Subcellular localization of the 65-kDa heat shock protein in mycobacteria by immunoblotting and immunogold ultracytochemistry.

The 65-kDa heat shock protein (hsp65) is an immunodominant antigen in mycobacterial infections and also the key etiologic factor in mycobacteria-induced autoimmune arthritis. Because the subcellular distribution of hsp65 in the mycobacteria may be relevant to understand its immunoreactivity, we have investigated the presence of hsp65 in the envelope and cytoplasmic compartments of the bacilli. Anti-hsp65 antibodies were used in western blottings to investigate the presence of hsp65 in cell fractions (membrane, envelope and cytosol) of Mycobacterium avium and M. smegmatis, and also to label hsp65 in situ by the immunogold method on thin-sectioned mycobacteria, including the non-cultivable M. leprae, that were studied by transmission electron microscopy. All of the three subcellular mycobacterial fractions showed significant labelling by anti-hsp65 antibodies. Immunogold ultracytochemistry revealed the presence of hsp65 in both the cytoplasm and the envelope of mycobacteria. The data indicate that hsp65 molecules are commonly present not only in the cytoplasm but also in the envelope of mycobacteria. The latter topography of hsp65 may contribute to the strong immunogenicity of hsp65 since it may correspond to export hsp65 molecules captured before being secreted into the extracellular milieu, thus making hsp65 a mycobacterial antigen readily available for presentation to the immune system of infected hosts.

New data on the ultrastructure of the membrane of Mycobacterium leprae.

In previous reports on the ultrastructure of Mycobacterium leprae, we described the occurrence of symmetric membranes in normal-looking bacilli from fresh or frozen samples primarily fixed with aldehydes. In those reports we admitted that such a symmetric profile, which is not found in the other normal mycobacteria, would not represent the structure of the normal membrane of the leprosy bacillus. We, therefore, re-analyzed the ultrastructure of the membrane of M. leprae. In the present work the micromorphology of the M. leprae membrane was studied by transmission electronmicroscopy after the fixation of fresh samples by OsO4 plus calcium followed by glutaraldehyde plus formaldehyde and calcium followed by uranyl acetate. The study of samples from two patients with lepromatous (LL) leprosy, three armadillos with natural leprosy, and one nude mouse with experimental leprosy showed that normal-looking bacilli present in lead-stained sections had asymmetric membranes with a thickness of 6.49 +/- 0.36 nm. These membranes showed periodic acid-Schiff (PAS)-positive components exclusively located in the outer half of the bilayer. We demonstrated that the symmetric profile of the M. leprae membrane described in our previous reports corresponds, as admitted in those reports, to an abnormal membrane structure. Such an abnormality was now found to result from the use of primary fixation with aldehydes or of samples stored frozen before fixation. These results indicate that, although ultrastructurally similar to that of the other mycobacteria, the membrane of M. leprae has a peculiar sensitivity to fixation by aldehydes. Such a characteristic, which was not found in M. lepraemurium, M. aurum, M. avium, and M. tuberculosis H37Ra, must reflect a unique membrane molecular structure, which is presently unknown.

New data onthe ultrastructure of the membrane of Mycobacterium leprae

In previous reports on the ultrastructure of Mycobacterium leprae, we described the occurrence of symmetric membranes in normal-looking bacilli from fresh or frozen samples primarily fixed with aldehydes. In those reports we admitted that such a symmetric profile, which is not found in the other normal mycobacteria, would not represent the structure of the normal membrane of the leprosy bacillus. We, therefore, re-analyzed the ultrastructure of the membrane of M. leprae. In the present work the micromorphology of the M. leprae membrane was studied by transmission electronmicroscopy after the fixation of fresh samples by OsO4 plus calcium followed by glutaraldehyde plus formaldehyde and calcium followed by uranyl acetate. The study of samples from two patients with lepromatous (LL) leprosy, three armadillos with natural leprosy, and one nude mouse with experimental leprosy showed that normal-looking bacilli present in lead-stained sections had asymmetric membranes with a thickness of 6.49 +/- 0.36 nm. These membranes showed periodic acid-Schiff (PAS)-positive components exclusively located in the outer half of the bilayer. We demonstrated that the symmetric profile of the M. leprae membrane described in our previous reports corresponds, as admitted in those reports, to an abnormal membrane structure. Such an abnormality was now found to result from the use of primary fixation with aldehydes or of samples stored frozen before fixation. These results indicate that, although ultrastructurally similar to that of the other mycobacteria, the membrane of M. leprae has a peculiar sensitivity to fixation by aldehydes. Such a characteristic, which was not found in M. lepraemurium, M. aurum, M. avium, and M. tuberculosis H37Ra, must reflect a unique membrane molecular structure, which is presently unknown

Methionine as methyl-group donor in the synthesis of Mycobacterium avium envelope lipids, and its inhibition by DL-ethionine, D-norleucine and DL-norleucine.

