Abstract
Despite the availability of effective drug treatment, Mycobacterium tuberculosis (Mtb), the causative agent of TB disease, kills ~1. 5 million people annually, and the rising prevalence of drug resistance increasingly threatens to worsen this plight. We previously showed that sublethal exposure to the frontline anti-TB drug, rifampicin, resulted in substantial adaptive remodeling of the proteome of the model organism, Mycobacterium smegmatis, in the drug-sensitive mc2155 strain [wild type (WT)]. In this study, we investigate whether these responses are conserved in an engineered, isogenic mutant harboring the clinically relevant S531L rifampicin resistance-conferring mutation (SL) and distinguish the responses that are specific to RNA polymerase β subunit- (RpoB-) binding activity of rifampicin from those that are dependent on the presence of rifampicin alone. We verified the drug resistance status of this strain and observed no phenotypic indications of rifampicin-induced stress upon treatment with the same concentration as used in WT (2.5 μg/ml). Thereafter, we used a cell wall-enrichment strategy to focus attention on the cell wall proteome and observed 253 proteins to be dysregulated in SL bacteria in comparison with 716 proteins in WT. We observed that decreased abundance of ATP-binding cassette (ABC) transporters and increased abundance of ribosomal machinery were conserved in the SL strain, whereas the upregulation of transcriptional machinery and the downregulation of numerous two-component systems were not. We conclude that the drug-resistant M. smegmatis strain displays some of the same proteomic responses observed in WT and suggest that this evidence supports the hypothesis that rifampicin exercises effects beyond RpoB-interaction alone and that mycobacteria recognise rifampicin as a signaling molecule in an RpoB-independent manner at sublethal doses. Taken together, our data indicates mixed RpoB-independent and RpoB-dependent proteomic remodeling in WT mycobacteria, with evidence for RpoB-independent ABC transporter downregulation, but drug activity-based transcriptional upregulation and two-component system downregulation.
Highlights
Mycobacterium tuberculosis (Mtb) causes TB disease, which accounts for ∼1.5 million deaths annually despite the existence of effective drug treatments for ∼80 years
While it might be naively assumed that antibiotics applied in the treatment of disease are foreign to the target microbe and exercise their activity using the designated primary mechanism with few other ramifications, in reality, a number of antibiotics are natural products, which were first discovered in microorganisms [10, 11] and at low concentrations can behave as cell signaling molecules, triggering significant changes in gene transcription and often in gene subsets capable of counteracting the applied antibiotic [12, 13]
We previously reported that a subinhibitory dose of rifampicin in the treatment of drug-susceptible (WT) M. smegmatis mc2155 resulted in increased abundance of both transcriptional and translational cellular machinery, decreased abundance of numerous ABC transporters, and eventual upregulation of the M. smegmatis-specific rifampicin inactivating enzyme, ADP-ribosyl transferase [24, 25]
Summary
Mycobacterium tuberculosis (Mtb) causes TB disease, which accounts for ∼1.5 million deaths annually despite the existence of effective drug treatments for ∼80 years. Their base mutation rate is low, they possess means by which this can be increased Drugs, such as the fluoroquinolones, can activate damage-induced mutagenesis pathways by which error-prone replication machinery, such as DnaE2, can enable both survival of the bacterium through translesion synthesis, and increased mutation frequency—allowing for the evolution of drug resistance [7, 8]. Another mechanism by which bacteria can acquire resistance is through initial phenotypic adaption to sublethal drug exposure. While it might be naively assumed that antibiotics applied in the treatment of disease are foreign to the target microbe and exercise their activity using the designated primary mechanism with few other ramifications, in reality, a number of antibiotics are natural products, which were first discovered in microorganisms [10, 11] and at low concentrations can behave as cell signaling molecules, triggering significant changes in gene transcription and often in gene subsets capable of counteracting the applied antibiotic [12, 13]
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