Molecular studies of three novel bacteriophages of shigella flexneri

Abstract

Temperate bacteriophages play a crucial role in the pathogenesis of Shigella flexneri. They modify the O-antigen of the host, leading to increased virulence and immune evasion. Characterization of S. flexneri phages is therefore essential for understanding the antigenicity and pathogenicity of S. flexneri. In this study, three novel bacteriophages of S. flexneri were isolated and characterized. The first was a serotype converting bacteriophage SfIV isolated from a serotype 4av strain of S. flexneri. The complete genome sequence of phage SfIV was determined. Protein level comparison of SfIV with other serotype converting phages of S. flexneri revealed that SfIV is similar to phage SfII and SfV. Nevertheless, SfIV phage contained five proteins (a tail fiber assembly protein and four proteins of unknown function) which were not found in any other phages of S. flexneri. The second phage, Sf101, was obtained from a serotype 7a strain. Complete genome sequence of Sf101 phage was determined and found to contain the gene encoding 3/4-O-acetylation modification (oacB). The comparative genomic analysis of Sf101 to 12 other lambdoid phages revealed that bacteriophage Sf101 has a highly mosaic genome and Sf6-like genome organisation. Sf101 was found to integrate into the sbcB locus representing a new integration site for serotype converting phages of S. flexneri. Additionally, this study also identified oacB gene in several serotype 7a isolates from different regions, providing first evidence of 3/4-O-acetylation modification in serotype 7a of S. flexneri. Unlike all the other phages of S. flexneri known thus far, the third phage isolated was a Mu-like phage from a serotype 4a strain of S. flexneri. The complete genome of phage SfMu was sequenced, characterized, and compared with bacteriophage Mu and other Mu-like phages. Results of comparative analysis revealed that SfMu is closely related to phage Mu. Moreover, analysis of phage SfMu cell wall receptor revealed that bacteriophage SfMu recognises LPS O-antigen as its primary receptor for adsorption. This study also reported presence of complete or cryptic SfMu prophage in various serotypes of S. flexneri, thereby indicating that transposable phages in S. flexneri are not uncommon. This study also identified the role of phage SfIV unique genes (orf35, 36, 48 and 49) and phage Sf101 oacB in the virulence of S. flexneri, using a knockout approach. Results of in vitro and in vivo virulence studies using orf 35-36, 48-49 and oacB disruption mutants indicated that the oacB does not affect the virulence of serotype 7a host. However, orf35-36 and orf48-49 gene clusters increased the virulence of its serotype 4av host, thus unveiling for the first time that S. flexneri phage genes other than the O-antigen modification genes contribute to the virulence of the host. Taken together, identification and genomic characterization of phages SfIV, Sf101 and SfMu advances our current knowledge of S. flexneri phages and their evolution. The phage-encoded virulence factors identified in this study provides novel insight into the role of bacteriophages in the pathogenesis of S. flexneri and will also facilitate the development of new therapeutic measures to control S. flexneri infections. Show more