Acción del agua del Volcán Copahue (Neuquén, Argentina) sobre micobacterias / Action of the Copahue volcano waters on mycobacteries

Se efectúa un estudio de la acción del agua del Volcán Copahue (AVC) sobre 14 cepas de micobacterias. Los tres ensayos utilizados para determinar resistencia o sensibilidad al agua dieron resultados coincidentes. Las Micobacterias no tuberculosas presentaron distintos grados de resistencia, en cambio M.tuberculosis, M. bovis y M.marinum mayor sensibilidad. Se complementa con breves estudios de la acción del AVC sobre el efecto pH, destrucción del "factor cuerda" de los bacilos, y pérdida de su actividad enzimática (catalasa y peroxidasa)

Acción del agua del Volcán Copahue (Neuquén, Argentina) sobre micobacterias / Action of the Copahue volcano waters on mycobacteries

Se efectúa un estudio de la acción del agua del Volcán Copahue (AVC) sobre 14 cepas de micobacterias. Los tres ensayos utilizados para determinar resistencia o sensibilidad al agua dieron resultados coincidentes. Las Micobacterias no tuberculosas presentaron distintos grados de resistencia, en cambio M.tuberculosis, M. bovis y M.marinum mayor sensibilidad. Se complementa con breves estudios de la acción del AVC sobre el efecto pH, destrucción del "factor cuerda" de los bacilos, y pérdida de su actividad enzimática (catalasa y peroxidasa)

Location of the mycolyl ester substituents in the cell walls of mycobacteria.

The question of the precise location of mycolic acids, the single most distinctive cell wall entity of members of the Mycobacterium genus, has now been addressed. The free hydroxyl functions of the arabinogalactan component of the mycobacterial cell wall were O-methylated under conditions in which the mycolyl esters were not cleaved. Subsequent replacement of the mycolyl functions with O-ethyl groups resulted in an acid- and base-stable differentially O-alkylated surrogate polysaccharide, more amenable to analysis. Complete hydrolysis, reduction, acetylation, and gas chromatography/mass spectrometry revealed the unexpected finding that the mycolyl substituents were selectively and equally distributed on the 5-hydroxyl functions of terminal- and 2-linked arabinofuranosyl (Araf) residues. Further analysis of the O-alkylated cell wall through partial acid hydrolysis, NaB[2H]4 reduction, pentadeuterioethylation, and gas chromatography/mass spectrometry demonstrated that the mycolyl units are clustered in groups of four on the previously recognized nonreducing terminal pentaarabinosyl unit [beta-Araf-(1----2)-alpha-Araf)2-3, 5-alpha-Araf. However, only about two-thirds of the available pentasaccharide units are so substituted. Thus, the antigenicity of the arabinan component of mycobacterial cell walls may be explained by the fact that about one-third of the pentaarabinosyl units are not mycolyated and are available for interaction with the immune system. On the other hand, the extreme hydrophobicity and impenetrability of the mycobacterial cell may be explained by the same motif also acting as the fulerum for massive esterified paraffin residues. New fundamental information on the structure of mycobacterial cell walls will aid in our comprehension of its impenetrability to antibiotics and role in immunopathogenesis and persistence of disease.

Immunotherapeutic potential in guinea-pig tumor model of deoxyribonucleic acid from Mycobacterium bovis BCG complexed with poly-L-lysine and carboxymethylcellulose.

In vivo antitumor activity of a deoxyribonucleic acid fraction obtained from Mycobacterium bovis BCG (named MY-1) increased when it was complexed with poly-L-lysine (poly LL) solubilized by addition of carboxymethylcellulose (CMC). The complex of MY-1 and poly LL/CMC induced interferon in vivo at a low dose of MY-1 which alone exerted no IFN induction. With Line 10 hepatoma (L10) which is syngeneic with strain 2 guinea pigs, it was demonstrated that repeated intralesional injections of the complex resulted in delay of tumor growth and complete cure of animals from L10 tumor inoculated. Similar treatment of the animals with the same amount of MY-1 or poly LL/CMC alone had little therapeutic effect on the tumor growth.

Extraction and localization by electron microscopy of an immunosuppressor fraction from Mycobacterium bovis Bacillus Calmette-Guérin (BCG).

BCG has been used all over the world to immunize against tuberculosis. Nevertheless in certain areas (South India) BCG vaccines failed to show any protective efficacy. Furthermore immunosuppressive cell populations have been reported in experimental mycobacterial infection in mice. The present work reports the localization and isolation of an immunosuppressor fraction from BCG. This lipid fraction called WDB inhibited the skin reactivity of delayed-type hypersensitivity (DTH) to the test antigen CEWA (crystalline egg white albumin) in guinea pigs and depressed the production of immune antibody to SRBC (sheep red blood cells) in mice. WDB is a glycolipid with an approximate mol.wt. of 62,000. By electron microscopy, WDB was located among the BCG extracellular metabolic products (ECMP) surrounding the BCG cell wall.

