Structural elucidation of the O-antigen of the Shigella flexneri provisional serotype 88-893: structural and serological similarities with S. flexneri provisional serotype Y394 (1c)

The structure of the repeating unit of the O-antigen polysaccharide from Shigella flexneri provisional serotype 88-893 has been determined. ¹H and ¹³C NMR spectroscopy as well as 2D NMR experiments were employed to elucidate the structure. The carbohydrate part of the hexasaccharide repeating unit is identical to the previously elucidated structure of the O-polysaccharide from S. flexneri prov. serotype Y394. The O-antigen of S. flexneri prov. serotype 88-893 carries 0.7 mol O-acetyl group per repeating unit located at O-2 of the 3-substituted rhamnosyl residue, as identified by H2BC and BS-CT-HMBC NMR experiments. The O-antigen polysaccharide is composed of hexasaccharide repeating units with the following structure: →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap2Ac-(1→3)[α-d-Glcp-(1→2)-α-d-Glcp-(1→4)]-β-d-GlcpNAc-(1→. Serological studies showed that type antigens for the two provisional serotypes are identical; in addition 88-893 expresses S. flexneri group factor 6 antigen. We propose that provisional serotypes Y394 and 88-893 be designated as two new serotypes 7a and 7b, respectively, in the S. flexneri typing scheme.     

Structural and immunochemical studies of the lipopolysaccharide from a new provisional serotype of Shigella flexneri

The chemical structure of the O-antigen of a proposed new provisional serotype of Shigella flexneri has been determined. Methylation analysis, GLC-MS, 1H-NMR and 13C-NMR showed that the linear O-antigenic polysaccharide is the same as for all S. flexneri [Kenne, L., Lindberg, B., Petersson, K. & Romanowska, E. (1977) Carbohydr. Res. 56, 363-370]. A novel structural feature is that the disaccharide alpha-D-Glcp-(1----2)-alpha-D-Glcp is linked to O4 of the N-acetyl-glucosamine residue. (Formula: see text) Western blotting of the lipopolysaccharide with an E. coli R3 core-specific monoclonal antibody, suggested the presence of an E. coli R3 core.     

O-Acetylation in the O-specific polysaccharide isolated from Shigella flexneri serotype 2a

Shigella flexneri causes diarrheal diseases especially in infants and children in developing countries. Modifications of the lipopolysaccharide (LPS) molecule, like bacteriophage-mediated glucosylation and acetylation of the O-specific chain (O-SP), are important for the LPS antigenicity and consequently for the immunogenicity of the polysaccharide-based vaccines against shigellosis. Here, we report the degree of O-acetylation and the localisation of O-acetyl groups and side-chain glucose substitution in the O-SP (scheme) in different preparations of S. flexneri type 2a LPS. [structure: see text]     

The isolation and immunologic study of ribosomal preparations from Shigella flexneri

S. flexneri ribosomal preparations were isolated by differential centrifugation or by fractionation with polyethylene glycol-6000. Their chemical composition and spectrophotometric properties were characteristic of ribosomes, and, as shown by the results of the serological assay, the content of O-specific component was, on the average, 1.4%. The ribosomal preparations were nontoxic for mice when injected intraperitoneally and intravenously in large doses and induced systemic O-antibody response in mice and rabbits. The parenteral administration of ribosomes to guinea pigs led to the increase of resistance to Shigella keratoconjunctivitis. The results of different tests with the use of this model greatly varied. According to the summary data of several tests, the ribosomal vaccine enhanced the resistance of the eyes from 11.3% to 48.5% and the effectiveness coefficient of immunization was 42 +/- 6. Ribosomes isolated from S. flexneri avirulent strain 2a 51.6 M (Iu. A. Belaia's vaccine) showed the same activity as those isolated from virulent strains. The results obtained in this study suggest the expediency of further experimental study of ribosomal preparations obtained from S. flexneri as potential vaccine.     

Complete genome sequence of Shigella flexneri 5b and comparison with Shigella flexneri 2a

Background

On most common microarray platforms many genes are represented by multiple probes. Although this is quite common no one has systematically explored the concordance between probes mapped to the same gene.

Results

Here we present an analysis of all the cases of multiple probe sets measuring the same gene on the Affymetrix U133a GeneChip and found that although in the majority of cases both measurements tend to agree there are a significant number of cases in which the two measurements differ from each other. In these cases the measurements can not be simply averaged but rather should be handled individually.

Conclusion

Our analysis allows us to provide a comprehensive list of the correlation between all pairs of probe sets that are mapped to the same gene and thus allows microarray users to sort out the cases that deserve further analysis. Comparison between the set of highly correlated pairs and the set of pairs that tend to differ from each other reveals potential factors that may affect it.     

