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.

La citoestructura de Mycobacterium leprae en pellet y vacuolas fagocíticas del nódulo hanseniano activo / Structure of Mycobacterium leprae in pellet and phagocitic vacuoles of active hansen node

Se hace estudio ultraestructural del bacilo de Hansen a 20.000 x en pellet y en vacuola del nódulo hanseniano activo. Se observa su pared celular, membrana plasmática mesosoma y nucleoide. En su citoestructura no se diferencia de otras bacterias ya sean éstas Gram positivas o Gram negativas. Solo las diferencia su ácido resistencia la cual le da sus características tintoriales especiales

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.

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.

Electron microscopic observations of intracytoplasmic inclusions in human and murine leprosy bacilli.

The intracytoplasmic inclusions of Mycobacterium leprae in human lepromata and M. lepraemurium in murine lepromata were studied in ultrathin serial sections at the electron microscopic level. The inclusions were mostly homogeneous and spherical, and did not exist uniformly throughout the bacillary cells. They did not appear to be delimited by membranous structures and apparently had no internal structure. There seemed to be fundamentally little difference between M. leprae and M. lepraemurium in the fine structure of these inclusions. However, the large diffuse inclusions observed in the cells of M. lepraemurium may be a special feature of murine bacilli.

Morphology of bacteriophages from Mycobacterium lepraemurium.

The two phages obtained from mycobacterium lepraemurium strain "Douglas" by using M. segmatis ATCC 607 as a non-lysogenic indicator (kindly supplied by L. Sula and named as AL1 and 1/1) were adsorbed on M. Smegmatis ATCC 607 with a P 45'/P0 of 0.046 and 0.01, respectively. Ultrastructural studies showed that these phages could be classified as Bradley type B1.

Ultrastructural features of the multiplication of human and murine leprosy bacilli in macrophages of nude mice.

Ultrastructural features of the growth of M. leprae and M. lepraemurium in nude mouse macrophages were studied by ultrathin sectioning and freeze-etching. In nude mouse macrophages, M. leprae produced spherical droplets (foamy structures) similar to those in human lepra cells. On the other hand, M. lepraemurium produced typical crystalline material in nude mouse macrophages, which is quite the same as that observed in the C3H strain mouse, Spherical droplets in the form of foamy structures seem to be made up of a specific substance produced by the multiplication of M. leprae in suitable host cells (human, nude mouse, and armadillo macrophages).

Growth of Mycobacterium lepraemurium in cell-free liquid medium containing soluble starch: evaluation by generation time and morphological observation.

A stimulatory effect of soluble starch on the growth of M. lepraemurium in vitro cell-free culture system has been noted. In this medium bacteria elongated gradually without manifesting any significant peak in elongation as has been noted in NC-5 medium. The maximum average elongation of bacteria was 2.84 mu on the 24th day in NCS-5 medium. In contrast, in NC-5 bacteria elongated maximum on the 6th day (2.32 mu). All the stages of bacterial cell division were noted. Hypothesis has been suggested for continuous multiplication of M. lepraemurium in NCS-5 medium.

A relationship between the length of bacilli and the log phase of growth of Mycobacterium lepraemurium in cell-free liquid medium.

A relationship between the early phase of growth of M. lepraemurium in vitro in NC-5/ND-5 media and elongation of bacteria has been noted. The doubling time in NC-5 is 3.06 days whereas it is 1.97 days in ND-5 medium. In ND-5 medium, M. lepraemurium elongates earlier to that in NC-5. Furthermore, in ND-5 the peaks in elongation of bacteria are more frequently noted than in NC-5 medium. Morphological observations have shown all stages of bacterial cell-division. At later stages of cultivation (21st and 24th day) M. lepraemurium often shows terminal swelling in ND-5 medium which also revealed stages of cell-division.

Partial inhibition of the growth of Mycobacterium lepraemurium in C3H mice immunized with cell wall skeletons.

C3H mice stimulated with M. lepraemurium cell wall skeletons (LM-CWS) were challenged with viable M. lepraemurium, and delayed-type hypersensitivity (DTH) to picryl chloride was measured. In one type of experiment the mice were challenged at the time when stimulation of cell-mediated resistance by means of LM-CWS was undertaken. The major purpose was to investigate principles pertaining to immunotherapy. In contrast to the loss of a detectable DTH response to picryl chloride, development of murine leprosy was partially suppressed. In the second type of experiment the mice were stimulated with oil-attached LM-CWS seven weeks before challenge with M. lepraemurium. Findings were: a) that nonspecific DTH as measured by sensitization and challenge with picryl chloride was activated before the infection with M. lepraemurium and b) that the DTH which developed was associated with partial protection against the growth of lepromata. The murine leprosy which developed in C3H mice stimulated with LM-CWS was progressive after some delay.

