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.     

A new ethanolamine phosphate-containing variant of the O-antigen of Shigella flexneri type 4a

The O-specific polysaccharide (O-antigen) structure of a Shigella flexneri type 4a strain from the Dysentery Reference Laboratory (London, UK) was elucidated in 1978 and its characteristic feature was found to be α-d-glucosylation of GlcNAc at position 6, which defines O-factor IV. Our NMR spectroscopic studies of the O-specific polysaccharides of two other strains belonging to S. flexneri type 4a (G1668 from Adelaide, Australia, and 1359 from Moscow, Russia) confirmed the carbohydrate backbone structure but revealed in both strains an additional component, ethanolamine phosphate (EtnP), attached at position 3 of one of the rhamnose residues:

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Phosphorylation has not been hitherto reported in any S. flexneri O-antigen. Reinvestigation of the O-specific polysaccharide of S. flexneri type 4b showed that it is not phosphorylated and confirmed its structure established earlier.     

Lipopolysaccharide with an altered O-antigen produced in Escherichia coli K-12 harbouring mutated, cloned Shigella flexneri rfb genes

Cloning of the rfb genes of Shigella flexneri 2a into Escherichia coli K-12 strain DH1 results in the synthesis of lipopolysaccharides (LPS) with an O-antigen chain having type antigen IV and group antigens 3,4. During genetic studies of these rfb genes in E. coli K-12, we observed that strains harbouring plasmids with certain mutations (inversion and transposon insertions) which should have blocked O-antigen synthesis nevertheless still produced LPS with O-antigen chains. These LPS migrated differently on silver-stained SDS—polyacrylamide gels, compared with the LPS produced by wild-type rfb genes, and the group 3,4 antigens were barely detectable, suggesting that the O-antigen was altered. Investigation of the genetic determinants for production of the altered O-antigen/LPS indicated that: (i) these LPS are produced as a result of mutations which are either polar on rfbF or inactivate rfbF; (ii) the rfbX gene product (or a similar protein in the E. coli K-12 rfb region) is needed for production of the altered O-antigen in the form of LPS; (iii) the rfbG gene product is required for the production of both the parental and altered LPS; (iv) the dTDP-rhamnose biosynthesis genes are required. Additionally, an E. coli K-12 gene product(s) encoded outside the rfb region also contributes to production of the O-antigen of the altered LPS. An antiserum raised to the altered LPS from strain DH1(pPM2217 (rfbX::Tn1725)) was found to cross-react with nearly all S. flexneri serotypes, and with the altered LPS produced by other DH1 strains harbouring plasmids with different rfb mutations, as described above. The reactivity of the altered LPS with a panel of monoclonal antibodies specific for various S. flexneri O-antigen type and group antigens demonstrated that their O-antigen components were closely related to that of S. flexneri serotype 4. The RfbF and RfbG proteins were shown to have similarity to rhamnose transferases, and we identified a motif common to the N-termini of 6-deoxy-hexose nucleotide sugar transferases. We propose that the E. coli K-12 strains harbouring the mutated S. flexneri rfb genes produce LPS with a hybrid O-antigen as a consequence of inactivation of RfbF and complementation by an E. coli K-12 gene product. Analysis of the genetic and immunochemical data suggested a possible structure for the O-antigen component of the altered LPS.     

Structure elucidation of the O-antigen of <Emphasis Type="Italic">Salmonella enterica</Emphasis> O51 and its structural and genetic relation to the O-antigen of <Emphasis Type="Italic">Escherichia coli</Emphasis> O23

The O-polysaccharide (O-antigen) of Salmonella enterica O51 was isolated by mild acid degradation of the lipopolysaccharide and its structure was established using sugar analysis and NMR spectroscopy. The O-antigen of Escherichia coli O23, whose structure was elucidated earlier, possesses a similar structure and differs only in the presence of an additional lateral α-D-Glcp residue at position 6 of the GlcNAc residue in the main chain. Sequencing of the O-antigen gene clusters of S. enterica O51 and E. coli O23 revealed the same genes with a high-level similarity. By comparison with opened gene databases, all genes expected for the synthesis of the common structure of the two O-antigens were assigned functions. It is suggested that the gene clusters of both bacteria originated from a common ancestor, whereas the O-antigen modification in E. coli O23, which, most probably, is induced by prophage genes outside the gene cluster, could be introduced after the species divergence.     

