Construction And Characterization Of The Gene Vca0107of T6SS Deleted Mutants Of Swine Extraintestinal Pathogenic Escherichia Coli Strain

Extraintestinal pathogenic Escherichia coli strain(ExPEC) is a pathogen which can causing the infection of the intestinal tissues and organs of the human and animal, via its specific pathogenic virulence factors (pili, adhesins, iron-related proteins, etc.) invading and colonizing the different tissues and organs of intestinal tissues, inducing the host produce meningitis, sepsis and urinary tract infections and other symptoms. Following the harm of the pig industry by ExPEC and the huge fiscal effort of the global medical industry, and studies have found the harm be from ExPEC even higher than E.coli0157: H7which is wide concerned, so which should be attention on a wide range of public health significance. Following the research of ExPEC is more and more, which can be spread through the food sources, and pollution of the drinking water and food, result in the outbreaks of disease, seriously leading to the death of human and animals. To stock farming, ExPEC can induse livestock produce pneumonia, nephritis, meningitis, sepsis.et. ExPEC is one of the important pathogenic factor causing swine pneumonia and meningitis in newborns.Many secretory proteins and toxins of bacteria are translated into the host cell via secretion system, and then interact with the host cell playing its toxicity. So far at least six secretion system(T1SS-T6SS) were found, and T6SS is a new type of secretion system found in recent years, but the research of T6SS can go back over a decade. Through the analysis of the genomics found that25%of the sequenced of gram-negative bacteria contain gene coding T6SS, and its sequence is highly conserved. The gene cluster of T6SS is usually made up of12-25genes, partial function of some proteins has been confirmed, e.g.VgrG, Hcp and VAS et al. Nevertheless, the function of a majority of proteins are not clear.We discover the T6SS gene cluster in the virulent strain PCN033but not in the attenuated strain PCN061after the analysis of the genomics and comparative genomics. Indicating T6SS may play an important roal in the pathogenesis of PCN033. We choose the virulent strain PCN033separated from swine as parental bacteria, whose serotype is O11and belong to D group. On the basis mentioned above, we deleted the gene vca-0107of T6SS, and study its fuction and biological characteristics. The reaserch mainly contents as follows:1. Construction of recombination suicide plasmids According to the PCN033genome sequences, the two flanks of the gene vca0107were amplified and cloned from PCN033genome. The upstream and downstream of the gene vca0107were respectively subcloned into intermediate transfer plasmid pBluescript Ⅱ SK(+). After the verification of PCR and double digestion, the fragment connected of upstream and downstream homologous arm of the gene vca-0107was cloned into suicide plasmid pRE112which contained chloramphenicol resistance (Cm) gene.The recombination suicide plasmid pREAvca-0107contained501bp-deleted the gene vca-0107.2. Transconjugation and identification of the mutants PCN33Δvca0107The E.coil donor strain X7213transformed with recombinant plasmid PRE112Avca-0107, was conjugated with the recipient PCN033. After transconjugation, Chloramphenicol-resistant (CmR) transconjugants were analyzed for the presence of a first crossover event by PCR. This first step selected for clones in which the whole plasmid had been incorporated into the recipient chromosome. Colonies with the correct PCR profile were incubated in LB didn’t contained NaCl. Following this,The sensitive colonies of CmS were identified using PCR to determine the presence of the second crossover. Then we got the gene deleted strain PCN033Avca-0107.3. Biological characteristics of the mutantsThe OD600of the bacterial cultures was determined at intervals of1h. No obvious difference was observed in the in vitro growth curves of PCN033and mutant strain, indicating that deletion of the gene vca-0107has not significant influence on the growth of PCN033.Female KM mice were used to compare the pathogencity of PCN033and the mutant strain. The result showed the mutant PCN033Avca-0107has no change comparing with the parent PCN033. The adhesive ability to the cell PK-15and the phagocytic ability to the cell RAW are significant differences between PCN033Avca-0107and PCN033.