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

Bi-Wei Wang,Babak Javid,Jun-Hao Zhu

doi:10.1038/s41598-020-65308-2

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.

Highlights

All wild-type bacterial populations exhibit antibiotic tolerance, bacterial mutants with higher or lower tolerant subpopulation sizes have been described
We have previously focused on tolerance in actively growing cells to the first-line anti-tuberculous antibiotic rifampicin, which inhibits RNA polymerase (RNAP), and which we termed rifampicin-specific phenotypic resistance (RSPR)
We identify that deletion of the putative translation elongation factor LepA mediates RSPR via perturbation of the physiological transcriptional response of rpoB, and show that mutations in lepA identified in clinical Mycobacterium tuberculosis (Mtb) isolates phenocopy lepA deletion in mediating rifampicin tolerance

Summary

Introduction

All wild-type bacterial populations exhibit antibiotic tolerance, bacterial mutants with higher or lower tolerant subpopulation sizes have been described. We observed that mycobacteria can survive, but actively grow in bulk-lethal concentrations of rifampicin Both specific translational errors involving the indirect tRNA aminoacylation pathway, as well as a paradoxical upregulation of rpoB in response to rifampicin mediated RSPR7,9,10. Both mechanisms of RSPR were confirmed in clinical isolates of Mycobacterium tuberculosis (Mtb), corroborating the potential clinical relevance of this form of antibiotic tolerance[7,9]. We identify that deletion of the putative translation elongation factor LepA mediates RSPR via perturbation of the physiological transcriptional response of rpoB, and show that mutations in lepA identified in clinical Mtb isolates phenocopy lepA deletion in mediating rifampicin tolerance Given these www.nature.com/scientificreports mutations are in conserved sites between M. smegmatis and M. tuberculosis, it is likely their phenotypes will be conserved

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