The role of TypeA in the virulence of enteropathogenic Escherichia coli
thesisposted on 15.12.2014, 10:33 by Stuart Charles. Clarke
Enteropathogenic Escherichia coli (EPEC) remain an important cause of diarrhoeal disease in developing countries. EPEC adhere to cultured epithelial cells in a localised pattern resulting in the effacement of microvilli and subversion of the host cell signalling pathways leading to rearrangement to the cytoskeleton. This results in the 'attaching and effacing (AE) lesion'. This project is concerned with the characterisation of a novel gene, typA (tyrosine phosphoprotein A), and its role in virulence.;TypA and its flanking regions were cloned form EPEC strain E2348/69 strain E2348/69 and sequenced to determine differences between EPEC typA and E. coli K-12 typA. Few differences were found between the TypA predicted amino acid sequences. The sequence upstream of typA in EPEC was identical to that in E. coli K-12 but the first 1.3 kb of the downstream sequence showed no homology with E. coli K-12. The remaining 422 bp showed homology with ORF o300 in E. coli K-12. ORF o300 is similar to members of the phosphofructokinase B family of carbohydrate kinases.;A frameshift mutation was constructed in typA of EPEC E2348/69, which was also introduced into other EPEC strains. Phenotypic assays were performed on the E2348/69 typA mutant and its parent. Growth rate and Bfp production of the typA mutant were very similar. However, adherence and invasion ability was reduced 3.5-fold. FAS tests indicate that the E2348/69 typA mutant was still able to cause AE lesions. FAS tests were also performed on the EAF plasmid-negative JPN15 typA mutant and its parent; the mutant did not cause AE lesion formation. The EAF plasmid of the per genes were introduced into the JPN15 typA mutant resulting in restoration of the AE lesion phenotype. This indicated that typA acted as a regulator in the absence of the per genes and may therefore be considered a virulence gene.