Genetic transformation of BCG.

Two substrains of BCG, the Pasteur and Japanese, were successfully transformed with E. coli-mycobacteria shuttle plasmids, constructed from the E. coli plasmid, pIJ666 and the M. fortuitum plasmid, pAL5000. Individual plasmids (pYUB13, pYUB14) were obtained that contain selectable antibiotic resistance markers for kanamycin and chloramphenicol resistance that can replicate in both E. coli and BCG. Transformation of two substrains of BCG was successfully accomplished in 8/14 experiments by means of electroporation, and assessed by the growth of kanamycin-resistant colonies. The E. coli plasmid pIJ666 alone was unable to effect transformation. The results suggest that the M. fortuitum sequences required for transformation function as an origin of replication in BCG. The introduction, persistence and the identity of the plasmids were monitored by re-isolation from consecutive subcultures and restriction analysis. The variables associated with transformation, including the age, viability, and glycine pretreatment of BCG cultures, as well as the electroporation parameters on transformation frequencies are analysed. Consecutive transformations of BCG with plasmid DNA isolated from a BCG transformant increased the efficiency from the level of 10(1)-10(2) obtained with the initial library to 10(3)-10(4) colonies/micrograms DNA with functional pYUB plasmids. The hybrid plasmids were genetically stable and maintained expression of kanamycin resistance in continuous subcultures containing kanamycin for 250 generations. The introduction and stable expression of foreign DNA in BCG on a plasmid vector establishes a basis for the construction of polyvalent recombinant BCG vaccine vehicles expressing not only putative protective mycobacterial antigens, but also antigens for other infectious and malignant diseases.

New-found phenolic glycolipids in Mycobacterium bovis BCG. Presence of a diglycosylated glycolipid.

A crude phenolic glycolipid extract from Mycobacterium bovis bacille Calmette-Guerin (BCG) was fractionated until homogeneity at the intact level into four phenolic glycolipids called B, B-1, B-2, and B-3 according to their polarity. The apolar one, which is the most abundant was assigned to the well-known mycoside B. The B-2 and B-3 phenolic glycolipids were purified by direct-phase high performance liquid chromatography using a 5 micron Spherisorb column but were only recovered in small amounts (3 mg). A linear gradient of 0-20% methanol in chloroform was used. The B-1, B-2, and B-3 glycolipids were subjected to suitable modern analytical techniques selected for their potential to elucidate the structure at the intact level. Desorption chemical ionization-mass spectrometry allowed the molecular mass of B-3 to be determined as 1652 Da for the major homolog establishing the molecular formula as C103H192O14. Thus, the B-3 polar phenolic glycolipid contained two deoxyhexoses, one molecule of phenolphthiocerol esterified by two molecules of mycocerosic acid. Using two-dimensional 1H NMR (correlated chemical shift and nuclear Overhauser effect spectroscopy) at the intact level the B-3 oligosaccharide structure was determined as an alpha-L-Rhap-(1----3)-2-O-Me-alpha-L-Rhap. This is the first report of a diglycosylated phenolic glycolipid in a nonpathogenic mycobacteria. The disaccharide unit, the antigenic determinant, appears to be characteristic of M. bovis BCG. This polar glycolipid B-3 and the apolar ones, B-1 and B-2, were reactive in enzyme-linked immunosorbent assay against serum from rabbit hyperimmunized with M. bovis BCG.

[Identification of Mycobacteria and measurement of whole cell fatty acids using a gas chromatography computer system].

The technique of combination gas chromatography with computer used in measuring fatty acids of 28 species standard Mycobacteria was introduced in this study. The data of components of fatty acids in this genus and GC graphs with satisfaction were gained. Several peaks with good reproducibility were distinguished automatically, based on which, M. tuberculosis, M. bovis and other comment atypical Mycobacteria may be separated respectively. It was showed that this method have the characteristics of accurate, sensitive and rapid by means of the identification of clinical strains, also have certain practical value in clinical laboratory.

Phenolic glycolipids of Mycobacterium bovis: new structures and synthesis of a corresponding seroreactive neoglycoprotein.

