Antigenic polysaccharides of bacteria: 41. Structures of the O-specific polysaccharides of Shigella dysenteriae types 4 and 5 revised by NMR spectroscopy

The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of nonstoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, the position of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by 13C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner. [Formula: see text].     

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->     

Production of Shiga toxin and a cytolethal distending toxin (CLDT) by serogroups of Shigella spp.

Abstract 56 strains of Shigella including 12 Shigella dysenteriae (serotypes 1, 2, 9, 11 and 12), 23 Shigella flexneri (serotypes 1, 2, 3, 4, 6, var. X and var. Y), 19 Shigella boydii (serotypes 1, 2, 4, 5, 7, 11, 13, 14, 15 and 18), and 2 Shigella sonnei were screened for their ability to produce both classic Shiga toxin and a new heat-labile cytolethal distending toxin (CLDT). Whereas extracellular Shiga toxin was only detectable in filtrates of five S. dysenteriae type 1 strains, CLDT was produced by four strains of S. dysenteriae type 2 and an isolate of S. boydii type 7. No cytotonic enterotoxins similar to Escherichia coli LT were observed in this study. None of the S. flexneri or S. sonnei isolates tested were found to produce extracellular cytotoxic factors. The Shiga toxin produced by the S. dysenteriae type 1 was neutralizable by anti-toxin to verotoxin 1 of E. coli O157 : H7. The Shigella CLDT was neutralizable by antisera prepared to a CLDT-producing E. coli O55 : H4.     

Antigenic polysaccharides of bacteria: 40. The structures of O-specific polysaccharides from <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> types 3 and 9 and <Emphasis Type="Italic">S. boydii</Emphasis> type 4 revised by NMR spectroscopy

The reported structures of O-specific polysaccharides from three type strains of Shigella bacteria were corrected by modern NMR techniques. The revisions concerned the configuration of the O-glycoside linkage (S. dysenteriae type 3, structure 1), the positions of monosaccharide residue glycosylation and acetalation by pyruvic acid (S. dysenteriae type 9, structure 2), and the attachment position of the side monosaccharide chain (S. boydii type 4, structure 3).

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Lipopolysaccharide core structures and their correlation with genetic groupings of Shigella strains. A novel core variant in Shigella boydii type 16

Bacteria Shigella, the cause of shigellosis, evolved from the intestinal bacteria Escherichia coli. Based on structurally diverse O-specific polysaccharide chains of the lipopolysaccharides (LPSs; O-antigens), three from four Shigella species are subdivided into multiple serotypes. The central oligosaccharide of the LPS called core is usually conserved within genus but five core types called R1-R4 and K-12 have been recognized in E. coli. Structural data on the Shigella core are limited to S. sonnei, S. flexneri and one S. dysenteriae strain, which all share E. coli core types. In this work, we elucidated the core structure in 14 reference strains of S. dysenteriae and S. boydii. Core oligosaccharides were obtained by mild acid hydrolysis of the LPSs and studied using sugar analysis, high-resolution mass spectrometry and two-dimensional NMR spectroscopy. The R1, R3 and R4 E. coli core types were identified in 8, 3 and 2 Shigella strains, respectively. A novel core variant found in S. boydii type 16 differs from the R3 core in the lack of GlcNAc and the presence of a D-glycero-D-manno-heptose disaccharide extension. In addition, the structure of an oligosaccharide consisting of the core and one O-antigen repeat was determined in S. dysenteriae type 8. A clear correlation of the core type was observed with genetic grouping of Shigella strains but not with their traditional division to four species. This finding supports a notion on the existing Shigella species as invalid taxa and a suggestion of multiple independent origins of Shigella from E. coli clones.     

New sugars from antigenic lipopolysaccharides of bacteria: identification and synthesis of 3-O-[(R)-1-carboxyethyl]-L-rhamnose, an acidic component of Shigella dysenteriae type 5 lipopolysaccharide.

