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.     

Construction of a multivalent vaccine strain of Shigella flexneri and evaluation of serotype-specific immunity

Shigella flexneri causes more fatalities by shigellosis than any other Shigella species. There are 13 different serotypes of S. flexneri and their distribution varies between endemic geographical regions. The immune response against S. flexneri is serotype-specific, so current immunization strategies have required the administration of multiple vaccine strains to provide protection against multiple serotypes. In this study, we report the construction of a multivalent S. flexneri vaccine strain, SFL1425, expressing the O-antigen structure specific for serotypes 2a and 5a. This combination of type antigens has not previously been reported for S. flexneri. The multivalent vaccine strain, SFL1425 was able to induce a specific immune response against both serotypes 2a and 5a in a mouse pulmonary model.     

Cloning and analysis of the glucosyl transferase gene encoding type I antigen in Shigella flexneri

The O-antigen of most Shigella flexneri serotypes contains an identical tetrasaccharide repeating unit. Apart from serotype Y, the O-antigen is modified by addition of a glucosyl and/or O-acetyl residue to a specific position in the O-unit. In this study the glucosyl transferase gene from a serotype 1a has been cloned and identified. The bacteriophage SfV integrase (int) gene was used to probe a S. flexneri Y53 (serotype 1a) cosmid library and 18 unique clones were identified. Southern hybridisation of these clones indicated two unlinked regions of the chromosome contained the int homologue. When expressed in a live candidate vaccine strain of S. flexneri serotype Y (SFL124), clones with one region produced type I antigen, whereas clones containing the other region produced mainly type Y antigen. One of the cosmid clones positive for type I antigen by agglutination and Western blotting was selected for further study. Genes involved in O-antigen glucosyl modification were mapped on a 5.8 kb fragment and subclones were produced which fully or partially expressed the type I antigen, depending on the extent of the clone. Fully and partially expressing clones may be useful vaccine candidate strains for protection against disease caused by two serotypes of S. flexneri.     

Molecular characterization of the O-acetyl transferase gene of converting bacteriophage SF6 that adds group antigen 6 to Shigella flexneri

Bacteriophage SF6 antigenically converts Shigella flexneri serotype Y strains (-;3,4) to type 3b carrying group antigen 6,3,4 by means of an O-acetylation of the O-antigenic polysaccharide chain. The gene for O-acetyl transferase of bacteriophage SF6 has been cloned, identified and sequenced. The predicted O-acetyl transferase protein encoded by this gene was found to consist of 333 amino acids, (37,185 daltons) and to have some similarity with the galactose-1-phosphate uridylyltransferase protein of Escherichia coli. The gene has been shown to function in a live vaccine strain of S. flexneri Y type (delta aroD), making it a 3b type. The converted type 3b strain, SFL1104, was found to elicit significant protection against challenge by both wild-type serotypes 3b and Y in a guinea-pig keratoconjunctivitis model.     

Immunogenicity of the Shigella flexneri Serotype Y (SFL124) Vaccine Strain Expressing Cloned Glucosyl Transferase Gene of Converting Bacteriophage SfX

The glucosyl transferase gene (gtr) from bacteriophage phage X (SfX) caused partial conversion of serotype Y (group antigen 3, 4) to X (group antigen 7, 8) when introduced into a candidate vaccine strain of Shigella flexneri serotype Y (SFL124). The gtr gene caused conversion of O-antigens but did not eliminate the adsorption of the corresponding phage SfX. The hybrid strain expressing both group antigens 7, 8 and 3, 4 showed 75% protection when immunized guinea pigs were challenged with a wild-type S. flexneri serotype X strain. No protection was observed against serotype Y challenge, although group antigen 3, 4 was detected in the LPS of the hybrid strain. This suggests the importance of O-antigen immunity in the host defense against shigellosis.     

Genome sequence of Shigella flexneri serotype 5a strain M90T Sm

Bacteria of the genus Shigella are a major cause of death worldwide (L. von Seidlein et al., PLoS Med. 3:e353, 2006). We sequenced the genome of Shigella flexneri strain M90T Sm (serotype 5a) and compared it to the published genome sequence of S. flexneri strain 8401 (serotype 5b).     

Genetic analysis of the rfb region of Shigella flexneri encoding the Y serotype O-antigen specificity

The gene cluster (rfb region) which determines the biosynthesis of the Shigella flexneri O-antigen of the Y serotype specificity was cloned from a S. flexneri serotype 2a strain. Two plasmids, pPM2212 and pPM2213, which conferred O-antigen biosynthesis were generated from separate cosmid clones by deletion with Clal. These plasmids expressed O-antigen in Escherichia coli K12 like that of the parental strain, as assessed by reactions to antisera in colony and Western immunoblots, sensitivity to bacteriophage Sf6, and by silver staining of lipopolysaccharides separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These plasmids also mediated O-antigen expression in an E. coli K12 rfb-delete background, indicating that all the necessary genes have been cloned. A detailed restriction map of the region has been constructed and analysis of various subclones has allowed the limits of the coding region for O-antigen biosynthesis to be defined to a maximum of 11 kb. Expression of these plasmids demonstrates a novel phenotype associated with control of lipopolysaccharide chain length. The gene(s) responsible maps adjacent to, but separate from, those associated with the biosynthesis of the O-antigen unit. Analysis of plasmid-encoded proteins in minicells and maxicells has facilitated the construction of a physical map. Finally, plasmid pPM-2212 was used to probe a collection of S. flexneri serotypes by Southern hybridization. With the exception of serotype 6, which appears to be unrelated, a similar pattern was found in all serotypes.     

