Partial characterization of the cell walls ofMycobacterium leprae

Cell walls were prepared fromMycobacterium leprae (separated and purified from experimentally infected armadillo),M. tuberculosis, M. smegmatis, andMicrococcus lysodeikticus. The purity of the above wall preparations was confirmed after negative staining and shadow-casting and subsequent observation under the electron microscope. As judged from the electron microscopic observations, the bacteria were of different fragility in the following increasing order:M. tuberculosis, M. smegmatis, M. leprae, andMicro. lysodeikticus. The cell walls were hydrolyzed with 6N HCl, and the amino acids were identified by thin-layer chromatography compared with the authentic standards. With the same purification procedures, it was not possible to obtain satisfactorily pure peptidoglycan fromM. leprae. In leprosy bacilli,meso-DAP was found to be present, ín the walls; however, contamination by nonwall amino acids did not allow the confirmation of previous results, a finding that suggests that glycine completely replaced L-alanine inM. leprae cell walls.     

Ultrastructure of “Gordona aurantiaca” Tsukamura 1971

Ultrastructure of Gordona aurantiaca^* M 296 (8128) was studied after the lead citrate coloration, whereas the cell envelope architecture was investigated by ruthenium red staining for outer wall acidic polysaccharides and the periodic-acid-thiocarbohydrazide-silver-proteinate cytochemical procedure (Thiéry method) for the detection of 1-2 glycol bond containing polysaccharides. The ultrastructural morphology of bacteria was distinct from both the mycobacteria and nocardia. The bacilli had a typical gram-positive cell wall that contained a thin, uniformly distributed, polysaccharide outer layer (POL) at its surface. The Thiéry cytochemical method stained only the cytoplasmic membrane, but not the cell wall, a feature that is common to the mycolic acid containing theCorynebacterium-Mycobacterium-Nocardia (CMN) group of organisms. The negative staining of the unfixed preparations of bacilli showed ribbonlike surface structures, common to the CMN group of organisms. The electron-microscopic preparations showed numerous lysing bacilli with bacteriophages indicating that the strain used was lysogenic.     

Comparative modeling of UDP-N-acetylmuramoyl-glycyl-D-glutamate-2, 6-diaminopimelate ligase from Mycobacterium leprae and analysis of its binding features through molecular docking studies

Leprosy is an infectious disease caused by Mycobacterium leprae. The increasing drug and multi-drug resistance of M. leprae enforce the importance of finding new drug targets. Mycobacterium has unusually impermeable cell wall that contributes to considerable resistance to many drugs. Peptidoglycan is an important component of the cell wall of M. leprae. UDP-N-acetylmuramoyl-glycyl-D-glutamate-2, 6-diaminopimelate ligase (MurE) plays a crucial role in the peptidoglycan biosynthesis and hence it could be considered as a potential drug target for leprosy. Structure of this enzyme for M. leprae has not yet been elucidated. We modeled the three-dimensional structure of MurE from M. leprae using comparative modeling methods based on the X-ray crystal structure of MurE from E. coli and validated. The 3D-structure of M. leprae MurE enzyme was docked with its substrates meso-diaminopimelic acid (A2pm) and UDP-N-acetyl muramoyl-glycyl-D- glutamate (UMGG) and its product UDP-N-acetyl muramoyl-glycyl-D-glu-meso-A₂pm (UTP) and also with ATP. The docked complexes reveal the amino acids responsible for binding the substrates. Superposition of these complex structures suggests that carboxylic acid group of UMGG is positioned in proximity to γ-phosphate of the ATP to facilitate the formation of acylphosphate intermediate. The orientation of an amino group of A₂pm facilitates the nucleophilic attack to form the product. Overall, the proposed model together with its binding features gained from docking studies could help to design a truly selective ligand inhibitor specific to MurE for the treatment of leprosy.     

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.     

Ultrastructural Visualization of Glucide Determinants in Membranes of Mycoplasma-like Organisms

Periodic acid-thiocarbohydrazide-silver proteinate (PATAg) reaction for poiysaccharide localization was performed on ultrathin sections of Spurr resin-embedded tissues of Physostegia virginina and Catharanthus roseusknown to be infected by mycoplasma-like organisms (MLO). Electron microscopy of PATAg-treated sections revealed heavy silver deposition on hmiting membranes of all MLO. It was not possible to detect differences m silver deposition within the multi stratified membrane structure because of masking by silver-grain deposition. Silver deposition was not found in the cytoplasmic region of the MLO. The results indicate the presence of glucide determinants only in the limiting membrane of the MLO. The significance of this finding is discussed.     

Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae

Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an‘up-regulated’promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The avaibility of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients.     

Carbohydrate-Dependent Binding of Langerin to SodC,a Cell Wall Glycoprotein of Mycobacterium leprae

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [K_D] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (K_D = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells.     