The glycolipid that characterizes the majority of isolates of Mycobacterium bovis and that has come to be known as M. bovis-identifying lipid is the phenolic glycolipid mycoside B described in the literature by others. However, when mycoside B obtained from M. bovis BCG, field isolates, and infected tissues was examined in detail, it was shown to be different from that described in the literature in some important respects. In particular, the glycosyl substituent is 2-O-methyl-alpha-L-rhamnopyranose rather than 2-O-methyl-beta-D-rhamnopyranose. With this information, a seroreactive neoglycoprotein (neoantigen) containing the 2-O-methyl-alpha-L-rhamnopyranosyl substituent suitable for the serodiagnosis of bovine tuberculosis was synthesized. M. bovis also contains other minor seroreactive phenolic glycolipids, one of which is a deacylated form of mycoside B and another of which contains an alpha-L-rhamnopyranosyl unit rather than 2-O-methyl-alpha-L-rhamnopyranose.

Efficient mapping and characterization of a T cell epitope by the simultaneous synthesis of multiple peptides.

Prediction, identification and analysis of T cell epitopes in protein antigens has become a central theme in fundamental and applied immunology. However, while for the characterization of linear B cell epitopes the so-called Pepscan procedure was found to be extremely effective, no such technique has so far been available for T cell studies. Recently, we described the identification and localization of a T cell epitope in a mycobacterial 65-kDa shock protein in the model of adjuvant arthritis. This was done by molecular cloning and conventional solid-phase synthesis techniques. We now show that the delineation of such a T cell epitope and its further characterization can be accomplished in a much more rapid and efficient manner by a modification of the existing Pepscan technique. We show for the first time that several hundreds of peptides, simultaneously synthesized in an automated way on activated polyethylene rods, can be easily recovered from these rods in adequate quantities, enabling a systematic analysis of T cell epitopes. Synthesis of sequentially overlapping peptides along the 65-kDa protein revealed that the adjuvant arthritis T cell clones are fully stimulated by peptides that comprise a minimal sequence of seven residues, corresponding to positions 180-186 in the sequence of the 65-kDa protein of M. bovis Bacillus Calmette Guerin (BCG). Detailed examination of the epitope by peptides containing a single amino acid substitution showed that, apart from one conservative replacement (Glu----Asp), the requirement for the native residue at all positions in peptide 180-186 was absolute for full T cell stimulation. Their indispensability was confirmed with deletion and insertion peptides. It is concluded that the occurrence of indifferent or spacer residues in a minimal stimulatory sequence, as observed by others, is not a general feature of T cell epitopes.

Viability of freeze-dried Tice-substrain BCG by bioluminescent measurement of adenosine triphosphate.

The ATP content of viable cells in a single lot of freeze-dried Tice-substrain Mycobacterium bovis-BCG vaccine was determined after washing of organisms with isotonic buffer, using four extraction methods: boiling in 0.2 M potassium phosphate buffer at pH 7.4 for 12 min; boiling in 0.1 M Tris-EDTA buffer at pH 7.75 for 2 min; n-butanol/1.9% sodium glutamate-0.01 M sodium arsenate buffer, pH 7.0; and n-butanol/0.01 M Tris-EDTA buffer, pH 7.0. Liberated ATP was assayed with a Lumac biocounter by integrated measurement of light produced by firefly luciferin/luciferase. The dose response of internal standards paralleled that of ATP standards in water, and the response of endogenous ATP in BCG was not significantly different from a composite linear internal standard curve above 10 pg ATP (corresponding to about 10(5) viable organisms per ml), the sensitivity of the assay. When the ATP content of BCG was calculated from the composite curve, the n-butanol/Tris-EDTA method was found to be the most precise (CV less than 10%). Butanol extraction procedures were about twice as efficient as boiling methods and yielded an ATP value of about 3.4 fg/CFU, similar to ATP/CFU factors previously reported for other BCG substrains. However, when results were corrected for quench and ATP recovery, which varied with extraction method, the conversion factor increased nearly threefold.

Local adoptive transfer of delayed-type hypersensitivity to tuberculin from M. bovis (BCG) infected mice.

Immunological properties of the cells mediating delayed-type hypersensitvity to tuberculin and genetic requirements of this reaction have been studied by the method of local adoptive transfer. Peritoneal cells from BCG-immunized mice transferred the reaction into unprimed recipients without any genetic restriction. In contrast, nonadherent peritoneal cells transferred the reaction only in H-2-compatible donor-recipient strain combinations. The use of H-2 recombinant strains has shown that delayed-type hypersensitivity to tuberculin in mice is restricted by the I-A locus. Monoclonal antibody against I-Ak beta abrogated (without complement) the transfer of the reaction to CBA recipients. The phenotype of nonadherent cells, transferring the reaction has been shown to be Thy-1+, Lyt-1+, 2-, L3T4+.

Fatty acids of Mycobacterium bovis BCG.

Three substrains of Mycobacterium bovis BCG which appeared to differ in biological activity could not be differentiated on the basis of fatty acids less than C20, but could be differentiated from six nonpathogenic mycobacterial species.