O-Antigen Protects Gram-Negative Bacteria from Histone Killing

Beyond their traditional role of wrapping DNA, histones display antibacterial activity to Gram-negative and -positive bacteria. To identify bacterial components that allow survival to a histone challenge, we selected resistant bacteria from homologous Escherichia coli libraries that harbor plasmids carrying pieces of the chromosome in different sizes. We identified genes required for exopolysaccharide production and for the synthesis of the polysaccharide domain of the lipopolysaccharide, called O-antigen. Indeed, O-antigen and exopolysaccharide conferred further resistance to histones. Notably, O-antigen also conferred resistance to histones in the pathogens Shigella flexneri and Klebsiella pneumoniae.     

Passive immunity with multi‐serotype heat‐killed Shigellae in neonatal mice

The short‐ and long‐term passive protective efficacy of a mixture of heat‐killed cells of six serogroups/serotypes of Shigella strains (Shigella dysenteriae 1, S. flexneri 2a, S. flexneri 3a, S. flexneri 6, S. boydii 4, and S. sonnei) were studied in neonatal mice. Neonatal mice from immunized dams exhibited significant short‐ and long‐term passive protection against individual challenge by each of the six Shigella strains. High IgG and IgA titers against the lipopolysaccharide from each of the six Shigella strains were observed in sera from immunized dams.     

Identification of an O-Acyltransferase Gene (oacB) That Mediates 3- and 4-O-Acetylation of Rhamnose III in Shigella flexneri O Antigens

O antigen (O polysaccharide) is an important and highly variable cell component present on the surface of cells which defines the serospecificity of Gram-negative bacteria. Most O antigens of Shigella flexneri, a cause of shigellosis, share a backbone composed of →2)-α-l-Rhap^III-(1→2)-α-l-Rhap^II-(1→3)-α-l-Rhap^I-(1→3)-β-d-GlcpNAc-(1→ repeats, which can be modified by adding various substituents, giving rise to 19 serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha^I, and phosphorylation with phosphoethanolamine on Rha^II or/and Rha^III. Recently, two new O-antigen modifications, namely, O-acetylation at position 3 or 4 of Rha^III and position 6 of GlcNAc, have been identified in several S. flexneri serotypes. In this work, the genetic basis for the 3/4-O-acetylation on Rha^III was elucidated. Bioinformatic analysis of the genome of S. flexneri serotype 2a strain Sf301, which carries 3/4-O-acetylation on Rha^III, revealed an O-acyltransferase gene designated oacB. Genetic studies combined with O-antigen structure analysis demonstrated that this gene is responsible for the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not serotype 6, which has a different O-antigen backbone structure. The oacB gene is carried by a transposon-like structure located in the proA-adrA region on the chromosome, which represents a novel mechanism of mobilization of O-antigen modification factors in S. flexneri. These findings enhance our knowledge of S. flexneri O-antigen modifications and shed light on the origin of new O-antigen variants.     

Identification of essential loops and residues of glucosyltransferase V (GtrV) of Shigella flexneri

Lipopolysaccharide (LPS), particularly the O-antigen component, is one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen modification is mediated by glucosyltransferase (gtr) genes encoded by temperate serotype-converting bacteriophages. The gtrV and gtrX genes encode the GtrV and GtrX glucosyltransferases, respectively. These are integral membrane proteins, which catalyze the transfer of a glucosyl residue via an α1,3 linkage to rhamnose II and rhamnose I of the O-antigen unit. This mediates conversion of S. flexneri serotype Y to serotype 5a and X, respectively. Essential regions in the topology of GtrV protein were identified by in vivo recombination and a PCR-mediated approach. A series of GtrX-GtrV and GtrV-GtrX chimeric proteins were constructed based on the fact that GtrV and GtrX share sequence similarity. Analysis of their respective serotype conversion abilities led to the identification of two important periplasmic loops: loops No 2 and No 10 located in the N- and C-termini, respectively. Within these two loops, three conserved motifs were identified; two in loop No 2 and one in loop No 10. These conserved motifs contain acidic residues which were shown to be critical for GtrV function.     

