Trehalose-containing lipooligosaccharides. A new class of species-specific antigens from Mycobacterium

Mycobacterium kansasii is characterized by the presence of seven species-specific neutral lipooligosaccharide antigens. All react with hyperimmune anti-M. kansasii serum in enzyme-linked immunosorbent assays, and the more glycosylated members also react by gel diffusion. Both the native glycolipids and their inherent oligosaccharides were purified and the major features of their unique structures determined by acetolysis, partial acid cleavage, 1H-NMR and 13C-NMR, and chemical ionization and electron impact mass spectrometry of the permethylated products. They have in common a tetraglucose "core," beta-D-Glcp-(1 leads to 3)-beta-D-Glcp-(1 leads to 4)- alpha-D-Glcp(1 leads to 1)-alpha-D-Glcp where Glcp is glucopyranose, distinguished by the presence of an alpha,alpha-trehalose substituent. Variable residues of xylose, 3-O-methylrhamnose, fucose, and a novel N-acyl aminosugar, (4,6-dideoxy-2-O-methyl-3-C-methyl-4-(2'-methoxypropionamido)hexose; proof of this structure is not given) are linked to the core and the resulting oligosaccharides are acylated with 2,4-dimethyltetradecanoyl and acetyl functions to present a familial arrangement. Related but species-specific lipooligosaccharides typify Mycobacterium szulgai and apparently a host of other atypical mycobacteria. Thus, the antigenicity, and perhaps other features such as pathogenesis and drug resistance, of most atypical mycobacteria may be rationalized in terms of these new found cell wall lipooligosaccharides and the previously described glycopeptidolipids.     

N-acylkansosamine. A novel N-acylamino sugar from the trehalose-containing lipooligosaccharide antigens of Mycobacterium kansasii

A novel N-acylamino sugar was isolated from the antigenic trehalose-containing lipooligosaccharides IV-VII of Mycobacterium kansasii. The native reducing sugar, its O-acetyl derivative, the methylglycoside, the O-acetylated alditol, and the de-N-acylated N-, O-acetylated alditol were all examined by high resolution 1H NMR, 13C NMR, direct probe and gas-liquid chromatography-mass spectrometry in both the chemical ionization and electron impact modes, and by high resolution mass spectrometry. The dideoxy sugar had a formula weight of 277, an empirical formula of C12H23NO6, C- and O-methyl substituents, and a N-methoxypropionyl branch. Upon alkaline hydrolysis, methoxypropionic acid was released and shown to correspond to the synthetic compound by gas chromatography and chemical ionization and electron impact mass spectrometry. The structure 4,6-dideoxy-2-O-methyl-3-C-methyl-4-(2'-methoxypropionamido)-alpha and beta-L-manno-hexopyranose, with the trivial name N-acylkansosamine, is proposed. The sugar is present in the more polar, highly antigenic lipooligosaccharides and is regarded as exclusive to M. kansasii and as its primary cell wall immunodeterminant.     

Mineralization of phenanthrene by a Mycobacterium sp.

A Mycobacterium sp., designated strain BG1, able to utilize the polycyclic aromatic hydrocarbon phenanthrene as the sole carbon and energy source was isolated from estuarine sediment following enrichment with the hydrocarbon. Unlike other phenanthrene degraders, this bacterium degraded phenanthrene via 1-hydroxy-2-naphthoic acid without accumulating this or other aromatic intermediates, as shown by high-performance liquid chromatography. Degradation proceeded via meta cleavage of protocatechuic acid. Different nonionic surfactants (Tween compounds) solubilized the phenanthrene to different degrees and enhanced phenanthrene utilization. The order of enhancement, however, did not correlate perfectly with increased solubility, suggesting physiological as well as physicochemical effects of the surfactants. Plasmids of approximately 21, 58, and 77 megadaltons were detected in cells grown with phenanthrene but not in those which, after growth on nutrient media, lost the phenanthrene-degrading phenotype. Given that plasmid-mediated degradations of aromatic hydrocarbons generally occur via meta cleavages, it is of interest that the addition of pyruvate, a product of meta cleavage, supported rapid mineralization of phenanthrene in broth culture; succinate, a product of ortho cleavage, supported growth but completely repressed the utilization of phenanthrene. The involvement of plasmids may have given rise to the unusual degradation pattern that was observed.     

