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ABSTRACT 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 ∼3,500 identified transposon mutants for hypothesis-driven research. The majority (96%) of the genes we identified as essential for MAH in vitro had a mutual ortholog in the related and highly virulent Mycobacterium tuberculosis (Mtb). 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 Mtb. 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. IMPORTANCE 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.

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ΔmycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems. IMPORTANCE Mycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growth in vitro. We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute.

Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the Pseudomonas putida-derived positively regulated XylS/Pm expression system to control inducible gene expression in Mycobacterium smegmatis and Mycobacterium tuberculosis, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/Pm-regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer m-toluate, we achieve a robust, time- and dose-dependent reversible induction of Pm-mediated expression in mycobacteria, with low background expression levels. XylS/Pm is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance.