Deciphering the proteomic profile of Mycobacterium leprae cell envelope.

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high-throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.

Modulation of the stability of rabbit skeletal muscle myosin light chain kinase through the calmodulin-binding domain.

The binding of Ca2+(4).calmodulin (CaM) to rabbit skeletal muscle myosin light chain kinase (MLCK) is required for expression of the enzyme's activity. While both MLCK and CaM were stable at 30 degrees C, their complex was not. The binding of CaM to MLCK resulted in a time- and temperature-dependent inactivation that reflected an intrinsic instability of the complex. Separation of the components of the inactive complex yielded functional CaM, but catalytically inert MLCK, indicating that the site of the inactivating event was confined to MLCK. The behavior of proteolytic fragments further localized this event to the C-terminal 60% of the 603-residue protein. Changes in the tryptophan fluorescence and proteolytic susceptibility of MLCK-CaM indicated that a conformational change accompanied, and thus may have caused, inactivation. Substrates protected against inactivation, as did millimolar concentrations of Mg2+, Mn2+, and Ca2+. These metals appeared to bind to a site on MLCK distinct from that which recognized Mg2+.ATP. A proteolytic fragment of MLCK lacking the ability to bind CaM, C beta 35 (residues 255-584; Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. G. (1985) J. Biol. Chem. 260, 11275-11285), was unstable at 30 degrees C, whereas a similar fragment which does bind CaM, T beta 40 (residues 236-595; Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. (1985) J. Biol. Chem. 260, 11275-11285), was unstable only when CaM was bound.

An electron microscopic study of alterations in the morphology and permeability of purified Mycobacterium leprae.

This communication reports the association of changes in ultrastructure of Mycobacterium leprae with alterations in its permeability. To study morphologic changes of the organisms under different conditions (of temperature and exposure to NaOH and trypsin), ultrathin sections of the bacteria were cut and examined in an electron microscope. In the untreated bacilli and those washed with trypsin, the cytoplasmic membrane and the cell wall (peptidoglycan layer) remained intact; dapsone showed little effect on diphenoloxidase of the bacteria. M. leprae is unique among mycobacteria in possessing an unusual form of the enzyme diphenoloxidase. The antileprosy drug dapsone is a potent inhibitor of the enzyme, but it does not readily penetrate the bacteria where the cell envelope remains intact. The cell wall of M. leprae exposed to -80 degrees C or washed with NaOH was partially detached from the cell membrane; dapsone readily penetrated these organisms and inhibited the bacterial enzyme. In the above preparations, the cytoplasmic membrane appeared undamaged and the bacteria remained viable, as evidenced by multiplication in mouse foot pads. At 50 degrees C, the peptidoglycan layer became completely separated from the membrane and the cytoplasm was partially denatured. These organisms were permeable to dapsone, but were no longer viable. At 100 degrees C, the structural organization of the bacilli was completely destroyed, and of course, they lost their enzyme activity as well as viability. Evidently, the intact cell wall layer mediates the exclusion of dapsone from M. leprae, and there is no correlation between its viability and permeability. The ultrathin sections also reveal the internal organization and cytoplasmic inclusions of M. leprae, as never before seen.

Trypsin-treated and coomassie blue-stained epimastigote antigen in a microagglutination test for Chagas' disease.

A microagglutination test using trypsin-treated and Coomassie blue-stained Trypanosoma cruzi epimastigote antigen was adapted for the diagnosis of Chagas' disease. When incorporated in the test, 2-mercaptoethanol treatment of chagasic sera had no influence on antibody titer. In contrast, titers in sera from patients with visceral leishmaniasis, African trypanosomiasis, and autoimmune disorders, subjected to similar treatment, showed remarkable decline. Accordingly, a lower cut-off point for Chagas' disease serological negativity could be taken resulting in a higher sensitivity (95.6%); the specificity was 94.7%. Similar specificities were obtained with Leishmania donovani chagasi and L. d. donovani antigens applied to homologous visceral leishmaniasis and heterologous Chagas' sera. Of 316 nonchagasic sera, only 3 with leptospirosis and 1 with leprosy showed seropositive titers prior to and after 2-mercaptoethanol treatment.

Temperature-induced changes in viability, diphenoloxidase and permeability of Mycobacterium leprae.

Among mycobacteria secretion of the enzyme diphenoloxidase has been established as a property of Mycobacterium leprae. The antileprosy drug dapsone (DDS), which completely inhibits the enzyme from plant and mammalian sources, does not readily penetrate intact M. leprae. When the drug is complexed with polylysine, it easily permeates the bacteria and produces 100% inhibition of its diphenoloxidase, suggesting a permeability barrier of the cytoplasmic membrane of M. leprae to dapsone. In this study: (1) when the organisms, purified from fresh tissues of experimentally infected armadillos, were treated with dilute alkali or exposed to warmer temperatures, DDS penetrated the bacteria and inhibited the diphenoloxidase. Washing with trypsin had no effect. Dapsone easily permeated the bacilli, purified from tissues stored at 0 degrees C or at -80 degrees C. (2) Diphenoloxidase of freshly-prepared M. leprae was stimulated when the bacteria were exposed to 50 degrees C for 10 min; at 60 degrees C the activity decreased, and at 100 degrees C the enzyme was completely inactivated. When the enzyme was assayed at temperatures below 37 degrees C, the activity was considerably lower, indicating that M. leprae may not be a psychrophilic organism in this respect. (3) The bacteria exposed to 50 degrees C failed to multiply in mouse footpads. M. leprae remained viable in tissues stored at 0 degrees C or -80 degrees C; but when the bacteria purified from these tissues were frozen, they lost their viability. On the other hand, the organisms separated from fresh tissues remained viable when frozen at -80 degrees C. The inhibition of diphenoloxidase of M. leprae by dapsone could serve as an indirect method to assess the integrity of the bacterial cell membrane and to predict whether the bacteria would retain their viability on freezing.

Antigen specific macrophage-lymphocyte interaction in lepromatous leprosy.

Peripheral blood derived macrophages from lepromatous leprosy patients were unable to interact with lymphocytes in the presence of M. leprae. This lack of interaction is probably not associated with membrane HLA-Dr antigens since trypsin and colchicine restored M. leprae induced depression in the latter but were unable to bring about a positive interaction. Two possible defects exist therefore in the lepromatous macrophage. These are an innate inability to process and present M. leprae antigens to lymphocytes and an induced inability to express some membrane receptors, an event detrimental to the normal functioning of a macrophage.

Alterations in the membrane of macrophages from leprosy patients.

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.

Permeability of Mycobacterium leprae to dapsone: alteration by purification procedures.

Permeability of Mycobacterium leprae to dapsone in vitro was determined by the ability of the drug to inhibit o-diphenoloxidase of the bacilli. Dapsone showed little effect on the enzyme activity of the intact organisms. When the M. leprae preparations were washed with trypsin, NaOH, or acetone and ether, DDS penetrated the bacillus to inhibit its o-diphenoloxidase. The method might be useful in studying the utilization of added metabolites by purified M. leprae suspensions.