The radioactivity from 3H-methyl methionine was rapidly incorporated into the surface lipids of Mycobacterium avium. The transmethylation reaction was efficiently inhibited by DL-ethionine, D-norleucine and DL-norleucine. The structure of the outerlayer of the M. avium envelope was profoundly altered in the bacteria treated with DL-norleucine.

Macrophage interaction with mycobacteria including M. leprae.

Resistance properties of pathogenic mycobacteria to macrophage bactericidal activity seems to be due mostly to the composition and constitution of their cell walls. In the case of Mycobacterium tuberculosis, sulfatides and polyglutamic acid could be implicated in the phenomenon of fusion inhibition between phagosomes and lysosomes. M. leprae and M. lepraemurium, which do not seem to inhibit fusions are protected by a thick electron transparent zone (ETZ) that seems to be composed of mycosides. This layer would inhibit lysosomal enzyme diffusion inside phagosomes. As ETZ does not exist in mycobacteria before their phagocytosis, we have tried to see when and how it is formed inside macrophages. We have compared ETZ formation in M. leprae and M. avium which both contain mycosides. These two species were allowed to be phagocytized by mouse bone-marrow derived macrophage and samples were taken for electron microscopy during the first hours of phagocytosis and also during several weeks of incubation. In M. avium ETZ appeared within 1 to 2 hours after phagocytosis. It seems to be formed by a sort of swelling of the thin electron transparent layer of the bacterial cell wall. This swelling occurs only in regions where the external polysaccharide layer of M. avium starts to disappear. After 1 to 3 hours, this layer was completely absent and all bacteria were enveloped in a thick ETZ. In M. leprae, the ETZ is also formed within one hour after ingestion. However, the presence in some bacteria of a very thin dense layer located at the original place of the outer dense layer of the cell wall does not fit well with the idea of ETL swelling. In addition, the appearance of a thick dense layer located between the ETZ and the phagosome membrane is not yet understood. The ETZ formed also rapidly in macrophages infected with heat killed cells of M. avium or M. leprae. This shows that its formation does not require the active participation of the bacterium. As already proposed ETZ seems to lessen considerably the diffusion of lysosomal enzymes towards the bacterium in both species. In M. leprae it seems especially efficient because despite acid phosphatase activity found in many phagosomes, neither the number of bacteria per macrophage nor their state of degradation changed during 3 and a half months of macrophage culture.(ABSTRACT TRUNCATED AT 400 WORDS)

Freeze-etching and freeze-fracture structural features of cell envelopes in mycobacteria and leprosy derived corynebacteria.

The structural properties of the cell wall and cell membrane of several mycobacteria and of Leprosy Derived Corynebacteria are investigated by freeze-etching and freeze-fracture. In all cases the freeze-fracture split the cell wall in two asymmetric halves. The cell wall fracture faces of the mycobacteria are characterized by a filamentous network which vary with respect to the amount and complexity among microorganism of the same species and even more of different species. In LDC the structure organization of the cell wall and cell membrane differs from that of mycobacteria. The most stricking difference is the presence on the fracture faces of the LDC cell wall of different classes of particulated entities of yet unknown nature. In the mycobacteria and LDC the periseptal annuli likely provide a potential frame for cell envelope and cell membrane assembly.

Cytochemical characterization of mycobacterial outer surfaces.

A cytochemical study of mycobacterial outer surfaces was carried out on both pathogenic (M. leprae, M. avium) and non pathogenic (M. aurum) strains. Different cytochemical markers were used: Ruthenium Red, Concanavalin A, Wheat Germ Agglutinin, Colloidal Iron and Cationized Ferritin. The cytochemical staining pattern varied according to the species studied. The relationship between outer surface properties of mycobacteria and their capacity of adhesion to or ingestion by bone marrow macrophages was also considered.