Serotype-Converting Bacteriophage SfII Encodes an Acyltransferase Protein That Mediates 6-O-Acetylation of GlcNAc in Shigella flexneri O-Antigens,Conferring on the Host a Novel O-Antigen Epitope

Shigella flexneri O-antigen is an important and highly variable cell component presented on the outer leaflet of the outer membrane. Most Shigella flexneri bacteria share an O-antigen backbone composed of →2)-α-l-Rhap^III-(1→2)-α-l-Rhap^II-(1→3)-α-l-Rhap^I-(1→3)-β-d-GlcpNAc-(1→ repeats, which can be modified by adding various chemical groups to different sugars, giving rise to diverse O-antigen structures and, correspondingly, to various serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha^I or/and Rha^III, and phosphorylation with phosphoethanolamine on Rha^II or/and Rha^III. Recently, a new O-antigen modification, namely, O-acetylation at position 6 of N-acetylglucosamine (GlcNAc), has been identified in S. flexneri serotypes 2a, 3a, Y, and Yv. In this study, the genetic basis of the 6-O-acetylation of GlcNAc in S. flexneri was elucidated. An O-acyltransferase gene designated oacD was found to be responsible for this modification. The oacD gene is carried on serotype-converting bacteriophage SfII, which is integrated into the host chromosome by lysogeny to form a prophage responsible for the evolvement of serotype 2 of S. flexneri. The OacD-mediated 6-O-acetylation also occurs in some other S. flexneri serotypes that carry a cryptic SfII prophage with a dysfunctional gtr locus for type II glucosylation. The 6-O-acetylation on GlcNAc confers to the host a novel O-antigen epitope, provisionally named O-factor 10. These findings enhance our understanding of the mechanisms of the O-antigen variation and enable further studies to understand the contribution of the O-acetylation to the antigenicity and pathogenicity of S. flexneri.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O60

An O-polysaccharide (O-antigen) was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O60 and studied by sugar and methylation analyses as well as ¹H and ¹³C NMR spectroscopy, including 2D ROESY and ¹H,¹³C HMBC experiments in D₂O and a ROESY experiment in a 9:1 H₂O–D₂O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear pentasaccharide repeating units containing an amide of d-glucuronic acid with l-serine and has the following structure:The O-antigen studied is structurally and serologically closely related to the O-antigen of Proteus vulgaris O44.     

Identification and Molecular Characterisation of a Novel Mu-Like Bacteriophage,SfMu, of Shigella flexneri

S. flexneri is the leading cause of bacillary dysentery in the developing countries. Several temperate phages originating from this host have been characterised. However, all S. flexneri phages known to date are lambdoid phages, which have the ability to confer the O-antigen modification of their host. In this study, we report the isolation and characterisation of a novel Mu-like phage from a serotype 4a strain of S. flexneri. The genome of phage SfMu is composed of 37,146 bp and is predicted to contain 55 open reading frames (orfs). Comparative genome analysis of phage SfMu with Mu and other Mu-like phages revealed that SfMu is closely related to phage Mu, sharing >90% identity with majority of its proteins. Moreover, investigation of phage SfMu receptor on the surface of the host cell revealed that the O-antigen of the host serves as the receptor for the adsorption of phage SfMu. This study also demonstrates pervasiveness of SfMu phage in S. flexneri, by identifying complete SfMu prophage strains of serotype X and Y, and remnants of SfMu in strains belonging to 4 other serotypes, thereby indicating that transposable phages in S. flexneri are not uncommon. The findings of this study contribute an advance in our current knowledge of S. flexneri phages and will also play a key role in understanding the evolution of S. flexneri.     

A Novel Plasmid-Encoded Serotype Conversion Mechanism through Addition of Phosphoethanolamine to the O-Antigen of Shigella flexneri

Shigella flexneri is the major pathogen causing bacillary dysentery in developing countries. S. flexneri is divided into at least 16 serotypes based on the combination of antigenic determinants present in the O-antigen. All the serotypes (except for serotype 6) share a basic O-unit containing one N-acetyl-d-glucosamine and three l-rhamnose residues, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation. Serotype Xv is a newly emerged and the most prevalent serotype in China, which can agglutinate with both MASF IV-1 and 7,8 monoclonal antibodies. The factor responsible for the presence of MASF IV-1 (E1037) epitope has not yet been identified. In this study, we analyzed the LPS structure of serotype Xv strains and found that the MASF IV-1 positive phenotype depends on an O-antigen modification with a phosphoethanolamine (PEtN) group attached at position 3 of one of the rhamnose residues. A plasmid carried gene, lpt-O (LPS phosphoethanolamine transferase for O–antigen), mediates the addition of PEtN for serotype Xv and other MASF IV-1 positive strains. These findings reveal a novel serotype conversion mechanism in S. flexneri and show the necessity of further extension of the serotype classification scheme recognizing the MASF IV-1 positive strains as distinctive subtypes.     

