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PubMed1977

[Differentiation of mycobacteria by the Jones-Mote reaction (author's transl)].

The specificity of the Jones-Mote reaction to the mycobacterial species was investigated in mycobacteria-free guinea pigs. To the sensitization and the sensitine production the following type strains (Trudeau Institute Saranac Lake) were used: M. avium, M. borstelense, M.chelonei, M. flavescens, M. fortuitum, M. gastri, M. gordonae, M.kansaii, M. marinum, M nonchromogenicum, M. phlei, M. scrofulaceum, M. smegmatis, M. terrae, M. triviale and M. bovis strain Vallee as well as M. intracellulare serotyp Davis ATCC 23435. Eight days after intracutaneous sensitization with 0,2 mg of bacterial mass (wett weight) in Al(OH)3 as adjuvans the groups of 10 animals for each species were tested intracutaneously with all 18 sensitins in a concentration of 5 mug. The animals reacted positive with the homologous sensitin only; (tab. 1) a cross-reaction was stated between M. avium and M. intracellulare (closely related) and between M. borstelense and M. chelonei (the same species). The homogeneity of the Jones-Mote reaction in some rapidly growing Mycobacteria was investigated with 11 strains of M. diernhoferi, 10 strains of M. smegmatis, M. phlei, M. fortuitum and 3 strains of M. borstelense. The positive reaction was stated with species-specific sensitin only. It should be possible to identify the primocultures using mycobacteria-free guinea pigs and suitable sensitins.

Animals↗
PubMedDate not supplied

[Nucleotide makeup of the DNA of fast-growing mycobacteria and related microorganisms].

The melting points of DNA were used to determine the nucleotide composition of DNA from the following microorganisms with a high growth rate: Mycobacterium phlei, M. smegmatis, M. fortuitum, Nocardia asteroides, the organism of the "rhodochrous"--N. opaca group, and organisms related to strain "Mycobacterium" rhodochrous ATCC 13808. M. fortuitum had a higher mol% G+C (67.5--69.5) than M. phlei (66.9--67.5) and M. smegnatis (64.4--68.1); the strains of M. smegmatis were characterized by the most heterogeneous nucleotide composition of DNA. The organisms of the "rhodochrous" complex (mol% G+C=69.5--72.2) were closer to N. asteroides (mol% G+C=72.5--73.5) than to the mycobacteria with a high growth rate.

DNA, Bacterial↗
PubMed1979

Differentiation of rapidly growing mycobacteria with trimethoprim (Tmp).

Inhibition of Mycobacterium smegmatis, M. vaccae, and M. diernhoferi by Trimethoprim (Tmp) in both liquid and solid media is described. Other mycobacteria were not inhibited by the same concentrations. The selective inhibition of the above strains, particularly M. smegmatis, by Tmp could be used for differentiation of these species from other fast-growing acid-fast bacteria.

Culture Media↗
PubMed1977

Synthesis of vitamin B12 by various species of mycobacteria.

The synthesis of Vitamin B12 by five reference strains of species of mycobacteria, the influence of cobalt upton the synthesis and the correlation between the phase of growth and quantitative biosynthesis of Vitamin B12 were studied. Assays were performed on cell extracts and culture filtrates using Lactobacillus leichmannii ATCC7830 (tube method). All five species synthesized Vitamin B12, which could be deomonstrated both in cell extracts and in the culture filtrates. An optimum accumulation of Vitamin B12 by BCG and M. phlei could be observed on the 30th day of cultivation. Addition of cobalt did not influence the growth of BCG and M. phlei, but markedly enhanced the production of Vitamin B12. Cells contained much more Vitamin B12 than culture filtrates did. Vitamin B12 levels in M. smegmatis, M. fortuitum and M. asiaticum were lower than those in BCG and M. phlei.

BCG Vaccine↗
PubMed1978

[Multiplication of mycobacteria in the gray layer of sphagnum vegetation (author's transl)].

