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Mol Microbiol ; 31(5): 1561-72, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10200973


The complex mycobacterial cell envelope is recognized as a critical factor in our failure to control tuberculosis, leprosy and other non-tuberculous pathogens. Although its composition has been extensively determined, many details regarding the organization of the envelope remain uncertain. This is particularly so for the non-covalently bound lipids, whose natural distribution may be disrupted by conventional biochemical or cytological techniques. In order to study the native organization of lipid domains in the mycobacterial envelope, we have applied a range of fluorescent lipophilic probes to live mycobacteria, including Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium gadium and Mycobacterium aurum, and analysed the resultant signals by fluorescence microscopy and digital image processing. Five key features were observed: (i) the presence of both envelope and intracellular lipid domains; (ii) differential localization of probes into these domains influenced predominantly by their hydrophobicity, as modelled by their calculated octanol:water partition coefficients and by their amphiphilicities; (iii) uneven distribution of lipophilic material in the envelope; (iv) selective labelling of septal regions of the envelope; and (v) modification of labelling patterns by additional treatments such as fluorescence quenching antibodies, detergents and solvents. Using this last approach, a coherent cell envelope lipid domain was demonstrated outside the cytoplasmic membrane and, for the first time, the proposed covalently linked mycolyl-arabinogalactan-peptidoglycan macromolecular complex was imaged directly. The use of fluorescent probes and high-resolution fluorescence microscopy has enabled us to obtain a coherent view of distinct lipid domains in mycobacteria. Further application of this approach will facilitate understanding of the role of lipids in the physiology of these organisms.

Corantes Fluorescentes/química , Metabolismo dos Lipídeos , Mycobacterium/metabolismo , Membrana Celular/metabolismo , Técnicas Microbiológicas/instrumentação , Microscopia Confocal , Mycobacterium/imunologia , Mycobacterium avium/citologia , Mycobacterium avium/metabolismo , Mycobacterium smegmatis/citologia , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/metabolismo , Polissorbatos/farmacologia , Tensoativos/farmacologia
Indian J Lepr ; 57(4): 739-49, 1985.
Artigo em Inglês | MEDLINE | ID: mdl-3915006


Improvement of the Redox System for growth of M. leprae as brought about by modification in the concentration and mode of preparation of individual media constituents, and by addition of newer substances, is being reported. A structural modification in the construction of the Thunberg's tubes and flasks that are used as culture vessels, has been introduced for ease of handling. Vitamin E (alpha-tocopherol) has been found to be useful. Concentrations of Liposomes and Gelatin in the medium could be reduced by at least five folds, considerably easing thereby smearing and harvesting of cultures. Dimercaptopropanol British Anti-lewisite or BAL) has been used, but its usefulness or otherwise is yet to be determined conclusively. The basis of intracellular parasitism of M. leprae has been discussed.

Meios de Cultura , Mycobacterium leprae/crescimento & desenvolvimento , Trifosfato de Adenosina , Alcanos/farmacologia , Animais , Sangue , Colesterol/farmacologia , Dimercaprol/farmacologia , Gelatina/metabolismo , Humanos , Lipossomos , Oxirredução , Penicilina G/metabolismo , Fosfatidilcolinas/farmacologia , Polissorbatos/farmacologia , Vitamina E/farmacologia , Vitamina K/farmacologia
Infect Immun ; 41(1): 121-7, 1983 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-6345387


Macrophage cultures pulsed with viable Mycobacterium leprae were assessed for erythrocyte rosetting in three groups of individuals, i.e., normal subjects, and tuberculoid and lepromatous patients. Of these, only the lepromatous group showed a reduction in rosetting ability after infection with M. leprae. The specificity of such a reduction pattern was confirmed by using various mycobacteria to infect the macrophages. A threshold effect was noted in all three groups. Although a reduction was obtained in the amount of rosetting of macrophages from lepromatous patients with 10(4) acid-fast bacilli per culture, tuberculoid and normal macrophages resisted such an effect with as large a dose as 20 X 10(6) to 30 X 10(6) and 30 X 10(6) bacilli per culture, respectively. The M. leprae-caused alterations in macrophages from lepromatous patients were reversible by treatment with trypsin and colchicine. Cytochalasin B and Tween 80 were unable to alter the pattern. Treatment of cells with neuraminidase was inconclusive since it enhanced rosetting values of both control and infected cultures. These manipulations were significant in elucidating the target point of the host (macrophage) and parasite (M. leprae) interaction and in delineation of the external and internal effects upon the macrophages. Both M. leprae and macrophages were participants in Fc reduction, as treatment of the former with rifampicin and of the latter with cyclocheximide significantly augmented the rosetting ability. In conclusion, it appears that M. leprae, upon entering a lepromatous macrophage, initiates the production of a protein which acts via the microtubules to alter membrane topography. It is possible that the altered membrane prevents effective macrophage-lymphocyte interaction. This could be one of the mechanisms by which cell-mediated immunity is suppressed in lepromatous leprosy.

Hanseníase/imunologia , Macrófagos/imunologia , Mycobacterium leprae/fisiologia , Animais , Células Cultivadas , Colchicina/farmacologia , Cicloeximida/farmacologia , Citocalasina B/farmacologia , Humanos , Macrófagos/microbiologia , Neuraminidase/farmacologia , Polissorbatos/farmacologia , Receptores Fc/fisiologia , Rifampina/farmacologia , Formação de Roseta , Tripsina/farmacologia
Int J Lepr Other Mycobact Dis ; 44(3): 294-300, 1976.
Artigo em Inglês | MEDLINE | ID: mdl-789261


The radiometric method has been applied for studying the metabolism of M. lepraemurium and the conditions which might force or inhibit its metabolic activity in vitro. These organisms assimilate and oxidize (U-14C) glycerol, and (U-14C) acetate, but are unable to oxidize (U-14C) glucose, (U-14C) pyruvate, (U-14C) glycine and 14C-formate. When incubated at 30 degrees C M. lepraemurium oxidizes (U-14C) acetate to 14CO2 faster than 37 degrees C. The smae effect was observed with increasing concentrations of polysorbate 80 (Tween 80), or the 14C-substrate. No change in metabolic rate was observed when the organisms were kept at -20 degrees C for 12 days. Although tried several times, it was not possible to demonstrate any "inhibitors" of bacterial metabolism in the reaction system. The radiometric method seems to be an important tool for studying metabolic pathways and the influence of physical and biochemical factors on the metabolism of M. lepraemurium in vitro.

Mycobacterium lepraemurium/metabolismo , Acetatos/metabolismo , Animais , Dióxido de Carbono/biossíntese , Radioisótopos de Carbono , Feminino , Congelamento , Glucose/metabolismo , Glicerol/metabolismo , Glicina/metabolismo , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos , Ácidos Oleicos/farmacologia , Polissorbatos/farmacologia , Piruvatos/metabolismo