Dataset Information

Mutational analysis of the Shigella flexneri O-antigen polymerase Wzy: identification of Wzz-dependent Wzy mutants.

ABSTRACT: The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on the wzySf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySf proteins in the wzySf-deficient (?wzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of WzySf and WzzSf was investigated by transforming these mutated wzySf plasmids into a wzySf- and wzzSf-deficient (?wzy ?wzz) strain. Comparison of the LPS profiles in the ?wzy and ?wzy ?wzz backgrounds identified WzySf mutants whose polymerization activities were WzzSf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the ?wzy and ?wzy ?wzz backgrounds identified a role for WzzSf in WzySf stability. Hence, in addition to its role in regulating Oag modal chain length, WzzSf also affects WzySf activity and stability.

SUBMITTER: Nath P

PROVIDER: S-EPMC4288684 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Mutational analysis of the Shigella flexneri O-antigen polymerase Wzy: identification of Wzz-dependent Wzy mutants.

Nath Pratiti P Tran Elizabeth Ngoc Hoa EN Morona Renato R

Journal of bacteriology 20141013 1

The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on the wzySf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySf protei ...[more]

PMID: 25313393

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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

Mutational analysis of the Shigella flexneri O-antigen polymerase Wzy: identification of Wzz-dependent Wzy mutants.

Publications

Mutational analysis of the Shigella flexneri O-antigen polymerase Wzy: identification of Wzz-dependent Wzy mutants.

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