Lipopolysaccharides ofVibrio fluvialis 181-86 Kobe possessing an antigenic factor in common with O1Vibrio cholerae

The chemical and serological characteristics of lipopolysaccharides (LPS) isolated fromVibrio fluvialis 181-86 Kobe were compared with those of LPS isolated from O1Vibrio cholerae and Vibrio bio-serogroup 1875 (Original and Variant), a marine vibrio possessing an antigenic factor in common with O1Vibrio cholerae. Two kinds of LPS (S-type LPS and R-type LPS) were extracted fromV. fluvialis 181-86 Kobe. The S-type LPS is different from LPS of the other serotypes (O1–O18) ofV. fluvialis in that the former was found to contain the pair perosamine and quinovosamine, a characteristic component of O1V. cholerae LPS. Furthermore, the sugar composition of the S-type LPS is close to that of Vibrio bio-serogroup 1875 LPS because both LPS contained mannose and the same two unidentified neutral sugars. Definite serological cross-reactivity in the passive hemolysis and the passive hemolysis inhibition tests were demonstrated between LPS fromV. fluvialis 181-86 Kobe and that from Vibrio bioserogroup 1875 Variant.     

Vibrio fluvialis: a new serogroup (19) possessing the Inaba factor antigen of Vibrio cholerae O1

A serogroup of Vibrio fluvialis possessing the C (Inaba) antigen but not the B (Ogawa) nor A antigen of V. cholerae O1 is described. The O-antigen of this serogroup was identical with that of bioserogroup 1875-variant of a marine Vibrio species. As the O-antigen of this serogroup was not agglutinated by any of O-antisera for the 18 serogroups of V. fluvialis already recognized, it was designated O-serogroup 19 of this species.     

A bioserogroup of marine vibrios possessing somatic antigen factors in common with Vibrio cholerae O1

A bioserogroup of halophilic vibrios, tentatively labelled bioserogroup 1875, strongly agglutinated by O1 antiserum of Vibrio cholerae is described. Cross-agglutination and agglutinin-absorption tests showed that these vibrios had antigens identical with the Ogawa and Inaba factors of V. cholerae O1, although they possessed their own specific antigen distinctive from the A factor that is the specific major antigen of the latter species. In addition, quantitative antigen variation similar to that of the Ogawa-to-Inaba type with V. cholerae O1 was recognized in this halophilic group. They were isolated from estuarine water and are considered to inhabit coastal aquatic environments. Phenotypically, however, this group is not identifiable with any of the species already recognized in the genus Vibrio.     

A bioserogroup of marine vibrios possessing somatic antigen factors in common with Vibrio cholerae O1

A bioserogroup of halophilic vibrios, tentatively labelled bioserogroup 1875, strongly agglutinated by O1 antiserum of Vibrio cholerae is described. Cross-agglutination and agglutinin-absorption tests showed that these vibrios had antigens identical with the Ogawa and Inaba factors of V. cholerae O1, although they possessed their own specific antigen distinctive from the A factor that is the specific major antigen of the latter species. In addition, quantitative antigen variation similar to that of the Ogawa-to-Inaba type with V. cholerae O1 was recognized in this halophilic group. They were isolated from estuarine water and are considered to inhabit coastal aquatic environments. Phenotypically, however, this group is not identifiable with any of the species already recognized in the genus Vibrio .     

O-antigenic lipopolysaccharides isolated from a marine Vibrio, bio-serogroup 1875, possessing an antigenic factor in common with O1 Vibrio cholerae

The chemical and serological characteristics of lipopolysaccharides (LPS) isolated from Vibrio bio-serogroup 1875 were compared with those of O1 Vibrio cholerae LPS. Vibrio bio-serogroup 1875 LPS contained all the component sugars which were found in O1 V. cholerae LPS, i.e. glucose, L-glycero-D-manno-heptose, fructose, glucosamine, perosamine and quinovosamine, though the amount of perosamine, a characteristic component of O1 V. cholerae LPS, was very low compared with that of O1 V. cholerae LPS. Their LPS additionally contained mannose and two unidentified neutral sugars which are not regular constituents of O1 V. cholerae LPS. Definite serological cross-reactivity in the passive haemolysis test between LPS from Vibrio bio-serogroup 1875 and LPS from O1 V. cholerae was demonstrated.     

