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Identification and characterization of a novel Shigella flexneri serotype Yv in China.

ABSTRACT: 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.

SUBMITTER: Sun Q

PROVIDER: S-EPMC3728103 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification and characterization of a novel Shigella flexneri serotype Yv in China.

Sun Qiangzheng Q Lan Ruiting R Wang Jianping J Xia Shengli S Wang Yiting Y Wang Yan Y Jin Dong D Yu Bo B Knirel Yuriy A YA Xu Jianguo J

PloS one 20130730 7

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 conv ...[more]

PMID: 23936172

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Project description:BackgroundShigella 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 RhaIII, 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.ResultsIn 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.ConclusionsThis 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.

Project description:BackgroundShigella flexneri is the major cause of shigellosis in the developing countries. The O-antigen component of the lipopolysaccharide is one of the key virulence determinants required for the pathogenesis of S. flexneri. The glucosyltransferase and/or acetyltransferase genes responsible for the modification of the O-antigen are encoded by temperate serotype converting bacteriophage present in the S. flexneri genome. Several serotype converting phages have previously been isolated and characterized, however, attempts to isolate a serotype converting phage which encodes the modification genes of serotypes 4a strain have not been successful.ResultsIn this study, a novel temperate serotype converting bacteriophage SfIV was isolated. Lysogenisation of phage SfIV converted serotype Y strain to serotype 4a. Electron microscopy indicated that SfIV belongs to Myoviridae family. The 39,758 bp genome of phage SfIV encompasses 54 open reading frames (orfs). Protein level comparison of SfIV with other serotype converting phages of S. flexneri revealed that SfIV is similar to phage SfII and SfV. The comparative analysis also revealed that SfIV phage contained five proteins which were not found in any other phages of S. flexneri. These proteins were: a tail fiber assembly protein, two hypothetical proteins with no clear function, and two other unknown proteins which were encoded by orfs present on a moron, that presumably got introduced in SfIV genome from another species via a transposon. These unique proteins of SfIV may play a role in the pathogenesis of the host.ConclusionsThis study reports the isolation and complete genome sequence analysis of bacteriophage SfIV. The SfIV phage has a host range significantly different from the other phages of Shigella. Comparative genome analysis identified several proteins unique to SfIV, which may potentially be involved in the survival and pathogenesis of its host. These findings will further our understanding on the evolution of these phages, and will also facilitate studies on development of new phage vectors and therapeutic agents to control infections caused by S. flexneri.

Project description:A significant cause of shigellosis in Bangladesh and other developing countries is Shigella flexneri serotype 6. This serotype has been subtyped, on the basis of the absence or presence of a group-specific antigen, E1037, into S. flexneri 6a and 6b, respectively. Here, we provided rationales for the subclassification, using several phenotypic and molecular tools. A set of S. flexneri 6a and 6b strains isolated between 1997 and 2015 were characterized by analyzing their biochemical properties, plasmid profiles, virulence markers, pulsed-field gel electrophoresis (PFGE) results, and ribotype. Additionally, the genomic relatedness of these subserotypes was investigated with global isolates of serotype 6 using publicly available genomes. Both subserotypes of S. flexneri 6 agglutinated with monoclonal antiserum against S. flexneri (MASF) B and type VI-specific antiserum (MASF VI) and were PCR positive for O-antigen flippase-specific genes and virulence markers (ipaH, ial, sen, and sigA). Unlike S. flexneri 6a strains, S. flexneri 6b strains seroagglutinated with anti-E1037 antibodies, MASF IV-I. Notably, these two antigenically distinct subserotypes were clonally diverse, showing two distinct PFGE patterns following the digestion of chromosomal DNA with either XbaI or IceuI. In addition, hybridization of a 16S rRNA gene probe with HindIII-digested genomic DNA yielded two distinguishing ribotypes. Genomic comparison of S. flexneri subserotype 6a and 6b strains from Bangladesh indicated that, although these strains were in genomic synteny, the majority of them formed a unique phylogroup (PG-4) that was missing for the global isolates. This study supports the subserotyping and emphasizes the need for global monitoring of the S. flexneri subserotypes 6a and 6b. IMPORTANCE Shigella flexneri serotype 6 is one of the predominant serotypes among shigellosis cases in Bangladesh. Characterization of a novel subserotype of S. flexneri 6 (VI:E1037), agglutinated with type 6-specific antibody and anti-E1037, indicates a unique evolutionary ancestry. PFGE genotyping supports the finding that these two antigenically distinct subserotypes are clonally diverse. A phylogenetic study based on single-nucleotide polymorphism (SNP) data revealed that these two subserotypes were in genomic synteny, although their genomes were reduced. Interestingly, a majority of the S. flexneri 6 strains isolated from Bangladesh form a novel phylogenetic cluster. Therefore, this report underpins the global monitoring and tracking of the novel subserotype.

Project description:BackgroundThirteen serotypes of Shigella flexneri (S. flexneri) have been recognised, all of which are capable of causing bacillary dysentery or shigellosis. With the emergence of the newer S. flexneri serotypes, the development of an effective vaccine has only become more challenging. One of the factors responsible for the generation of serotype diversity is an LPS O-antigen modifying, integral membrane protein known as O-acetyltransferase or Oac. Oac functions by adding an acetyl group to a specific O-antigen sugar, thus changing the antigenic signature of the parent S. flexneri strain. Oac is a membrane protein, consisting of hydrophobic and hydrophilic components. Oac bears homology to several known and predicted acetyltransferases with most homology existing in the N-terminal transmembrane (TM) regions.ResultsIn this study, the conserved motifs in the TM regions and in hydrophilic loops of S. flexneri Oac were targeted for mutagenesis with the aim of identifying the amino acid residues essential for the function of Oac. We previously identified three critical arginines-R73, R75 and R76 in the cytoplasmic loop 3 of Oac. Re-establishing that these arginines are critical, in this study we suggest a catalytic role for R73 and a structural role for R75 and R76 in O-acetylation. Serine-glycine motifs (SG 52-53, GS 138-139 and SYG 274-276), phenylalanine-proline motifs (FP 78-79 and FPV 282-84) and a tryptophan-threonine motif (WT141-142) found in TM segments and residues RK 110-111, GR 269-270 and D333 found in hydrophilic loops were also found to be critical to Oac function.ConclusionsBy studying the effect of the mutations on Oac's function and assembly, an insight into the possible roles played by the chosen amino acids in Oac was gained. The transmembrane serine-glycine motifs and hydrophilic residues (RK 110-111, GR 269-270 and D333) were shown to have an affect on Oac assembly which suggests a structural role for these motifs. The phenylalanine-proline and the tryptophan-threonine motifs affect Oac function which could suggest a catalytic role for these amino acids.

Dataset Information

Identification and characterization of a novel Shigella flexneri serotype Yv in China.

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Identification and characterization of a novel Shigella flexneri serotype Yv in China.

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