The World Health Organization (WHO) reports that tuberculosis (TB) is one of the top 10 leading causes of global mortality. The increasing incidence of multidrug-resistant TB highlights the urgent need for an intensified quest to discover innovative anti-TB medications In this study, we investigated four new derivatives from the quinoxaline-2-carboxylic acid 1,4-dioxide class. New 3-methylquinoxaline 1,4-dioxides with a variation in substituents at positions 2 and 6(7) were synthesized via nucleophilic aromatic substitution with amines and assessed against a Mycobacteria spp. Compound 4 showed high antimycobacterial activity (1.25 μg/mL against M. tuberculosis) and low toxicity in vivo in mice. Selection and whole-genomic sequencing of spontaneous drug-resistant M. smegmatis mutants revealed a high number of single-nucleotide polymorphisms, confirming the predicted mode of action of the quinoxaline-2-carboxylic acid 1,4-dioxide 4 as a DNA-damaging agent. Subsequent reverse genetics methods confirmed that mutations in the genes MSMEG_4646, MSMEG_5122, and MSMEG_1380 mediate resistance to these compounds. Overall, the derivatives of quinoxaline-2-carboxylic acid 1,4-dioxide present a promising scaffold for the development of innovative antimycobacterial drugs.

The emergence of drug resistance in pathogens leads to a loss of effectiveness of antimicrobials and complicates the treatment of bacterial infections. Quinoxaline 1,4-dioxides represent a prospective scaffold for search of new compounds with improved chemotherapeutic characteristics. Novel 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides with alteration of substituents at position 2 and 6 were synthesized via nucleophilic substitution with piperazine moiety and evaluated against a broad panel of bacteria and fungi by measuring their minimal inhibitory concentrations. Their mode of action was assessed by whole-genomic sequencing of spontaneous drug-resistant Mycobacterium smegmatis mutants, followed by comparative genomic analysis, and on an original pDualrep2 system. Most of the 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides showed high antibacterial properties against Gram-positive strains, including mycobacteria, and the introduction of a halogen atom in the position 6 of the quinoxaline ring further increased their activity, with 13c being the most active compound. The mode of action studies confirmed the DNA-damaging nature of the obtained quinoxaline 1,4-dioxides, while drug-resistance may be provided by mutations in redox homeostasis genes, encoding enzymes potentially involved in the activation of the compounds. This study extends views about the antimicrobial and antifungal activities of the quinoxaline 1,4-dioxides and can potentially lead to the discovery of new antibacterial drugs.

The m ulti-copy p hage resistance gene ( mpr ) of M. smegmatis is a major factor in resistance to the lytic mycobacteriophage D29. Mpr is a membrane-bound exonuclease that cleaves phage DNA post injection, hence blocking downstream stages in the phage infection cycle. The mechanism of resistance allows for adsorption, is non-abortive and independent of any mutation in the gene. Rather, it depends on overexpression of a wild type copy of the gene. However, the underlying factor behind mpr overexpression in spontaneous D29-resistant mutants of M. smegmatis remained elusive. Here, we report that D29 infection triggers insertion sequence (IS) rearrangements, including the transposition and integration of IS6120 directly upstream of mpr . Mutants with IS6120 integration upstream of mpr show highly elevated Mpr expression. Whole genome sequence analysis reveals that IS6120 introduced a putative transcription factor-binding site and a canonical -35 promoter element at the integration site, hence reconstituting a fuller promoter ( rcp ) than the original promoter ( wtp ) at the site. Promoter reporter assays suggest that rcp is a far stronger promoter than wtp , implying that elevated mpr expression in D29-resistant mutants with this transposition event could be due to promoter reconstitution. While strains with this transposition event appear to grow normally, rcp -driven, vector-borne Mpr overexpression appears to be toxic as it barely allows for colony formation on agar plates. This study reports a previously unknown factor likely behind mpr regulation in M. smegmatis , adding to the existing knowledge of mycobacterial anti-phage defense mechanisms and guiding rational phage engineering efforts for therapeutic applications.

