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Identification of a divergent O-acetyltransferase gene oac 1b from Shigella flexneri serotype 1b strains.

ABSTRACT: Shigella flexneri is a leading cause of bacterial dysentery in developing countries. Among the 15 known serotypes, four (1b, 3a, 3b and 4b) contain a group 6 epitope due to an acetyl group connected to the O-2 position of rhamnose III on the tetrasaccharide structure of the lipopolysaccharide. O-acetyltransferase encoded by a bacteriophage, Sf6, mediates the acetylation reaction. We found that the oac gene in serotype 1b strains was very different from that in serotypes 3a, 3b and 4b strains and is herein after referred to as oac 1b which shares with oac 88%-89% identity at the DNA level and 85% identity at the protein level. Considering that S. flexneri strains of serotypes 1-5 share a recent common ancestry, the divergent oac 1b is more likely to have been obtained from outside S. flexneri than to have undergone rapid divergence from the oac gene in the other serotypes (3a, 3b and 4b) within S. flexneri. The cloned oac 1b gene was found to perform the same acetylation function as oac. Analysis of the genomic regions flanking oac 1b showed that it was present in a prophage on the chromosome and the organizational structure is different from that of phage Sf6. Additionally, phage conversion assay showed that serotype 1b cannot be generated by infecting serotype 1a strains with Sf6. We conclude that oac 1b was carried by a non-Sf6 phage and is uniquely present in serotype 1b.

SUBMITTER: Sun Q

PROVIDER: S-EPMC3630932 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Identification of a divergent O-acetyltransferase gene oac 1b from Shigella flexneri serotype 1b strains.

Sun Qiangzheng Q Lan Ruiting R Wang Yan Y Wang Jianping J Xia Shengli S Wang Yiting Y Zhang Jin J Yu Deshan D Li Zhenjun Z Jing Huaiqi H Xu Jianguo J

Emerging microbes & infections 20120905 9

Shigella flexneri is a leading cause of bacterial dysentery in developing countries. Among the 15 known serotypes, four (1b, 3a, 3b and 4b) contain a group 6 epitope due to an acetyl group connected to the O-2 position of rhamnose III on the tetrasaccharide structure of the lipopolysaccharide. O-acetyltransferase encoded by a bacteriophage, Sf6, mediates the acetylation reaction. We found that the oac gene in serotype 1b strains was very different from that in serotypes 3a, 3b and 4b strains and ...[more]

PMID: 26038428

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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.

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: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:BackgroundShigellosis is an acute gastrointestinal disease caused primarily by the bacterium Shigella flexneri. Upon ingestion, S. flexneri initiates a serotype-specific immune response that targets the O-antigen of the pathogen's lipopolysaccharide. O-antigen subunits are modified by the addition of chemical moieties, which give rise to new serotypes of S. flexneri. Nineteen different serotypes of S. flexneri have been recognized. A recently identified O-antigen-modifying enzyme, O-acetyltransferase B (OacB), which adds an acetyl residue at either position 3 or 4 of RhamnoseIII (3/4-O-acetylation) in serotypes 1a, 1b, 2a, 5a, 7a, Y, and 6 and position 6 of N- acetylglucosamine (6-O-acetylation) in serotypes 2a, 3a, Y and Yv of the O-antigen subunits. Critical residues in other proteins involved in O-antigen modifications such as glucosyltransferases (Gtrs) and acetyltransferase (Oac) of S. flexneri have been identified, whereas identification of important amino acids in OacB function is yet to be determined.ResultsHydrophobicity analysis showed that OacB is a transmembrane protein with 11 transmembrane segments, 12 loops, and periplasmic N- and cytoplasmic C- termini. Bioinformatics analyses revealed that OacB contains acetyltransferase-3 domain and several conserved residues. Using site-directed mutagenesis, selected amino acids were mutated to alanine to elucidate their role in the mechanism of action of OacB. Seven amino acids R47, H58, F98, W71, R116, R119, and S146 were found critical for the OacB function.ConclusionIn the absence of a three-dimensional structure of the serotype converting enzyme, O-acetyltransferase B (OacB), a clear role of important residues in the mechanism of action is precluded. Therefore, in this study, using site-directed mutagenesis, seven residues critical to the function of OacB were identified. The lack of agglutination of cell expressing mutant OacB in the presence of the antiserum indicated the functional role of the corresponding residues. Hence, this study provides significant information about key residues in OacB which might be involved in forming the catalytic sites of this O-antigen modifying enzyme of S. flexneri.

Dataset Information

Identification of a divergent O-acetyltransferase gene oac 1b from Shigella flexneri serotype 1b strains.

Publications

Identification of a divergent O-acetyltransferase gene oac 1b from Shigella flexneri serotype 1b strains.

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