Molecular mechanics of the mycobacterial cell wall: from horizontal layers to vertical scaffolds.

Current models depicting the structural organization of the mycobacterial cell wall assume peptidoglycan and galactan strands to run in parallel to the cytoplasmic membrane forming several horizontal layers beneath perpendicularly oriented mycolic acids. Following a thorough re-evaluation of the currently available chemical, biochemical and electron microscopical data, we propose a fundamentally distinct principle of the physical organization and biosynthesis of the mycobacterial cell wall skeleton. According to this new concept, the solid and elastic matrix that makes the mycobacterial cell wall a formidably impermeable barrier is the direct consequence of cross-linked glycan strands which all run in a direction perpendicular to the cytoplasmic membrane.

Layered murein revisited: a fundamentally new concept of bacterial cell wall structure, biogenesis and function.

The classical concept of the architecture of microbial murein assumes cross-linked glycan chains to be arranged in horizontal layers outside of the plasma membrane. It necessitates elaborate hypotheses to explain processes such as the biosynthesis, growth and division of the bacterial cell wall and provides no explanation for transenvelope macromolecular transport. Moreover, this model is difficult to reconcile with a number of basic chemical and electron microscopical data. According to a fundamentally distinct concept which is presented here, glycan strands in the microbial wall run perpendicular to the plasma membrane, each strand being cross-linked by peptide bridges with four other strands. This arrangement allows the formation of a structured matrix pierced with ordered ionophoric channels potentially harboring either lipoprotein or teichoic (lipoteichoic) acid molecules in Gram-negative and Gram-positive bacteria, respectively. New wall structures are synthesized in toto emerging from the cytoplasmic membrane as a condensed gel-like network below the old wall without being covalently attached to it, expanding due to inherent elasticity as the old wall is lyzed. This model reflects published genetic and biochemical data and offers a simple explanation for peptidoglycan biogenesis. As the biosynthesis is terminated by enzymic cleavage of all glycan strands, murein is irreversibly released from the membrane. The murein detachment prepares the membrane for de novo assembly of both the new wall synthesis machinery and the multicomponent factory for protein, DNA and phospholipid transfer. Being assembled in parallel, both new murein and the traffic complexes grow from the membrane together. This concept eliminates the necessity for the traffic complexes to penetrate intact murein. In the process of simultaneous assembly, the expanding murein functions as a lifting platform driven by the force of turgor pressure, transporting macromolecules through the perisplasmic space.

Structure of the O-specific polysaccharide chain of Shigella boydii type 5 lipopolysaccharide: a repeated study.

An acidic, partially O-acetylated O-specific polysaccharide was obtained by mild acid degradation of Shigella boydii type 5 lipopolysaccharide and studied by 1H and 13C NMR spectroscopy, including 2D COSY, 13C-1H heteronuclear COSY, 1D NOE, and 2D ROESY experiments, and chemical methods (sugar and methylation analysis, O-deacetylation, carboxyl reduction, solvolysis with anhydrous HF, partial acid hydrolysis. Smith degradation). It was concluded that the polysaccharide has a hexasaccharide repeating unit of the following structure: [Formula: See Text] with the degree of O-acetylation varying over 30-50%. The established structure differs from that proposed recently for the O-specific polysaccharide of the same S. boydii serotype [M.J. Albert et al., Carbohydr. Res., 265 (1994) 121-127].

[The immunological activity of Neisseria meningitidis lipo-oligosaccharide incorporated into liposomes]. / Immunologicheskaia aktivnost' lipooligosakharida Neisseria meningitidis, vkliuchennogo v liposomy.

Immune response to lipooligosaccharide (LOS), isolated from group B N. meningitidis cell wall, was studied after its incorporation into liposomes. The adjuvant effect produced by liposomal LOS, more pronounced in comparison with that produced by native LOS, was observed after the injection of the preparation by different routes (intravenous, intraperitoneal and subcutaneous injections), but the highest level of response was achieved after the intravenous and intraperitoneal injections of the preparation. Experiments aimed at the study of the dynamics of plaque-forming cells and the level of antibodies after intraperitoneal immunization of CBA mice with LOS revealed that immune response to liposomal LOS was more intense and prolonged than that to the free antigen. Liposomal LOS induced genetically nonrestricted and T-independent immune response. This fact was confirmed in experiments on mice having different haplotypes, such as CBA (k), C57BL/6 (b), BALB/c (d) and in nude mice with congenital thymic aplasia. In addition, liposomal LOS induced mainly the accumulation of IgM and IgG and did not produce the effect of immunological memory.

Structure of the O-specific polysaccharide of the lipopolysaccharide from Yersinia kristensenii O:25.35.

