Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient‐derived mycobacteria species were found to translocate to the cytosol, while non‐pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX‐1 (type VII) secretion system into the non‐virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C‐terminus of the early‐secreted antigen ESAT‐6. The C‐terminal truncation of ESAT‐6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.

To characterize the genetic determinants of resistance to antituberculosis drugs, we performed a genome-wide association study (GWAS) of 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries. A GWAS approach within a mixed-regression framework was followed by a phylogenetics-based test for independent mutations. In addition to mutations in established and recently described resistance-associated genes, novel mutations were discovered for resistance to cycloserine, ethionamide and para-aminosalicylic acid. The capacity to detect mutations associated with resistance to ethionamide, pyrazinamide, capreomycin, cycloserine and para-aminosalicylic acid was enhanced by inclusion of insertions and deletions. Odds ratios for mutations within candidate genes were found to reflect levels of resistance. New epistatic relationships between candidate drug-resistance-associated genes were identified. Findings also suggest the involvement of efflux pumps (drrA and Rv2688c) in the emergence of resistance. This study will inform the design of new diagnostic tests and expedite the investigation of resistance and compensatory epistatic mechanisms.

Summary: ESX‐5 is one of the five type VII secretion systems found in mycobacteria. These secretion systems are also known as ESAT‐6‐like secretion systems. Here, we have determined the secretome of ESX‐5 by a proteomic approach in two different strains of Mycobacterium marinum. Comparison of the secretion profile of wild‐type strains and their ESX‐5 mutants showed that a number of PE_PGRS and PPE‐MPTR proteins are dependent on ESX‐5 for transport. The PE and PPE protein families are unique to mycobacteria, are highly expanded in several pathogenic species, such as Mycobacterium tuberculosis and M. marinum, and certain family members are cell surface antigens associated with virulence. Using a monoclonal antibody directed against the PGRS domain we showed that nearly all PE_PGRS proteins that are recognized by this antibody are missing in the supernatant of ESX‐5 mutants. In addition to PE_PGRS and PPE proteins, the ESX‐5 secretion system is responsible for the secretion of a ESAT‐6‐like proteins. Together, these data show that ESX‐5 is probably a major secretion pathway for mycobacteria and that this system is responsible for the secretion of recently evolved PE_PGRS and PPE proteins.

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC₅ prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria. Mycobacteria have a thick protective outer membrane that helps them to withstand adverse conditions both outside and within the host. However, in order to cause disease, the bacterium also needs to secrete proteins across this outer membrane. To achieve this, mycobacteria possess so-called type VII secretion systems. One of these systems, the ESX-5 secretion system, is only present in the group of slow-growing mycobacteria, which contains most pathogenic species. In this study, we show that the ESX-5 system is essential for growth of mycobacteria. We found that when we generated a ‘leaky’ outer membrane, by interfering in the construction of the outer membrane, or by introducing an outer membrane porin, the ESX-5 system was no longer essential for growth. We additionally show that ESX-5 mediates uptake of fatty acids, which suggests that ESX-5 substrates can form specific transport systems or pores in the outer membrane required for the uptake of crucial nutrients. Understanding the role of ESX-5 in outer membrane permeability helps us to understand a fundamental difference between fast-growing and slow-growing mycobacteria. Since most pathogenic mycobacteria are slow-growing this helps us to understand the mycobacterial requirements for pathogenesis in more detail.