Pulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by the Mycobacterium avium complex (MAC), whereof >90% are due to 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. 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 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.

Drosophila melanogaster (Drosophila), the common fruit fly, is one of the most extensively studied animal models we have, with a broad, advanced, and organized research community with tools and mutants readily available at low cost. Yet, Drosophila has barely been exploited to understand the underlying mechanisms of mycobacterial infections, including those caused by the top-killer pathogen Mycobacterium tuberculosis (Mtb). In this study, we aimed to investigate whether Drosophila is a suitable host model to study mycobacterial virulence, using Mycobacterium marinum (Mmar) to model mycobacterial pathogens. First, we validated that an established mycobacterial virulence factor, EccB1 of the ESX-1 Type VII secretion system, is required for Mmar growth within the flies. Second, we identified Mmar virulence factors in Drosophila in a high-throughput genome-wide manner using transposon insertion sequencing (TnSeq). Of the 181 identified virulence genes, the vast majority (91%) had orthologs in Mtb, suggesting that the encoded virulence mechanisms may be conserved across Mmar and Mtb. Finally, we validated one of the novel Mmar virulence genes we identified, a putative ATP-binding protein ABC transporter encoded by mmar_1660, as required for full virulence during both Drosophila and human macrophage infection. Together, our results show that Drosophila is a powerful host model to study and identify novel mycobacterial virulence factors relevant to human infection.

27 Nontuberculous mycobacterial infections caused by the opportunistic pathogen 28 Mycobacterium avium subsp. hominissuis (MAH) are currently receiving renewed 29 attention due to increased incidence combined with difficult treatment. Insights into 30 the disease-causing mechanisms of this species have been hampered by difficulties in 31 genetic manipulation of the bacteria. Here, we identified and sequenced a highly 32 transformable, virulent MAH clinical isolate susceptible to high-density transposon 33 mutagenesis, facilitating global gene disruption and subsequent investigation of MAH 34 gene function. By transposon insertion sequencing (TnSeq) of this strain, we defined 35 the MAH genome-wide genetic requirement for virulence and in vitro growth, and 36 organized ~3500 identified transposon mutants for hypothesis-driven research. The 37 majority (71 %) of the genes we identified as essential for MAH in vitro had a 38 growth-essential mutual ortholog in the related and highly virulent M. tuberculosis 39 (Mtb). However, passaging our library through a mouse model of infection revealed a 40 substantial number (54% of total hits) of novel virulence genes. Strikingly, > 97 % of 41 the MAH virulence genes had a mutual ortholog in Mtb. Two of the three virulence 42 genes specific to MAH (i.e. no Mtb mutual orthologs) were PPE proteins, a family of 43 proteins unique to mycobacteria and highly associated with virulence. Finally, we 44 validated novel genes as required for successful MAH infection; one encoding a 45 probable MFS transporter and another a hypothetical protein located in immediate 46 vicinity of six other identified virulence genes. In summary, we provide new, 47 fundamental insights into the underlying genetic requirement of MAH for growth and 48 host infection. 49 50 3 51 Author summary 52 Pulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. 53 The majority of these infections are caused by the M. avium complex (MAC), 54 whereof >90% arise from Mycobacterium avium subsp. hominissuis (MAH).55 Treatment of MAH infections is currently difficult, with a combination of antibiotics 56 given for at least 12 months. To control MAH by improved therapy, prevention and 57 diagnostics, we need to understand the underlying mechanisms of infection. While 58 genetic manipulation of pathogens is crucial to study pathogenesis, M. avium (Mav) 59 has been found notoriously hard to engineer. Here, we identify an MAH strain highly 60 susceptible to high-density transposon mutagenesis and transformation, facilitating 61 genetic engineering and analysis of gene function. We provide crucial insights into 62 this strain's global genetic requirements for growth and infection. Surprisingly, we 63 find that the vast majority of genes required for MAH growth and virulence (96% and 64 97%, respectively) have mutual orthologs in the tuberculosis-causing pathogen M.65 tuberculosis (Mtb). However, we also find growth and virulence genes specific to 66 MAC species. Finally, we validate novel mycobacterial ...