The effect of exogenous peroxidase on the evolution of murine leprosy.

Mycobacterium lepraemurium (MLM) is a successful parasite of murine macrophages; in vitro, this microorganism infects macrophages without triggering these cells' ability to produce either the reactive oxygen intermediaries (ROI) or the reactive nitrogen intermediaries (RNI), and ends up lodging within these cells, that, in addition, do not contain myeloperoxidase (MPO). In this study, we analyzed the effect of exogenous peroxidase on the evolution of murine leprosy. Bacilli were intraperitoneally injected, either alone (MLM) or precoated with horseradish peroxidase (MLM-PO), into two different groups of mice. At two-week intervals, the groups were blood-sampled to measure the levels of antibodies to protein- or lipid-MLM antigens. The extent of the disease was also assessed by looking at the histopathologic changes that occurred both in the liver and the spleen of the infected animals. We found that the animals injected with MLM-PO developed a disease that evolved at a slower pace than the disease that occurred in the animals injected with intact MLM. The difference between groups, both in terms of antibody levels and histological changes, was clearly evident at the intermediate stages of the disease (2 to 2.5 months), but was not so obvious at the more advanced stage of 3 months. Several possibilities to explain how the PO-coated bacilli might have regained their infectiousness are discussed. Lowering the infective dose of MLM and MLM-PO from 5 x 10(7) bacilli to 5 x 10(6) bacilli would, probably, have resulted in a different outcome of the disease: more extended in the MLM-group than in the MLM-PO group.

Evaluation of methods for isolation of DNA from slowly and rapidly growing mycobacteria.

Mycobacteria generally have thick cell walls and contain large amounts of lipid, making them resistant to DNA extraction. Five methods, namely, extensive enzymic digestion method (M1), 2-min mechanical glass-bead disruption method (M2), thermal shock method (M3), modified conventional enzymic digestion method (M4), and manual disruption with modified conventional enzymic digestion method (M5), were used to compare their effectiveness and simplicity in extracting DNA from slowly growing mycobacteria (Mycobacterium leprae, M. lepraemurium and M. bovis BCG), and a rapidly growing mycobacterium (M. phlei). The highest DNA yield was obtained by M2 from M. lepraemurium which produced 2.82 micrograms DNA/mg wet weight of cells, representing a theoretical yield of 78%. M3 gave the lowest DNA yield; 0.01 microgram DNA/mg wet weight of cells of M. lepraemurium was obtained. M4, in which proteinase K was used, is more effective than M1, in which subtilisin and pronase were used. M5 yielded a higher amount of DNA, but it required more manipulations to extract DNA as compared to M4. Extraction of DNA of M. leprae from nude mice is more difficult than that of M. leprae from armadillos by all of the methods used. These results suggest that the biosynthetic capabilities of these two forms of M. leprae may vary, depending on their cultural conditions and/or strain differences. Our results have shown that both M2 and M4 are the simplest, most effective and time-saving methods which are suitable for every routine laboratory to extract DNA from slowly and rapidly growing mycobacteria.

[Specific odor component produced by Mycobacterium lepraemurium on Ogawa yolk medium].

When Mycobacterium lepraemurium is grown on the 1% Ogawa yolk medium, it produces a specific odor. This odor was not observed in other easily cultivable acid-fast bacilli. Therefore, identification of the components responsible for the specific odor produced by M. lepraemurium was attempted. The odor components were extracted for overnight with sterilized and distilled water from the Ogawa yolk medium on which M. lepraemurium had been cultivated for two months. The odor components in the extract was adsorbed on refined charcoal. After washing with distilled water for three times, the charcoal was dried. Then the odor components were eluted from the charcoal with ethanol and the eluate was condensed under nitrogen gas flow at 40 degrees C. The condensate was analyzed by Gas-Chromatography-Mass-Spectrum (GC-MS). Phenylethanol and phenylacetic acid were identified as major odor components. A mixture of authentic phenylacetic acid, its methyl and ethyl esters, smelled similar to the odor of cultivated medium of M. lepraemurium. Thus, phenylacetic acid was identified as the key odor component produced by M. lepraemurium. When initial isolation culture of M. lepraemurium from murine leproma was cultivated on the Ogawa yolk medium by adding phenylacetic acid, growth inhibition was brought by the compound.

