Construction Of F18Ab-fimbriaed E. Coli Hcp Gene Mutant And Its Effection On The Secretion Of Virulance Facters

[Background] Enterotoxigenic Escherichia coli(STEC) strain F18ab frequently cause severe watery diarrhoea and are responsible for significant economic losses in the pig industry worldwide. Virulence factors of STEC strain F18ab include adhesins such as type I fimbriae, F18ab fimbriae, flagellum, AIDA (adhesin involved in diffuse adherence) and Shiga like toxin2e, which are secreted to its outer membrane or surrounding envirenment by secretion system. Thus far, seven secretion systems have been identified, named Type I to Type Ⅶ, which carry out the process of translocation, Type Ⅵ and Ⅶ being the most recent discovery.[Purpose] To study the role of hcp gene of Type VI secretion system in the secretion of virulance factors, and find new ways to prevent and control the STEC-caused disease.[Methods and Procedures]Based on the original sequences of hcp operon gene clusters in the GenBank, the STEC F18ab was selected as the prototype and constructed the hcp mutant by Red recombination system. First, we generated PCR products by primer and sequenced of hcp gene from STEC F18ab. Second, we generated PCR products using primers with the homologies extension of hcp gene to be deleted and template plasmid pKD3carrying selectable antibiotic chloramphenicol resistance (cat) gene that is flanked by FRT (FLP recognition target) sites. And then the PCR products were introduced into STEC F18ab by electroporation, with the help of λ Red-mediated recombination system, hcp gene was replaced by the cat gene, the strains expressing chloramphenicol resistance gene were selected, the chloramphenicol resistance gene was then eliminated by using a helper plasmid, pCP20, encoding the Flp recombinase. Using this system, hcp gene in chromosome of Escherichia coli was deleted. STEC F18ab△hcp mutant obtained were confirmed by PCR amplification and sequencing. And then we compared the STEC F18ab and△hcp mutant with their biological characteristics, including growthrhythm, motor ability, the capability of adhering and invading epithelial cells in vitro and vitro, and its activity of killing Vero cells.[Results] By comparing the STEC F18ab and△hcp mutant, the results showed that the motility of△hcp mutant reduced about18.8%; and its ability of adherence and invasion on epithelial cells decreased about40%and28%respectively; in cytotoxicity test, the Vero cells viability of△hcp mutant group was7time more than the wild group; and experiments on living mouse also showed tremendously decreased adherence in vitro. However, Real Time-PCR tests showed that there was no significant difference in the transcribed level among type I fimbriae, F18ab fimbriae, flagellum, AIDA and stx2e between the wild type strain and△hcp mutant. From the above, we made a conclusion that hcp gene of T6SS may be not associated to the transcription of the five virulence factors above, that is to say, but does relate to their secretion。...