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.

Mgm1 is a member of the dynamin family of GTP-binding proteins. Mgm1 was first identified in yeast, where it affects mitochondrial morphology. The human homologue of Mgm1 is called OPA1. Mutations in the OPA1 gene are the prevailing cause of dominant optic atrophy, a hereditary disease in which progressive degeneration of the optic nerve can lead to blindness. Here we investigate the properties of the Mgm1/OPA1 protein in mammalian cells. We find that Mgm1/OPA1 is localized to the mitochondrial intermembrane space, where it is tightly bound to the outer surface of the inner membrane. Overexpression of wild type or mutant forms of the Mgm1/OPA1 protein cause mitochondria to fragment and, in some cases, cluster near the nucleus, whereas the loss of protein caused by small interfering RNA (siRNA) leads to dispersal of mitochondrial fragments throughout the cytosol. The cristae of these fragmented mitochondria are disorganized. At early time points after transfection with Mgm1/OPA1 siRNA, the mitochondria are not yet fragmented. Instead, the mitochondria swell and stretch, after which they form localized constrictions similar to the mitochondrial abnormalities observed during the early stages of apoptosis. These abnormalities might be the earliest effects of losing Mgm1/OPA1 protein.

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, α-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins. The genus Mycobacterium contains a number of important pathogens, such as Mycobacterium tuberculosis. The highly characteristic cell envelope of these bacteria plays a crucial role in the infection process. The most apparent difference with other bacteria is the recently described outer membrane composed of unique (glyco)lipids. However, on top of this membrane mycobacteria also have an ill-defined capsular layer. In this paper, we studied this capsular layer using different electron microscopy techniques and mass spectrometry. Using close to native state preparation method, we show that both pathogenic and non-pathogenic mycobacteria have a labile capsular layer that covers the outer membrane. This capsular layer, in addition to containing arabinogalactan, glycan and mannose-containing glyco-lipids, also surprisingly contains a large amount of ESX-1-secreted proteins in Mycobacterium marinum. Furthermore, we also show that the capsule plays a role in the binding of macrophages and the induction of cytokines. Collectively, these results show for the first time that the capsule can be visualized on both pathogenic and non-pathogenic mycobacteria. In addition, growing mycobacteria under standard laboratory conditions in the presence of detergent with agitation promotes capsular shedding and influences the biological characteristics of the bacteria.