Insight into the antibiotic resistance in tuberculous and non-tuberculous mycobacteria

Antimicrobial resistance has become one of the most urgent health threats of the 21st century (Ahmed et al., 2024; Murray et al., 2022). The progressive loss of efficacy of existing antibiotics against bacterial infections risks undermining decades of medical advances and highlights the pressing need for alternative therapeutic strategies. Among the microorganisms of greatest concern, mycobacteria occupy a prominent position due to their intrinsic resistance to a wide spectrum of antimicrobial agents, to their remarkable ability to acquire additional resistance mechanisms and to persist in hostile environments, including those shaped by host immune responses (Jumei Zeng and Xingyan Tan and Chao, 2024). While Mycobacterium tuberculosis (Mtb) remains the causative agent of tuberculosis - still one of the world’s deadliest infectious diseases - non-tuberculous mycobacteria (NTM) are increasingly recognized as opportunistic pathogens responsible for chronic and difficult-to-treat infections (Burzyńska et al., 2025). A central feature of their resilience against antimicrobial agents is the mycobacterial cell envelope. This highly complex, multilayered structure acts as a formidable permeability barrier against antimicrobials. In addition to the conserved mycolyl-arabinogalactan-peptidoglycan complex, many NTM species produce an outermost layer enriched in glycopeptidolipids (GPLs). These glycolipids, composed of a conserved lipopeptidyl core variably substituted with glycosyl and acetyl groups, are non-covalently bound constituents of the envelope’s outer leaflet (Pang et al., 2013). GPLs strongly influence bacterial physiology, shaping colony morphology, sliding motility, biofilm formation, and host–pathogen interactions (Daher et al., 2022; Gutiérrez et al., 2018). Given these functions, GPLs have recently attracted growing attention as potential modulators of both pathogenicity and drug resistance. Alterations in their biosynthesis or export can affect surface hydrophobicity, envelope permeability, and immune recognition, thereby influencing susceptibility to antimicrobials (Daher et al., 2022; Tatham et al., 2012). Building on this background, the first aim of the thesis was the functional characterization of MSMEG_0394, a gene located within the GPL biosynthetic cluster of M. smegmatis. Notably, MSMEG_0394 shares 75% amino acid sequence identity with the uncharacterized MAB_4102c gene of M. abscessus, a clinically relevant NTM. Despite its annotation within the GPL locus, the specific function of MSMEG_0394 had remained unexplored. The working hypothesis was that this gene might contribute either directly to the enzymatic machinery responsible for GPL biosynthesis or indirectly to their modification, transport, or regulation. To test this hypothesis, a combination of genetic and biochemical approaches was applied, enabling the dissection of its role in cell envelope physiology. Beyond structural barriers, antimicrobial resistance in mycobacteria is also strongly shaped by regulatory mechanisms that adjust gene expression in response to environmental cues. Among the various transcriptional regulator families, the TetR family of transcriptional regulators (TFTRs) is particularly abundant and functionally diverse. First identified as repressors of tetracycline resistance, TFTRs are now known to control a wide range of processes, including the activity of efflux pumps, transport systems, lipid metabolism, and stress responses (Singh et al., 2024). Their ability to sense diverse ligands and rapidly reprogram transcriptional outputs makes them crucial players in bacterial adaptation and survival under antimicrobial stress. Within this family, TetR-3765 was selected as a transcriptional regulator of interest in M. smegmatis. Previous studies showed that TetR-3765 represses the ABC-type efflux pump MSMEG_3762/63 and its ortholog Rv1687/86 in M. tuberculosis, an efflux system linked to resistance against rifampicin and ciprofloxacin, key first- and second-line anti-TB drugs (De Siena et al., 2020; Perrone et al., 2017). Building on these findings, the second aim of this thesis was to define the broader regulon of TetR-3765 and to assess its potential role in additional resistance-associated pathways. A multifaceted strategy was adopted, combining bioinformatics and transcriptomic analyses to identify additional stress-related genes potentially under its control. Taken together, the studies presented in this thesis were designed to elucidate both structural determinants of resistance, through the characterization of GPL biosynthetic genes, and regulatory determinants, through the analysis of TetR-3765. By integrating these complementary perspectives, this work contributes to a deeper understanding of the molecular mechanisms that underpin the extraordinary resilience of mycobacteria against antimicrobial agents.