The capillary moisture of the gray layer of the Sphagnum magellanicum ass. contains nutrients for the growth of mycobacteria. Tested in vitro significant multiplication was stated in M. intracellulare, serotyp Davis, M. avium, M. scrofulaceum, M. fortuitum, M. xenopi, M. kansasii, M. borstelense, M. flavescens, M. gaxtri, M. gordonae, M. marinum, M. terrae, M. smegmatis, M. spec. "S" and M. nonchromogenicum (Fig. 1 and 2). There was no evident multiplication in M. phlei, M. triviale, M. tuberculosis, M. bovis, and M. ulcerans in vitro. Using the cultivation of mycobacteria in the hollow fibers (pore size 100,000 mol. weight) inserted directly in the gray layer of the Sph. magellanicum ass. a significant growth of all 18 species investigated was stated (the same species as above, excl. M. bovis and M. tuberculosis; Fig. 3). A rapid multiplication was detected also in M. ulcerans. The sphagnum vegetation spreads over all continents. It develops very good under wet conditions surrounding springs and brooklets in the moutain region. Accumulation of solar heat directly under the surface of this vegetation makes possible a temperature 15-28 degrees C higher than the air temperature. In this way favorable conditions for mesophilic mycobacteria arise over an extend period of time. The close contact to water contributes to the wide distribution of mycobacteria.

Mycobacterium↗
PubMed1976

The specificity of the fluorescent antibody test using the sera of rabbits inoculated with strains of myocobacteria.

Sera from experimentally infected rabbits were used to test the specificity of the fluorescent antibody test. It was possible using mono-specific sera to differentiate antigenically Mycobacterium phlei, M fortuitum, M smegmatis, M avium, M intracellulare, M bovis (BCG) and M johnei. The cross-reactivity within the M avium and M intracellulare group was such that one antigen from these groups would detect infection within that group and exclude M johnei infection. The M phlei growth factor independent strain M johnei 316F was shown to be antigenically distinct from a M phlei dependent strain 9N96. There was loss of specificity when M avium infection was superimposed on a previous M johnei infection and when M johnei infection was superimposed on M avium infection.

Animals↗
PubMed1978

[Atypical mycobacteria isolated from the water of the Simeto River at Catania].

From 25 samples of water taken in the course of 1975 from different points of the river Simeto have been isolated 11 strains of atypical mycobacteria, classifiable in Runyon's II, III and IV group. M. fortuitum has been most frequently found (6 strains), while M. gordonae, M. smegmatis, M. chelonei, M. engbaekii have been isolated only once. An unclassifiable strain belonging to Runyon's 3rd group has also been isolated. The importance of research of atypical mycobacteria in the environment from an epidemiological and epizootological point of view has been discussed.

Italy↗
PubMed1979

Identification of a Mycobacterium leprae specific protein antigen(s) and its possible application for the serodiagnosis of leprosy.

Acetone-killed Mycobacterium leprae separated from infected armadillo liver tissue without the use of proteases were treated with 0.2 M lithium acetate, 20 mM EDTA, pH 8.8 solution, and the concentrated antigen extract was analyzed by Ouchterlony immunodiffusion. The antigen extract gave a single immunoprecipitate when reacted with pooled lepromatous leprosy (LL) patients sera made highly specific for M. leprae by adsorption. Apparently identical precipitates were produced by reacting the antigen extract with sera of each of 15 treated LL patients, 5 of 7 patients with tuberculoid leprosy, and 3 of 4 M. leprae infected armadillos. Serum from 1 of 16 persons immunized with BCG and from none of 15 patients with chlamydial urethritis or brucellosis reacted with the antigen. Identically prepared extracts of M. smegmatis, M. phlei, M. vaccae, M. duvali and M. diernhoferi gave no immunoprecipitates with sera from LL patients or infected armadillos. Preliminary characterization indicates the antigen is protein since antigenicity was destroyed by pronase and/or heat treatment. The relative specificity of the protein antigen for M. leprae and the presence of antibody to this antigen in patients with leprosy suggest a possible role for this antigen in the serodiagnosis of leprosy.

