Isolation, characterization and comparative genomics of bacteriophage SfIV: a novel serotype converting phage from Shigella flexneri

BACKGROUND Shigella flexneri is the major cause of shigellosis in the developing countries. The O-antigen component of the lipopolysaccharide is one of the key virulence determinants required for the pathogenesis of S. flexneri. The glucosyltransferase and/or acetyltransferase genes responsible for the modification of the O-antigen are encoded by temperate serotype converting bacteriophage present in the S. flexneri genome. Several serotype converting phages have previously been isolated and characterized, however, attempts to isolate a serotype converting phage which encodes the modification genes of serotypes 4a strain have not been successful. RESULTS In this study, a novel temperate serotype converting bacteriophage SfIV was isolated. Lysogenisation of phage SfIV converted serotype Y strain to serotype 4a. Electron microscopy indicated that SfIV belongs to Myoviridae family. The 39,758 bp genome of phage SfIV encompasses 54 open reading frames (orfs). Protein level comparison of SfIV with other serotype converting phages of S. flexneri revealed that SfIV is similar to phage SfII and SfV. The comparative analysis also revealed that SfIV phage contained five proteins which were not found in any other phages of S. flexneri. These proteins were: a tail fiber assembly protein, two hypothetical proteins with no clear function, and two other unknown proteins which were encoded by orfs present on a moron, that presumably got introduced in SfIV genome from another species via a transposon. These unique proteins of SfIV may play a role in the pathogenesis of the host. CONCLUSIONS This study reports the isolation and complete genome sequence analysis of bacteriophage SfIV. The SfIV phage has a host range significantly different from the other phages of Shigella. Comparative genome analysis identified several proteins unique to SfIV, which may potentially be involved in the survival and pathogenesis of its host. These findings will further our understanding on the evolution of these phages, and will also facilitate studies on development of new phage vectors and therapeutic agents to control infections caused by S. flexneri

Serotype-conversion in Shigella flexneri: identification of a novel bacteriophage, Sf101, from a serotype 7a strain

BACKGROUND Shigella 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. RESULTS In 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. CONCLUSIONS This 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.This work was supported by grants from the Swedish Research Council and The Knut and Alice Wallenberg Foundation to GW

Molecular studies of three novel bacteriophages of shigella flexneri

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

Identification and Molecular Characterisation of a Novel Mu-Like Bacteriophage, SfMu, of Shigella flexneri.

S. flexneri is the leading cause of bacillary dysentery in the developing countries. Several temperate phages originating from this host have been characterised. However, all S. flexneri phages known to date are lambdoid phages, which have the ability to confer the O-antigen modification of their host. In this study, we report the isolation and characterisation of a novel Mu-like phage from a serotype 4a strain of S. flexneri. The genome of phage SfMu is composed of 37,146 bp and is predicted to contain 55 open reading frames (orfs). Comparative genome analysis of phage SfMu with Mu and other Mu-like phages revealed that SfMu is closely related to phage Mu, sharing >90% identity with majority of its proteins. Moreover, investigation of phage SfMu receptor on the surface of the host cell revealed that the O-antigen of the host serves as the receptor for the adsorption of phage SfMu. This study also demonstrates pervasiveness of SfMu phage in S. flexneri, by identifying complete SfMu prophage strains of serotype X and Y, and remnants of SfMu in strains belonging to 4 other serotypes, thereby indicating that transposable phages in S. flexneri are not uncommon. The findings of this study contribute an advance in our current knowledge of S. flexneri phages and will also play a key role in understanding the evolution of S. flexneri

Morphology of bacteriophage SfMu.

<p>A) Electron micrograph of phage SfMu, negatively stained with 2% phosphotungstenic acid. Scale bar 100 nm. B) Plaques of phage SfMu.</p

Genome of bacteriophage SfMu.

<p>The genome of phage SfMu is shown with a scale in bp. Predicted <i>orfs</i> are designated with arrows, pointing in the direction of transcription. Putative functional modules are indicated above the arrows. Black knobs indicate the position of predicted terminators. Comparison of SfMu encoded proteins to their counterparts in phage Mu and D108 is shown below the scale. Different colours show the level of amino acid identity: red: 90–100% identity, yellow: 80–89% identity, orange: 70–79% identity, green: 60–69% identity, blue: 50–59% identity and grey: 10–50% identity. The purple arrows in the SfMu map represent SfMu proteins which have no homologues in phage Mu or D108.</p