Structure of the causative agents of shigellosis in areas of the USSR 1979-1981

Summarized data on the specific structure of Shigella strains isolated in different territories of the USSR in 1979-1981 are presented. These data indicate that in the intra-specific structure of S. flexneri strains the proportion of serological subvariant 2a increased and that of serological variant 6 and subvariants 16 and 4a decreased during this period. Using one of the base territories of the All-Union Shigellosis Center as an example, the spread of an unusual serological variant of S. flexneri (IV:7,8) in the USSR is shown. The increase of the proportion of S. boydii strains, mainly S. boydii 4, is noted.     

Structural and genetic characterization of Shigella boydii type 17 O antigen and confirmation of two new genes involved in the synthesis of glucolactilic acid

Shigella strains are human pathogens and normally identified based on their O antigens. The chemical structure and gene cluster of Shigella boydii type 17 O antigen were studied. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen of S. boydii type 17 has a linear trisaccharide O unit, which consists of two residues of N-acetylgalactosamine (GalNAc) and a 4-O-[(R)-1-carboxyethyl]-d-glucose (glucolactilic acid). The O antigen gene cluster of S. boydii type 17 was sequenced and genes encoding UDP-N-acetylglucosamine C4 epimerase for GalNAc synthesis, O unit flippase, O antigen polymerase, and glycosyltransferases were putatively identified based on sequence similarities and the presence of conserved motifs. Two genes, whose functions could not be clearly indicated by homology search, were confirmed to be involved in the synthesis of glucolactilic acid by mutation and structural verification of the O antigens from the mutants. To our knowledge, this is the first time that genes involved in the synthesis of glucolactilic acid have been reported. Two genes specific to S. boydii type 17 were also identified.     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

鲍氏志架氏菌的一个新血清型

Two strains which belong to the same serotype of Shigella were isolated from the bloody-pus stool of two patients (in 1986) and is reported in this paper. The results were identical both showing agglutination in low titer with serotype 8 of S. dysenteriae and serotype 4 of S. boydii when the two strains were checked well with all kinds of diagnostic antisera and vice versa, ie the antisera produced by the two strains were also checked well with sera prepared with the representative strains of all Shigella spp. No cross agglutination with O6, O7, and O150 of E. coli were found. Consequently, It appears to be a new serotype of Shigella. These two strains possess the ability of causing keratitis in guinea-pigs as well as invading epithelial cells, the DNA of both strains in agarose-electrophoresis showed a large plasmid, indicating that they are virulent strains possessing invasive ability. It was concluded that these two strains belonged to Shigella boydii as they fermented mannitol and non-related antigenically with Shigella flexneri. Since serotype 1-18 of S. boydii have been reported recently, we propose that this new serotype should be serotype 19 of Shigella boydii.     

Structural and molecular characterization of Shigella boydii type 16 O antigen

Shigella is a well-known human pathogen causing dysentery and their typing is solely based on the O antigens. We investigated the chemical structure and gene cluster of Shigella boydii type 16 O antigen. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen has an O-acetylated branched pentasaccharide repeating O unit, which consists of two D-mannose residues (D-Man), one residue each of d-glucuronic acid (D-GlcA), N-acetylglucosamine (D-GlcNAc) and D-galactose (D-Gal), and the structure of the O unit was established. The O antigen gene cluster of S. boydii type 16 was identified and shown to contain putative genes for the synthesis of GDP-D-Man, genes encoding sugar transferases, O unit flippase (Wzx) and O antigen polymerase (Wzy) as expected. The function of the wzy gene was characterized by mutation test. Genes specific to S. boydii type 16 O antigen gene cluster were identified by screening 186 Escherichia coli and Shigella type strains, and can be used to develop PCR assays for detection of type 16 strains.     

The importance of Shigella boydii in shigellosis morbidity in the USSR

The etiological role of S. boydii in Shigella infections registered at different territories of the USSR in 1986-1987 was analyzed. As established by this analysis, S. boydii infection occurred mainly at the territories with unsatisfactory water supply of the population; from these territories the infection spread to ther territories of the USSR. The dominating serovars causing S. boydii dysentery, as well as Shigella infections, at different territories of the USSR was shown to be identical, which was indicative of the fact that the immunological factor had no influence on the etiological structure of Shigella infections, determined by the activity of the main routes of the transfer of infection. On the whole, S. boydii serovars 2, 4 and 1 were found to prevail in the USSR.     