A new acidic sugar, 3-O-[(R)-1-carboxyethyl]-L-rhamnose (1), has been identified as a constituent of the O-antigenic lipopolysaccharide of Sh. dysenteriae type 5. The structure of 1 has been established by physico-chemical methods and by synthesis. Alkylation of methyl 2,5-di-O-benzyl-alpha-L-rhamnofuranoside (6) with (S)- or (R)-2-chloropropionic acids, followed by removal of the protecting groups, afforded 3-O-[(R)-1-carboxyethyl]-L-rhamnose (9) and 3-O-[(S)-1-carboxyethyl]-L-rhamnose (10), respectively. The properties of 1 coincide with those of 9.     

Methionine uptake and cytopathogenicity of viable but nonculturable Shigella dysenteriae type 1.

A pathogenic strain of Shigella dysenteriae type 1 was selected for study to elucidate the physiology and potential pathogenicity of organisms in the viable but nonculturable (VBNC) state in the environment. Studies in our laboratory have shown that S. dysenteriae type 1 survives in laboratory microcosms in the VBNC state for long periods of time, i.e., more than 6 months. VBNC cells of S. dysenteriae type 1 were found to retain cytopathogenicity for cultured HeLa cells. To determine whether VBNC S. dysenteriae type 1 expressed protein after loss of culturability, 35S-labelled methionine was added to suspensions of VBNC cells. Total cellular proteins were extracted and examined by autoradiography. Results indicate that VBNC S. dysenteriae type 1 is capable of both active uptake of methionine and incorporation of methionine into protein. Amino acid uptake and protein synthesis substantiate the viability of cells of S. dysenteriae type 1 in the VBNC state, i.e., although the cells are unable to be cultured on laboratory media by standard bacteriological methods, the cells remain metabolically active. Furthermore, VBNC cells of S. dysenteriae type 1 may pose a potential public health hazard that has not yet been recognized.     

Potential virulence of viable but nonculturable Shigella dysenteriae type 1.

We examined a virulent strain of Shigella dysenteriae type 1 after induction into the viable but nonculturable (VBNC) state for its ability to (i) maintain the Shiga toxin (stx) gene; (ii) maintain biologically active Shiga toxin (ShT); and (iii) adhere to intestinal epithelial cells (Henle 407 cell line). PCR was used to amplify the stx gene from VBNC cells of S. dysenteriae type 1, thereby establishing its presence even when cells are in the VBNC state. VBNC S. dysenteriae type 1 ShT was monitored by the enzyme-linked immunosorbent assay with mouse monoclonal antibodies against the B subunit of ShT and affinity-purified rabbit polyclonal antibodies against ShT. We used the Henle 407 cell line to study the adhesive property of VBNC S. dysenteriae type 1 cells in a series of tissue culture experiments. Results showed that VBNC S. dysenteriae type 1 not only maintained the stx gene and biologically active ShT but also remained capable of adhering to Henle 407 cells. However, S. dysenteriae type 1 cells lost the ability to invade Henle 407 cells after entering the VBNC state. From results of the study, we conclude that VBNC cells of S. dysenteriae type 1 retain several virulence factors and remain potentially virulent, posing a public health problem.     

Somatic antigens of shigella. Structural investigation on the O-specific polysaccharide chain of Shigella dysenteriae type 1 lipopolysaccharide.

The O-specific polysaccharide obtained from the lipopolysaccharide of Shigella dysenteriae type 1 (Shigella shiga) by mild acid hydrolysis followed by fractionation on Sephadex G-50 was found to be identical to that desribed by Morgan's group and was composed of L-rhamnose, D-galactose and N-acetyl-D-glycosamine in a ratio 2:1:1. On the basis of methylation analysis data the polysaccharide was proved to be a linear chain of monosaccharide residues in pyranose forms substituted at position 3, except for that of galactose substituted at position 2. Selective cleavage, based on the N-deacetylation reaction of the polymer, together with determination of linkage configurations by chromic anhydride oxidation showed that the O-specific polysaccharide is built up of repeating tetrasaccharide units whose proposed structure is given below -3)-alpha-L-Rhap (1-3)-alpha-L-Rhap(1-2)-alpha-D-Galp(1-3)-alphapD-GlcNAcp(1- where RHAP = rhamnopyranose, Galp = galactopyranose, and GlcNAcp = N-acetyl-glucosamine. The present findings confirmed the considerations of Heidelberger on the substitution patterns of L-rhamnose and D-galactose residues from the results of serological studies.     