Mechanism of bacteriophage SfII-mediated serotype conversion in Shigella flexneri

We have isolated the lysogenic bacteriophage SfII, which mediates glucosylation of Shigella flexneri O-antigen, resulting in expression of the type II antigen. SfII belongs to group A of the Bradley classification and has a genome size of 42.3 kb. DNA sequencing of a 4 kb BamHI subclone identified four open reading frames (ORFs), of which only two were found to be necessary for serotype conversion. These genes were named bgt, which encodes a putative bactoprenol glucosyl transferase, and gtrII, encoding the putative type II antigen determining glucosyl transferase. These genes are adjacent to the integrase gene (int ) and attachment site (attP ), which are highly homologous to those of Salmonella bacteriophage P22. Another ORF encoded a highly hydrophobic protein of 120 amino acids with homologues in Escherichia coli, Salmonella bacteriophage P22 and S. flexneri. Previous studies identified gtrX, the glucosyl transferase gene, of bacteriophage SfX, which also glucosylates the O-antigen specifically. We determined that gtrX-mediated expression of the group 7,8 antigen also requires bgt. This allowed us to identify gtrII as being the serotype antigen II determining glucosyl transferase. Southern hybridization and polymerase chain reaction (PCR) analyses indicated that bgt homologues exist in the genomes of all S. flexneri serotypes and in E. coli K-12, whereas gtrII was only detected in strains of serotype 2. Transposon TnphoA-derived chromosomal mutations of bgt and gtrII in S. flexneri serotype 2a were isolated and characterized. [³⁵S]-methionine labelling and the use of a T7 RNA polymerase expression system identified a protein of 34 kDa corresponding to Bgt. However, GtrII, which has a predicted molecular weight of 55 kDa, was not detected. We propose that the function of Bgt is to transfer the glucose residues from the UDP-glucose onto bactoprenol and GtrII then transfers the glucose onto the O-antigen repeat unit at the rhamnose III position. The chromosomal organization of these serotype-converting genes, when compared with their homologues in E. coli K-12 chromosome and the P22 bacteriophage genome, were very similar. This suggests that the regions encode similar functions in these organisms and have a similar evolutionary origin.     

Serotype-conversion in Shigella flexneri: identification of a novel bacteriophage,Sf101, from a serotype 7a strain

Background

Shigella flexneri is the major cause of bacillary dysentery in the developing countries. The lipopolysaccharide (LPS) O-antigen of S. flexneri plays an important role in its pathogenesis and also divides S. flexneri into 19 serotypes. All the serotypes with an exception for serotype 6 share a common O-antigen backbone comprising of N-acetylglucosamine and three rhamnose residues. Different serotypes result from modification of the basic backbone conferred by phage-encoded glucosyltransferase and/or acetyltransferase genes, or plasmid-encoded phosphoethanolamine transferase. Recently, a new site for O-acetylation at positions 3 and 4 of Rha^III, in serotypes 1a, 1b, 2a, 5a and Y was shown to be mediated by the oacB gene. Additionally, this gene was shown to be carried by a transposon-like structure inserted upstream of the adrA region on the chromosome.

Results

In this study, a novel bacteriophage Sf101, encoding the oacB gene was isolated and characterised from a serotype 7a strain. The complete sequence of its 38,742 bp genome encoding 66 open reading frames (orfs) was determined. Comparative analysis revealed that phage Sf101 has a mosaic genome, and most of its proteins were >90% identical to the proteins from 12 previously characterised lambdoid phages. In addition, the organisation of Sf101 genes was found to be highly similar to bacteriophage Sf6. Analysis of the Sf101 OacB identified two amino acid substitutions in the protein; however, results obtained by NMR spectroscopy confirmed that Sf101-OacB was functional. Inspection of the chromosomal integration site of Sf101 phage revealed that this phage integrates in the sbcB locus, thus unveiling a new site for integration of serotype-converting phages of S. flexneri, and determining an alternative location of oacB gene in the chromosome. Furthermore, this study identified oacB gene in several serotype 7a isolates from various regions providing evidence of O-acetyl modification in serotype 7a.