Molecular analysis of Shigella boydii O1 O-antigen gene cluster and its PCR typing

Shigella is an important human pathogen and is closely related to Escherichia coli. O-antigen is the most variable part of the lipopolysaccharide on the cell surface of Gram-negative bacteria and plays an important role in pathogenicity. The O-antigen gene cluster of S. boydii O1 was sequenced. The putative genes encoding enzymes for rhamnose synthesis, transferases, O-unit flippase, and O-unit polymerase were identified on the basis of homology. The O-antigen gene clusters of S. boydii O1 and E. coli O149, which share the same O-antigen form, were found to have the same genes and organization by adjacent gene PCR assay. Two genes specific for S. boydii O1 and E. coli O149 were identified by PCR screening against E. coli- and Shigella-type strains of the 186 known O-antigen forms and 39 E. coli clinical isolates. A PCR sensitivity of 103 to 104 CFU/mL overnight culture of S. boydii O1 and E. coli O149 was obtained. S. boydii O1 and E. coli O149 were differentiated by PCR using lacZ- and cadA-based primers.     

Differentiation of Shigella by esterase electrophoretic polymorphism

The electrophoretic mobilities of four esterases (A, B, C, and I) of 182 strains of Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei were compared to those of 636 strains of Escherichia coli from various origins, including the Alkalescens Dispar group and enteroinvasive strains. Discriminant analysis of the distribution of esterases among the strains revealed that Shigella could be distinguished from E. coli by differences in the distribution of allozymes of esterases C and I. Principal components analysis distinguished four major clusters of Shigella strains corresponding to the following: S. dysenteriae serotype 1; S. flexneri serotypes 1 to 5; S. flexneri serotype 6 and S. boydii serotypes 2 and 4; and S. sonnei. The last three were characterized by distinct electrophoretic variants of carboxylesterase B, as judged by the two-dimensional electrophoretic profile and titration curves. The distinct esterase pattern obtained for the strains of S. boydii serotype 13 substantiates the view that this serotype may constitute a new species.     

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.     

Antigenic polysaccharides of bacteria: 40. The structures of O-specific polysaccharides from Shigella dysenteriae types 3 and 9 and S. boydii type 4 revised by NMR spectroscopy

The reported structures of O-specific polysaccharides from three standard 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 acetylation by pyruvic acid (S. dysenteriae type 9, structure 2), and the attachment position of the side monosaccharide chain (S. boydii type 4, structure 3) [struxture in text].     

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.     

Structural and genetic characterization of the Shigella boydii type 10 and type 6 O antigens

Comparison of the O antigens of Shigella boydii types 10 and 6 by chemical analysis and nuclear magnetic resonance spectroscopy showed that their structures are similar, with the only difference being the presence or absence of d-ribofuranose, which is the immunodominant sugar in S. boydii type 10. In S. boydii type 6, a residue previously reported as alpha-d-GlcpA, was shown to be beta-d-GlcpA as in S. boydii type 10. S. boydii types 10 and 6 are reported not to cross-react serologically, and the role of d-ribofuranose in the specificity of S. boydii was confirmed by making a mutant of type 10 that lacked d-ribofuranose. However, S. boydii type 11, which has a d-ribofuranose but with different linkage does show cross-reaction with type 10. The O-antigen gene loci of S. boydii types 10 and 6 were shown to be virtually identical except that orf8 (wbaM), which was confirmed as the ribofuranosyltransferase gene, is interrupted by IS629 in type 6. Therefore, it is proposed that the O-antigen gene cluster of S. boydii type 6 was derived from type 10 by an IS element insertion.     

Growth of Allescheria boydii in anitbiotic-containing media

Cazin, John, Jr. (University of Iowa, Iowa City), and David W. Decker. Growth of Allescheria boydii in antibiotic-containing media. J. Bacteriol. 90:1308-1313. 1965.-Thirteen isolates of Allescheria boydii were surveyed for their ability to grow and sporulate on media containing cycloheximide and chloramphenicol. The fungus grew well in the presence of 4 to 8 mg/ml of cycloheximide, whereas ascocarps and coremia were always inhibited at a concentration of the drug lower than that required to inhibit growth and conidial production. In certain strains, ascocarp production was inhibited by concentrations of cycloheximide (0.4 mg/ml) normally used in selective media; however, two strains produced mature ascocarps at a concentration of 1 mg/ml of the antibiotic, and two strains produced immature ascocarps at a concentration of 4 mg/ml. Growth inhibition was not observed with concentrations of chloramphenicol as high as 1,000 mg per liter in any of eight strains tested.     