Energy generation mechanisms in the in vitro-grown Mycobacterium lepraemurium.

Mycobacterium lepraemurium was cultivated on Ogawa egg-yolk medium and its energy coupling mechanisms were investigated. Cell-free extracts prepared from in vitro-grown cells catalyzed phosphorylation coupled to the oxidation of generated NADH, added NADH, and succinate-yielding ratios of phosphorus moles incorporated into high-energy bonds to oxygen atoms utilized (P/O ratios) of 0.75, 0.52, and 0.36, respectively. Ascorbate oxidation alone or in the presence of tetramethyl-p-phenyline-diamine (TMPD) did not yield any adenosine triphosphate (ATP). However, ascorbate in the presence of added cytochrome c was coupled to ATP synthesis and yielded a P/O ratio of 0.12. The oxidative phosphorylation was uncoupled by all of the uncouplers used without any inhibition of oxygen consumption. ATP generation coupled to NADH oxidation was completely inhibited by the flavoprotein inhibitors, such as rotenone and amytal; these inhibitors had no effect, however, on ATP synthesis associated with succinate oxidation. Antimycin A or 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO) and cyanide inhibited markedly the oxidations of NADH and succinate as well as the coupled ATP generation. The phosphorylation coupled to ascorbate plus cytochrome c was not affected by either of the flavoprotein inhibitors or by antimycin A or HQNO, but was completely inhibited by cyanide. The thiol-bearing agents p-chloromercuribenzoate (PCMB) and N-ethylmaleimide were the potent inhibitors of the phosphorylation associated with the oxidation of NADH and succinate. The results indicate that the three energy-coupling sites are functional in the respiratory chain of in vitro-grown M. lepraemurium.

Palmitate oxidation by in vivo and in vitro grown Mycobacterium lepraemurium.

Oxidation of palmitate by Mycobacterium lepraemurium isolated from C3H mice lepromata (in vivo) and also grown on Ogawa egg-yolk medium (in vitro) was investigated. Palmitate was found to be oxidized, after a lag period of about 8 h, by both the in vivo and in vitro grown bacilli. Cell-free extracts prepared from in vivo and in vitro grown cells catalysed an active oxidation of palmitate after a lag period of 3-4 h. The amount of ATP increased, with the increase in time during oxidation of palmitate by the cell-free extracts. The generation of ATP was strongly inhibited by the inhibitors rotenone, antimycin A and cyanide as well as by the uncouplers 2,4-dinitrophenol and 2,6-dibromophenol. These results indicated that oxidation of palmitate by the in vivo and in vitro grown M. lepraemurium is mediated through the respiratory chain using oxygen as the terminal electron acceptor.

[Energy production in Mycobacterium lepraemurium cultured in vitro]. / Production d'énergie chez Mycobacterium lepraemurium cultivé in vitro.

Cell-free extracts prepared from in vitro cultured Mycobacterium lepraemurium catalysed phosphorylation coupled to the oxidation of NADH and succinate, yielding P/O ratios of 0.52 and 0.34, respectively. No ATP synthesis occurred during oxidation of ascorbate. Oxidative phosphorylation was uncoupled by dinitrophenol and dibromophenol. Oxidation of NADH and coupled phosphorylation was markedly inhibited by rotenone, whereas this inhibitor had no effect on succinate oxidation and associated ATP synthesis. Oxidative phosphorylations and coupled oxidations of NADH and succinate were strongly inhibited by antimycin A and cyanide.

Cytochrome system in cultivated Mycobacterium lepraemurium.

The respiratory pigments of cell suspensions of Mycobacterium lepraemurium cultivated on Ogawa egg-yolk medium were investigated spectrophotometrically. The results obtained showed that whole cell suspensions of both Kumato and Hawaiian strains contained flavins, cytochromes of the a2 and b type, as well as a CO-binding pigment similar to cytochrome o. The whole cell suspensions of M. lepraemurium did not show detectable quantities of c type cytochrome. However, cytochrome c was present in small amounts, and its presence became evident in the dithionite-reduced minus oxidized difference spectra of pyridine haemochromogens prepared from in vitro grown cells of M. lepraemurium.

Propane and tetradecane as carbon sources for in vitro cultivation of Mycobacterium lepraemurium in a liquid medium (a preliminary communication).

