Yusuke Minato

Nontuberculous mycobacteria (NTM) are emerging pathogens for which genetic tools remain limited. Here, we developed an arabinose-inducible gene expression system based on a modified pBAD24 vector adapted for mycobacterial hosts. The vector carries replication origins for mycobacteria and Escherichia coli, as well as selectable markers compatible with NTM. In Mycobacterium abscessus (Mycobacteroides abscessus), the system enabled dose-dependent induction of target gene expression by arabinose, as demonstrated by increased antibiotic resistance and quantitative RT-PCR analysis. Although basal expression was observed in the absence of arabinose, expression levels were tunable across arabinose concentrations. The system was also functional in Mycobacterium smegmatis (Mycolicibacterium smegmatis) and Mycobacterium bovis BCG, although the degree of basal expression varied among host species. These results establish a tunable inducible expression system for mycobacteria and provide a useful genetic tool for studies of NTM biology.

Abstract Mycobacterium abscessus exhibits intrinsic resistance to many antimicrobial agents, including rifampicin, a frontline anti-tuberculosis drug, severely limiting treatment options. Here, we used transposon insertion sequencing (Tn-Seq) to perform a genomewide screen to identify genes required for intrinsic rifampicin resistance in M. abscessus . We uncovered candidate genes that confer intrinsic resistance to the rifamycin-class antibiotics, rifampicin and rifabutin. The genes we identified included previously reported genes such as arr , helR, and MAB_2807 . By comparing our results with the rifampicin intrinsic resistance gene in Mycobacterium tuberculosis , we found that the mechanisms underlying rifampicin intrinsic resistance were distinct between the two species. The contribution of seven representative candidate genes to rifampicin resistance was confirmed by characterizing targeted gene deletion or transposon insertion mutants. Among these determinants, MAB_2807 was identified as a major efflux-based contributor to rifamycin resistance, and its disruption increased intracellular rifampicin accumulation. In addition to known resistance determinants, Tn-Seq revealed contributions from many genes involved in cell envelope processes to rifamycin resistance. Guided by this genetic signature, we evaluated the combined effects of cell wall-targeting antimicrobial agents with rifamycins. We found that rifamycins displayed selective synergistic interactions with specific β-lactam antibiotics. Interestingly, we found that rifamycin exposure altered cell envelope ultrastructure and increased the accumulation of the peptidoglycan precursor UDP-N-acetylglucosamine, suggesting that rifampicin perturbs envelope-associated metabolic homeostasis. These findings highlight rifamycin-induced vulnerable cellular processes that may inform rational combination strategies. Importance Rifamycins are cornerstone antibiotics for the treatment of mycobacterial infections, yet they are ineffective against Mycobacterium abscessus . Although several key resistance mechanisms have been described, the full genetic basis of intrinsic rifamycin resistance remains to be elucidated. Here, we used transposon insertion sequencing to systematically identify genes that contribute to intrinsic rifamycin resistance in M. abscessus . Because recent studies have shown that chemical modification of rifamycins can overcome specific intrinsic resistance mechanisms, a comprehensive understanding of these determinants could facilitate the development of next-generation rifamycins with improved activity against M. abscessus . In addition, we unexpectedly found that rifamycin exposure alters cell envelope structure, the intracellular levels of a key metabolic intermediate in peptidoglycan biosynthesis, and susceptibility to certain β-lactams in M. abscessus . These findings may help guide the rational development of combination therapeutic strategies with next-generation rifamycins.

Abstract Pulmonary disease caused by nontuberculous mycobacteria (NTM-PD) is an emerging global health concern. Among NTM, Mycobacterium avium subsp. hominissuis (MAH) is the major causative agent of NTM-PD. Similar to Mycobacterium tuberculosis ( Mtb ), MAH exhibits lineage-specific geographical distributions and host adaptations. Here, we characterized three MAH strains from the residential bathrooms of MAH-PD patients in Japan. A genetic population clustering analysis revealed that the three strains belong to the East Asia (EA) lineages that are predominant in Japan and Korea. Pan-genome analysis using the publicly available complete genome sequences of MAH and the newly sequenced MAH strains identified 3,313 core genes that are conserved among distinct MAH lineages. Identification of essential genes in the three strains was conducted using transposon insertion sequencing (Tn-Seq), and their gene essentiality profiles were compared to those of a previously studied SC3 lineage strain, MAC109. Despite their genetic diversity, nearly all essential genes were derived from the core gene set. In addition, we identified a set of common essential genes for the EA and SC3 lineages, as well as lineage-specific essential genes. Our results highlight the evolutionary and clinical importance of lineage-specific adaptations in MAH. Importance By integrating transposon insertion sequencing with pan-genome analysis, we provide the first systematic comparison of essential genes across multiple Mycobacterium avium subsp. hominissuis (MAH) strains. Although MAH strains exhibit remarkable genetic diversity, we found that MAH essential genes are primarily confined to the core genome of MAH. This essential plasticity highlights the evolutionary strategies that underpin MAH survival across diverse environments and patient populations. Recognizing this interplay provides a foundation for identifying robust drug targets and developing lineage-informed therapies for MAH infection.

Solid-phase total synthesis of atratumycin (1) and its analogues is described. The linear depsipeptide was synthesized by the standard Fmoc solid-phase peptide synthesis, on-resin esterification, and epimerization-free macrolactamization. A structure-activity relationship study of 1 led to the identification of the amino acid residue that preserves its antituberculosis activity. Conformational analyses of 1 and its analogues revealed that the solution structure of 1 closely resembles the crystal structure of 1, suggesting the structural rigidity of its unique conformation.