Mild hydrolysis of the lipopolysaccharide (LPS) from Yersinia kristensenii serovar O:25.35 with acid afforded the O-specific polysaccharide (PS) which contained D-glucose, D-galactose, 2-acetamido-2,6-dideoxy-L-galactose, 2-acetamido-2-deoxy-D-glucose, glycerol, and phosphate in the ratios 3:1:1:1:1. On the basis of 31P and 13C NMR spectroscopy, hydrolysis, methylation studies, Smith degradation, and dephosphorylation, the repeating unit of PS was shown to have the following structure. [formula: see text]

Glycopeptidolipids--a new class of artificial antigens with carbohydrate determinants. Synthesis of artificial antigen with type-specific oligosaccharide hapten from Neisseria meningitidis group B.

Synthetic lipopeptide N-palmitoyltyrosyl-seryl-seryl-asparaginyl-alanine, an analogue of B-mitogenic tripalmitoyl-pentapeptide from Escherichia coli lipoprotein, was coupled with an oligosaccharide hapten from Neisseria meningitidis lipooligosaccharide to give a glycopeptidolipid conjugate--the artificial antigen of a new type processing the type-specific microbial determinant.

Structural characterization of the lipid A component of pathogenic Neisseria meningitidis.

The lipid A component of meningococcal lipopolysaccharide was structurally characterized by using chemical modification methods, methylation analysis, 31P nuclear magnetic resonance, and laser desorption mass spectroscopy. It was shown that Neisseria meningitidis lipid A consists of a 1,4'-bisphosphorylated beta(1'----6)-linked D-glucosamine disaccharide (lipid A backbone), both phosphate groups being largely replaced by O-phosphorylethanolamine. This disaccharide harbors two nonsubstituted hydroxyl groups at positions 4 and 6', the latter representing the attachment site of the oligosaccharide portion in lipopolysaccharide. In addition, it is substituted by up to six fatty acid residues. In the major lipid A component, representing a hexaacyl species, the hydroxyl groups at positions 3 and 3' carry (R)-3-hydroxydodecanoic acid [12:0(3-OH)], whereas the amino groups at positions 2 and 2' are substituted by (R)-3-(dodecanoyloxy)tetradecanoic acid [3-O(12:0)-14:0]. A minor portion was present as a tetraacyl lipid A component lacking either dodecanoic acid (12:0) or 12:0 and 12:0(3-OH). N. meningitidis lipid A, therefore, significantly differs from Escherichia coli lipid A by the nature and locations of fatty acids and the substitution of O-phosphorylethanolamine for the nonglycosyl (4'-P) and glycosyl phosphate.

Protein antigens of genetically related Rickettsia prowazekii strains with different virulence.

The protein antigens of two distinct lines of genetically related strains, namely the nonpathogenic strain E and its virulent revertant EVir and of the standard virulent strain Breinl were compared in SDS-PAGE and immunoblot assay using typhus patient sera and immune rabbit sera. No differences in the polypeptide pattern as detected in SDS-PAGE were found between strain E and EVir; the Breinl strain differed in a 30 kD protein. The high immunogenicity of the protein antigens of E, EVir and Breinl strains was demonstrated by immunoblot assay with human sera, which did not show any differences between the strains studied. Immunoblot analysis with immune rabbit sera to the strain E, EVir, and Breinl showed differences in immunological response to the 70 kD and 60 kD polypeptides of low virulent strain E and those of virulent strains EVir and Breinl.

Structural characterization of the lipid A component of Pseudomonas aeruginosa wild-type and rough mutant lipopolysaccharides.

The structure of the lipid A component of lipopolysaccharides isolated from two wild-type strains (Fisher 2 and 7) and one rough mutant (PAC 605) of Pseudomonas aeruginosa was investigated using chemical analysis, methylation analysis, combined gas-liquid chromatography/mass spectrometry, laser-desorption mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of a pyranosidic beta 1,6-linked D-glucosamine disaccharide [beta-D-GlcpN-(1----6)-D-GlcpN], phosphorylated in positions 4' and 1. Position 6' of the beta-D-GlcpN-(1----6)-D-GlcpN disaccharide was identified as the attachment site of the core oligosaccharide and the hydroxyl group at C-4 was not substituted. Lipid A of the three P. aeruginosa strains expressed heterogeneity with regard to the degree of acylation: a hexaacyl as well as a pentaacyl component were structurally characterized. The hexaacyl lipid A contains two amide-bound 3-O-acylated (R)-3-hydroxydodecanoic acid groups [12:0(3-OH)] at positions 2 and 2' of the GlcN dissacharide and two ester-bound (R)-3-hydroxydecanoic acid groups [10:0(3-OH)] at positions 3 and 3'. The pentaacyl species, which represents the major lipid A component, lacks one 10:0(3-OH) residue, the hydroxyl group in position 3 of the reducing GlcN residue being free. In both hexa- and pentaacyl lipid A the 3-hydroxyl group of the two amide-linked 12:0(3-OH) residues are acylated by either dodecanoic (12:0) or (S)-2-hydroxydodecanoic acid [12:0(2-OH)], the lipid A species with two 12:0(2-OH) residues, however, being absent. The presence of only five acyl residues in the major lipid A fraction may account for the low endotoxic activity observed with P. aeruginosa lipopolysaccharide.

