of RNA, bridging the mRNA (or tmRNA) template and the head of the 30S ribosome, and is terminated at the C terminus by a 117–amino acid segment. The E. coli RpsA protein contains six repeating S1 domains, with the two N-terminal domains required for ribosome binding. The C-terminal domains have been shown to be specifically involved in trans-translation; thus the deletion of Ala438 observed in one clinical isolate is consistent with POA exerting an effect on ribosome rescue. The deletion occurs within a region homologous (35% identical) to the protein Xrcc4 involved in illegitimate DNA recombination that forms an extensive α helix (28). Homology modeling of the M. tuberculosis C terminus based on the Xrcc4 structure (Protein Data Bank identification code 1FU1) within the highly homologous region suggests that Ala438 lies within several turns in an α helix connecting a small globular domain to a long helical stalk potentially involved in dimerization. This region has been proposed to be the primary site of nucleic acid interaction in Xrcc4. There are numerous basic residues along the helical face and particularly in the short linker between this helix and the globular domain potentially involved in DNA binding, especially Arg423, Arg424, His425, and Lys426, that may interact directly with POA and disrupt the site of tmRNA interaction.

Trans-translation is dispensable during active growth conditions but becomes important for bacteria in managing stalled ribosomes or damaged mRNA and proteins under stress conditions (29, 30). It is required for stress survival and pathogenesis in some bacteria (26). The levels of RpsA are known to correlate with growth rate and stress conditions that halt bacterial growth and down-regulate RpsA in bacteria (31). When POA binds to RpsA, it prevents binding of tmRNA, which therefore cannot rescue stalled ribosomes. PZA inhibition of the trans-translation process may therefore interfere with survival under stressful, nonreplicating conditions in M. tuberculosis. The finding that POA binds to RpsA and inhibits the trans-translation process helps to explain how diverse stress conditions, such as starvation, acid pH, hypoxia, and energy inhibitors and other drugs could all potentiate PZA activity (15, 32). On the basis of our current and previous studies, we propose a revised model for the mode of action of PZA that can better explain all the peculiar features of this intriguing drug (fig. S2). Trans-translation may provide a valuable pathway for developing new drugs to sterilize infection more rapidly.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1208813/DC1

Materials and Methods

Figs. S1 and S2

Table S1

References

References and Notes

  1. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; and V, Val.
  2. Acknowledgments: This work was supported in part by NIH grant AI44063, in part by the intramural research program of the NIAID, NIH, and by National Key Technologies Research and Development Program of China (2008ZX10003003). We thank D. E. Griffin and M. J. Klag for encouragement and support. Johns Hopkins University has filed a patent on detection of RpsA mutation as a marker for pyrazinamide resistance and use of trans-translation pathway as a target for development of new antibiotics against TB persister bacteria.
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