Three strains of host grown Mycobacterium lepraemurium and five strains of Mycobacterium lepraemurium, grown on egg yolk medium, were inoculated into propane-tetradecane media. The media contained in one litre distilled water: KH2PO4, 7 g, Na2HPO4, 0.5 g, (NH4)2SO4, 2 g, MgSO4, 0.1 g, ferric ammonium citrate, 20 mg, and yeast extract (Difco), 0.1 g. Tetradecane 0.1 ml was added to each tube containing 20 ml of the medium. Media were sterilized in the autoclave. Following inoculation with the bacilli, the cultures were bubbled aseptically with 99% purity propane gas for 10 s. When incubated at 32 degrees C, logarithmic growth rate was counted in the cultures. Bacilli were strongly acid-fast. Growth occurred at the interface of the tetradecane oil and water as a thin weil, developing into a one to three millimeter thick emulsion in two to three months. Cultures were transferred into fresh media at two to three month intervals. Growth pattern in the subcultures were indistinguishable from the growth in the primary cultures. The cultures did not grow on Löwenstein-Jensen or in Dubos media, but produced the characteristic disease of murine leprosy when injected subcutaneously into mice. Bacilli isolated from the subcutaneous lepromas of mice were again cultivable in the propane-tetradecane medium, but not on Löwenstein-Jensen or in Dubos.

Respiratory activities of in vitro grown Mycobacterium lepraemurium.

Mycobacterium lepraemurium was cultivated on Ogawa egg-yolk medium and its respiratory activities using several substrates were investigated. Glycerol and succinate were oxidized at a slow rate by the cell-free extracts prepared from in vitro grown Hawaiian and Keishicho strains of M. lepraemurium. None of the other intermediates of the glycolysis cycle as well as of the tricarboxylic acid cycle was oxidized by the whole cell suspensions or cell-free extracts. Likewise, many sulfur compounds such as cystine, mercaptosuccinate, monothioglycerol, thioacetate, etc., were inactive. However, sulfhydryl compounds such as L-cysteine, D-cysteine, DL-cysteine, dithioerythritol, dithiothritol, and DL-penicillamine were actively oxidized. Yeast extract was also readily oxidized by cell suspensions of in vitro grown M. lepraemurium. Tween 80 was very poorly oxidized by whole cell suspensions but the cell-free preparations catalyzed an active oxidation of Tween 80. While bovine serum albumin was oxidized at a slow rate by cell-free extracts, egg albumin was inactive. The thiol-binding agents, p-hydroxymercuribenzoate and N-ethylmaleimide were effective inhibitors of succinate and NADH oxidation, thus indicating the involvement of sulfhydryl compounds in the metabolism of M. lepraemurium.

Taxonomic studies on Mycobacterium leprae.

M. leprae harvested from livers and spleens of infected armadillos showed close similarity in biochemical characteristics with M. vaccae than with any other cultivable mycobacteria. Further more. M. lepraemurium was least similar to M. leprae. Thus, M. leprae can be linked with fast-growing groups of acid-fast bacilli, and perhaps most closely with the M. vaccae complex.

Metabolism of carbon sources by Mycobacterium leprae: a preliminary report.

Glucose was established in Mycobacterium leprae by glycolysis and the hexose monophosphate pathway (30 %)-pentose phosphate pathway. Glycerol was also catabolised to CO2 at a similar rate to glucose. Key in cell-free extracts as enzymes for M. leprae.

Mycobacterial lipids in infected tissue samples.

Mycobacteria synthesize characteristic lipids which can readily be distinguished from those synthesized by the host cells which they infect during pathogenesis. This fact can be exploited to provide information about mycobacteria growing in infected tissue samples: (a) qualitative analysis of lipid extracts from lepromatous leprosy skin biopsies reveals the presence of several mycobacterial lipids including an M. leprae-specific glycolipid, phthiocerol dimycocerosate and mycolic acids; (b) quantitative analysis shows that the amount of mycobacterial lipids present in lepromatous lesions is much greater than that expected on the basis of the number of acid-fast bacilli present; (c) incorporation of 14C-acetate into cell wall mycolic acids can be used to monitor the growth of intracellular mycobacteria.

Radiometric measurement of differential metabolism of fatty acid by mycobacteria.

