Research On Function Of Quorum Sensing LuxS Gene In F18-fimbriaed E. Coli

F18-fimbriaed E. coli is the main pathogen of edema disease (ED) and post-weaning diarrhea (PWD) in piglets. ED and PWD are important diseases which cause significant economic losses in swine industry. The virulence factors of E. coli are related closely with each other and damage the host cells together. Virulence factors are strictly regulated, and Quorum Sensing system is involved in the regulation process. In this study, E. coli107/86strain was chosen as the reference strain.In growth of bacteria, the bacteria can utilize Quorum Sensing system to monitor cell population density in surrounding environment, therefore to regulate gene expression. This regulation mechanism is called Quorum Sensing, while the signaling molecules in Quorum Sensing are known as autoinducer. In Quorum Sensing Ⅱ system, luxS and pfs play significant roles in the synthesis of AI-2. To determine whether the QS Ⅱ system exists in107/86strain or not, two primer pairs were designed based on the sequences of luxS and pfs genes of E. coli K12deposited in GenBank, and PCR amplication technique was used to get the two genes from107/86strain. The sequences of luxS and pfs are highly homologous with genes from K12strain, which means QSⅡ system exists in107/86strain. AI-2molecules can induce bioluminescence reaction in report strain BB170, thus researchers employ this method to measure expression level of AI-2. This method was employed to detect that107/86supernatant can induce bioluminescence, while DH5a supernatant can not induce the same reaction. The results proved that107/86strain has QS II system, and DH5a does not have the ability to synthesize AI-2. To further verify the significance of luxS gene in synthesis of AI-2, we transformed luxS-complementary plasmid into QS II negative strain DH5a, then we found DH5a obtain the capacity to induce BB170bioluminescence and AI-2synthesis. At pH7.5and37℃,107/86strain possesses highest expression level of AI-2, the higher or lower changes of pH and temperature would result in lower expression level of AI-2.0.5%glucose added into LB medium led to two-fold increase of AI-2expression, while added0.5%NaCl and0.5%sucrose can not get the same result. AI-2levels increase rapidly in exponential phase for107/86strain, but decrease dramatically in stationary phase. LuxS protein sequences from E. coli, Salmonella, Yersinia, Pasteurella multocida and so on were compared and analyzed, and a high homology in LuxS is found between these species. This high homology may explain that Quorum Sensing Ⅱ system could be an intra-species regulation mechanism, which means AI-2secreted by one species can regulate other species.Based on the original sequences of luxS gene in the previous study, the wild type E. coli107/86was selected to construct the luxS mutant with Red recombination system. First, we construct a pair of PCR primers-5’end of each primer has homologous sequences with the luxS gene of wild type, while3’end of each primer has homologous sequences with cat gene, which is the chloramphenicol resistant gene existed in pKD3plasmid. Therefore the PCR product which has chloramphenicol resistance and homologous sequences was obtained. The PCR product was transformed into107/86, which can generate three kinds of homologous enzymes with the help of pKD46plasmid. After the homologous recombination reaction between homologous sequences of PCR product and wild type107/86, we can select the positive strains according to their Ampicillin and Chloramphenicol resistance. In order to induce the FRT site mutate reaction, then we transform the pCP20plasmid into the first-mutant-strain, to delete the cat gene and generate the second-mutate strain. After PCR amplification and sequencing steps, we can confirm that the107/86△luxS strain had been constructed correctly. This mutant provides a basis for the further study about the molecular mechanisms of relationship between Quorum Sensing system and virulence factor of bacteria, and pathogenesis mechanisms.To further study, the regulation of Quorum Sensing system to virulence factors of E. coli, luxS was cloned into pBR322plasmid and pBR-luxS complementary plasmid was constructed. Then luxS-complementary plasmid was transformed into luxS mutant107/86△luxS. After constructing complementary strain, we use wild type, deletion mutant, complementary strain as a whole system to study luxS function. Supernatant of complementary strain can induce bioluminescence of BB170, which verifies that complementary strain restored the AI-2activity. In the growth curve, we found that wild type strain grows a little faster in exponential phase compared with deletion mutant and complementary strain. The results of routine biochemical tests, such as IMVIC, glucose fermentation experiment, sucrose fermentation experiment, arabinose fermentation experiment and so on, between wild type, deletion mutant, complemented strain have few differences. F18-fimbriaed E. coli use F18fimbriae to adhere to intestinal epithelial cells, then colonize and multiply there. So F18fimbriae is primary virulence factor in F18-fimbriaed E. coli. Data shows adhesion of mutant strain to porcine small intestinal epithelial cell line decreases37%compared with wild type and complementary strain. Stx2e is an important virulence factor in F18-fimbriaed E. coli, which reacts with Gb5receptor in intestinal epithelial cells then induce cell pathogenic effect. Vero cell is specifically sensitive to Stx2e. In this study, crystal violet cytotoxicity assay was used to quantify survival Vero cells after adding Stx2e produced by three strains into Vero cells. In this test, the mutant team has20%more survival Vero cells compared with wild type and complementary strain. Outer membrane protein locates in the outer membrane surface of gram-negative bacteria, and plays important role in maintenance of physiological functions. In this research, we extracted OMP from three strains through Triton X-114method. Although the difference of SDS-PAGE bands between OMP from three strains is hard to find, after BCA quantification method, we found production of OMP from mutant strain is only two thirds compared with OMP from wild type and complementary strain.