Mycobacterium confluentis sp. nov.

A new rapidly growing mycobacterium was isolated from human sputum. This organism grew at 22, 31, 37, and 41 degrees C and possessed catalase, acid phosphatase, acetamidase, urease, nicotinamidase, pyrazinamidase, and nitrate reductase activities. It did not produce nicotinic acid, hydrolyze Tween, or have benzamidase, isonicotinamidase, succinidamidase, and arylsulfatase activities. A mycolic acid analysis revealed a simple, unique pattern. The organism is susceptible to antituberculotic drugs. A comparative 16S rRNA sequence analysis placed this organism within the confines of the genus Mycobacterium, most closely related to the thermotolerant rapidly growing species. On the basis of the pattern of enzymatic activities and metabolic properties, as well as the unique 16S rRNA sequence, we propose that our single strain represents a new species, for which we propose the name Mycobacterium confluentis. The type strain is strain 1389/90; a culture of this strain has been deposited in the German Collection of Microorganisms and Cell Cultures as strain DSM 44017.     

Mycobacterium alvei sp. nov.

A new species of rapidly growing, nonphotochromogenic mycobacteria, Mycobacterium alvei, is described. The inclusion of this organism in the genus Mycobacterium is based on its acid fastness, its mycolate pattern, and its G + C content. A study of six strains showed that they form a homogeneous group with an internal phenotypic similarity value of 97 +/- 2.22%. DNA relatedness studies showed that the six M. alvei strains which we studied form a single DNA hybridization group which is less than 49% related to 14 other species of the genus Mycobacterium; the deltaTm values determined for the strains which exhibited higher levels of DNA homology were all greater than 7.9 degrees C. A lipid analysis showed that tuberculostearic acid was present. Docosanoic and tetracosanoic acid methyl esters were detected as mycolic acid cleavage products. All six isolates which we tested contained alpha-mycolic acids and relatively large amounts of a new kind of mycolic acid containing a methoxy group of omega-1 position, a characteristic that has not been described previously in mycobacteria. Strain CR-21 is the type strain; a culture of this strain has been deposited in the Collection Nationale de Cultures de Microorganismes de l'Institut Pasteur, Paris, France, as strain CIP 103464.     

Mycobacterium rhodesiae sp. nov.

A number of rapid-growing scotochromogenic mycobacteria were isolated from the sputum specimen of Rhodesian patients with pulmonary disease and recognized as a new species. This was given the name Mycobacterium rhodesiae sp. nov. A comparison between M. rhodesiae, M. parafortuitum, M. aurum and M. vaccae was done, and distinguishing characters serving for differentiation between these 4 species of rapid-growing scotochromogenic mycobacteria were described.     

Synthesis of a novel pentasaccharide core component from the lipooligosaccharide of Moraxella catarrhalis

The novel pentasaccharide [p-(trifluoroacetamido)phenyl]ethyl 3-O-β-d-glucopyranosyl-4-O-β-d-glucopyranosyl-6-O-[2-O-(α-d-glucopyranosyl)-β-d-glucopyranosyl]-α-d-glucopyranoside (1), which includes a linker moiety to enable facile coupling to an antigenic protein, was synthesised as a component of a potential vaccine candidate against the Gram-negative bacterium Moraxella catarrhalis. This microorganism is one of three principal causative agents of otitis media in children. The pentasaccharide represents a common cross-serotype (A, B and C) structure from the lipooligosaccharides of Moraxella catarrhalis.     

