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)

Heat shock proteins and antigens of Mycobacterium tuberculosis.

The heat shock response of Mycobacterium tuberculosis has been characterized in detail by one- and two-dimensional polyacrylamide gel electrophoresis after metabolic labeling with [35S]methionine and 14C-amino acids. A temperature increase from 37 to 42 degrees C induced elevated synthesis of three major proteins corresponding to the DnaK, GroEL, and GroES proteins of M. tuberculosis previously identified as prominent antigens. At higher temperatures (45 to 48 degrees C), synthesis of GroEL decreased and novel heat shock proteins with molecular masses of 90, 28, 20, and 15 kDa were observed. These new proteins did not comigrate with known antigens during two-dimensional gel electrophoresis. The heat shock response is discussed with regard to the possible importance of transcriptional regulation of mycobacterial genes in vivo.

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.

Novel type-specific lipooligosaccharides from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (strain Canetti) is characterized by the presence of two novel glycolipids of the alkali-labile, trehalose-containing lipooligosaccharide class. Their structures were established by permethylation, partial acid hydrolysis, infrared and high-field NMR spectroscopy, and electron-impact and fast atom bombardment mass spectrometry of the native glycolipids and hydrolysis products. The trehalose substituent is unique in that it is methylated at the 6'-position. The structure of the simpler of the two glycolipids is 2-O-Me-alpha-L-Fucp(1----3)-beta-D-Glcp(1----3)-2-O-Me- alpha-L-Rhap(1----3)-2-O-Me-alpha-L- Rhap(1----3)-beta-D-Glcp(1----3)-4-O-Me-alpha-L-Rhap(1----3) -6-O-Me-alpha-D- Glc. Further glycosylation of the octaglycosyl unit of this nonantigenic glycolipid by an incompletely defined N-acyl derivative of a 4-amino-4,6-dideoxy-Galp residue results in the second, highly antigenic nonasaccharide-containing glycolipid. Application of two-dimensional proton correlation spectroscopy demonstrated that the fatty acyl substituents are located on the 2,3,6 and 3,4,6 hydroxyl groups of the terminal glucosyl unit in the proportions of 2:3. Gas chromatography/mass spectrometry and optical rotation measurement allowed identification of the fatty acyl esters as primarily 2L-, 4L-dimethylhexadecanoate, 2L-,4L-,6L-,8L-tetramethyloctadecanoate, and 2-methyl-3-hydroxyeicosanoate. The relationship of these glycolipids to different morphological forms of M. tuberculosis and to virulence is discussed.

[Purified protein derivatives prepared from Mycobacterium tuberculosis (PPDs) and M. intracellulare (PPD-B) in differential diagnosis of mycobacteriosis].

To reveal the possibility of differentiating diseases caused by M. tuberculosis and M. intracellulare, simultaneous tuberculin testing by PPDs and PPD-B was carried out among X-ray suspects of tuberculosis and health persons. PPD-B was prepared by Dr. Tasaka (Department of Bacteriology, Hiroshima University) from M. intracellulare (ATCC 13950). For tuberculin testing, 0.05 micrograms of PPDs from M. tuberculosis (Nihon BCG Co.) and 0.1 microgram of PPD-B were used. The study included 61 patients with disease caused by M. tuberculosis (TB), 23 patients with that of M. avium complex (MAC) and 40 healthy persons with no roentgenological abnormality (H). Forty healthy persons had been vaccinated with BCG. Statistical analysis of the diameter of reaction (redness) in each antigen in each group has been done by Boxplotting method. The results were as follows: (75% upper quartile point, median, 25% lower quartile point/mean +/- S. D.): PPDs in TB (41.8, 30.0, 19.0/32.0 +/- 17.7); PPD-B in TB (15.0, 10.5, 5.0/10.9 +/- 8.1); PPDs in MAC (26.0, 10.0, 7.0/16.4 +/- 13.9); PPD-B in MAC (20.5, 17.5, 12.5/19.1 +/- 11.4); PPDs in H (18.0, 12.0, 6.0/13.5 +/- 10.9); PPD-B in H (7.0, 2.8, 0.0/4.4 +/- 5.4). Mean of PPDs in TB patients and PPD-B in MAC patients were significantly (P less than 0.01) larger than those in other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobacterium tuberculosis.

