A tRNA-Acetylating Toxin and Detoxifying Enzyme in Mycobacterium tuberculosis

Abstract

The global rise in antibiotic-resistant tuberculosis has prompted an urgent search for new drugs. Toxin-antitoxin (TA) systems allow bacteria to adapt rapidly to environmental changes, and Mycobacterium tuberculosis encodes more TA systems than any known pathogen.

Peptidyl tRNA hydrolase is required for robust prolyl-tRNA turnover inMycobacterium tuberculosis

“…Both types of strains had growth defects that were proportional to the level of depletion (Figure 1), confirming that pth is required for normal growth of Mtb . Due to the technical ease of validating pth knockdown levels and our use of the tightly-regulated transcriptional repression strain in published work [22], we used the CRISPRi Pth depletion strain for subsequent experiments in this work.…”

“…We previously characterized a second function for Pth in Mtb . In addition to its peptidyl tRNA hydrolase activity, Pth is a detoxifying enzyme for a tRNA-acetylating toxin TacT that is part of a newly-identified toxin-antitoxin system [22]. We found that this function does not contribute to pth essentiality in Mtb since TacT was not active during normal laboratory growth conditions and a tacAt knockout strain was equally susceptible to pth depletion by CRISPRi [22].…”

“…In addition to its peptidyl tRNA hydrolase activity, Pth is a detoxifying enzyme for a tRNA-acetylating toxin TacT that is part of a newly-identified toxin-antitoxin system [22]. We found that this function does not contribute to pth essentiality in Mtb since TacT was not active during normal laboratory growth conditions and a tacAt knockout strain was equally susceptible to pth depletion by CRISPRi [22]. Thus, we focused our efforts on studying the effects of Pth depletion in Mtb on peptidyl tRNA pools and tRNA turnover following translation errors.…”

“…Pth-knockdown constructs: Transcriptional knockdown of pth was accomplished using mycobacterial CRISPRi-interference (CRISPRi) as described with the same strain used in Chapter Two [22]. Proteolytic degradation strains were built using the plasmids and primers listed in Table S3.…”

“…CuSO 4 -tRNA sequencing (Cu-tRNAseq) distinguishes N-acetylated aminoacyl tRNA from aminoacyl or uncharged tRNA. Cu-tRNAseq was carried out on a previously described M. smegmatis (Msmeg) strain with inducible Mtb TacT overexpression [22]. The fraction of peptidyl tRNA is plotted as a ratio of the number of 3'CCA-ending reads to the sum of 3'CCA-and 3'CC-ending reads for each tRNA species.…”