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 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.

Mycobacterium abscessus exhibits high intrinsic drug resistance, requiring combination therapy. We developed a silkworm (Bombyx mori) infection model as a whole-organism, in vivo-based platform for quantitative, outcome-based evaluations of antimicrobial combinations. The system, examined using clarithromycin-amikacin and imipenem-cefoxitin combinations, showed interaction profiles that were qualitatively consistent with those observed in vitro. This rapid, reproducible, and ethical assay enables reliable phenotypic assessments of synergistic or antagonistic effects and may facilitate the evaluation and prioritization of antimicrobial combination regimens in preclinical studies.