Background: Shigellosis, an acute gastrointestinal disease is caused primarily by the bacterium Shigella flexneri. Upon ingestion, S. flexneri initiates a serotype-specific immune response that targets the O-antigen of pathogen’s lipopolysaccharide. O-antigens 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 gene, O-acetyltransferase B (oacB), which adds an acetyl residue at either position 3 or 4 of Rhamanose^III (3/4-O-acetylation) in serotypes 1a, 1b, 2a, 5a, 7a, Y, and 6 and at position 6 of N- acetylglucosamine (6-O-acetylation) in serotypes 2a, 3a , Y and Yv of the O-antigen subunits. Topologies of other serotype converting proteins like glucosyltransferases (Gtr) and acetyltransferase (Oac) of S. flexneri have been solved experimentally whereas the topological analysis of OacB is yet to be determined.

Materials/methods: Topology prediction of OacB was performed using membrane protein prediction programs such as DAS, HMMTOP, TMHMM and TopPred .Identification of conserved critical residues and domains was carried out using side-directed mutagenesis and functionality of the protein was determined by slide agglutination and western blots using specific antibodies to acetyl modification.

Results: Hydrophobicity analysis showed that OacB similar to its close homologue O-acetyltransferase (Oac) from serotype 3b is a transmembrane protein with 10 transmembrane segments, 11 loops, and cytoplasmic N and C termini. Bioinformatics analysis revealed that OacB has several motifs. Mutants were created by mutating conserved amino acids within and outside these motifs. Amino acids critical to function were identified via slide agglutination test and western blots using O-acetyl specific antisera. Five mutants showed a negative agglutination reaction hence turned protein nonfunctional.  

Conclusions: The importance of a number of amino acid residues within the conserved domain and the homologous region with regard to function of OacB was determined. Identified amino acids may play an important role in forming the catalytic domain within the acetyltransferase homologous region. The study furthers our understanding of the structure, function of a serotype converting protein, and will contribute to vaccine development research against shigellosis.