M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells.

M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.

Subcellular localization of mycobacteria in tissues and detection of lipid antigens in organelles using cryo-techniques for light and electron microscopy.

The survival of intracellular pathogens within a host is determined by microbial evasion, which can be partially attributed to their subcellular trafficking strategies. Microscopic techniques have become increasingly important in understanding the cell biology of microbial infections. These recently developed techniques can be used for the subcellular localization of antigens not only in cultured cells but also within tissues such as Mycobacterium tuberculosis in lung and Mycobacterium leprae in skin. High-resolution immunofluorescence microscopy can be used in combination with cryo-immunogold electron microscopy using consecutive cryo-sections on the same tissue block forming a direct connection between the two microscopy techniques. The detection of mycobacterial lipid antigens in situ at an ultrastructural level is currently a challenge, but new modifications can be used to address this. These methods might be of interest to microbiologists and cell biologists who study host-pathogen interactions.

Comparative studies of the cell structures of Mycobacterium leprae and M. tuberculosis using the electron microscopy freeze-substitution technique.

The cell envelope and cytoplasmic architecture of the Mycobacterium leprae Thai-53 strain were examined using the freeze-substitution technique of electron microscopy and compared with those of the M. tuberculosis H37Rv strain. Both strains had similarly multilayered envelope architectures composed of an electron-translucent layer, a peptidoglycan layer and the plasma membrane, from outside to inside. A comparison of the structures of these two mycobacteria revealed that the M. leprae cell was smaller in size and had a thinner peptidoglycan layer than the M. tuberculosis cell. The cell widths measured on electron micrographs were 0.44 microm for M. tuberculosis and 0.38 microm for M. leprae. The peptidoglycan layer of M. leprae was 4-5 nm, while the corresponding layer of M. tuberculosis was 10-15 nm.

Systemic dissemination in tuberculosis and leprosy: do mycobacterial adhesins play a role?

More than one century after the discovery of their etiological agents, tuberculosis and leprosy remain as major health threats for humans, and the molecular mechanisms that lead to the development of both diseases are poorly understood. The elucidation of these mechanisms, and especially those allowing for the mycobacteria to systemically disseminate, should facilitate the development of new prophylactic and/or therapeutic strategies. This review is focused on the routes that Mycobacterium tuberculosis and Mycobacterium leprae may use to disseminate within the human body, and the potential roles played by recently characterized adhesins in this process.

Immunolocalization of cell-wall-deficient forms of Mycobacterium tuberculosis complex in sarcoidosis and in sinus histiocytosis of lymph nodes draining carcinoma.

In sarcoidosis, pleomorphic chromogens (PCs) occur as multivariate pigmented elements within sinusoids of lymph nodes (sinusoidal phase) and as tiny "round bodies" detectable in granulomas (generalized phase). The sinusoidal phase occurs in other conditions as well and characteristically contains yeastlike bodies also known as H-W bodies. To elucidate the antigenic profile of all variant forms, 28 cases of sarcoidosis (series A) and 14 cases of malignancy associated sinus histiocytosis (series B) were studied immunohistochemically with panels of various antibodies, including antimycobacterial MAbs specific for M tuberculosis complex (TB68, TB71), for M. leprae (MMP-I-3C3) and for cross-reactive mycobacterial antigens (F24-2-3 and F116-5, the latter recognizing superoxide dismutase). Results for series A indicate that: 1) PCs are cell-wall-deficient (CWD) mycobacterial forms belonging to M. tuberculosis complex (over 95%); 2) both phases are antigenically identical parts of the L-cycle; 3) "round bodies" of the "infective" phase have an endolysosomal evolution; 4)uncommon vacuolated forms represent a labile spheroplast stage; 5) the yeastlike bodies are specialized sinusoidal large bodies of unknown function. Results for series B show that in roughly two thirds of cases the pigmented forms are also CWD mycobacteria, have the same immunophenotype as sarcoid PCs in 35.7% of cases, have a much higher incidence of labile vacuolated forms and, finally, that malignancy associated "pseudosarcoid" granulomas do not differ antigenically from genuine sarcoid granulomas. Unlike conventional mycobacteria, PCs do not express cytoskeletal proteins consistently. Their general reactivity for HBcAg raises the possibility of phage interactions being responsible for the L-cycle since it may reflect shared epitopes between unrelated virus entities.

Mycobacteria and human autoimmune disease: direct evidence of cross-reactivity between human lactoferrin and the 65-kilodalton protein of tubercle and leprosy bacilli.

We document here by Western immunoblotting and immunogold ultracytochemistry that polyclonal antibodies against human lactoferrin (Lf) bind to tubercle and leprosy bacilli. In situ immunogold labeling of Mycobacterium leprae (present in armadillo liver and in human skin) and of Mycobacterium tuberculosis indicated that receptors for anti-Lf antibodies were present both on the cytoplasm and on the envelope of the bacilli. We found by immunoblotting that the 65-kDa heat shock protein is the major component of M. leprae and M. tuberculosis that is responsible for the binding of the anti-Lf probe. Furthermore, we show that anti-Lf immunoglobulin G eluted from the nitrocellulose-transferred mycobacterial 65-kDa protein band did bind back to Lf. Ultracytochemistry of biopsy samples of human lepromas showed that dead or severely damaged M. leprae was strongly marked by the anti-Lf antibodies; a similar pattern of immunogold marking was observed on M. leprae when antibodies against the 65-kDa mycobacterial protein were used. Our results offer direct evidence that the 65-kDa protein of leprosy and tubercle bacilli is recognized with specificity by antibodies against the human protein Lf. The Lf-65-kDa protein antigenic cross-reactivity may contribute to the formation of autoantibodies and immune complexes as well as to other autoimmune events that are frequent in tuberculosis and leprosy. Our immunocytochemical data also suggest that the cross-reactivity may persist for some time after the death of mycobacteria in infected hosts.

Ultrastructural characterization of normal and damaged membranes of Mycobacterium leprae and of cultivable mycobacteria.

Microdensitometry showed that the membrane profiles of normal cultivable mycobacteria were very asymmetric (outer layer denser and thicker than the inner layer), while the profiles of normal-looking M. leprae in lepromatous patients, in experimentally infected armadillos and in nude mice were approximately symmetric; moreover, the membrane of M. leprae was thicker than that of cultivable species. Using two cytochemical methods for the ultrastructural detection of periodic acid-Schiff (PAS)-positive molecules (the Thiéry procedure, and staining with phosphotungstic acid at low pH) we found that the membrane of cultivable mycobacteria, growing in vitro or in vivo, had PAS-positive components exclusively in the outer layer, while the normal-looking M. leprae in patients and in armadillos had membranes with PAS-positive components in both layers. The membranes of damaged cultivable mycobacteria, in vivo or in vitro, and of damaged M. leprae, in patients or armadillos, were PAS-negative.

Ultrastructure of Mycobacterium leprae and other acid-fast bacteria as influenced by fixation conditions.

A procedure using aldehydes, OsO4, Ca++ and uranyl acetate was selected for study of the fixation of Mycobacterium leprae in skin biopsies of leprosy patients. The ultrastructural pattern of recognized normal M. leprae cells fixed by the above procedure was characterized, and was found to be similar to that of other acid-fast bacteria fixed by the same procedure, except for the geometry of the membrane profile. Under such fixation conditions, this profile is always asymmetric in in vitro-cultured normal Nocardia asteroides, M. aurum and M. tuberculosis, whereas in skin biopsies no M. leprae cells with asymmetric membranes have been found so far. The implications of this observation for the interpretation of the ultrastructure of damaged M. leprae cells found in skin biopsies are discussed.