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Nontuberculous mycobacterial infections caused by the opportunistic pathogen subsp. (MAH) are currently receiving renewed attention due to increased incidence combined with difficult treatment. Insights into the disease-causing mechanisms of this species have been hampered by difficulties in genetic manipulation of the bacteria. Here, we identified and sequenced a highly transformable, virulent MAH clinical isolate susceptible to high-density transposon mutagenesis, facilitating global gene disruption and subsequent investigation of MAH gene function. By transposon insertion sequencing (TnSeq) of this strain, we defined the MAH genome-wide genetic requirement for virulence and growth and organized ∼3,500 identified transposon mutants for hypothesis-driven research. The majority (96%) of the genes we identified as essential for MAH had a mutual ortholog in the related and highly virulent (). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. More than 97% of the MAH virulence genes had a mutual ortholog in Finally, we validated novel genes required for successful MAH infection: one encoding a probable major facilitator superfamily (MFS) transporter and another encoding a hypothetical protein located in the immediate vicinity of six other identified virulence genes. In summary, we provide new, fundamental insights into the underlying genetic requirement of MAH for growth and host infection. Pulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by the complex (MAC), whereof >90% are due to subsp. (MAH). Treatment of MAH infections is currently difficult, with a combination of antibiotics given for at least 12 months. To control MAH by improved therapy, prevention, and diagnostics, we need to understand the underlying mechanisms of infection. Here, we provide crucial insights into MAH's global genetic requirements for growth and infection. We find that the vast majority of genes required for MAH growth and virulence (96% and 97%, respectively) have mutual orthologs in the tuberculosis-causing pathogen (). However, we also find growth and virulence genes specific to MAC species. Finally, we validate novel mycobacterial virulence factors that might serve as future drug targets for MAH-specific treatment or translate to broader treatment of related mycobacterial diseases.