Animals↗
PubMed1977

Antibody response in rabbits to immunization with Mycobacterium leprae.

Mycobacterium leprae purified from liver tissue of an infected armadillo (the A/10 preparation) was tested for antigenic composition by immunization of rabbits and characterization of the antibody response by crossed immunoelectrophoresis. The rabbit antisera detected seven distinct components in the M. leprae preparation. This number is far lower than in similar experiments with other mycobacteria. The M. leprae sonic extract gave far fewer lines after polyacrylamide gel electrophoresis and staining with Coomassie brillant blue than sonic extracts prepared from BCG, M. smegmatis, and M. phlei adjusted to the same protein concentration based on the Folin assay. The seven components detected in M. leprae cross-reacted extensively with M. avium, BCG, M. lepraemurium, M. smegmatis, and Nocardia asteroides. The seven components are involved in immune reactions in leprosy; antibodies against all of them were demonstrated in sera from patients with lepromatous leprosy, but the specificity of the antibodies varied from patient to patient. The reason for the demonstration of so few antigenic components and some of the implications of these findings for the use of armadillo-grown M. leprae to develop specific skin test reagents and in other aspects of leprosy research are discussed.

Animals↗
PubMed1979

Immunochemical and structural integrity of surface protein antigens of mycobacteria during separation from armadillo liver tissue.

Surface proteins of Mycobacterium smegmatis were iodinated using the lactoperoxidase method. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated two major surface proteins in the radiolabelled M. smegmatis. Both surface proteins were released from M. smegmatis using the nonionic detergent Triton X-100. The major surface component was sensitive to pronase digestion and contained no detectable carbohydrate. The second radiolabelled component was found to be of low molecular weight, resistant to pronase digestion and stained positive for carbohydrate by the periodic acid/Schiff method. Triton X-100 solubilized radiolabelled surface proteins were antigenic as assessed by a radioimmune precipitation test. When surface labelled M. smegmatis was mixed with armadillo liver tissue and separated from tissue using a method formerly employed by the World Health Organization Immunology of Leprosy Program for the purification of M. leprae, as much as 50% of the surface proteins of M. smegmatis was either released or destroyed. In addition, another twenty distinct proteins were released from M. smegmatis after treatment with Triton X-100. Similar losses of proteins from M. leprae may also occur using this procedure for M. leprae purification. Separation techniques employing surfactants and enzymatic treatment should be carefully evaluated since proteins lost during these procedures may prove relevant to human immune responses to M. leprae.

Animals↗
PubMed1978

Turnover of lipids in Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354.

The rates of breakdown and renewal of individual lipids in cultures of Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354 were investigated by means of a pulse labelling technique using palmitate-1-14C. The results indicated that in growing cultures of both strains phospholipids were broken down, and cardiolipin had a very rapid turnover. In chase experiments, almost 45% and 40% of the radioactivity of this component were lost respectively from M. smegmatis and M phlei during one generation time of the cell. The other two major components, phosphatidyl ethanolamine and phosphatidylinositol mannosides showed relatively low turnover. The loss of radioactivity from phosphatidylinositol mannosides was greater in M. phlei than in M. smegmatis but the loss of radioactivity from phosphatidyl ethanolamine was higher in M. smegmatis. The pattern of loss of radioactivity from lipids was almost the same in both strains, the difference being only in the extent of loss. The differences in the cellular localization of the phospholipids indicate their different roles within the cell. Results obtained with the glyceride fraction indicated a very rapid turnover of triglycerides in both strains.

Glycerides↗
PubMed1977

Common antigen of Mycobacterium leprae, M. lepraemurium, M. avium, and M. fortuitum in comparative studies using two different types of antisera.