The recent availability (Hunter, S.W., Gaylord, H., and Brennan, P.J. (1986) J. Biol. Chem. 261, 12345-12351) of the well known arabinomannan of Mycobacterium leprae and Mycobacterium tuberculosis as the pure native lipoarabinomannan has resulted in its implication in key aspects of the immunopathogenesis of leprosy and tuberculosis. We had indicated that the lipid moiety of lipoarabinomannan is probably based on a diacylglycerol unit in that glycerol and the two fatty acids, hexadecanoate and 10-methyloctadecanoate, were identified. In addition, lipoarabinomannan was also shown to contain myo-inositol 1-phosphate. Evidence is now presented, based on selective radiolabeling and analysis of various cleavage fragments, that the inositol phosphate exists as both an alkalilable phosphodiester and as part of a phosphatidylinositol "membrane anchor." The mannan of M. tuberculosis was also isolated as the native lipomannan. It also apparently contains a phosphatidylinositol unit but is devoid of the alkali-labile inositol phosphate residues. These lipopolysaccharides are apparently multiglycosylated versions of the well known myocobacterial mannosyl phosphatidylinositols and are prokaryotic versions of the growing list of phosphatidylinositol-anchored macromolecules. Immunogold labeling demonstrates that lipoarabinomannan is a true antigenic capsular or extracellular product of M. tuberculosis. The presence of a phosphatidylinositol residue on lipoarabinomannan may explain its interaction with macrophage membranes and role in mycobacterial pathogenesis.

Ornithine lipid of Mycobacterium tuberculosis: its distribution in some slow- and fast-growing mycobacteria.

An ornithine-amide lipid is present in Mycobacterium tuberculosis. Its structure was established by a combination of chemical analysis and mass spectrometry. 3-Hydroxyoctadecanoic and 3-hydroxyeicosanoic acids (and homologues) were found to be linked through an amide bond to the alpha-amino group of L-ornithine, the hydroxyl group of the fatty acid being esterified mainly by tuberculostearic acid (10-methyloctadecanoic acid). This ornithine-amide lipid was detected in several other slow-growing pathogenic mycobacteria by thin layer chromatography, but not in an avirulent strain (H37 Ra) of M. tuberculosis. In each case mass spectrometry showed that all the structures were identical, thus revising an earlier reported structure for the lipid from M. bovis.

Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses.

The peptidoglycan-bound arabinogalactan of a virulent strain of Mycobacterium tuberculosis was per-O-methylated, partially hydrolyzed with acid, and the resulting oligosaccharides reduced and O-pentadeute-rioethylated. The per-O-alkylated oligoglycosyl alditol fragments were separated by high pressure liquid chromatography and the structures of 43 of these constituents determined by 1H NMR and gas chromatography/mass spectrometry. The arabinogalactan was shown to consist of a galactan containing alternating 5-linked beta-D-galactofuranosyl (Galf) and 6-linked beta-D-Galf residues. The arabinan chains are attached to C-5 of some of the 6-linked Galf residues. The arabinan is comprised of at least three major structural domains. One is composed of linear 5-linked alpha-D-arabinofuranosyl (Araf) residues; a second consists of branched 3,5-linked alpha-D-Araf units substituted with 5-linked alpha-D-Araf residues at both branched positions. The non-reducing terminal region of the arabinan was characterized by a 3,5-linked alpha-D-Araf residue substituted at both branched positions with the disaccharide beta-D-Araf-(1----2)-alpha-D-Araf. 13C NMR of intact soluble arabinogalactan established the presence of both alpha- and beta-Araf residues in this domain. This non-reducing terminal motif apparently provides the structural basis of the dominant immunogenicity of arabinogalactan within mycobacteria. A rhamnosyl residue occupies the reducing terminus of the galactan core and may link the arabinogalactan to the peptidoglycan. Evidence is also presented for the presence of minor structural features involving terminal mannopyranosyl units. Models for most of the heteropolysaccharide are proposed which should increase our understanding of a molecule responsible for much of the immunogenicity, pathogenicity, and peculiar physical properties of the mycobacterial cell.