An assay system has been developed based on automated radiometric quantification of 14CO2 produced through oxidation of [1-14C] fatty acids by mycobacteria. Two stains of M. tuberculosis (H37Rv and Erdman) and one of M. bovis (BCG) in 7H9 medium (ADC) with 1.0 microCi of one of the fatty acids (butyric, hexanoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic and linolenic) were studied. Results previously published on M. lepraemurium (Hawaiian) were also included for comparison. Both strains of M. tuberculosis had maximum 14CO2 production from hexanoic acid. Oxidation of butyric and avid oxidation of lauric acids were also found with the H37Rv strain but not with Erdman. In contrast, 14CO2 production by M. bovis was greatest from lauric and somewhat less from decanoic acid. M. lepraemurium showed increasing oxidation rates from myristic, decanoic and lauric acids. Assimilation studies of M. tuberculosis H37Rv confirmed that most of the oxidized substrates were converted into by-products with no change in those from which no oxidation was found. These data suggest that the radiometric measurement of differential fatty acid metabolism may provide a basis of strain identification of the genus Mycobacterium.

Isolation and identification of mycolic acids in Mycobacterium leprae and Mycobacterium lepraemurium.

Mycolic acids with a characteristic structure were isolated by high performance-liquid-chromatography (HPLC) and mass-spectrometry from a foot pad of a nude mouse inoculated with Mycobacterium leprae. Mycolic acids with the same structure were also obtained from mycobacteria collected from the liver of an armadillo with experimental leprosy. Mycolic acids were isolated from Mycobacterium lepraemurium grown both in vivo and in vitro and these mycolic acids had different structures from those of M. leprae. Mycolic acid structures have great taxonomical significance. The methods used for isolating and analyzing mycolic acids appear applicable for the rapid identification of M. leprae in samples containing at least 10(9)-10(10) mycobacterial cells. Using our method, mycolic acids with the same structure were found in mycobacteria from armadillos experimentally infected with M. leprae and from armadillos with naturally acquired leprosy-like disease. It is likely, therefore, that the pathogenic mycobacteria of the naturally acquired disease are the same as, or at least closely related to, M. leprae. The present work suggests that M. leprae has a special position in mycobacterial phylogeny.

Energy coupling mechanisms in host-grown Mycobacterium lepraemurium.

Energy coupling mechanisms of Mycobacterium lepraemurium isolated from Sprague-Dawley rats lepromata were investigated. Cell-free extracts catalyzed phosphorylation coupled to the oxidation of generated NADH, added NADH, and succinate yielding P/O ratios of approximately 0.8, 0.6, and 0.4, respectively. Ascorbate oxidation alone or in the presence of cytochrome c or N,N,N',N'-tetramethyl-p-phenylenediamine was not coupled to ATP synthesis. The oxidative phosphorylation was completely uncoupled by 2,4-dinitrophenol, 2,6-dibromophenol, pentachlorophenol, m-chlorocarbonylcyanide phenylhydrazone, dicumarol, and gramicidin at concentrations which did not cause any inhibition of oxygen uptake. While the NADH oxidation and associated phosphate esterification was markedly sensitive to rotenone and other flavoprotein inhibitors, these inhibitors had no effect, however, on the phosphorylation coupled to succinate oxidation. The respiratory chain inhibitors such as antimycin A or 2-n-heptyl-4-hydroxyquinoline-N-oxide, and cyanide were the potent inhibitors of the phosphorylation associated with the oxidation of NADH and succinate. The ATP formation coupled to the oxidation of NADH and succinate was also inhibited by oligomycin as well as by the thiol-binding agents, p-hydroxymercuribenzoate and N-ethylmaleimide. The results indicated that NADH and succinate oxidation by in vivo grown M. lepraemurium was mediated by oxidative enzymes involving first and second energy coupling sites.

Role of sulfhydryls in in vitro growth of Mycobacterium lepraemurium.