Location and identification of substituents with free amino group in lipopolysaccharides of gram-negative bacteria with the use of chromophore labelling.

Interaction of ten different lipopolysaccharides (LPS) with 2,4-dinitrofluorobenzene yielded quantitatively yellow dinitrophenyl derivatives (DNP-LPS) to show the presence of substituents with free amino group. The DNP-LPS samples were degraded with 1% acetic acid, and after removal of lipid A precipitates the supernatants were separated on a Sephadex G-25 column to give coloured polysaccharide, oligosaccharide and monomeric fractions monitored at lambda DNP = 365 nm. The coloured materials, including DNP-derivative of lipid A, were dephosphorylated with hydrofluoric acid followed by identification of the released DNP-amines by thin layer chromatography (TLC) on silica gel. Subsequently, the dephosphorylated materials were hydrolysed with hydrochloric acid followed by TLC analysis. The approach allowed to detect, locate and identify the substituents with free amino group within the LPS molecules. Moreover, two types of core structures within LPS preparation from one strain were discovered for five microorganisms.

[Antigenic polysaccharides of Shigella bacteria. Structure of the polysaccharide chain of the lipopolysaccharide from Shigella boydii, type 11]. / Antigennye polisakharidy bakterii roda Shigella. Struktura polisakharidnoi tsepi lipopolisakharida Shigella boydii, tip 11.

On mild acid degradation of the Shigella boydii, type 11 lipopolysaccharide, the corresponding O-specific polysaccharide composed of D-glucuronic acid, 2-acetylamino-2-deoxy-D-glucose, D-ribose and L-rhamnose residues in the ratio 1:1:1:3 was obtained. Methylation, partial acid hydrolysis and 13C-NMR spectral data for the polysaccharide led to the structure of the oligosaccharide repeating unit as a branched hexasaccharide: [formula: see text]. Numerous O-acetyl groups attached non-stoichiometrically to the residues of D-glucuronic acid, L-rhamnose and 2-acetylamino-2-deoxy-D-glucose were located with the use of 13C-NMR spectroscopy.

FITC-labeled lipopolysaccharide: use as a probe for liposomal membrane incorporation studies.

FITC-labeled LPS from Neisseria meningitidis can be used as a probe to follow the process of LPS incorporation into liposomal membrane and to study its interaction with a bilayer. The incorporation of FITC-LPS into the bilayer was proved by physicochemical methods as well as by liposomal LPS toxicity decrease in actinomycin D-sensitized mice. Fluorescence intensity increase was observed upon the insertion of FITC-LPS into the membrane of dehydration/rehydration vesicles and vesicles obtained by co-sonication of lipid suspension and FITC-LPS. Following FITC-LPS fluorescence polarization it was shown that the substance seems to be clusterized in the liposomal membrane starting from FITC-LPS/lipid molar ratio 1:800.

[The immunological properties of the main outer membrane protein of Legionella]. / Immunologicheskie svoistva glavnogo belka vneshnei membrany legionell.

The immunogenic properties of Legionella outer membrane main protein (OMMP) were studied by its effect on the proliferative activity of lymphocytes in guinea pigs. Preliminary immunization with OMMP activated only the specific and nonspecific proliferation of spleen cells. After infection with Legionella, secondary immune response developed in the spleen and lungs of previously immunized animals, in contrast to intact ones, and the nonspecific proliferative activity of lymphocytes in the spleen and lungs of previously immunized animals considerably increased. These results are indicative of the fact that Legionella OMMP, similarly to other Legionella antigens and immunomodulators, may be used for the formation of protective immunity.

[The protective properties of different Legionella antigens in experimental Legionella infection]. / Izuchenie protektivnykh svoistv razlichnykh antigenov legionell pri éksperimental'noi legionelleznoi infektsii.

The protective properties of Legionella antigenic preparations were studied on guinea pigs with experimental Legionella infection. Preliminary immunization of guinea pigs with serotypic antigen, cytolysin, as well as live or formalin-treated Legionella cells, did not protect the animals from the subsequent aerogenic infection with 10(5) colony-forming units of virulent L. pneumophila. Immunization with the main outer membrane protein ensured the survival of 70% of the animals and inhibited the proliferation of the infective agent in the lungs of guinea pigs subjected to aerogenic infection with 10(5) colony-forming units of virulent L. pneumophila. The data obtained in this study indicate that the main outer membrane protein of L. pneumophila is capable of stimulating protective immunity.

