Abstract
Uropathogenic E. coli (UPEC) is a subtype of extraintestinal pathogenic E. coli, and many virulence genes have been identified for the uropathogenicity. In this regard, the gene constitution of the E. coli genome is very important. Because DNA methylation of specific sequence on the genome has been identified to be involved in pathogenicity, DNA methyltransferases (DNMT) can be a virulence factor. The single molecular real time (SMRT) sequencing is available to identify methylated sequence. However, phenotypical approach is also necessary to assign the recognition sequence of DNMT. In this study, we develop a suicide-donor-conjugation method to screen a combination of DNMT and restriction enzyme, which recognize overlapping sequences. A type IV restriction enzyme (McrBC), which specifically recognizes methylated site, was used to carry out the screening in UPEC as a model.A plasmid expressing McrBC was transferred to 83 of clinically isolated UPEC and 50 of fecal E. coli isolates through the suicide donor conjugation. When a recipient harbors a cytosine-5-DNMT which recognizes a sequence overlapping with McrBC, its genome DNA will be methylated and restricted, and then the cell shows a conflict phenotype (lethal). Twenty-nine strains were identified as shown the conflict from the 133 strains. Frequency of the conflict in the UTI isolates (24/83) showed significantly higher than that in the fecal isolates (5/50), suggested that the DNMTs conflicted with McrBC were associated with uropathogenicity. Nine C5-DNMT genes covering 24 of the 29 strains were identified as determinants of the conflict. Their clone showed the conflict and overlapping of recognition sequence with McrBC in K-12 strain. We also challenged whether the DNMTs associated with higher pathogenicity. Caenorhabditis elegans model was adopted for the survey, however, there are no significant association between the existence of DNMT and pathogenicity level in the model. These results implied that the DNMTs involved in horizontal gene transfer to acquire virulence genes, but did not involved to increase the pathogenicity.
| Date of Award | 2018 |
|---|---|
| Original language | English |
| Supervisor | Masayuki Hashimoto (Supervisor) |