Size characterization of Mycobacterium bovis BCG (Bacillus Calmette Guérin) vaccine, Tice substrain.

Reconstituted, lyophilized, attenuated Mycobacterium bovis, Bacillus Calmette Guérin (BCG) vaccine, Tice substrain, was characterized using a Coulter Multisizer and a HIAC/Royco counter. The primary organism has an equivalent spherical diameter approximating 1 micron but the BCG cell suspension is heavily aggregated. The cumulative size distribution of the suspension fits a log-probit plot and this information can be used to determine the total number of particles per ampoule. The instrumental count may be related to the viable count. The state of dispersion was unaffected by mild shear (syringe aspiration or ultrasound) and only slightly affected by the addition of cetylpyridinium chloride or sodium tauroglycolate.

Restriction endonuclease analysis of total deoxyribonucleic acid of Mycobacterium tuberculosis H37RV (ATCC 27294) and of M. bovis (ATCC 19210).

Total DNA from two slowly-growing pathogenic mycobacterial species propagated in vitro was isolated, digested with each of 34 restriction endonucleases and analyzed by agarose gel electrophoresis. The most distinct profiles for M. tuberculosis (ATCC 27294) and for M. bovis (ATCC 19210) were obtained respectively using (BamHI, DraI, ClaI, EcoRI, EcoRV, HindIII, HpaI, SalI, SmaI, XbaI, and XmaI). The patterns produced for these strains were reproducible and distinguishable from each other. However, with several enzymes the patterns for M. tuberculosis and M. bovis were similar. Evidence was obtained for the presence of dam and dcmI methylations in the DNA of each mycobacterial species.

In vitro augmentation of natural killer cell activity and production of interferon-alpha/beta and -gamma with deoxyribonucleic acid fraction from Mycobacterium bovis BCG.

A nucleic acid-rich fraction extracted and purified from BCG (MY-1) augmented natural killer (NK) cell activity of mouse spleen cells in vitro, and produced factor(s) which showed anti-viral activity and rendered normal macrophages cytotoxic towards tumor cells. These cellular responses were induced by the MY-1 digested preliminarily with RNase, but not by the MY-1 digested with DNase, indicating that DNA contained in MY-1 was essential for the responses. The function of the factor to activate macrophages was destroyed by treatment with a small amount of anti-interferon (IFN)-gamma antiserum or under acidic conditions (pH 2), but not by treatment with anti-IFN-alpha/beta antiserum, while the anti-viral activity was destroyed almost completely by treatment with anti-IFN-alpha/beta antiserum. It appears that DNA from BCG stimulated mouse spleen cells in vitro, resulting in augmentation of NK activity and production of IFN-alpha/beta and -gamma.

Distribution of phthiocerol diester, phenolic mycosides and related compounds in mycobacteria.

Among 28 mycobacterial species studied, only Mycobacterium tuberculosis, M. bovis, M. africanum, M. marinum, M. kansasii, M. gastri and M. ulcerans produced waxes yielding long-chain beta-diol components (called phthiocerol and companions) and polymethyl-branched fatty acids on saponification. The same mycobacterial species also produced diesters of phenol phthiocerol and companions. Fatty acids esterifying these fatty alcohols in M. marinum and M. ulcerans were found to belong to the phthioceranic series (dextrorotatory fatty acids), in contrast to those of the other species (laevorotatory fatty acids called mycocerosic acids), both groups having the same chain length and methyl-branched positions. M. kansasii and M. gastri contained the same waxes with identical structures, as did M. tuberculosis, M. bovis and M. africanum. Neither the type strain of M. tuberculosis, nor that of M. bovis or M. marinum accumulated the strain-specific phenolic glycolipids.

Monoglycosyldiacylphenol-phthiocerol of Mycobacterium tuberculosis and Mycobacterium bovis.

The structure of a minor glycolipid of M. tuberculosis (strain Canetti) is shown to be 2-O-methyl-alpha-L-rhamnosyldiacylphenol-phthiocerol. A similar compound with non-methylated rhamnose as sugar moiety was also detected. In the course of this work, the structure of mycoside B from Mycobacterium bovis was reexamined, and was shown to be identical to that of the 2-O-methylrhamnosyldiacylphenol-phthiocerol of the Canetti strain, while it was described as a 2-O-methyl-beta-D-rhamnosyl derivative in the literature. This result is in agreement with the known close relationship between M. tuberculosis and M. bovis. Careful examination of chromatographic fractions containing the above mentioned lipids showed that the occurrence of mycoloyl residues in some phenol-phthiocerol glycolipids, postulated in the literature, was likely to be due to the presence of glycerol monomycolate contaminants.