[Isolation and primary characteristics of the common species-specific outer membrane protein of Rickettsia prowazekii]. / Vydelenie i pervichnaia kharakteristika obshchevidovogo belka naruzhnoi membrany Rickettsia prowazekii.

Common species-specific protein is isolated for the first time in the chromatographically pure state from the outer membrane of Rickettsia prowazekii, and its amino acid composition is determined. As revealed by chromatofocusing technique, the protein possesses pI 4.18 +/- 0.03. Three basic ninhydrin-positive compounds, differing from usual amino acids, were discovered in the protein hydrolyzate. Data suggesting the subunit structure of the isolated protein are presented.

[The structure of a repetitive unit of the glycerolphosphate- containing O-specific polysaccharide chain from Yersinia kristensenii strain 103 (0:12,26) lipopolysaccharide]. / Struktura povtoriaiushchegosia zvena glitserofosfatsoderzhashchei O-spetsificheskoi polisakharidnoi tsepi iz lipopolisakharida Yersinia kristensenii, shtamm 103 (0:12,26).

Mild acid hydrolysis of the lipopolysaccharide from Yersinia kristensenii strain 103 (0:12.26) afforded teichoic acid-like polysaccharide. From the results of methylation, dephosphorylation, partial Smyth degradation, and 13C and 31P NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [formula: see text] The structure was confirmed by complete interpretation of polysaccharide 13C NMR spectrum.

[Protective artificial polysaccharide-protein antigens made from the O-specific polysaccharides of Pseudomonas aeruginosa bacteria of the immunotypes 1, 2 and 7]. / Protektivnye iskusstvennye polisakharid-belkovye antigeny iz O-spetsificheskikh polisakharidov bakterii Pseudomonas aeruginosa immunotipov 1, 2 i 7.

O-specific polysaccharide (PS) obtained from P. aeruginosa lipopolysaccharide (LPS), immunotypes 1, 2 and 7, were condensed in the presence of aminated bovine serum albumin (amBSA). As a result, artificial polysaccharide-protein antigens were synthesized and their protective properties were studied by the subcutaneous immunization of mice with their subsequent challenge with the cells of the homologous strain injected intraperitoneally in a dose of 3-5 LD50. The most effective immunization was achieved by using homologous LPS, taken in a subimmunogenic dose, as adjuvant. Heterologous LPS, taken in the same doses, showed no stimulating effect. The protective properties of the conjugates were also found to depend on the length of the PS chain. The conjugate of amBSA and PS of immunotype 2 with a molecular weight of about 10 KD was found to possess the highest protective potency. The decrease of the molecular weight of PS of immunotype 7 from 50 KD to 10 KD by selective acid hydrolysis led to obtaining the conjugate with high protective potency. The possibility of using the structurally linked pair antigen-adjuvant for the development of artificial vaccines is discussed.

Somatic antigens of Shigella: structure of the O-specific polysaccharide chain of the Shigella dysenteriae type 7 lipopolysaccharide.

4-(N-Acetylglycyl)amino-4,6-dideoxy-D-glucose has been identified as a component of the Shigella dysenteriae type 7 O-specific polysaccharide, in addition to the previously reported 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galacturonic acid. On the basis of selective cleavage with anhydrous hydrogen fluoride and analysis by 1H- and 13C-n.m.r. spectroscopy and f.a.b.-mass spectrometry, it was concluded that the tetrasaccharide repeating-unit of the polysaccharide has the following structure: (structure; see text) where D-GalNAcAN is 2-acetamido-2-deoxy-D-galacturonamide and D-Qui4N is 4-amino-4,6-dideoxy-D-glucose.

[Identification of 5-aminovaleric acid as a characteristic product of metabolism of various Clostridium species]. / Identifikatsiia 5-aminovalerianovoi kisloty kak kharakteristicheskogoprodukta metabolizma nekotorykh predstavitelei roda klostridii.

An unusual ninhydrin-positive compound has been isolated from feces of accidentally contaminated Sprague-Dawley autbred rats and identified by mass spectrometry and 1H-NMR spectroscopy techniques as 5-aminovaleric acid. The described procedure of isolation, purification and structure determination can be recommended as a general method of identification of unusual ninhydrin-positive compounds in complex mixtures of biological origin. The 5-aminovaleric acid was found to be produced by an anaerobic bacterium Clostridium bifermentans. It is shown that not all Clostridium spp. excrete his amino acid and that the majority of microorganisms, normally inhabiting intestine of rats, simians and man, do not possess this ability. On the basis of data obtained, the test for 5-aminovaleric acid is proposed to be included into the taxonomy of bacteria.