An N-[(R)-(-)-2-Hydroxypropionyl]-α-L-Perosamine Homopolymer Constitutes the O Polysaccharide Chain of the Lipopolysaccharide from Vibrio cholerae O144 Which Has Antigenic Factor(s) in Common with V. cholerae O76

Chemical and serological studies were performed with the lipopolysaccharide (LPS) from Vibrio cholerae O144 (O144). The LPS of O144 contained D -glucose, D -galactose, L -glycero-D -manno-heptose, D -fructose, D -quinovosamine (2-amino-2,6-dideoxy-D -gluco-pyranose) and L -perosamine (4-amino-4,6-dideoxy-L -manno-pyranose). The perosamine, a major component sugar of the LPS from O144, was in an L -configuration, as is also the case in the LPS from V. cholerae O76 (O76), in contrast to the D -configuration of the perosamine in the LPS of V. cholerae O1. A structural analysis revealed that the O polysaccharide chain of the LPS from O144 is an α(1 → 2)-linked homopolymer of (R)-(-)-2-hydroxypropionyl-L -perosamine. The serological cross-reactivity between O144 and O76 was clearly revealed by cross-agglutination and cross-agglutinin absorption tests with whole cells, as well as by passive hemolysis tests with sheep red-blood cells that had been sensitized with the LPS from O144 and O76. In contrast, in passive hemolysis tests, the LPS of O144 did not cross-react serologically with the LPSs from other strains such as V. cholerae O1 (Ogawa and Inaba), V. cholerae O140, Vibrio bio-serogroup 1875 (Original and Variant) and Yersinia enterocolitica O9. The LPSs from these strains consist of O polysaccharide chains composed of α(1 → 2)-linked homopolymers of D -perosamine with various N-acyl groups, and they share the Inaba antigen factor C of V. cholerae O1 in common. The results obtained in this study demonstrate that the absolute configuration of the perosamine residue in homopolymers plays a very important role in the expression of the serological specificity of the Inaba antigen factor C of V. cholerae O1.     

Structure of the O-polysaccharide from the lipopolysaccharide from Vibrio cholerae O6

The O-polysaccharide from Vibrio cholerae O6 was isolated from the LPS by mild-acid hydrolysis and has been investigated by sugar and methylation analysis and NMR spectroscopy. The polysaccharide was also depolymerized with aqueous hydrofluoric acid to give the repeating unit and multiples thereof. The O-polysaccharide had the following tetrasaccharide repeating unit. Two O-acetyl groups are present, one of them making the GlcNAc residue fully substituted and the steric crowding considerable at the branching residue.     

Lipopolysaccharides of Escherichia coli K12 Strains That Express Cloned Genes for the Ogawa and Inaba Antigens of Vibrio cholerae O1: Identification of O-Antigenic Factors

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O-polysaccharide chain composed of an α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine (4-amino-4,6-dideoxy-D -manno-pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N-(3-deoxy-L -glycero-tetronyl)-2-O-methyl-D -perosamine at the non-reducing terminus of the O-polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co-existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O-antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O-forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2-O-methyl group at the non-reducing terminus of the α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine homopolymer that forms the O-polysaccharide chain of LPS of V. cholerae O1 (Ogawa).     

O139霍乱弧菌LPS基因在大肠杆菌中的克隆和表达

利用粘粒载体pCOS5构建了国内分离的O139霍乱弧菌的基因组文库,并从文库中筛选获得可以表达O139霍乱弧菌脂多糖的重组克隆株E.coliJM109(pMG310)。重组粘粒pMG310经酶切分析,所克隆的外源DNA片段大小为37kb。实验证明：重组克隆株E.coliJM109(pMG310)所表达的脂多糖具有良好的免疫原性及反应原性。     

Chemical and serological properties of lipopolysaccharides from Vibrio parahaemolyticus O-untypeable strains isolated from patients

Chemical and serological studies have been carried out on the O-antigenic lipopolysaccharides (LPS) of six strains, U-6443, W-90144, X-3972, AD-7999, 90A-6611 and KX-V212, of Vibrio parahaemolyticus isolated from patients. The O-serotypes of these strains have not been identified because they were not agglutinated by any diagnostic antisera against known O-serotype strains. A compositional sugar analysis of their LPS revealed that out of the six O-untypeable (OUT) strains, U-6443, W-90144 and AD-7999 strains belonged to chemotype II (chemotype of O2), 90A-6611 and KX-V212 strains to chemotype III (chemotype of O3, O5, O11 and O13) and X-3972 strain to chemotype IV (chemotype of O4). A structural analysis of LPS isolated from KX-V212 revealed that the inner core region of the LPS consisted of only one mole of 2-keto-3-deoxy-D-manno-octonic acid, which carried a phosphate group at position C4 and the outer core at position C5. In passive hemolysis tests performed by using LPS as the antigen to sensitize sheep red blood cells (SRBC), and diagnostic antisera (O1 to O11) or anti-whole-cell rabbit antisera raised against O12, O13 and the six OUT strains, strong cross-reactivity was observed among LPS derived from the strains belonging to chemotype II (U-6443, W-90144, AD-7999 and O2). Strong cross-reactivity was also observed between X-3972 (chemotype IV) and O4 LPS. In contrast, LPS from two of the strains belonging to chemotype III (90A-6611 and KX-V212) did not react with any of the antisera raised against known O-serotypes. Cross-absorption tests showed that the O-antigens of U-6443, W-90144 and AD-7999 were identical to that of O2, and the O-antigen of X-3972 to that of O4. On the other hand, after the absorption of antisera raised against 90A-6611 and KX-V212 with O2 cells, the hemolytic activities against SRBC sensitized with homologous LPS were still retained at a high titer, whereas the hemolytic activities against SRBC sensitized with LPS from other O-serotype strains were completely eliminated. A cross-absorption test revealed that the O-antigens of these two strains were identical to each other. Thus, it was demonstrated that the O-serotype of OUT strains 90A-6611 and KX-V212 was not involved in the known O-serotypes; rather it represented a novel serotype which has not hitherto been reported.     

