Global assessment ofMycobacterium aviumsubspecieshominissuisgenetic requirement for growth and virulence

Nontuberculous mycobacterial infections caused by the opportunistic pathogenMycobacterium aviumsubsp.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 andin vitrogrowth, and organized ~3500 identified transposon mutants for hypothesis-driven research. The majority (71 %) of the genes we identified as essential for MAHin vitrohad a growth-essential mutual ortholog in the related and highly virulentM. 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 inMtb. Two of the three virulence genes specific to MAH (i.e. noMtbmutual 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.Author summaryPulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by theM. aviumcomplex (MAC), whereof >90% arise fromMycobacterium aviumsubsp.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 strain’s 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 pathogenM. 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.