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..     

Large plasmid in Shigella boydii is responsible for epithelial cell penetration

A large plasmid in a virulent Shigella boydii 5 strain was transferred to plasmid-cured avirulent strains of S. boydii 5, S. boydii 12, S. sonnei form II, and Escherichia coli K12. The transconjugants acquired the ability to invade tissue culture cells, which indicated that the large plasmid in S. boydii is responsible for epithelial cell invasiveness.     

Microbial transformation of (+)-nootkatone and the antiproliferative activity of its metabolites

Six metabolites were obtained as a result of microbial transformation of (+)-nootkatone (1) by the fungal strains: Botrytis, Didymosphaeria, Aspergillus, Chaetomium and Fusarium. Their structure were established as (+)-(4R,5S,7R,9R)-9α-hydroxynootkatone (2), (+)-(4R,5S,7R)-13-hydroxynootkatone (3) and (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9α-hydroxynootkatone (4), (+)-(4R,5S,7R,11S)-11,12-epoksynootkatone (5), (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6) and (+)-(4R,5S,7R)-7,11,12-trihydroxynootkatone (7) on the basis of their spectral data. Two products: (4) and (7) were not previously reported in the literature. The antiproliferative activity of (+)-nootkatone (1) and isolated metabolites (2-7) of its biotransformation has been evaluated.     

Somatic antigens of Shigella and Escherichia coli. Determination of the structure of O-specific polysaccharide from Shigella boydii type 12 and its serological properties compared with the O-specific polysaccharide from Escherichia coli

The structure of the O-specific polysaccharide of the somatic antigen (lipopolysaccharide) of Shigella boydii, type 12, was established by 1H- and 13C-NMR, methylation analysis and partial acid hydrolysis methods. The polysaccharide consists of pentasaccharide repeating units of the following structure: (formula; see text) The amount of O-acetyl groups was far less than stoichiometric, only about 2 for 3-4 repeating units. Nevertheless, the results of serological studies revealed 3-O-acetyl-alpha-L-rhamnose residue to be the major immunodominant group. In spite of the presence of similar trisaccharide fragments, the lipopolysaccharide and polysaccharide from Shigella boydii type 12 gave no crossreaction with lipopolysaccharide and polysaccharide from Escherichia coli 07. The possible reasons of the absence of serological relatedness between the Sh. boydii, type 12, and E. coli 07 cells were discussed.     

Occurrence and characteristics of the cytolysin A gene in Shigella strains and other members of the family Enterobacteriaceae

Cytolysin A (ClyA, HlyE, SheA) is a hemolytic pore-forming toxin found in Escherichia coli and Salmonella enterica serovars Typhi and Paratyphi A. In the present study, analysis of several Shigella strains revealed that they harbor only nonfunctional clyA gene copies that have been inactivated either by the integration of insertion sequence (IS) elements (Shigella dysenteriae, Shigella boydii, and Shigella sonnei strains) or by a frameshift mutation (Shigella flexneri). Shigella dysenteriae and S. boydii strains also exhibited IS-associated deletions at the clyA locus. PCR and Southern blot analyses as well as database searches indicated that clyA-related DNA sequences are completely absent in strains belonging to various other genera of the family Enterobacteriaceae. According to these data, ClyA may play a role only for a rather small subset of the enteric bacteria.     

Antigenic polysaccharides of bacteria of the genus Shigella. The structure of the polysaccharide chain of Shigella boydii type 14

A specific acidic polysaccharide has been isolated from the Shigella boydii type 14 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of the D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose and D-galactose residues in the ratio 1:1:3. From the results of methylation analysis and partial acid hydrolysis, the structure of the repeating unit of the specific polysaccharide was deduced as follows: (-6DGalp alpha 1-4DGlcAp beta 1-6DGalp beta 1-4DGalp beta 1-4DGlcNAcp beta 1-)n. The 13C NMR spectra of native and carboxyl-reduced polysaccharides, as well as of oligosaccharides produced by partial acid hydrolysis fully confirmed the proposed structure. The approach was suggested to determine the type of substitution of uronic acid moieties in polysaccharide chain by use of chromato-mass-spectrometry of acetylated methyl esters of partially methylated aldonic acids. Serological characteristics of Sh. boydii LPS type 14 and its modified derivatives are discussed.