Phosphate regulon in members of the family Enterobacteriaceae: comparison of the phoB-phoR operons of Escherichia coli, Shigella dysenteriae, and Klebsiella pneumoniae.

The structure and function of the phoB and phoR genes of Shigella dysenteriae strains and Klebsiella pneumoniae, which are involved in regulation of the phosphate regulon, were analyzed. Complementation tests among the genes of Escherichia coli, S. dysenteriae strains, and K. pneumoniae for production of alkaline phosphatase indicate that S. dysenteriae serotype 2 and serotype 3 strains and K. pneumoniae are phoA+ phoB+ phoR+ but S. dysenteriae Sh and serotype 1 strains are phoA phoB+ phoR. Nucleotide sequences of phoB and phoR of S. dysenteriae Sh and K. pneumoniae are highly homologous to those of E. coli, except for a single base insertion found in phoR of S. dysenteriae Sh.     

Molecular epidemiology of plasmid patterns in Shigella dysenteriae type I obtained from an outbreak in West Bengal (India)

Abstract Multiple antibiotic-resistant Shigella dysenteriae type 1 isolates from a recent epidemic in West Bengal (India) showed identical plasmid patterns. All isolates were resistant to ampicillin (Am), chloramphenicol (Cm), tetracycline (Tc), streptomycin (Sm) and trimethoprim (Tp) and contained 6 plasmids, ranging from 2.5–120 kb. The Am resistance determinant was located on the 120 kb plasmid. This plasmid was unstable when the S. dysenteriae strains were grown above 37°C. The Bangladesh strains of S. dysenteriae type 1 showed identical plasmid patterns, except that many isolates were Am-sensitive and lacked the 120 kb plasmid. In strains from both Bangladesh and West Bengal, predominantly group-B plasmids conferred resistance to Cm and Tc. Comparisons of Eco R1 fragments generated from the total plasmid DNA content of each strain support the view that the plasmids present in the S. dysenteriae type 1 strains isolated from all recent epidemics in India and Bangladesh were identical.     

Monoclonal antibodies against the surface antigens of Shigella flexneri serotype 1b and Shigella dysenteriae serotype 1

Monoclonal antibodies against the surface antigens of Shigella flexneri 1b and S. dysenteriae 1 were prepared. The specificities of the antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), and quantitative agglutination using microtiter plate. Monoclonal antibodies against S. flexneri 1b, designated Sf2B2 and Sf2G4, belonged to IgG2a and IgG1 subclass, respectively. The former was specific for S. flexneri 1b, whereas the latter was reactive not only to S. flexneri 1b, but also weakly to 3a and 4b. Monoclonal antibody against S. dysenteriae 1, Sd5E1 (IgM), reacted with S. dysenteriae 1, 3, 6, 7, and S. boydii 2.     

Influence of different rol gene products on the chain length of Shigella dysenteriae type 1 lipopolysaccharide O antigen expressed by Shigella flexneri carrier strains.

Introduction of the rol genes of Shigella dysenteriae 1 and Escherichia coli K-12 into Shigella flexneri carrier strains expressing the heterologous S. dysenteriae type 1 lipopolysaccharide resulted in the formation of longer chains of S. dysenteriae 1 O antigen. In bacteria producing both homologous and heterologous O antigen, this resulted in a reduction of the masking of heterologous O antigen by homologous lipopolysaccharide and an increased immune response induced by intraperitoneal immunization of mice by recombinant bacteria. The rol genes of S. dysenteriae 1 and E. coli K-12 were sequenced, and their gene products were compared with the S. flexneri Rol protein. The primary sequence of S. flexneri Rol differs from both E. coli K-12 and S. dysenteriae 1 Rol proteins only at positions 267 and 270, which suggests that this region may be responsible for the difference in biological activities.     