Peptidoglycan-associated polypeptides of Mycobacterium tuberculosis.

Important protein-based immunoreactivities have long been associated with the cell wall core of mycobacteria. In order to explore the molecular basis of such activities, purified cell walls of Mycobacterium tuberculosis were extracted with sodium dodecyl sulfate to produce an insoluble residue composed of the mycolylarabinogalactan-peptidoglycan complex and about 2% of unextractable protein. Treatment of the product from an avirulent strain of M. tuberculosis with trifluoromethanesulfonic acid released a single polypeptide with a molecular size of 23 kilodaltons, accounting for all of the insoluble cell wall protein. Extensive purification and then analysis of the 23-kilodalton protein demonstrated the absence of diaminopimelic acid, muramic acid, or other peptidoglycan components, pointing to either a novel linkage between protein and peptidoglycan or a noncovalent but tenacious association. The released 23-kilodalton protein showed amino acid homology and other similarities to the outer membrane protein OmpF of Escherichia coli. Although a similar product was released in small quantities from cell walls of the virulent M. tuberculosis Erdman and H37Rv by lysozyme treatment, the cell walls of virulent bacilli were dominated by the presence of poly-alpha-L-glutamine, accounting for as much as 10% of their weight. The poly-alpha-L-glutamine was successfully separated from the cell wall proper, demonstrating again the absence of a covalent association between peptidoglycan and the polymer. The antigenicity of these products is demonstrated, and their roles vis-a-vis analogous polypeptides from other bacteria in immunogenicity, pathogenicity, and bacterial physiology are discussed.

[Glycolipid-patterns of Mycobacterium tuberculosis as an aid for sub-typing].

Total lipids were extracted from Mycobacterium tuberculosis with chloroform-methanol (2:1), applied on a silica-gel thin-layer plate, and developed with chloroform-methanol-acetone (90:10:5). Glycolipids were detected by spraying Anthrone-reagent and heating. Strain H37Rv of M. tuberculosis showed four Anthrone-positive spots, namely trehalose-monomycolate, unidentified glycolipid, trehalose-dimycolate and GL-Rv, and strain H37Ra showed only two spots corresponding to trehalose-monomycolate and trehalose-dimycolate. Other 4 laboratory-stock strains of M. tuberculosis showed glycolipid-pattern identical with either of these two patterns. One hundred and fifty-eight strains of M. tuberculosis, isolated clinically from tuberculosis patients, were classified into 7 types according to their glycolipid-pattern. Twenty-seven strains contained one more Anthrone-positive spot other than those of strain H37Rv. Pattern II was most frequently observed (60 strains), and then pattern I (33 strains), VI (29 strains), IV (13 strains), V (9 strains), VII (8 strains), and III (6 strains). Pattern I corresponded to that of strain H37Ra and pattern VI corresponded to that of strain H37Rv. Glycolipid-pattern did not correlate to clinical features of patients from whom the bacilli had been isolated. A glycolipid, which moved to just under the solvent front, was a new glycolipid which has been found by us and designated as GL-Rv. Chemical structure of GL-Rv was clarified by us as trehalose-polyacyl derivatives (no mycolic acid as the acyl residue). Glycolipid-pattern was very stable and reproducible for each strain of M. tuberculosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Scalar, dipolar-correlated and J-resolved 2D-NMR spectroscopy of the specific phenolic mycoside of Mycobacterium tuberculosis.

