Global assessment of Mycobacterium avium subspecies hominissuis genetic requirement for growth and virulence.
Nontuberculous mycobacterial infections caused by the opportunistic pathogen Mycobacterium avium subsp. hominissuis (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 in vitro growth, and organized ~3500 identified transposon mutants for hypothesis-driven research. The majority (71 %) of the genes we identified as essential for MAH in vitro had a growth-essential mutual ortholog in the related and highly virulent M. tuberculosis (Mtb). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. Strikingly, > 97 % of the MAH virulence genes had a mutual ortholog in Mtb. Two of the three virulence genes specific to MAH (i.e. no Mtb mutual orthologs) were PPE proteins, a family of proteins unique to mycobacteria and highly associated with virulence. Finally, we validated novel genes as required for successful MAH infection; one encoding a probable MFS transporter and another a hypothetical protein located in 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.\n\nAuthor summaryPulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by the M. avium complex (MAC), whereof >90% arise from Mycobacterium avium subsp. hominissuis (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. While genetic manipulation of pathogens is crucial to study pathogenesis, M. avium (Mav) has been found notoriously hard to engineer. Here, we identify an MAH strain highly susceptible to high-density transposon mutagenesis and transformation, facilitating genetic engineering and analysis of gene function. We provide crucial insights into this strains global genetic requirements for growth and infection. Surprisingly, 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 M. tuberculosis (Mtb). 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.