No. 21 mycobacterial antigens of Mycobacterium lepraemurium, M. avium, M. fortuitum, and M. leprae were compared in crossed immunoelectrophoresis using two different antibody sources, a serum pool from lepromatous leprosy patients (LSII) and a rabbit anti-M. smegmatis antiserum. M. lepraemurium, like M. avium, was found to contain the 21 A and 21 C determinants. M. fortuitum contained in addition a new type of determinant, 21 D.M. leprae antigen no. 21 carried the A as well as the B dertminants, the latter found so far only in the leprosy bacillus. The separate taxonomic position of M. leprae, suggested by earlier studies of the no. 21 antigen, is further supported by the present results, which also demonstrate the potential use of submolecular heterogeneity for such investigations.

Animals↗
PubMedDate not supplied

Acid-fast properties and pyridine extraction of M. leprae.

The reportedly unique pyridine extractability of acid-fastness as an identifying characteristic for M. leprae was examined in the leprosy bacilli and in eight other strains of mycobacteria. The initial findings were, in general, in accord with previous reports except that M. smegmatis and M. phlei likewise demonstrated two hour pyridine extractability of acid-fastness. Perhaps, more significantly, it was found that this characteristic in M. leprae is related to aged, probably nonviable bacilli. Some other strains of mycobacteria when tested in aged cultures showed the same phenomenon while M. leprae cultivated in vitro in a recently developed medium resisted pyridine extraction up to three weeks of growth, but thereafter as the culture aged pyridine extractability became characteristic. It is concluded that this pyridine extractability of acid-fastness is a characteristic of aging or nonviable bacilli. As such it is not definitive in the determination of whether or not in vitro cultivation of M. leprae has been achieved.

Bacteriological Techniques↗
PubMed2025

Rv2741 Promotes Mycobacterium Survival by Modulating Macrophage Function via the IL-1α-MAPK Axis.

One of the primary healthcare problems in the world today is tuberculosis (TB), a chronic infectious illness brought on by Mycobacterium tuberculosis (M. tuberculosis). A distinct family of PE_PGRS proteins, encoded by the M. tuberculosis genome, has attracted more attention because of their involvement in immune evasion and bacterial pathogenicity. Nevertheless, the specific functions and mechanisms of action for the majority of PE_PGRS proteins remain largely unexplored. This study focuses on the Rv2741 (PE_PGRS47) gene, which is exclusively present in pathogenic mycobacteria. To examine the function of Rv2741 in host-pathogen interactions, we created recombinant strains of Mycobacterium smegmatis (M. smegmatis) that expressed the M. tuberculosis Rv2741 gene. IL-1α was found to be a key mediator of host response modulation by Rv2741. Rv2741 downregulates the secretion of IL-1α and inhibits the MAPK signaling pathway, particularly the p38 and ERK1/2 pathways, thereby cooperatively inhibiting macrophage autophagy and apoptosis. Meanwhile, the decrease in IL-1α secretion directly leads to changes in the cytokine secretion pattern and a reduction in nitric oxide (NO) production. This multifaceted regulatory mechanism ultimately favors the survival of M. smegmatis in macrophages. This research significantly expands our understanding of Rv2741 function, revealing its crucial role as a multifunctional virulence factor in the immune evasion of M. tuberculosis.

Interleukin-1alpha↗
PubMedDate not supplied

Fatty acid synthesizing enzyme activity of cultured Mycobacterium lepraemurium.

In comparing the specific activity of enzymes pertaining to the biosynthesis of fatty acids in crude extracts of cultivated M. lepraemurium and M. smegmatis, it was found that: 1. The activity of acetyl CoA carboxylase of the former organism was undetectable and that of de novo fatty acid synthetase was too weak to measure exactly, under the condition used, whereas both activities of the latter organism were comparable to those already reported by other authors. 2. The activity of acetyl CoA dependent acyl CoA elongation system of M. lepraemurium was relatively high and close to that of M. smegmatis. 3. The activities of acetyl CoA synthetase and acyl CoA synthetase of M. lepraemurium were moderately lower than those of M. smegmatis. The relation between this peculiar fatty acid synthesizing enzyme system of M. lepraemurium and its extremely sluggish growth is discussed.