Level and nature of the drug resistance of Shigella flexneri]

Characteristics of antibiotic resistance of 300 strains of Shigella flexner 2a isolated from patients within 1976--1977 in the regions where these bacteria were very rare for a long period of time were studied. It was shown that most of the isolates were resistant to chloramphenicol (88.1 per cent), tetracycline (94.8 per cent), streptomycin (89.1 per cent), polymyxin M (82.4 per cent) and others. 46.5--61.6 per cent of the isolates were resistant to neomycin antibiotics. A high level of the resistance was also noted: the bactericidal effect was registered in 52.0-74.0 per cent of the cultures at a dose of 500--1000 microgram/ml. 91.4 per cent of the strains possessed multiple dug resistance, 78.8 per cent of them being simultaneously resistant to 4--7 drugs. Transmissive R-plasmids were found in 68.8 per cent of the isolates. After exposure to acridine dyes the plasmid nature of the resistance was confirmed in 72.3 per cent of the cultures. Variability of the r-determinant sets in r-plasmids was noted. Strains (64.9 per cent) carrying r-determinants Tc, Cm, Sm and Tc, Cm were more frequent. Strains with one transmissive r-determinant were usually solitary.     

福氏志贺氏2a及Y变种保护性抗原的研究

福氏志贺菌Y变种曾经作为一种痢疾疫苗的候选株,其特有的抗原结构在疫苗的有效性抗原研究中起主要作用。以Y变种毒株与无毒株、野生型F2a株与T32株及失去Ⅱ型抗原结构的T32-1株之间分别进行了各种毒力表型的检测、四种外膜侵袭蛋白表达、菌株的外膜蛋白提取物(OMPs)分析、质粒DNA图谱和小鼠主动免疫、被动保护试验的对比分析,了解其抗原特性。结果显示：细菌外膜蛋白抗原和具有完整型特异性抗原结构的福氏菌LPS在动物机体免疫中都发挥着重要的作用。这些抗原物质的共同存在似乎能达到更好的免疫效果。     

Genetic and physical characterization of trimethoprim resistance plasmids from Shigella sonnei and Shigella flexneri

Analysis of six Shigella flexneri and four S. sonnei isolates with trimethoprim (Tp) resistance from clinical cases in Ontario has shown that, in all isolates, the Tp resistance is mediated by gene(s) on conjugative, multiple antibiotic-resistance plasmids. The physical and genetic characterization of these plasmids revealed that there are three different Tp resistance plasmids. One group, composed of all six S. flexneri plasmids, consists of plasmids which are about 70 megadaltons (MDa) and inhibit the fertility of an Escherichia coli Hfr strain (Fi+). A representative member of this group, pPT4, demonstrates a weak incompatibility reaction with IncFl plasmid R455-2. Another group, three of the four S. sonnei plasmids, contains plasmids which are about 43 MDa, Fi-, and mediate propagation of phage PRD1. The third group, the remaining S. sonnei plasmid, is 53 MDa, fi+, mediates propagation of phages fd and MS2, and is incompatible with IncFII plasmid R100. These plasmids also have been differentiated by restriction endonuclease fragment profiles. Analysis of pPT4 has revealed that the Tp resistance of this plasmid is transposable. The transposon, Tn536, is different from previously described Tp resistance transposons; it is 16 MDa, and in addition to Tp, it encodes resistance to mercuric chloride ions, spectinomycin, streptomycin, and sulfonamides.     

Characteristics of the action of the biologically active factor from virulent Shigella flexneri strains in a study on mice and a cell culture]

The authors subjected to further study the biologically active factor revealed by them earlier in the virulent Sh. flexneri cultures by using the genetically bound triad of Sh. flexneri 5a-222 cultures and the corresponding couple of Sh. flexneri 2a-516. There was shown correlation of the strains virulence determined by the keratoconjunctival test, with the presence of genetically-determined production of the biologically active factor detectable in the culture filtrate, which produced toxic action of the continuous cell cultures in the virulen Sh. flexneri strains of different serovars (2a and 5a), and lethal action in intravenous injection to mice. Comparative study of toxicity of the preparations of the endotoxin, free endotoxin, and neurotoxin types showed the biologically active factor to resemble the neurotoxin, differing from it in the toxic action and thermolability. Filtrates of the virulent and genetically characterised avirulent strains differed in the protein and lipids content, this permitting to suggest participation of the protein and lipid complex in the toxic action of the biologically active factor.     