Fine structure ofMicrococcus denitrificans andM. halodenitrificans in relation to their taxonomy

A study of ultrathin sections ofMicrococcus denitrificans andM.halodenitrificans has shown similar cell structures. The cell wall consists of several layers corresponding to those of the cell wall of gram-negative bacteria. The thickness of the cell wall is 250 – 350 Å; that of the cytoplasmic membrane 70 Å. The cytoplasm in both species contains ribosomes and inclusions of polymetaphosphate. Comparison with ultrathin sections ofThiobacillus novellus shows too much difference to consider the former two species to be identical with the latter one. The taxonomic position ofM.denitrificans andM.halodenitrificans is discussed.Deceased 3 July 1967.     

Superoxide production from macrophages of leprosy patients after stimulation withMycobacterium leprae

The macrophages from peripheral blood of normal healthy individuals respond to live or killedMycobacterium leprae by producing superoxide. On the other hand, the macrophages from bacteriologically positive (B +LL) or long term treated bacteriologically negative (B -LL) and tuberculoid leprosy patients are unable to produce superoxide when stimulated with liveMycobacterium leprae. While killedMycobacterium leprae induce superoxide with the cells from tuberculoid andB(-)LL patients, cells fromB(+)LL patients fail to respond. The deficiency inB(-)LL patients to produce superoxide appears to be specific withMycobacterium leprae and the defect can be counteracted by the addition of colchicine. These observations indicate a preexisting membrane disposition which does not favour superoxide production. A similar situation is seen in the cells from tuberculoid leprosy patients. Thus it appears that both cured and active lepromatous leprosy patients have defective macrophages, unable to respond to liveMycobacterium leprae to produce superoxide anion, in contrast to the situation with the cells from normal healthy individuals.     

Iron-regulated envelope proteins of mycobacteria grown in vitro and their occurrence inMycobacterium avium andMycobacterium leprae grown in vivo

Summary Several iron-regulated envelope proteins (IREPs), 11–180 kDa, have been detected in preparations of walls and membranes ofMycobacterium smegmatis, in an armadillo-derived mycobacterium (ADM) and inM. avium. The same sized proteins fromM. vaccae appeared under both iron-deficient and iron-sufficient growth conditions. Two larger proteins, of 240 and 250 kDa, appeared in the membranes ofM. smegmatis andM. avium only when grown iron-sufficiently but were constitutively present in both ADM andM. vaccae. The IREPs fromM. smegmatis were not induced under zinc-deficient growth conditions. Three of the four IREPs (14, 21 and 29 kDa) recognized inM. avium grown in vitro were also recovered from membrane fractions of the same strain grown in mice. In addition, these membranes contained both the high-molecular-mass proteins associated with iron-sufficient growth conditions. Membranes ofM. leprae, recovered from infected armadillos, showed the faint presence of a possible IREP at 29 kDa and wall preparations showed the presence of a 21-kDa protein. Membranes also contained the two larger proteins at 240 and 250 kDa. An explanation for the simultaneous occurrence of both low-iron-regulated and high-iron-regulated proteins is offered.     

THE THICKENED LEPTOID (SIEVE ELEMENT) WALL OF DENDROLIGOTRICHUM (BRYOPHYTA): CYTOCHEMISTRY AND FINE STRUCTURE

Leptoids (sieve elements) of Dendroligotrichum exhibit unevenly thickened lateral walls. The thickened wall areas are predominantly confined to the radial walls. With the light microscope the thickened wall cannot be resolved into distinct layers, but rather is optically homogeneous. Standard histochemical tests reveal that these walls are rich in cellulose (birefringent; IKI-H₂SO₄-positive) with small amounts of polyuronides (toluidine blue-positive) and pectins (hydroxylamine-positive) and are non-lignified. They also contain abundant natural aldehydes as revealed by the Schiff, silver hexamine, and silver proteinate reagents. Aldehyde blockades (sodium borohydrate, sodium chlorite) confirmed the presence of aldehyde groups in the cell wall. At the ultrastructural level, the lateral walls of sieve elements react strongly with uranyl and lead salts and yield little fine structural information. Electron cytochemical localization of aldehydes with silver proteinate revealed three distinct wall regions: outer, middle and inner. The outer and middle regions appeared polylamellate while the inner region contained no reaction product. The nacreous sieve elements of vascular plants are compared to the thickened sieve elements in bryophytes.     

Correlation of bacterial viability with uptake of [14C] acetate into phenolic glycolipid-1 ofMycobacterium leprae within Schwannoma cells

The viability ofMycobacterium leprae, maintained within 33B Schwannoma cells, was estimated in terms of incorporation of [¹⁴C] acetate into its specific phenolic glycolipid-1. This measure of viability was correlated with two other assays,viz., fluorescein diacetate/ethidium bromide staining and mouse footpad growth. Observation of a 2-fold increase in the number of intracellularMycobacterium leprae over an experimental period of 12 days also corroborated this contention. Furthermore, on addition of anti-leprosy drugs to these intracellularMycobacterium leprae there was significant decrease in phenolic glycolipid-1 synthesis indicative of loss of viability of the organisms. This study also established the importance of the host cell for active bacillary metabolism, asMycobacterium leprae maintained in cell-free conditions showed no incorporation into phenolic glycolipid-1. Moreover, compromising the host’s protein synthesis capacity with cycloheximide, also led to reduction in bacillary metabolism. As this system measures the metabolic synthesis of a uniqueMycobacterium leprae component, it would be useful for development and screening of compounds acting against specific bacillary targets.     