Lipopolysaccharide Isolated from a New O-Antigenic Form (O13) of Vibrio parahaemolyticus

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L -glycero-D -manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.     

Alterations in lipopolysaccharide produced by chemostat-grown Escherichia coli O157:H7 as a function of growth rate and growth-limiting nutrient

Escherichia coli O157:H7 was grown in chemostats as continuous cultures at different controlled growth rates and under different nutrient limitations to determine the effects on lipopolysaccharide (LPS) structure. LPS from whole cells and extracted using the hot aqueous phenol method was examined by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration after hydrolysis with acetic acid. At low growth rates under glucose limitation (D = 0.1 h-1, doubling time (td), approx. 416 min; or D = 0.4 h-1, td, approx. 104 min), E. coli O157 produced high molecular weight LPS identical to that previously characterized from cells grown in batch culture. At a high growth rate (D = 0.8 h-1, td, approx. 52 min), the ratio of high molecular weight LPS to low molecular weight LPS produced greatly decreased. A small amount of high molecular weight LPS, containing O-polysaccharide which lacked amino sugars, and which thus was chemically different from that previously characterized, was produced by the cells at high growth rates. The predominant form of LPS from these cells was of slightly higher molecular weight than rough LPS, probably S-R LPS, and it consistently formed aggregates on SDS-PAGE. This form of LPS was also predominant when E. coli O157 was grown under Mg2+ limitation at an intermediate growth rate (D = 0.4 h-1, td, approx. 104 min).     

Structure of the O-polysaccharide and classification of Proteus mirabilis strain G1 in Proteus serogroup O3.

The O-chain polysaccharide of the lipopolysaccharide (LPS) of a previously nonclassified strain of Proteus mirabilis termed G1 was studied by sugar analysis and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, rotating-frame NOE (ROESY), H-detected 1H,13C HMQC, and heteronuclear multiple-bond correlation (HMBC) experiments. The following structure of the polysaccharide was established: [carbohydrate structure: see text] where D-GalA6(L-Lys) stands for N(alpha)-(D-galacturonoyl)-L-lysine. The structure of the O-polysaccharide of P. mirabilis G1 is similar, but not identical, to that of P. mirabilis S1959 and OXK belonging to serogroup O3. Immunochemical studies with P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera revealed close LPS-based serological relatedness of P. mirabilis G1 and S1959, and therefore it was suggested to classify P. mirabilis G1 in serogroup O3 as a subgroup. P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera also cross-reacted with LPS of P. mirabilis strains from two other serogroups containing D-GalA6(L-Lys) in the O-polysaccharide or in the core region.     

Sugar composition of the polysaccharide portion of lipopolysaccharides isolated from non-O1 Vibrio cholerae O2 to O41, O44, and O68

The sugar composition of the polysaccharide portion of lipopolysaccharides (LPS) was determined for 42 serovars of non-O1 Vibrio cholerae, i.e., from serogroups O2 to O41, O44, and O68. On the basis of their compositional sugar pattern, they were classified into 24 chemotypes. 2-Keto-3-deoxyoctonate (KDO) was totally undetectable by the conventional color test (Weissbach's reaction) under the conventional hydrolysis conditions. Instead, a kind of KDO-like substance, which was positive in the reaction but not identical to KDO, was found in serogroup O19. Fructose, a characteristic sugar constituent of O1 V. cholerae LPS, was found in 33 serogroups but was absent from nine serogroups, approximately 20% of the members of this group so far examined.     

