| Type Ⅵ secretion system (T6SS) is a kind of protein secretion system found recently in pathogens of Gram-negative proteobacteria, of which it is an important virulence factors. The proteins encoded by15-25genes forms a transmembrane composite structure throughout the "inner membrane-periplasmic space-outer membrane" structure of bacteria, and its tube can directly passes through the envelop of target cells and then exerts biological effect. The target cells can be prokaryotic cells or eukaryotic cells. There are13core components which are conserved proteins among all of the T6S components playing key roles. One of them is Hemolysin-Coregulated Protein (Hcp), which is the structure protein forming hollow passage of the T6S equipment, and also is an important effecter playing biological effect to target cells, so that it is considered as hallmark of functional T6SSs.Extraintestinal pathogenic Escherichia coli (ExPEC) is an important class of zoonotic infectious pathogen, including urethra pathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), avian pathogenic E. coli (APEC) et. al. The porcine ExPEC is a class of ExPEC causing lesions of variety of tissues or organs other than intestinal, such as meningitis, pneumonia, arthritis, sepsis, et.al. Moreover, the strains in pigs have strong residence ability, bringing to farms big problems in disease control and huge economic losses. The ExPEC strains have some special virulence factors. Some of them was identified possessing T6SS, which are reported having close relationship with pathogenesis.Here based on the analysis in comparative genomics of5porcine ExPEC, it was found that there is integral T6SS cluster containing all core components in chromatin of the high pathogenicity strains, and there are3hcp genes (hcp1-3) in the T6SS, so that it is predicted to have relevance to pathogenicity of this strain. Therefore this study was carried out by construction a mutant and to study its effection to phagocytic cells and epithelial cells, and its secreted protein, compared with its parental strain. The main research was described as follows:1. Construction of deletion mutant PCN033Ahcp3of porcine ExPECAccording to the gene sketch sequence of the porcine ExPEC strain PCN033, the two flanks of hcp3gene were amplified and cloned from PCN033genome, the upstream and downsteam of hcp3gene were subcloned into the suicide plasmid pRE112respectively. The recombination suicide plasmid was constructed and named pRE△hcp3, which was transformed into the E. coil donor strain X7213, then conjugated with the recipient PCN033. Chloramphenicol-resistant(Cmr) and sucrose-sensitive transconjugants were screened out by observation for colony morphology, and the correct colonies were incubated in LB contained no NaCl in order to facilitate the second crossover. The Chloramphenicol sensitivity(Cms) phenotype colonies were identified by PCR to confirm the loss of plasmid, the hcp3gene was deleted. The deletion mutant was named PCN033△hcp3.2. Preparation and characterization of the Hcpl polyclonal antibody to porcine ExPECAccording to the gene sketch sequence of PCN033, hcpl gene were amplified and cloned from PCN033genome, then subcloned into the prokaryotic expression plasmid. The recombinant plasmids were transformed into host strain E. coli BL21. The fusion protein His-Hcp1was expressed induced by IPTG, then was purified through nickel ion affinity chromatography column. The purified product as immunogen was inoculated subcutaneously into2months white rabbit. The second immunity was carried out2weeks later, and then after a week blood was draw off from ear vein. Reaction and specificity of the Hcp1polyclonal antibody were identified using western blot.3. Extraction and identification of secretion protein of porcine ExPECThe reactived stains PCN033Ahcp3and PCNO33were inoculated into M9medium, and were shaked24h in4℃, then the supernatant was remained to extraction secretion protein using trichloroacetic acid, It was identified existence of Hcp by western blot with Hcpl polyclonal antibody in result of the T6S equipment is functional. The different content of secreted Hcp1of these two strains was compared to find out the decrease of Hcpl secreted in the mutunt which hcp3was deleted.4. Virulence of the deletion mutant strain of porcine ExPECPCN033Ahcp3and PCN033were respectively inoculated4weeks female BalB/C mice intraperitoneally in106,107,108three gradient of the amount of bacteria to test virulence to mice, the result shows there was no different in virulence to mice between the two strains. It was spended3h for bacterial strains and mouse alveolar macrophages (RAW) co-incubating in CO2incubator at37℃, The ratio of cfu of bacterial strains and acoumt of cells is10:1. Then the number of strains survived in RAW were counted, and result showes that in mouse alveolar macrophages the replication ability of the mutant were consistent with that of the parental strain. However, in porcine alveolar macrophages (PAM)the replication ability of the mutant were decrease compared with the parental strain, especially at3h, the number of PCN033and PCN033Ahcp3survived in PAM respectively is3560cfu/mL and833cfu/mL, the ratio is4.27. And PCN033和PCN033Ahcp3were co-incubated with porcine kidney epithelial cellsthe (PK-15) in CO2incubator at37℃, after3h, the number of the two strains invaded into cells respectived is 1427cfu/mL and1317cfu/mL, and the number of them adhered to surface of cells respectived is5840cfu/mL and1256cfu/mL. The result shows deletion of hcp3gene presents decrease in its ability to adhesion of the pig kidneys epithelial cells, rather than the ability to invasion. |
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