Comparative genomics and phylogenetic analysis of S. dysenteriae subgroup

Shigella spp are the pathogen of bacillary dysentery, the leading intestinal contagious disease in China. According to the O antigen, Shigella spp can be sub-grouped into four species: S.dysenteriae, S. flexneri, S. boydii and S. sonnei, also known as Shigella sub-groups A, B, C and D. In developing countries, S. dysenteriae A1, one of the thirteen serotypes in A sub-group, resulted in extremely serious bacterial diarrhea with mortality of 7%[1]. Traditional classifications were unsucces…     

Antigenic bacterial polysaccharides. 23. The structure of the O-specific polysaccharide chain of Salmonella arizonae 059 lipopolysaccharide

The O-specific polysaccharide of Salmonella arizonae O59 (Arizona 19) is composed of D-galactose, N-acetyl-D-glucosamine, and N-acetyl-L-fucosamine (FucNAc, 2-acetamido-2,6-dideoxy-L-galactose) in the ratio 1:1:1. The computerized calculation of the 13C NMR spectrum of the polysaccharide, based on the monosaccharide composition, spectra of the free monosaccharides and glycosydation effects, together with the chemical analysis (methylation and Smith degradation) showed that the polysaccharide is built up of trisaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp(1----3)-beta-D-GlcNAcp-(1----2)-beta- D-Galp-1(----. The molecular basis of serological interrelations between S. arizonae O59 and Pseudomonas aeruginosa O7 (Lányi) is discussed.     

Expression of Shigella dysenteriae serotype 1 O-antigenic polysaccharide by Shigella flexneri aroD vaccine candidates and different S. flexneri serotypes.

The potential utility of Shigella flexneri aroD vaccine candidates for the development of bi- or multivalent vaccines has been explored by the introduction of the genetic determinants rfp and rfb for heterologous O antigen polysaccharide from Shigella dysenteriae serotype 1. The serotype Y vaccine strain SFL124 expressed the heterologous antigen qualitatively and quantitatively well, qualitatively in the sense of the O antigen polysaccharide being correctly linked to the S. flexneri lipopolysaccharide R3 core oligosaccharide and quantitatively in the sense that typical yields were obtained, with ratios of homologous to heterologous O antigen being 4:1 for one construct and 1:1 for another. Moreover, both polysaccharide chains were shown to be linked to position O-4 of the subterminal D-glucose residue of the R3 core. In contrast to the hybrid serotype Y SFL124 derivatives, analogous derivatives of serotype 2a vaccine strain SFL1070 did not elaborate a complete heterologous O antigen. Such derivatives, and analogous derivatives of rough, O antigen-negative mutants of SFL1070, formed instead a hybrid lipopolysaccharide molecule consisting of the S. flexneri lipid A R3 core with a single repeat unit of the S. dysenteriae type 1 O antigen. Introduction of the determinants for the S. dysenteriae type 1 O antigen into a second serotype 2a strain and into strains representing other serotypes of S. flexneri, revealed the following for the expression of the heterologous O antigen: serotypes 1a, 1b, 2a, and 5a did not produce the heterologous O antigen, whereas serotypes 2b, 3a, 3b, 4a, 4b, 5b, and X did.     

A monoclonal antibody to Shigella dysenteriae serotype 13 cross-reacting with Shiga toxin

Abstract A monoclonal antibody (mAb ICT6) was produced against the newly described Shigella dysenteriae serotype type 13. The mAb was of IgM isotype and recognized purified Shiga toxin in ELISA and immunoblot. It also recognized periplasmic extract S. dysenteriae type 13 in immunoblot as did an affinity-purified polyclonal rabbit antiserum and a previously described monoclonal antibody to the B subunit of Shiga toxin. The mAb ICT6 did not neutralize the cytotoxic effects or S. dysenteriae type 13, Shiga toxin or periplasmic extracts of S. dysenteriae type 1 for HeLa cells.     