Acetate-CoA Ligase↗
PubMed1978

Studies on transfer RNA from mycobacteria.

Active preparations of tRNA and aminoacyl-tRNA synthetases have been isolated from exponentially growing cells of Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv. Though the aminoacyl-tRNA synthetases of older cells retain their activity, the tRNAs seem to undergo modification and show poorer activity. The mycobacterial enzyme preparations catalyse homologous and heterologous aminoacylation between tRNA from the two species (M. smegmatis and M. tuberculosis H37Rv) or from Escherichia coli, with equal efficiency; tRNA samples from eukaryotic cells (yeast and rat liver) do not serve as substrates for the mycobacterial synthetases. The analytical separation of the different amino acid specific tRNAs from M. smegmatis resembles the pattern found in other bacteria. Purification of valine- (three species) and methionine-specific tRNA (two species) to 70-80% purity has been accomplished by using column-chromatographic techniques. Of the two species of tRNAMet, one can be formylated in the presence of formyl tetrahydrofolate and the transformylase from mycobacteria.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Purification and characterization of 3-hydroxyacyl-CoA dehydrogenase of Mycobacterium smegmatis.

3-Hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35] was purified 100-fold to homogeneity from crude extracts of Mycobacterium smegmatis, using ammonium sulfate fractionation, gel filtration, and chromatography on DEAE-cellulose, hydroxyapatite, and NAD-Sepharose 4B columns. Its molecular weight was estimated to be 50,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NADH acted twelve times more efficiently than NADPH as an electron donor for the reduction of 3-ketoacyl-CoA, and there was strict substrate stereospecificity (L form) in the oxidation of 3-hydroxyacyl-CoA. The pH optimum depended upon the direction of reaction, i.e., 6.0 for the oxidation of NADH and 9--10 for the reduction of NAD. The Km values for different thioesters of acetoacetate, i.e., esters of CoA, pantetheine, and acetyl-cysteamine were determined to be 0.036, 1.19, and 44.4 mM, respectively. Antibodies raised against the dehydrogenase of M. smegmatis strongly inhibited the enzyme activity, but did not affect the corresponding dehydrogenase of pig heart. The antibodies were found to inhibit the acetyl-CoA dependent elongation of fatty acids by the crude extract of M. smegmatis. These findings, together with those on the reconstitution of the elongation activity reported previously (Shimakata, T., Fujita, Y., & Kusaka, T. (1977) J. Biochem. 82, 725-732) indicate that 3-hydroxyacyl-CoA dehydrogenase is involved in the acetyl-CoA dependent elongation of fatty acids in M. smegmatis.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1979

Cross-reactions between mycobacteria. II. Crossed immunoelectrophoretic analysis of soluble antigens of BCG and comparison with other mycobacteria.

Cross-reactions between Mycobacterium bovis BCG and various other mycobacteria, Nocardia asteroides, Corynebacterium pyogenes and Listeria monocytogenes were studied by incorporating antibodies against these bacteria in the intermediate gel of a crossed immunoelectrophoretic system with BCG antigen and anti-BCG antibodies. In the BCG reference system forty-four distinct antigenic components were recorded, of which thiryt-three cross-reacted with Mycobacterium tuberculosis, twenty-five with M. avium, twenty-one with M. suvalii, eighteen with M. smegmatis, Fifteen with M. nonchromogenicum, twelve with M. phlei, eight with N. anteroides and two with C. pyogenes, whereas no cross-reaction was detected with L. monocytogenes. The value of the method for characterization of mycobacterial antigens is discussed. A taxonomic system based on this method appears particularly valuable for studies of non-cultivativable mycobacteria such as M. leprae. A majority of twenty-one patients with lepromatous leprosy had anti-BCG antibodies of restricted specificity, affecting only four or five BCG antigens, although one patient had twelve anti-BCG specificities. Most of these antibodies reacted with those BCG antigens that cross-react extensively with other mycobacteria.

Antibodies, Bacterial↗