Dataset Information

Clinically relevant mutations in mycobacterial LepA cause rifampicin-specific phenotypic resistance.

ABSTRACT: Although all wild-type bacterial populations exhibit antibiotic tolerance, bacterial mutants with higher or lower tolerant subpopulation sizes have been described. We recently showed that in mycobacteria, phenotypically-resistant subpopulations can grow in bulk-lethal concentrations of rifampicin, a first-line anti-tuberculous antibiotic targeting RNA polymerase. Phenotypic resistance was partly mediated by paradoxical upregulation of RNA polymerase in response to rifampicin. However, naturally occurring mutations that increase tolerance via this mechanism had not been previously described. Here, we used transposon insertional mutagenesis and deep sequencing (Tnseq) to investigate rifampicin-specific phenotypic resistance using two different in vitro models of rifampicin tolerance in Mycobacterium smegmatis. We identify multiple genetic factors that mediate susceptibility to rifampicin. Disruption of one gene, lepA, a translation-associated elongation factor, increased rifampicin tolerance in all experimental conditions. Deletion of lepA increased the subpopulation size that is able to grow in bulk-lethal rifampicin concentrations via upregulation of basal rpoB expression. Moreover, homologous mutations in lepA that are found in clinical Mycobacterium tuberculosis (Mtb) isolates phenocopy lepA deletion to varying degrees. Our study identifies multiple genetic factors associated with rifampicin tolerance in mycobacteria, and may allow correlation of genetic diversity of clinical Mtb isolates with clinically important phenotypes such as treatment regimen duration.

SUBMITTER: Wang BW

PROVIDER: S-EPMC7242378 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Clinically relevant mutations in mycobacterial LepA cause rifampicin-specific phenotypic resistance.

Wang Bi-Wei BW Zhu Jun-Hao JH Javid Babak B

Scientific reports 20200521 1

Although all wild-type bacterial populations exhibit antibiotic tolerance, bacterial mutants with higher or lower tolerant subpopulation sizes have been described. We recently showed that in mycobacteria, phenotypically-resistant subpopulations can grow in bulk-lethal concentrations of rifampicin, a first-line anti-tuberculous antibiotic targeting RNA polymerase. Phenotypic resistance was partly mediated by paradoxical upregulation of RNA polymerase in response to rifampicin. However, naturally ...[more]

PMID: 32439911

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Project description:Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampin tolerance. However, the mechanisms by which these clinically derived mutations in gatA impact GatCAB function were unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity. IMPORTANCE Most bacteria use a two-step indirect pathway to aminoacylate tRNAGln and tRNAAsn, despite the fact that the indirect pathway consumes more energy and is error prone. We have previously shown that the higher protein synthesis errors from this indirect pathway in mycobacteria allow adaptation to hostile environments such as antibiotic treatment through generation of novel alternate proteins not coded by the genome. However, the precise mechanisms of how translational fidelity is tuned were not known. Here, we biochemically and biophysically characterize the critical enzyme of the Mycobacterium tuberculosis indirect pathway, GatCAB, as well as two mutant enzymes previously identified from clinical isolates that were associated with increased mistranslation. We show that the mutants dysregulate the pathway via destabilizing the enzyme complex. Importantly, increasing stability improves translational fidelity in both wild-type and mutant bacteria, demonstrating a mechanism by which mycobacteria may tune mistranslation rates.

Dataset Information

Clinically relevant mutations in mycobacterial LepA cause rifampicin-specific phenotypic resistance.

Publications

Clinically relevant mutations in mycobacterial LepA cause rifampicin-specific phenotypic resistance.

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