Beyond Inhibition - Sub-lethal Rifampicin-induced molecular adaptations confer phenotypic drug tolerance in Mycobacteria

ABSTRACT: This study investigates how sub-lethal concentrations of rifampicin act as a signalling stimulus that induces phenotypic drug tolerance in mycobacteria. Using Mycobacterium smegmatis as a fast-growing model and validating findings in the attenuated Mycobacterium tuberculosis H37Ra strain, we examined growth dynamics and proteomic adaptations following exposure to half the minimum inhibitory concentration (MIC) of rifampicin. Growth assays demonstrated a transient growth deceleration followed by recovery, indicative of acquired phenotypic tolerance. Deep data-independent acquisition parallel accumulation–serial fragmentation (DIA-PASEF) proteomics was employed to capture early (45 min) and adaptive (180 min) molecular responses in M. smegmatis, with early validation in M. tuberculosis H37Ra. Proteomic analyses revealed rapid and dynamic remodelling of transcriptional, translational, metabolic, and stress-response pathways, including ribosomal perturbation, altered purine biosynthesis, activation of resistance-associated regulators, and modulation of predicted rifampicin influx and efflux systems. In M. tuberculosis H37Ra, conserved responses were accompanied by pronounced cell envelope remodelling via polyketide synthase pathways. Functional validation confirmed that sub-MIC rifampicin pre-exposure primes mycobacteria for enhanced tolerance to lethal drug concentrations. These findings highlight sub-lethal antibiotic exposure as a critical driver of phenotypic tolerance, with important implications for tuberculosis treatment efficacy and resistance evolution.

INSTRUMENT(S):

ORGANISM(S): Mycobacterium Smegmatis (strain Atcc 700084 / Mc(2)155) Bacteria Mycobacterium Tuberculosis H37ra

DISEASE(S): Tuberculosis

SUBMITTER: Blake Stuart  

LAB HEAD: Tariq Ganief

PROVIDER: PXD075873 | Pride | 2026-06-08

REPOSITORIES: Pride