Construction And Characterization Of Hcp Deleted Mutants Of Extraintestinal Pathogenic Escherichiacoli. Isolated From Swine

Type Ⅵ secretion system(T6SS) is a novel secretion system originally defined in Vibrio Choerae in 2006. T6 SS is widespread in Gram-negative proteobacteria consisting of 15-20 highly conserved core genes that assemble the apparatus, and can deliver effectors into target cell, including competitor bacteria or eukaryocytic cells. Owing to diversity of effectors, function of T6 SS distinct in different situation, T6 SS generally contributes to pathogenesis or bacterial competition in many pathogens. Hcp(Hemoysin-coregulated protein) and Vgr G(valine-glycine repeat protein G) are important components of T6 SS which show significantly homology to T4 bacteriophage tail tube. In T6 SS active pathogens, Hcp and Vgr G can be detected in supernatant, thus Hcp becomes hallmark of a functional T6 SS. It is convinced that individual Hcp can form homohexameric ring, crystal structure and transmission electron microscopy have shown that these hexameric rings are capable of stacking thus forming a nanotube that effector can bound to the inner surface thus Hcp can be secreted together with effector and keep it from degration. On the other hand, Hcp is considered as a secretion protein with multiple functions in different bacteria. In conclusion, Hcp is not only an important ingredient of T6 SS but also can be an effector or molecular chaperone.Pathogenic Escherichia coli(E coli.), an important opportunistic pathogen, has great impact on stock farming, the study on E coli. becomes a hotspot, especially extraintestinal pathogenic Escherichia coli(Ex PEC). In Ex PEC, research on T6 SS mainly focus on neonatal meningitis Escherichia coli(NMEC) strain RS218 as well as avain pathogenic Escherichia coli(APEC) strain SEPT362 and TW-XM, in those strains, T6 SS mostly influence pathogenicity. T6 SS in enteroaggregative Escherichia coli(EHEC) strain 042 involved in inter-bacteria competition. More interesting still that more than one integrated T6 SS exsits in bacteria chromosome or multiple copies of hcps and vgr Gs can be found in one T6 SS cluster in many cases, and they generally showed different functions.Our laboratory has sequenced Ex PEC strain PCN033 for whole genome, an integrated T6 SS has been identified. As with many other pathogenic Escherichia coli, three copies of hcps were encoded in the chromosome of Ex PEC PCN033, two of which were located in T6 SS cluster while the third one has a certain distance, named hcp1, hcp2, hcp3, resepactively. In this study, we set out to define the function of each hcp in PCN033. We constructed markerless deletion mutant of each hcp, including triple mutant and study on their biological characteristics comparing to parental strain, in order to determine their function in PCN033 bacterial competition and pathogenesis and made foundation of study on T6 SS in PCN033. Main contents of this study are as follows: 1. Construction of gene deletion mutants Δhcp1, Δhcp2, Δhcp1Δhcp2Δhcp3.Primers have been designed with reference to genome sequence of Ex PEC PCN033 strain(unpublished), hcp1, hcp2, hcp3 loci are PCN033-0227 、 PCN033-0245 、PCN033-4200, respectively. Sequence of upstream and downstream homology arms of hcp1, hcp2 were amplified from PCN033, respectively, digested then connected to suicide plasmid p RE112 to construct recombination plasmids. Gene deletion mutants have been constructed via homologous recombination. 2. Preparation and identification of polyclonal antibody against Hcp2, Hcp3.Primers have been designed by sequence of hcp2 and hcp3. Amplification product were connected to p ET30 a and the recombination plasmids were transformed into host strain E coli./BL21. The fusion protein Hcp2-His and Hcp3-His were purified through nickel ion affinity chromatography column. Purified protein were injected into rabbits hypodermically on neck and back with Freund’s complete adjuvant, followed by mixture with Freund’s incomplete adjuvant for the antiserum production. Specificity of the antibody were identified by western blot. 3. Bacterial competition assayGrowth competition assay was performed using PCN033 and its derivatives as predators, using the E coli. K12 strain W3110 as prey as described. After co-cultured with predators for 12 hours at 30℃, CFU of prey strain have been counted to calculate survival rate. Comparing to parental strain PCN33, Δhcp1Δhcp2Δhcp3 showed most obvious increase of W3110 survival rate, and Δhcp2 showed significant increase, indicating that hcp2 may play important role in bacterial competition. 4. Pathogenesis of mutant and parental strainsInteraction with cells was conducted to analysis their abilities of intracellular survival after phagocytosed as well as abilities of adhere and invase. On the one hand, in the porcine pulmonary alveolar macrophages(PAM) intracellular survival experiment, comparing to parental strain PCN033, Δhcp1Δhcp2Δhcp3 showed most obvious decrease in survival rate as hcp1 maybe more involved in this process. On the other hand, in the pig kidney cell line(PK-15) adherence and invasion assay, comparing to PCN033, Δhcp1Δhcp2Δhcp3 showed distinct decline in capability of incasion while adherence capability has no evident change, indicating that hcp play roles synergetically on intracellular survival and invasion abilities when interacting with host cells.Mutant and parental strains were intraperitoneal injected 4 week-old female Kuming mices in three gradient of 105, 106, 107 CFU individually. Mortality rate of each strain seems to be no distinct different. C57BL/6 mice were inoculated intravenous(i.v.) in a lateral tail vein with an infection dose of 107 CFU of bacteria individually. Reproduction ability of mutants showed significant decrease in blood comparing with parental strain and capabilities in colonization in organs were also weakened distinctly.