Comparative ultrastructure of Mycobacterium leprae and Mycobacterium lepraemurium cell envelopes.

The structural properties of the cell envelopes of Mycobacterium leprae and Mycobacterium lepraemurium were investigated by freeze-fracture, freeze-etching, and negative-staining techniques. Freeze-fracture split the cell wall and exposed the internal features of the peptidoglycolipid mycosidic filamentous network. The cell membrane was also split into two asymmetric faces. The external fracture face was characterized by linear arrays of intramembranous particles, whereas the protoplasmic fracture face showed randomly distributed clusters of particulate entities. Comparative analysis of the ultrastructural features observed in M. leprae and M. lepraemurium indicated that the organization of the cell envelope in these two species differed particularly with respect to the amount and complexity of the superficial peptidoglycolipid and mycosidic integument, which is poorly developed in the mycobacterium responsible for human disease.

Phagosome-lysosome fusion and cyclic adenosine 3':5'-monophosphate in macrophages infected with Mycobacterium microti, Mycobacterium bovis BCG or Mycobacterium lepraemurium.

When ingested by mouse peritoneal macrophage monolayers, live Mycobacterium microti caused a sustained increase in monolayer cyclic AMP content and fusion of lysosomes with the bacterium-containing phagosomes was impaired. Ingested live M. bovis BCG caused a transient increase in cyclic AMP and the defect in phagolysosome formation was less pronounced. Dead mycobacteria and live M. lepraemurium neither enhanced monolayer cyclic AMP content nor inhibited phagolysosome formation. Mycobacterium microti and BCG exceeded M. lepraemurium in cyclic AMP-synthesizing activity in vitro but the question of whether bacterial cyclic AMP contributed substantially to the increments in infected macrophages was not resolved. Antibody-coated BCG retained the ability to synthesize cyclic AMP and to enhance monolayer cyclic AMP but lost the ability to inhibit phagolysosome formation in macrophages, The observations are discussed in terms of possible control of phagolysosome formation by cyclic nucleotides.

Freeze-etching study of human and murine leprosy bacilli.

Morphologic features of the electron transparent zone (ETZ) material around human and murine leprosy bacilli were examined by a freeze-etching technic. The ETZ around human leprosy bacilli is composed of spherical droplets of hydrophobic material. These are always liquid at body temperature and they never show crystalline lamellar structure even at the temperature of liquid nitrogen. The ETZ around murine leprosy bacilli is composed of ribbon-like or membranous crystalline structures. This material is solid and crystalline at the body temperature of mice, and this solid material is the chief cause of the random arrangement of murine leprosy bacilli inside the cytoplasm of murine lepra cells. This crystalline structure has also been observed around murine leprosy bacilli grown on cell-free culture media.

Cytochemical evidence for aerobic pathways in Mycobacterium lepraemurium.

Three enzymes of aerobic pathways (cytochrome c oxidase, peroxidase and catalase) and one key enzyme of the tricarboxylic acid cycle (succinate dehydrogenase) were investigated for their ultrastructural localization in M. lepraemurium in infected mouse liver and in cultures of M. fortuitum as a control. All four enzymes were localized in M. fortuitum. To M. lepraemurium only cytochrome c oxidase and peroxidatic activity were detected. The localization of the latter enzyme activity was different compared with M. fortuitum. Succinate dehydrogenase was not detected in M. lepraemurium but rather surprisingly was found in the membrane of the phagosomes containing the bacteria. It is concluded that M. lepraemurium can function aerobically and has either a glyoxalate pathway or is an obligate autotroph.

Growth of Mycobacterium lepraemurinum in the mouse bone marrow: an ultrastructural study.

The ultrastruct of the mouse bone marrow during the first 8 weeks after intravenous inoculation of animals with 10(9) Mycobacterium lepraemurium is described. The bacteria were almost exclusively in macrophages, which became converted to epithelioid cells after 8 weeks, at which time they were very heavily infected. The nature of the exceptionally rapid increase in numbers of bacteria in the bone marrow compared with other tissues early in the infection is discussed. It is concluded that a short doubling time of bacteria situated in the marrow is a more probable explanation than recruitment from elsewhere in the animal.