Identification of the lipooligosaccharide biosynthetic gene cluster from Mycobacterium marinum

Lipooligosaccharides (LOSs) are antigenic glycolipids that are present in some species of Mycobacterium including the Canetti strain of M. tuberculosis. The core LOS structures from several mycobacterial organisms have been established, but the biosynthetic pathways of LOSs remain unknown. In this study, we describe two transposon insertion mutants of M. marinum that exhibit altered colony morphology. Cell wall analysis reveals that the MRS1271 mutant is defective in the synthesis of LOS-II, whereas the MRS1178 mutant accumulates an intermediate between LOS-I and -II. The genetic lesions were localized to two genes, MM2309 and MM2332. MM2309 encodes a UDP-glucose dehydrogenase that is involved in the synthesis of d-xylose. MM2332 is predicted to encode a decarboxylase. These two genes and a previously identified losA gene are localized in a gene cluster likely to be involved in the biosynthesis of LOSs. Our results also show that LOSs play an important role in sliding motility, biofilm formation, and infection of host macrophages. Taken together, our studies have identified, for the first time, a LOS biosynthetic locus. This is an important step in assessing the differential distribution of LOSs among Mycobacterium species and understanding the role of LOSs in mycobacterial virulence.     

The presence of novel glucuronic acid-containing, type-specific glycolipid antigens within Mycobacterium spp. Revision of earlier structures

Previously, we had described the structures of the haptenic oligosaccharides of the surface glycopeptidolipid antigens from serotypes 9 and 25 of the Mycobacterium avium complex and had synthesized these units as putative antigenic probes. The lack of chemical concordance between the synthetic products and the haptens has prompted a re-examination of these structures utilizing the instrumental techniques not previously available of fast atom bombardment-mass spectrometry, Fourier transform infra-red, and high resolution NMR spectroscopy. With the additional information thus available, more extensive chemical fragmentations by base degradation, followed by alkylation, have furnished supportive evidence to allow formulation of revised and novel structures, all of which contain glucuronic acid: serotype 9, 2,3-di-O-Me-L-Fucp(alpha 1----4)-D-GlcAp(beta 1----4)-2,3-di-O-Me-L-Fucp(alpha 1----3)-L-Rhap(alpha 1----2)-6dTal; and serotype 25, 4-acetamido-4,6-dideoxy-2-O-Me-hexosyl(alpha 1----4)-D- GlcAp(beta 1----4)2-O-Me-L-Fucp(alpha 1----3)-L-Rhap(alpha 1----2)6dTal. Glucuronic acid, acetamido sugars, and other novel sugars appear to be widespread in the glycopeptidolipid antigens of Mycobacterium spp. The revised structures will allow renewed synthesis of artificial antigen probes and rational approaches to preparing monoclonal antibodies, both necessary for the new diagnostics required to trace the sources of widespread infections due to M. avium and Mycobacterium intracellulare.     

Lack of correlation between colony morphology and lipooligosaccharide content in the Mycobacterium tuberculosis complex.

Rough and smooth colony variants of the Mycobacterium tuberculosis complex were compared with respect to their composition in trehalose-containing glycolipid antigens in view of the results of a recent investigation suggesting that the chemical basis of rough and smooth colony morphology in mycobacteria may reside in the occurrence of lipooligosaccharides. A careful chemical characterization of the individual glycolipids of the selected strains allowed the identification of the major glycolipids. The comparative study of the glycolipid content of the smooth Canetti strain, its spontaneous rough variant, and 16 additional strains of M. tuberculosis, M. bovis and M. africanum showed that the presence of lipooligosaccharides was not related to the morphology of the colonies.     

Mycobacterium valentiae sp. nov., a new species of the genus Mycobacterium isolated from soil

One strain of rapidly growing scotochromogenic mycobacteria was isolated. This study comprises 101 biochemical, cultural and morphological characteristics and reports the behavior towards several concentrations of the most commonly used antituberculous drugs. The organism is considered to belong to a new species of the genus Mycobacterium and has been deposited in the American Type Culture Collection, Rockville, Maryland, USA (ATCC 29356) and in the Czechoslovak Collection of Microorganisms, Czechoslovak National Collection of Type Cultures, Institute of Hygiene and Epidemiology, Srobárova 48, 100 42 Prague 10 under the My 220/77.     

