Beyond Inhibition: Sublethal Rifampicin-Induced Molecular Adaptations Confer Phenotypic Drug Tolerance in Mycobacteria

Tuberculosis (TB) remains a major global health threat, largely due to Mycobacterium tuberculosis (Mtb) resilience, which can be exacerbated by exposure to sublethal antibiotic concentrations arising from factors such as patient nonadherence and the granuloma structure which limits drug penetration. Within host granulomas, Mtb can exhibit both phenotypic tolerance and genotypic resistance, complicating curative treatments. This study aimed to determine whether sublethal rifampicin acts as a signaling molecule in Mycobacterium smegmatis (Msm) and the attenuated Mtb H37Ra strain, triggering phenotypic changes that promote tolerance to lethal drug levels. Msm exposed to half-MIC rifampicin showed an initial transient growth deceleration followed by a resumption of proliferation, indicating the acquisition of phenotypic tolerance. Deep data-independent acquisition (DIA) mass spectrometry-based proteomic profiling revealed that the early response (45 min) involved the upregulation of ribosomal proteins, DNA replication, and de novo purine biosynthesis. Proteins associated with phenotypic resistance (e.g., RpoZ, GidB, WhiB2) and efflux transporters were also upregulated. As Msm recovered (180 min), its proteome largely returned to baseline, but key resistance-associated pathways, including the Rifampin ADP-ribosyltransferase superfamily and certain efflux systems, remained dysregulated. Parallel studies on Mtb H37Ra also demonstrated a distinct proteomic shift, comprising conserved adaptive responses such as ribosomal perturbation and compensatory transcriptional activity, as well as species-specific dysregulation of drug influx/efflux pumps and cell envelope remodelling via the polyketide synthase family. These findings demonstrate that sublethal rifampicin exposure primes mycobacteria for enhanced tolerance to lethal drug concentrations, underscoring a significant challenge in current TB therapy.