Integrated analysis of Tn-Seq and pangenome reveals core and lineage-specific essential genes in Mycobacterium avium subsp. hominissuis

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.