Investigating The Function Of OI-172in Enterohaemorrhagic Escherichia Coli O157:H7Edl933

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7is a major cause of zoonotic food-and water-borne intestinal infections worldwide with clinical consequences ranging from mild diarrhoea to haemolytic uraemic syndrome (HUS). The genome of EHEC O157:H7contains many regions of unique DNA that are referred to as O islands including the Shiga toxin prophages and pathogenicity islands encoding key virulence factors. However, many of these O islands are of unknown function. In this study, genetic analysis was conducted on01-172which is a44,434bp genomic island with27open reading frames. Comparative genome analysis showed that complete OI-172island was only found in EHEC O157:H7strains. Comparative proteomic analysis performed previously identified flagellin that was down-regulated in the Z5898mutant (EHEC O157:H7EDL933AZ5898). In this study, motility assay showed that EDL933△Z5898migrated slower than the wild-type EDL933. Electron microscopy found reduced surface flagella present on most of the mutants. Quantitative reverse transcription PCR revealed that the fliC expression of EDL933△Z5898was significantly lower while the expression of its upstream regulator gene fliA, was not affected. Using a fliA and a fliC promoter- green fluorescent protein (GFP) fusion contruct, Z5898was found to affect only the fliC promoter activity. Therefore, Z5898regulates the flagella based motility by exerting its effect on fliC.We also compared global transcription profiles of the Z5898mutant and wild-type (WT) strain in exponential (2h) and stationary (10h) growth phase. Deletion of Z5898affected transcription of267genes in exponential growth phase and712genes in stationary growth phase.48(60.7%) of79upregulated genes in the Z5898mutant in exponential growth phase are identified in OI-45, which include genes encoding Stx2AB, the deadly Shiga toxin. While,51(91.1%) of56upregulated genes in the Z5898mutant in stationary growth phase are identified in OI-45, which include the gene encoding Stx2A. Decreased expression of12genes associated with flagella biosynthesis and assembly in the Z5898mutant occurred at stationary growth phase, which include fliD, fliK, fliO, fliS, fliZ, flgE, flgF, flgG, flgH,flgI,flgK and motA.In conclusion, our study indicates that OI-172is a motility associated O island and hereby name it the MAO island. And also, Z5898in MAO island plays a wide regulatory role in EHEC strain EDL933and involves in the regulation of transcription of genes encoding Stx2and genes associated with flagella biosynthesis and assembly.