一株有争议的志贺氏菌的抗原分析与血清型检定

对中国医学细菌保藏管理中心所存,1935年国外分离,国内检定结果不一的一株志贺氏菌51331进行了详细的抗原分析,确定为福氏志贺氏菌X变种。 该菌能与国内外出品的福氏3型特异血清发生交叉凝集的原因,主要是由于上述诊断用血清中交叉反应性抗体尚未吸收纯净,至少没用类似51331这样的菌株参与成品血清检定的缘故。因此,建议在生产福氏3型特异血清时,应用本菌种参与检定以提高制品质量。本菌种作为诊断血清检定时用的菌种是十分有价值的。     

Characterization of functional oligosaccharide mimics of the Shigella flexneri serotype 2a O-antigen: implications for the development of a chemically defined glycoconjugate vaccine

Protection against reinfection with noncapsulated Gram-negative bacteria, such as Shigella, an enteroinvasive bacterium responsible for bacillary dysentery, is mainly achieved by Abs specific for the O-Ag, the polysaccharide part of the LPS, the major bacterial surface Ag. The use of chemically defined glycoconjugates encompassing oligosaccharides mimicking the protective determinants carried by the O-Ag, thus expected to induce an efficient anti-LPS Ab response, has been considered an alternative to detoxified LPS-protein conjugate vaccines. The aim of this study was to identify such functional oligosaccharide mimics of the S. flexneri serotype 2a O-Ag. Using protective murine mAbs specific for S. flexneri serotype 2a and synthetic oligosaccharides designed to analyze the contribution of each sugar residue of the branched pentasaccharide repeating unit of the O-Ag, we demonstrated that the O-Ag exhibited an immunodominant serotype-specific determinant. We also showed that elongating the oligosaccharide sequence improved Ab recognition. From these antigenicity data, selected synthetic oligosaccharides were assessed for their potential to mimic the O-Ag by analyzing their immunogenicity in mice when coupled to tetanus toxoid via single point attachment. Our results demonstrated that induction of an efficient serotype 2a-specific anti-O-Ag Ab response was dependent on the length of the oligosaccharide sequence. A pentadecasaccharide representing three biological repeating units was identified as a potential candidate for further development of a chemically defined glycoconjugate vaccine against S. flexneri 2a infection.     

Interrelation of virulent and avirulent (vaccinal) strains of Shigella flexneri 2A in long-term bacterial carriage in gnotobiotic rats

The ability of S. flexneri 2a virulent and avirulent (vaccine) strains No. 516 and No. 516 m to displace one another during long-term carrier state in germ-free rats has been studied. The long-term persistence of the vaccine strain in the intestine of the rats has been shown to produce colonization resistance to subsequent infection with the virulent culture of these bacteria. During carrier state in rats progressive S--R dissociation of the bacteria occurs, type II antigen is lost with simultaneous retention of group 3, 4 antigens and the resulting transformation of S. flexneri, serotype 2a, into variant y; the virulence of Shigella S-forms is also lost.     

Serotype 1a O-Antigen Modification: Molecular Characterization of the Genes Involved and Their Novel Organization in the Shigella flexneri Chromosome

The factors responsible for serotype 1a O-antigen modification in Shigella flexneri were localized to a 5.8-kb chromosomal HindIII fragment of serotype 1a strain Y53. The entire 5.8-kb fragment and regions up- and downstream of it (10.6-kb total) were sequenced. A putative three-gene operon, which showed homology with other serotype conversion genes, was identified and shown to confer serotype 1a O-antigen modification. The serotype conversion genes were flanked on either side by phage DNA. Multiple insertion sequence (IS) elements were located within and upstream of the phage DNA in a composite transposon-like structure. Host DNA homologous to the dsdC and the thrW proA genes was located upstream of the IS elements and downstream of the phage DNA, respectively. The sequence analysis indicates that the organization of the 10.6-kb region of the Y53 chromosome is unique and suggests that the serotype conversion genes were originally brought into the host by a bacteriophage. Several features of this region are also characteristic of pathogenicity islands.     