In an attempt to evaluate various factors that influence the growth of Mycobacterium lepraemurium in NC-5 medium, the effects of sulfur and --SH compounds were investigated. Cysteine could be replaced by equimolar concentrations of other --SH compounds containing carboxyl group, and at lower concentrations by nonpolar sulfhydryl compounds. The oxidized form of sulfhydryls, as well as certain organic and inorganic reducing agents, did not support growth. The results suggest that the function of sulfhydryl compounds is to maintain low reducing potential in the medium and also to participate in metabolic or biosynthetic pathways or both. A combination of dithiothreitol and thioglycolate gave better results than when these compounds were incorporated individually in the medium. This suggests the protective action of dithiothreitol in preventing oxidation of monothiols.

[Oxidative phosphorylation in Mycobacterium lepraemurium]. / Phosphoryoation oxydative chez Mycobacterium lepraemurium.

The generation of ATP by cell-free extracts of Mycobacterium lepraemurium isolated from Sprague-Dawley rats was investigated. Cell-free preparations catalyzed phosphorylation coupled to the oxidation of NADH and succinate yielding P/O ratios of 0.6 and 0.4, respectively. Ascorbate oxidation did not result in ATP formation. The oxidative phosphorylation was uncoupled by 2,4- dinitrophenol and pentachlorophenol. Phosphate esterification coupled to NADH oxidation was inhibited by rotenone which had no effect on ATP synthesis associated with succinate oxidation. Antimycin A and cyanide completely inhibited phosphorylation coupled to the oxidation of NADH or succinate.

Phospholipid deacylating activities in murine leprosy bacilli.

1) The particulate fraction of cultivated murine leprosy bacilli (Mycobacterium lepraemurium, rough colonies of the Hawaiian-Ogawa strain) contained phospholipid deacylating activities with acidic pH optima. It hydrolyzed phosphatidylcholine and phosphatidylethanolamine at similar rates, and phosphatidylinositol oligomannosides more slowly. It also hydrolyzed 1-acyl- and 2-acyl-GPCs (sn-glycerol 3-phosphocholine) more rapidly than phosphatidylcholine. 2) Ca2+ did not stimulate either diacyl- or monoacyl-hydrolase activity. Triton X-100 and Emulgen 913 had little influence on the hydrolysis of phosphatidylcholine, but at rather high concentrations inhibited the hydrolyses of 1-acyl- and 2-acyl-GPCs. Iron ions strongly inhibited the hydrolysis of phosphatidylcholine, but caused little or no inhibition of the deacylations of 1-acyl- and 2-acyl-GPCs. 3) With 1-[stearoyl-14C]phosphatidylcholine and 2-[oleoyl-14C]phosphatidylcholine as substrates, both labeled fatty acid and lysophosphatidylcholine were produced. Labeled fatty acid appeared more rapidly from 2-[oleoyl-14C]phosphatidylcholine than labeled lysophosphatidylcholine, while labeled lysophosphatidylcholine was produced more than labeled fatty acid from 1-[stearoyl-14C]phosphatidylcholine in the early stage of incubation.

Phospholipid deacylating activities in murine leprosy bacilli

1) The particulate fraction of cultivated murine leprosy bacilli (Mycobacterium lepraemurium, rough colonies of the Hawaiian-Ogawa strain) contained phospholipid deacylating activities with acidic pH optima. It hydrolyzed phosphatidylcholine and phosphatidylethanolamine at similar rates, and phosphatidylinositol oligomannosides more slowly. It also hydrolyzed 1-acyl- and 2-acyl-GPCs (sn-glycerol 3-phosphocholine) more rapidly than phosphatidylcholine. 2) Ca2+ did not stimulate either diacyl- or monoacyl-hydrolase activity. Triton X-100 and Emulgen 913 had little influence on the hydrolysis of phosphatidylcholine, but at rather high concentrations inhibited the hydrolyses of 1-acyl- and 2-acyl-GPCs. Iron ions strongly inhibited the hydrolysis of phosphatidylcholine, but caused little or no inhibition of the deacylations of 1-acyl- and 2-acyl-GPCs. 3) With 1-[stearoyl-14C]phosphatidylcholine and 2-[oleoyl-14C]phosphatidylcholine as substrates, both labeled fatty acid and lysophosphatidylcholine were produced. Labeled fatty acid appeared more rapidly from 2-[oleoyl-14C]phosphatidylcholine than labeled lysophosphatidylcholine, while labeled lysophosphatidylcholine was produced more than labeled fatty acid from 1-[stearoyl-14C]phosphatidylcholine in the early stage of incubation.