O-Acetylation of the terminal N-acetylglucosamine of the lipooligosaccharide inner core in Neisseria meningitidis. Influence on inner core structure and assembly

O-Acetylation is a common decoration on endotoxins derived from many Gram-negative bacterial species, and it has been shown to be instrumental (e.g. in Salmonella typhimurium) in determining the final tertiary structure of the endotoxin and the immunogenicity of the molecule. Structural heterogeneity of endotoxins produced by mucosal pathogens such as Neisseria meningitidis is determined by decorations on the heptose inner core, including O-acetylation of the terminal N-acetylglucosamine (GlcNAc) attached to HepII. In this report, we show that O-acetylation of the meningococcal lipooligosaccharide (LOS) inner core has an important role in determining inner core assembly and immunotype expression. The gene encoding the LOS O-acetyltransferase, lot3, was identified by homology to NodX from Rhizobium leguminosarum. Inactivation of lot3 in strain NMB resulted in the loss of the O-acetyl group located at the C-3 position of the terminal GlcNAc of the LOS inner core. Inactivation of either lot3 or lgtG, which encodes the HepII glucosyltransferase, did not result in the appearance of the O-3-linked phosphoethanolamine (PEA) groups on the LOS inner core. Construction of a double mutant in which both lot3 and lgtG were inactivated resulted in the appearance of O-3-linked PEA groups on the LOS inner core. In conclusion, O-acetylation status of the terminal GlcNAc of the gamma-chain of the meningococcal LOS inner core is an important determinant for the appearance or exclusion of the O-3-linked PEA group on the LOS inner core and contributes to LOS structural diversity. O-Acetylation also likely influences resistance to complement-mediated lysis and may be important in LOS conjugate vaccine design.     

Identification of metabolites from degradation of naphthalene by a Mycobacterium sp.

A Mycobacterium sp. isolated from oil-contaminated sediments was previously shown to mineralize 55% of the added naphthalene to carbon dioxide after 7 days of incubation. In this paper, we report the initial steps of the degradation of naphthalene by a Mycobacterium sp. as determined by isolation of metabolites and incorporation of oxygen from ¹⁸O₂ into the metabolites. The results indicate that naphthalene is initially converted to cis- and trans-1,2-dihydroxy-1,2-dihydronaphthalene by dioxygenase and monooxygenase catalyzed reactions, respectively. The ratio of the cis to trans-naphthalene dihydrodiol isomers was approximately 25:1. Thin layer and high pressure liquid chromatographic and mass spectrometric techniques indicated that besides the cis- and trans-1,2-dihydroxy-1,2-dihydronaphthalene, minor amounts of ring cleavage products salicylate and catechol were also formed. Thus the formation of both cis and trans-naphthalene dihydrodiols by the Mycobacterium sp. is unique. The down-stream reactions to ring cleavage products proceed through analogous dioxygenase reactions previously reported for the bacterial degradation of naphthalene.     

An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis

The insertion sequence IS900 has been considered specific for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and has, therefore, been used as the target gene for diagnostic PCR of M. paratuberculosis. From a healthy dairy cow we have isolated and characterised a mycobacterium harbouring one copy of a sequence with 94% identity to IS900 at the nucleic acid level. The isolate was shown to be related to Mycobacterium cookii, as assessed by 16S rRNA sequencing. Strong amplifications were obtained with several PCR primers described for detection of IS900. This finding shows the need of alternative PCR systems based on other genes than IS900 to confirm the presence of M. paratuberculosis.     

Pyrene degradation by a Mycobacterium sp.: identification of ring oxidation and ring fission products.

The degradation of pyrene, a polycyclic aromatic hydrocarbon containing four aromatic rings, by pure cultures of a Mycobacterium sp. was studied. Over 60% of [14C]pyrene was mineralized to CO2 after 96 h of incubation at 24 degrees C. High-pressure liquid chromatography analyses showed the presence of one major and at least six other metabolites that accounted for 95% of the total organic-extractable 14C-labeled residues. Analyses by UV, infrared, mass, and nuclear magnetic resonance spectrometry and gas chromatography identified both pyrene cis- and trans-4,5-dihydrodiols and pyrenol as initial microbial ring-oxidation products of pyrene. The major metabolite, 4-phenanthroic acid, and 4-hydroxyperinaphthenone and cinnamic and phthalic acids were identified as ring fission products. 18O2 studies showed that the formation of cis- and trans-4,5-dihydrodiols were catalyzed by dioxygenase and monooxygenase enzymes, respectively. This is the first report of the chemical pathway for the microbial catabolism of pyrene.