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.
Bibliographical noteFunding Information:
This work was supported by grants from the NIH (AI123146) and Bill & Melinda Gates Foundation (Grand Challenges Explorations), and institutional startup funds from the University of Minnesota to ADB. K.C.K. and H.A.F. were supported by grant GM068720 from NIH. N.A.D was supported by an institutional training grant from the NIH (HL007741). We thank Dr. William R. Jacobs, Jr. for providing M. tuberculosis strains H37Ra and H37Rv, Dr. Yusuke Minato for assistance in analyzing mutant strains of M. tuberculosis, Joshua Thiede for help with analyzing transcriptional data, and Chris Rae who helped provide reagents for in vitro trans-translation assays.