Study of O-acetyltransferase B (oacB) and three novel orfs encoded by Sf101 bacteriophage of Shigella flexneri

Abstract

Shigella flexneri is the leading cause of bacillary dysentery in developing countries and is associated with significant morbidity and mortality. Bacteriophages are known to play important role in the pathogenesis of S. flexneri by encoding O-antigen-modifying genes involved in serotype conversion. Bacteriophage Sf101 encoded O-acetyltransferase B (oacB) is a serotype converting gene that adds an acetyl group at either 3/4 position of Rhamnose III of the O-antigen. In some serotype 1c strains oacB is carried by Sf101 bacteriophage integrated within sbcB gene on the host chromosome. In contrast, in several serotypes 1c strains, and other strains of other serotypes carrying 3/4-O-acetylation, the location of oacB gene is upstream of adrA gene within proA-adrA region, where all gtr-carrying phages integrated. The first part of the study deals with the investigation of the topological features of OacB and the identification of important residues in OacB. For this purpose, multiple alignments of OacB were performed with other acetyltransferases from related bacterial species, and several conserved domains and motifs were identified. Site-directed mutagenesis carried out on the selected residues of the OacB revealed seven amino acids that were critical for the function of OacB. In the second part of the study, the distribution of the oacB gene in the serotype 1c strains was investigated with reference to the Sf101 lysogenic strain (SFL1683). The complete genome sequence of SFL1683 was generated using MinION flow technology to develop a reference genome, and regions carrying oacB in 1c strains were investigated. To gain insight into the origin of oacB gene in serotype 1c strains, whole-genome sequences of strains collected from various geographical regions were inspected for the presence of Sf101 phage or its remnants. The results revealed that in only two lysogenic strains Sf101 phage integrated within sbcB gene, and in other strains within proA- adrA region. All the analysed strains carried conserved Sf101 attachment sites within the sbcB gene, and sbcB gene was flanked by similar insertion sequences (IS). The genetic arrangement upstream adrA gene in SFL1683 was different from other 1c strains due to the absence of IS elements flanking oacB gene. It was also identified that the SfI phage attachment site shared six base pairs (bp) homology with the Sf101 attachment site. In the Y394 strain, the SfI phage attachment site was identified at three different locations within the proA-adrA region which might have helped the Sf101 phage to integrate into this region. The abundance of IS elements in this region was indicative of insertion or deletion events and resulted in the deletion of Sf101 phage leaving behind oacB gene. In a previous study, the four novel orfs (oacB/orf16, orf17, orf41, and orf56) were found to have no phage-related functions and limited homologies with Shigella and E. coli proteins. The physiological effect of novel orfs encoded by Sf101 on the virulence of S. flexneri was also explored in the last part of the study. The lysogenic strain of Sf101 lacked a large virulence plasmid (VP) due to which virulence studies were performed in another serotype 1c strain harbouring VP. Three different virulence assays were employed involving C. elegans as an in vivo model and HeLa cells as an in vitro model. The results showed the presence of these orfs did not affect the virulence phenotype of the host. This study has provided a detailed characterization of oacB of serotype converting Sf101 phage of S. flexneri and opened avenues for the upcoming research to understand the serotype conversion in S. flexneri.