Monoclonal antibodies against Vibrio anguillarum O2 and Vibrio ordalii identify antigenic differences in lipopolysaccharide O-antigens

Abstract Monoclonal antibodies (mAbs) that recognize distinct species-specific antigenic epitopes in O-antigens from Vibrio anguillarum O2, O2a and certain O2b strains (mAb 7B4) and from Vibrio ordalii strains (mAbs A16 and 7D11) were generated. Western immunoblot analysis using these mAbs revealed that vibrio strains grown in the presence of fresh rainbow trout blood expressed lipopolysaccharide (LPS) with longer (high molecular mass) O-antigens and extracellular capsular layers when compared to strains grown without rainbow trout blood. We also generated mAbs that react with O-antigens from V. anguillarum serotype O1 (mAbs 7B8, 7B5 and 1C3) and serotype O3 (mAbs 13A1 and 14C5) strains. These mAbs provide rapid and accurate diagnostic reagents for serological differentiation of V. ordalii from serotype O2 strains of V. anguillarum , and for serotyping of these pathogenic vibrios.     

Vibrio cholerae hemolytic activity

Mechanisms of realization of Vibrio cholerae hemolytic activitywere analyzed using summarized own results and data from the literature. It has been shown that lectin receptor, which coded by hlyA gene, participates in lysis of sheep erythrocytes, but not of rabbit erythrocytes, as well as interact with D-galactose with selectivity to 3 anomers. Lectin nature of HlyA can determine formation of its complexes with lypopolysaccharides (LPS) and enzymes, which promote realization of hemolysis (by lipase, lecitinase, neuraminidase). It has been determined that lipase activity correlates with hemolytic activity of nonepidemic variants of V. cholerae. Lipase is considered as the enzyme marker of sheep erythrocytes hemolysis. It is assumed that LPS and lipase play shaperon-like role during interaction of HlyA with lipids, which promote denaturation of hemolytic active monomer in hemagglutinating oligomer.     

The effect of removal of D-fructose on the antigenicity of the lipopolysaccharide from a rough mutant of Vibrio cholerae Ogawa

The lipopolysaccharide (LPS) of a rough mutant (95R) of Vibrio cholerae Ogawa has been investigated chemically and serologically. D-Fructose was released from LPS under conditions (10mM trifluoroacetic acid, 60 degrees, 1 h) that liberated no other sugar constituent of the LPS (2-amino-2-deoxy-D-glucose, D-glucose, L-glycero-D-manno-heptose). Upon periodate oxidation, D-fructose and D-glucose were oxidised quantitatively, whereas approximately 50% of heptose was periodate-resistant. The data indicate that D-fructose does not link the polysaccharide and lipid A portion as proposed earlier, and suggest that D-fructose is present as a branch. By passive hemolysis inhibition, it was shown that the release of D-fructose paralleled the exposure of an antigenic determinant cryptic in LPS.     

Evaluation of polysaccharide antigens of Vibrio cholerae O139 of different origin by monoclonal antibodies

The epitope composition of O-polysaccharides in the lipopolysaccharide (LPS) of V. cholerae, serogroup O139, isolated from clinical material and water of surface reservoirs was analyzed with the use of monoclonal antibodies. The analysis demonstrated that these O-polysaccharides were similar in their structure and chemical composition. In LPS of V. cholerae O139 clinical strains O-polysaccharide determinants occurred more often. Among V. cholerae isolated from water strains on whose surface individual epitopes of O-polysaccharide occurred less frequently or were absent appeared to be more numerous. A decrease in the concentration of microbial cells in the process of their testing by immunological methods led to increased percent of negative reactions with specific antibodies. Some V. cholerae O139 strains isolated from water were similar in the epitope composition of their O-polysaccharide and binding activity to cultures isolated from humans. As indicated by the results of these studies, cholera vibrios Bengal and vibrios isolated from river water on the territory of Russia had quantitative differences due to a higher level of the production of O-polysaccharide determinants and their occurrence in V. cholerae of serogroup O139.     

Comparison of hemolysins of Vibrio cholerae non-O1 and Vibrio hollisae with thermostable direct hemolysin of Vibrio parahaemolyticus

Hemolysin (Vh-rTDH) produced by Vibrio hollisae and hemolysin (NAG-rTDH) produced by Vibrio cholerae non-O1 were characterized and compared with hemolysin (Vp-TDH) produced by Vibrio parahaemolyticus. These three hemolysins are each composed of two subunits and have similar, but not identical, molecular weights. The amino acid compositions of Vp-TDH and NAG-rTDH are similar, but are different from that of Vh-rTDH. The three hemolysins showed similar lethal toxicities to mice. The effects of temperature on hemolysis and the time dependencies of hemolysis by the three hemolysins were similar. The three were concluded to be immunologically related, but not identical, and to have common and also unique antigenic determinants.     

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.