Conclusions

This is the first report on the isolation of bacteriophage Sf101 which contains the S. flexneri O-antigen modification gene oacB. Sf101 has a highly mosaic genome and was found to integrate in the sbcB locus. These findings contribute an advance in our current knowledge of serotype converting phages of S. flexneri.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-742) contains supplementary material, which is available to authorized users.     

Identification and Characterization of a Novel Shigella flexneri Serotype Yv in China

Shigella flexneri is the major cause of bacterial shigellosis in developing countries. S. flexneri is divided into at least 19 serotypes, the majority of which are modifications of the same basic O-antigen by glucosylation and/or O-acetylation of its sugar residues by phage encoded serotype-converting genes. Recently, a plasmid encoded phosphoethanolamine (PEtN) modification of the O-antigen has been reported, which is responsible for the presence of the MASF IV-1 determinant and results in conversion of traditional serotypes X, 4a and Y to novel serotypes Xv, 4av and Yv, respectively. In this study, we characterized 19 serotype Yv strains isolated in China. A variant of the O-antigen phosphoethanolamine transferase gene opt (formerly called lpt-O) carried by a pSFxv_2-like plasmid was found in serotype Yv strains, which specifies the phosphorylation pattern on the O-antigen of this serotype. For the majority of the O-antigen units, the PEtN modification occurs on Rha^III, while for a minority, modifications occur on both Rha^II and Rha^III. Serotype-specific gene detection and PFGE analysis suggested that these serotype Yv isolates were originated from serotypes Y, Xv and 2a by acquisition of an opt-carrying plasmid and/or inactivation of serotype-specific gene gtrII or gtrX. These data, combined with those of serotypes Xv and 4av reported earlier, demonstrate that the plasmid-encoded PEtN modification is an important serotype conversion mechanism in S. flexneri, in addition to glucosylation and O-acetylation.     

Structural analysis and involvement in plant innate immunity of Xanthomonas axonopodis pv. citri lipopolysaccharide

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo₂Hex₆GalA₃Fuc3NAcRha₄ and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.     

Structural characterization of closely related O-antigen lipopolysaccharide (LPS) chain length regulators

The surface O-antigen polymers of Gram-negative bacteria exhibit a modal length distribution that depends on dedicated chain length regulator periplasmic proteins (polysaccharide co-polymerases, PCPs) anchored in the inner membrane by two transmembrane helices. In an attempt to determine whether structural changes underlie the O-antigen modal length specification, we have determined the crystal structures of several closely related PCPs, namely two chimeric PCP-1 family members solved at 1.6 and 2.8 Å and a wild-type PCP-1 from Shigella flexneri solved at 2.8 Å. The chimeric proteins form circular octamers, whereas the wild-type WzzB from S. flexneri was found to be an open trimer. We also present the structure of a Wzz^FepE mutant, which exhibits severe attenuation in its ability to produce very long O-antigen polymers. Our findings suggest that the differences in the modal length distribution depend primarily on the surface-exposed amino acids in specific regions rather than on the differences in the oligomeric state of the PCP protomers.     

Protective immunity by oral immunization with heat‐killed Shigella strains in a guinea pig colitis model

The protective efficacy of and immune response to heat‐killed cells of monovalent and hexavalent mixtures of six serogroups/serotypes of Shigella strains (Shigella dysenteriae 1, Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, Shigella boydii 4, and Shigella sonnei) were examined in a guinea pig colitis model. A monovalent or hexavalent mixture containing 1 × 10⁷ of each serogroup/serotype of heat‐killed Shigella cells was administered orally on Days 0, 7, 14 and 21. On Day 28, the immunized animals were challenged rectally with 1 × 10⁹ live virulent cells of each of the six Shigella serogroups/serotypes. In all immunized groups, significant levels of protection were observed after these challenges. The serum titers of IgG and IgA against the lipopolysaccharide of each of the six Shigella serogroups/serotypes increased exponential during the course of immunization. High IgA titers against the lipopolysaccharide of each of the six Shigella serogroups/serotypes were also observed in intestinal lavage fluid from all immunized animals. These data indicate that a hexavalent mixture of heat‐killed cells of the six Shigella serogroups/serotypes studied would be a possible broad‐spectrum candidate vaccine against shigellosis.     

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.