Effect of mutations in Shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance

This study shows that both length and distribution of lipopolysaccharide (LPS) are important for Shigella flexneri invasion and virulence. Mutants were generated in the chromosomal LPS synthesis genes rfa , rfb , and rol , and in a plasmid-encoded O-antigen chain-length regulator, cld _pHS-2. LPS analysis showed that mutations in rfb genes and in a candidate rfaL gene either eliminated the entire O-antigen side chains or produced chains of greatly reduced length. Mutation in a previously unidentified gene, rfaX , affected the LPS core region and resulted in reduced amounts of O-antigen. Mutants defective in cld _pHS-2 or rol had different distributions of O-antigen chain lengths. The results of tissue-culture cell invasion and plaque assays, the Serény test, and serum-sensitivity assay suggested roles for the different LPS synthesis genes in bacterial survival and virulence; rfaL, rfaX and rfb loci are required for serum resistance and intercellular spread, but not for invasion; cld _pHS-2 is required for resistance to serum killing and for full inflammation in the Serény test, but not for invasion or intercellular spread, while rol is required for normal invasiveness and plaque formation, but not for serum resistance. Thus, O-antigen synthesis and chain-length regulation genes encoded on both the chromosome and the small plasmid pHS-2 play important roles in S. flexneri invasion and virulence.     

Plasmid-mediated bacteriocin production by Shigella flexneri isolated from dysenteric diarrhoea and their transformation into Escherichia coli

AIMS: To determine the production of bacteriocin by Shigella flexneri strains, to relate their production to the presence of dysenteric diarrhoea and to asses the genetic determination of the bacteriocin. METHODS AND RESULTS: One hundred and sixteen strains of Sh. flexneri were isolated from patients with diarrhoea and 49 of them produced bacteriocin active against several Escherichia coli and abacteriocinogenic Sh. flexneri strains. The extrachromosomal DNA isolated from bacteriocinogenic Sh. flexneri strains were used as a substrate to transform E. coli HB-101 cells by means of electroporation. CONCLUSIONS: Only the Sh. flexneri strains isolated from dysenteric diarrhoea produced bacteriocin. It was demonstrated that a plasmid of approx. 3 kb was responsible for the genetic determination of these anti-bacterial substances. Significance and IMPACT OF THE STUDY: A 3-kb plasmid that harboured information for the production of bacteriocin by Sh. flexneri strains was described. The production of this bacteriocin may be related to dysenteric diarrhoea produced by these bacterial strains.     

Nucleotide sequence of the rhamnose biosynthetic operon of Shigella flexneri 2a and role of lipopolysaccharide in virulence.

N1308, a chromosomal Tn5 mutant of Shigella flexneri 2a, was described previously as a lipopolysaccharide (LPS) mutant with a short O side chain. N1308 formed foci, but not plaques, in LLC-MK2 cell monolayers and was negative in the Serény test. In this study, the wild-type locus inactivated in N1308 was cloned and further defined by means of complementation analysis. A 4.3-kb BstEII-XhoI fragment of S. flexneri 2a YSH6200 DNA was sufficient to restore both normal LPS and virulence phenotype to the mutant. DNA sequencing of this region revealed four genes, rfbA, rfbB, rfbC, and rfbD, encoding the enzymes required for the biosynthesis of activated rhamnose. The four genes were expressed in Escherichia coli, and the expected protein products were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N1308 was shown to have normal levels of surface IpaC and IpaD, while a Western blot (immunoblot) of whole-cell lysates or outer membrane fractions indicated an elevated level of appropriately localized VirG. An in vitro invasion assay revealed that N1308 had normal primary invasive capacity and was able to multiply and move normally within the initial infected cell. However, it exhibited a significant reduction in its ability to spread from cell to cell in the monolayer. A double immunofluorescence assay revealed differences between LLC-MK2 cells infected with the wild-type YSH6000 and those infected with N1308. The wild-type bacteria elicited the formation of the characteristic F-actin tails, whereas N1308 failed to do so. However, N1308 was capable of inducing deposition of F-actin, which accumulated in a peribacterial fashion with only slight, if any, unipolar accumulation of the cytoskeletal protein.     

Antibiotic sensitivity of Shigella isolated from patients from 1974-1985

Antibiotic sensitivity of 3524 Shigella cultures isolated from patients in 1974-1982 and 414 cultures isolated in 1983-1985 was assayed with standard paper disks. The isolates of 1974-1982 were mostly responsive to ampicillin, carbenicillin, kanamycin, gentamicin, monomycin, neomycin and chloramphenicol. Certain differences in the level of the antibiotic resistance were observed in the Shigella isolates belonging to diverse species. Polyresistant cultures of Shigella amounted to 96.5% and ranged from 88.5 to 99.4% in different years. The number of the cultures with multiple resistance among Shigella sonnei was somewhat higher than that among the Flexneri and Newcastle bacilli. The Shigella isolates of 1983-1985 were mostly responsive to gentamicin, carbenicillin, neomycin, kanamycin and monomycin. 55.5% of the Shigella isolates were responsive to chloramphenicol and only 3.1% to tetracycline. Almost all the causative agents of dysentery isolated within that period were polyresistant. Phenotypic characteristics of multiple resistance in the Shigella cultures were studied.