Characterising the mobile genome of Shigella

Shigella spp. are pathogenic variants of Escherichia coli that cause bacillary dysentery, resulting in over 1 million deaths per year. Across E. coli bacteria, the dynamic process of acquisition and loss of GIs, especially those associated with virulence (pathogenicity islands [PAIs]) is a driving force behind the emergence of new pathogenic strains. In Shigella only 5 GIs have been well characterised and there is currently no effective vaccine. Therefore the development of an efficient screen to detect GIs in unsequenced Shigella strains could be highly informative. Nineteen Shigella strains were probed for the presence of GIs using a high throughput PCR screen (tRIP), across 16 tRNA gene integration hotspots. Putative GIs were then investigated using a chromosome walking technique (SGSP-PCR). Representative PCR amplicons were sequenced to get a snapshot of the islands contents. Islands of particular interest were characterised further using allelic exchange and marker rescue to capture clones that harbour larger portions of the GI and subsequent sequencing and analysis. Using SGSP-PCR, 81% of the putative GI occupied tRNA loci were characterised, and sequencing analysis found they all contain island DNA, indicating that tRIP followed by SGSP-PCR is a robust strategy for GI discovery in unsequenced strains; also this method should be applicable to a broad range of microorganisms. At least 54% of the islands identified harbour phage-like integrase genes, strongly supporting the notion that many of these elements arose following acquisition of horizontally acquired integrative GIs. The frequent presence of integrase genes also highlights the potential role of bacteriophage in the original and/or ongoing dissemination of island DNA in Shigella. Only one novel GI was discovered; it has classic prophage-like features and contains completely novel DNA, indicating that while Shigella has a plastic genome, it is a highly specialised human pathogen that has undergone considerable pathoadaptive genome reduction. The major development from this study is evidence that a number of key Shigella virulence determinants are independently mobile and not only localised to a single family of islands; this significantly increases their potential to spread by HGT across Shigella and could contribute to the rapid emergence of new endemic strains.