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.

Project description:Wild birds including raptors can act as vectors of clinically relevant bacteria with antibiotic resistance. The aim of this study was to investigate the occurrence of antibiotic-resistant Escherichia coli in black kites (Milvus migrans) inhabiting localities in proximity to human-influenced environments in southwestern Siberia and investigate their virulence and plasmid contents. A total of 51 E. coli isolates mostly with multidrug resistance (MDR) profiles were obtained from cloacal swabs of 35 (64%, n = 55) kites. Genomic analyses of 36 whole genome sequenced E. coli isolates showed: (i) high prevalence and diversity of their antibiotic resistance genes (ARGs) and common association with ESBL/AmpC production (27/36, 75%), (ii) carriage of mcr-1 for colistin resistance on IncI2 plasmids in kites residing in proximity of two large cities, (iii) frequent association with class one integrase (IntI1, 22/36, 61%), and (iv) presence of sequence types (STs) linked to avian-pathogenic (APEC) and extra-intestinal pathogenic E. coli (ExPEC). Notably, numerous isolates had significant virulence content. One E. coli with APEC-associated ST354 carried qnrE1 encoding fluoroquinolone resistance on IncHI2-ST3 plasmid, the first detection of such a gene in E. coli from wildlife. Our results implicate black kites in southwestern Siberia as reservoirs for antibiotic-resistant E. coli. It also highlights the existing link between proximity of wildlife to human activities and their carriage of MDR bacteria including pathogenic STs with significant and clinically relevant antibiotic resistance determinants. IMPORTANCE Migratory birds have the potential to acquire and disperse clinically relevant antibiotic-resistant bacteria (ARB) and their associated antibiotic resistance genes (ARGs) through vast geographical regions. The opportunistic feeding behavior associated with some raptors including black kites and the growing anthropogenic influence on their natural habitats increase the transmission risk of multidrug resistance (MDR) and pathogenic bacteria from human and agricultural sources into the environment and wildlife. Thus, monitoring studies investigating antibiotic resistance in raptors may provide essential data that facilitate understanding the fate and evolution of ARB and ARGs in the environment and possible health risks for humans and animals associated with the acquisition of these resistance determinants by wildlife.

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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