Two-dimensional chemical shift correlated (COSY), nuclear Overhauser (NOESY) and J-resolved spectroscopy were used to determine the complete structure of the major triglycosyl dimycocerosyl phenol phthiocerol of the tubercle bacillus (strain Canetti) without any other analytical technique. The COSY spectrum of the native glycolipid allowed the composition of the trisaccharide and the location of one methoxyl group to be determined through the assignment of the resonances of the non-anomeric methine protons. Information with respect to the configuration of the sugar residues, the pyranose structure and the linkage sites of the trisaccharide were obtained by the analysis of the COSY spectrum of the peracetylated glycolipid. The NOESY spectrum confirmed the linkage sites through the inter-residue connectivity across glycosidic linkages and allowed the determination of the sequence of the oligosaccharide and the configuration of its sugar residues from the through-space connectivities. The J-resolved spectroscopy was used to elucidate the structure of the glycolipid and gave additional data showing the presence of 11 non-equivalent methyl groups and allowing the recognition of two other analogues of the phenol phthiocerol derivative in the mixture. Thus, the non-destructive 2D-NMR spectroscopy was found to be satisfactory for the analysis of mycobacterial mycosides.

Glycolipids of recent clinical isolates of Mycobacterium tuberculosis: chemical characterization and immunoreactivity.

Five distinct glycolipids were readily detected in isolates of Mycobacterium tuberculosis. Spectroscopic methods and chemical degradation techniques allowed the structural identification of four of these glycolipids. The specific phenolic glycolipid antigen previously characterized from the Canetti strain was found in all the strains examined, with identical structural features (triglycosyl phenol phthiocerol dimycocerosate). The other three glycolipids identified were acylated trehaloses: penta-acyl trehalose (containing phthienoyl substituents), tetra-acyl trehalose 2'-sulphate (with C40-C50 hydroxyphthioceranoyl substituents) and diacyl trehalose 2'-sulphate (with C16 and C18 substituents). The two latter glycolipids as well as the phenolic glycolipid immunoreacted with whole-cell antiserum, indicating their surface location. The occurrence of these glycolipid antigens in recent clinical isolates suggests their possible utilization in the serodiagnosis of tuberculosis and the rapid identification of M. tuberculosis with specific antisera.

Synthesis of galactofuranose disaccharides of biological significance.

Methyl beta-D-galactofuranoside was readily obtained by tin(IV) chloride-catalyzed glycosylation of penta-O-benzoyl-alpha,beta-D-galactofuranose, followed by debenzoylation with sodium methoxide. Glycosylation of 1 with 2,3,5-tri-O-benzoyl-D-galactono-1,4-lactone or with the 6-O-trityl-lactone derivative 5 gave the benzoylated beta-D-galactofuranosyl-(1----6)-D-galactono-1,4-lactone 6 in excellent yield. The structure of disaccharide 6 was confirmed by borohydride reduction to the glycosyl-alditol 7. A byproduct of the condensation reaction of 1 with 4 or 5 was identified as the benzoylated (1----1)-beta,beta'-D-galactofuranosyl disaccharide 8. Compound 8 was readily prepared (88% yield) by controlled addition of water to 1, in the presence of stannic chloride. O-Debenzoylation of 8 afforded crystalline beta'-D-galactofuranosyl-(1----1)-beta-D-galactofuranoside. The glycosyl-lactone 6 constitutes a key intermediate for the synthesis of a disaccharide derivative having both units in the furanoid form. Thus, diisoamylborane reduction of the lactone function of 6 led to the disaccharide derivative 10, from which the methyl glycoside 12 was prepared. O-Debenzoylation of 12 gave the corresponding methyl beta-D-galactofuranosyl-(1----6)-beta-D-galactofuranoside. The free disaccharide beta-D-Galf-(1----6)-D-Galp and its acetylated derivative were also synthesized from 10.

[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.

Further specific triglycosyl phenol phthiocerol diester from Mycobacterium tuberculosis.

Mono- and two-dimensional 1H-NMR allowed the structural elucidation of a glycolipid belonging to the phenolic mycosides series: 2,3,4-tri-0-methyl fucopyranosyl (alpha 1----3) rhamnopyranosyl (alpha 1----3) rhamnopyranosyl (alpha 1----dimycocerosyl) phenol phthiocerol. It shares with the major phenolic glycolipid the two terminal sugar residues, suggesting its potential antigenicity. The glycolipid may also represent an intermediate in the biosynthesis of the major one.

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.