Abstract
Pyrazinamide (PZA) is a first line anti-tubercular drug for which the mechanism of action remains unresolved. Recently, it was proposed that the active form of PZA, pyrazinoic acid (POA), disrupts the ribosome rescue process of trans-translation in Mycobacterium tuberculosis. This model suggested that POA binds within the carboxy-terminal domain of ribosomal protein S1 (RpsA) and inhibits trans-translation leading to accumulation of stalled ribosomes. Here, we demonstrate that M. tuberculosis RpsA interacts with single stranded RNA, but not with POA. Further, we show that an rpsA polymorphism previously identified in a PZA resistant strain does not confer PZA resistance when reconstructed in a laboratory strain. Finally, by utilizing an in vitro trans-translation assay with purified M. tuberculosis ribosomes we find that an interfering oligonucleotide can inhibit trans-translation, yet POA does not inhibit trans-translation. Based on these findings, we conclude that the action of PZA is entirely independent of RpsA and trans-translation in M. tuberculosis.
Highlights
Pyrazinamide (PZA) is a first line anti-tubercular drug that has enabled a reduction in tuberculosis (TB) treatment duration from 9 to 6 months and has played a critical role in lowering relapse rates[1, 2]
Since coenzyme A (CoA) is a critical cofactor in fatty acid synthesis, it is likely that disruption of CoA homeostasis explains the ability of PZA to interfere with this pathway
We further examined the role of RpsA in PZA susceptibility of M. tuberculosis and biochemically evaluated the interaction of pyrazinoic acid (POA) with RpsA and the trans-translation complex
Summary
Using isogenic M. tuberculosis laboratory strains we assessed the impact of the rpsA∆A438 polymorphism on PZA susceptibility. These data demonstrate that the purified RpsA is properly folded and has the expected biochemical properties Despite this robust interaction with polyC RNA and in contrast to that which was described previously[25], no signal was observed when POA was titrated with RpsA (Fig. 2B). These data demonstrate that M. tuberculosis RpsA and POA do not show detectable interaction. We find that when a saturated POA solution is titrated into 10 mM phosphate buffer, a robust signal due to proton dissociation is observed and is entirely independent of the presence of RpsA (Fig. 3A). Considering the unparalleled in vivo sterilizing activity of PZA and the increasing global burden of multidrug-resistant and extensively drug-resistant tuberculosis, elucidating the requirements for susceptibility of M. tuberculosis to key drugs such as PZA is of paramount importance for the optimization of impactful treatment regimens
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