Guanine plus cytosine content of theMycobacterium avium complex and other mycobacteria by high-performance liquid chromatography analysis of deoxyribonucleotides

DNA was extracted from 12 clinical isolates of theMycobacterium avium-M. intracellulare-M. scrofulaceum serocomplex (including serovars 1, 2, 3, 4, 8, 42A, and 43), as well as from nine reference strains of other mycobacterial species. Rapid nitrogen decompression was used to weaken the cells for subsequent DNA extraction by a modified Marmur procedure. The DNA was hydrolyzed with phosphodiesterase, and the concentration of released deoxyribonucleotides was determined by reversed-phase high-performance liquid chromatography. The data were used to calculate the mole percentage guanine plus cytosine. Values ranged from 58.6 forM. leprae to 71.5 forM. avium serovar 3. The lowest value for theM. avium-M. intracellulare-M. scrofulaceum serocomplex was 66.3 forM. scrofulaceum serovar 42A. The data indicate that there is greater genetic variability for this group of organisms than is suggested by diagnostic clinical tests and seroagglutination reactions.     

Expression of the 18kDa protein of Mycobacterium leprae in Saccharomyces cerevisiae

Summary Mycobacterium leprae, the etiologic agent of leprosy, until now has not been grown in vitro, resulting in exceedingly obstacles for the production of purified antigens. It is therefore of interest to clone the relevant M.leprae antigens in other easy to handle microbial hosts. Here, we describe two different systems for expressing the 18kDa antigen of M.leprae in S. cerevisiae. Each system was shown to be effective in antigen expression, but the secretion system provided easier purification. Working with different host strains under different growth conditions, large quantities of biologically active proteins were obtained.     

The karyotypes ofMicroseris bigelovii,M. douglasii,andM. pygmaea (Asteraceae)

The karyotypes of the three annuals,Microseris bigelovii, M. douglasii andM. pygmaea, consist of 2n = 18, small, submetacentric chromosomes. Length, centromere position, C-banding pattern, silver staining of NOR's, and the use of base specific fluorochromes, allow the identification of four of the nine chromosome pairs. The banding pattern ofM. bigelovii andM. pygmaea is identical, but intraspecific differences are found between strains ofM. douglasii.     

High resolution shadowing of Mycobacterium leprae

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.     

In vitro drug screening system using membrane alteration in macrophages byMycobacterium leprae

The observation that liveMycobacterium leprae on entry into macrophages from lepromatous leprosy patients reduced the number of EA rosetting macrophages, was extended to macrophages from Swiss white mice also. Further, the fact that deadMycobacterium leprae do not bring about such a change in macrophages from mice, allowed us to develop this into a bacterial viability testing system. Thus drug treated macrophages in the presence ofMycobacterium leprae showed normal rosetting ability ifMycobacterium leprae are inactivated by the drug, but showed reduced level of rosetting when bacteria were not susceptible to the drug. It was shown that a drug like dapsone, does act onMycobacterium leprae based on its permeability, quantity available inside the macrophages and inhibition of its action by Para amino benzoic acid. The inactivation ofMycobacterium leprae by sulphone and rifampicin was also proved by the flourescence diacetate method, which showed poorly viable bacteria after exposure to drugs. Thus it has been possible to develop a rapid drug screening method for testing the activity of unknown compound againstMycobacterium leprae.     

Cytochemical and ultrastructural studies of Candida albicans

Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid — thiocarbohydrazide silver proteinate technique (a polysaccharide detectron technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms.Abbreviations Con A Concanavalin A - Man-fer mannosyl ferritin - PATAg Periodic acid-thiocarbohydrazide-silver proteinate     

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis

Growth curves were determined for three strains each ofNocardia asteroides andNocardia brasiliensis. Two strains ofN. brasiliensis and one strain ofN. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours.The ultrastructure of the cell envelope of eachNocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains ofN. brasiliensis and one strain ofN. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degress of virulence as well as the divergent pathologies exhibited by these organisms.     

Cholesterol metobolism of macrophages in relation to the presence ofMycobacterium leprae

Macrophages phagocytoseMycobacterium leprae and live bacilli inside such macrophages alter the lipid metabolism. There is increased accumulation of cholesterol ester in the bacteria infected cells. This increase appears to be due to the decreased level of esterase enzyme that could hydrolyse cholesterol esters. Associated with decreased level of this enzyme is the reduced amount of protein synthesis. Increased cholesterol ester may be responsible for conversion of macrophages into foamy cells in the presence ofM. leprae.