Mycobacterium abscessus (Mab) is a pathogenic fast-growing mycobacterium with complex, poorly understood drug resistance mechanisms and limited treatment options. This warrants renewed efforts to dissect resistance mechanisms and identify novel targets. Here, we report that disruption of the putative sigma factor sigH in Mab results in hypersensitivity to levofloxacin, moxifloxacin, tigecycline, tetracycline, amikacin, vancomycin, and rifabutin. Mab SigH shares 84% peptide sequence identity with Mycobacterium tuberculosis (Mtb) SigH, a well-known stress-response protein and global transcriptional regulator. Complementation of the knockout strain (Δ sigH ) with Mab sigH or its Mtb homolog restored the drug resistance phenotype. Additionally, Δ sigH showed increased sensitivity to oxidative and heat stress compared to the wild-type (WT) Mab and complemented strains. Transcriptomic analysis revealed that the deletion of sigH disrupted the balance of gene expression, primarily elevating the expression of genes encoding YrbE and MCE family proteins and downregulating genes encoding ABC-type transporters, sigma and anti-sigma factors, and other genes associated with antimicrobial resistance. Notably, Δ sigH accumulated more ethidium bromide and displayed cell-elongation phenotypes, both partially reversed by complementation. In addition, a native sigH promoter driving green fluorescent protein (GFP) produced 10-fold higher fluorescence in WT than in Δ sigH , suggesting that the presence of SigH drives higher GFP expression . Collectively, our findings identify SigH as a key regulator of global gene expression that links stress adaptation, cell-envelope homeostasis, and intrinsic multidrug resistance in Mab, hence representing a promising target for the development of novel therapeutics against Mab infections. IMPORTANCE: Mycobacterium abscessus (Mab) causes severe acute and chronic lung infections. The intrinsic drug resistance mechanisms of Mab remain poorly understood, posing significant therapeutic challenges. Here, we examined the role of the putative sigma factor SigH in intrinsic multidrug resistance in Mab. Mab SigH shares 84% amino acid identity with M. tuberculosis . SigH is a well-known stress response regulator. Transcriptomic analysis revealed that the deletion of sigH disrupted the balance of gene expression, primarily elevating the expression of genes encoding YrbE and MCE family proteins and downregulating genes encoding ABC-type transporters, sigma and anti-sigma factors, and other genes associated with antimicrobial resistance. A native sigH promoter driving green fluorescent protein (GFP) produced 10-fold higher fluorescence in wild type than in Δ sigH , suggesting that the presence of SigH drives higher GFP expression. Thus, SigH emerges as a central regulator integrating stress adaptation, envelope homeostasis, and multidrug resistance, offering a promising target for novel anti-Mab therapies.

Impact statement: Fluoroquinolones (FQs) are important for treating fast‐growing mycobacterial infections; however, their efficacy can be limited by intrinsic resistance and the emergence of acquired resistance. FQs induce lethal DNA double‐strand breaks (DSBs) by targeting topoisomerases. Through a transposon screen in Mycobacterium abscessus , we identified that disruption of adnB , a homologous recombination (HR) gene, markedly sensitized the pathogen to FQs. Subsequent study showed that deleting core HR components ( adnB , recO , recA , recR , and ruvB ) increased FQs' susceptibility, while other DSB repair pathways (single‐strand annealing or non‐homologous end joining) had no effect, demonstrating a unique reliance on HR for FQs' tolerance. Complementation with native or its homolog genes restored the resistance phenotype, indicating a conserved HR‐dependent tolerance mechanism in M . abscessus . In a murine model, genetic HR abrogation significantly improved FQs' therapeutic efficacy. These results highlight HR repair as a potential therapeutic target to potentiate FQs' activity and combat drug resistance in M . abscessus infections.

Mycobacterium abscessus exhibits intrinsic resistance to conventional antibiotics, significantly limiting treatment options. Our previous studies established that MAB_2362 (SteA) is a key regulator of cell division that contributes to intrinsic resistance and virulence. Considering that SteA-like proteins often act alongside SteB counterparts, we hypothesized that the adjacent gene MAB_2363 encodes the corresponding SteB-like division regulator. In this study, we found that deletion of MAB_2363 significantly increased susceptibility to multiple antibiotics and disrupted cell wall permeability. Microscopy revealed pronounced cell division defects in the mutant, including elongated cell morphology and multiple septa. Subcellular localization of a GFP-MAB_2363 fusion protein demonstrated its enrichment at division septa, confirming its direct involvement in cell division. Furthermore, deletion of MAB_2363 led to attenuated virulence, as evidenced by reduced bacterial survival in macrophages and murine infection models. To assess its functional relation with MAB_2362, we compared the single-deletion mutant of MAB_2363 with the single-deletion mutant of MAB_2362 and the double-deletion mutant of MAB_2362-MAB_2363. Notably, the phenotypes of the MAB_2363 mutant, including cell division defects, antibiotic susceptibility, and virulence, were markedly milder than those of the other two mutants. Collectively, these findings indicate that MAB_2363 functions as a secondary but essential division-associated factor that operates during cell division, thereby influencing intrinsic resistance and virulence in M. abscessus.