Structure of the O-specific polysaccharide chain of Shigella dysenteriae type 7 lipopolysaccharide

The structure of the O-specific polysaccharide chain of the Shigella dysenteriae type 7 lipopolysaccharide has been established mainly by 13C NMR analysis of the intact and modified (acetylated and de-O-acetylated) polymers, as well as of products of its solvolysis with anhydrous hydrogen fluoride. The polysaccharide contains two unusual sugar derivatives. N-acetyl-D-galactosaminuronamide and 4-(N-acetylglycyl)amido-4,6-dideoxy-D-glucose (GalNAcAN and Qui4N----GlyAc, respectively) and is built up of tetrasaccharide repeating units of the following structure: (Formula: see text). Serological cross-reaction of S. dysenteriae type 7 and Pseudomonas aeruginosa O4 (Lányl) is accounted for by the similarity of their O-specific polysaccharides.     

Use of the polymerase chain reaction and fluorescent-antibody methods for detecting viable but nonculturable Shigella dysenteriae type 1 in laboratory microcosms.

Epidemiological studies of shigellosis in Bangladesh have demonstrated that surface-water sources can act as foci of infection. Studies of laboratory microcosms have shown that shigellae become nonculturable but remain viable when exposed to environmental samples of water. The present study was carried out to detect viable but nonculturable Shigella dysenteriae 1 from laboratory microcosms by the polymerase chain reaction and the fluorescent-antibody techniques. S. dysenteriae 1 was inoculated into laboratory microcosms consisting of water samples collected from ponds, lakes, rivers, and drains in Bangladesh. The survival of S. dysenteriae in microcosms was assessed by viable counting on MacConkey agar. After 2 to 3 weeks, S. dysenteriae 1 became nonculturable but remained viable. After 6 weeks, this nonculturable but viable S. dysenteriae 1 was detected by both the polymerase chain reaction and the fluorescent-antibody methods. The viable but nonculturable state of S. dysenteriae 1 demonstrated in this study may be important for understanding the epidemiology of shigellosis.     

Characterization of cryptic flagellin genes in Shigella boydii and Shigella dysenteriae.

Flagellin (fliC) genes of 12 Shigella boydii and five Shigella dysenteriae strains were characterized. Though these strains are nonmotile, the cryptic fliCSB gene, cloned from S. boydii strain C3, is functional for expression of flagellin. It consists of 1,704 bp, and encodes 568 amino acid residues (57,918 Da). The fliCSD gene from S. dysenteriae strain 16 consists of 1,650 bp encoding 549 amino acid residues (57,591 Da) and contains an IS1 element inserted in its 3' end. The two genes are composed of the 5'-constant, central variable and 3'-constant sequences, like other known fliC genes. The two genes share high homology in nucleotide and amino acid sequences with each other and also with the Escherichia coli fliCE gene, indicating that both genes are closely related to the fliCE gene. Comparison of the central variable sequences of six different fliC genes showed that the fliCSB and fliCSD genes share low homology in amino acid sequence with the other fliC genes, suggesting that they encode antigenic determinants intrinsic to respective subgroups. However, Southern blotting using as probes the central variable sequences of several fliC genes showed that four of 12 S. boydii strains have a fliC gene similar to that of Shigella flexneri, and that among five fliC genes from S. dysenteriae strains, one is similar to that of S. flexneri, two are similar to that of S. boydii, and only one is unique to S. dysenteriae. Some of these variant alleles were verified by immunoblotting with flagellins produced from cloned fliC genes. The presence of variant fliC alleles in S. boydii and S. dysenteriae indicates that subdivision into subgroups does not reflect the ancestral flagella H antigenic relationships. These data will be useful in considering the evolutionary divergence of the Shigella spp..