New animal model of shigellosis in the Guinea pig: its usefulness for protective efficacy studies

It has been difficult to evaluate the protective efficacy of vaccine candidates against shigellosis, a major form of bacillary dysentery caused by Shigella spp. infection, because of the lack of suitable animal models. To develop a proper animal model representing human bacillary dysentery, guinea pigs were challenged with virulent Shigella flexneri serotype 2a (strains 2457T or YSH6000) or S. flexneri 5a (strain M90T) by the intrarectal (i.r.) route. Interestingly, all guinea pigs administered these Shigella strains developed severe and acute rectocolitis. They lost approximately 20% of their body weight and developed tenesmus by 24 h after Shigella infection. Shigella invasion and colonization of the distal colon were seen at 24 h but disappeared by 48 h following i.r. infection. Histopathological approaches demonstrated significant damage and destruction of mucosal and submucosal layers, thickened intestinal wall, edema, erosion, infiltration of neutrophils, and depletion of goblet cells in the distal colon. Furthermore, robust expression of IL-8, IL-1beta, and inducible NO synthase mRNA was detected in the colon from 6 to 24 h following Shigella infection. Most importantly, in our new shigellosis model, guinea pigs vaccinated with an attenuated S. flexneri 2a SC602 strain possessing high levels of mucosal IgA Abs showed milder symptoms of bacillary dysentery than did animals receiving PBS alone after Shigella infection. In the guinea pig, administration of Shigella by i.r. route induces acute inflammation, making this animal model useful for assessing the protective efficacy of Shigella vaccine candidates.     

Single-molecule approach to bacterial genomic comparisons via optical mapping

Modern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species. To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them. Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery. Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species. Since whole-genome optical maps are ordered restriction maps, sequenced strains of Shigella flexneri serotype 2a (2457T and 301), Yersinia pestis (CO 92 and KIM), and Escherichia coli were aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequenced Shigella flexneri strain (serotype Y strain AMC[328Y]) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions. Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.     

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.     

Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells

Shigella spp. are a group of Gram-negative enteric bacilli that cause acute dysentery in humans. We demonstrate that Shigella flexneri has evolved the ability to regulate functional components of tight junctions after interaction at the apical and basolateral pole of model intestinal epithelia. In the regulation of tight junctional protein assemblies, S. flexneri can engage serotype-specific mechanisms, which targets not only expression, but also cellular distribution and membrane association of components of tight junctions. Distinct mechanisms resulting in the regulation of tight junction-associated proteins are initiated after either apical or basolateral interactions. S. flexneri serotype 2a has the ability to remove claudin-1 from Triton X-insoluble protein fractions upon apical exposure to T-84 cell monolayers. S. flexneri serotype 2a and 5, but not the non-invasive Escherichia coli strain F-18, share the ability to regulate expression of ZO-1, ZO-2, E-cadherin and to dephosphorylate occludin. The disruption of tight junctions is dependent on direct interaction of living Shigella with intestinal epithelial cells and is supported by heat-stable secreted bacterial products. Intestinal epithelial cells have the ability to compensate in part for S. flexneri induced regulation of tight junction-associated proteins.     

Identification of a putative pathogenicity island in Shigella flexneri using subtractive hybridisation of the S. flexneri and Escherichia coli genomes

The genetic differences between the human pathogen, Shigella flexneri, and the non-pathogenic Escherichia coli were investigated in an attempt to identify pathogenicity islands (PAIs) in the S. flexneri genome. Genomic subtraction identified a large unique region of DNA which was present in S. flexneri serotype 2a but absent from E. coli K-12. This 42-kb DNA segment was localised to the S. flexneri chromosome and was found to contain a number of elements often associated with PAIs including: insertion sequence elements, bacteriophage genes, and a previously identified Shigella virulence gene (criR). These findings indicate that this region may form a new PAI in the S. flexneri genome.     

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。     

Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its delta galU mutant: glycine residue location in the inner core of the lipopolysaccharide

Shigella flexneri is a gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections. Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multidisciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a L,D-Hep-(1 -->7)-L,D-Hep-(1 -->3)-L,D-Hep-(1 -->5)-[Kdo-(2 -->4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T delta galU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.     

Lipopolysaccharide O antigen chains mask IcsA (VirG) in Shigella flexneri

Shigella flexneri 2a strain 2457T lipopolysaccharide (LPS) has O antigen (Oag) chains with two modal lengths (S-type and VL-type), and has IcsA apparently located at one pole on its cell surface. Treatment of Y serotype derivatives of 2457T and RMA696 (2457T wzz(SF)) with Sf6 tailspike protein (TSP) resulted in hydrolysis of Oag chains, and an increase in detection of IcsA by indirect immunofluorescence staining on both the lateral and polar regions of the cell surface. Newly synthesised IcsA expressed from a pBAD promoter in a S. flexneri Y strain was also detected on both the lateral and polar regions of the cell when incubated with TSP prior to immunofluorescence staining. We conclude that IcsA is actually located on both lateral and polar regions of the S. flexneri cell surface, and that LPS Oag chains mask the presence of IcsA by hindering its detection with antibodies. These results have implications for the mechanism of IcsA export. They suggest that while IcsA export is predominantly targeted to the old cell pole, it can also occur on the lateral regions of the cell surface.     

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.