| Enterococci is an important member of commensal flora in human and animals’ gastrointestinal tracts,but it was proved to be pathogenic both to human and animal. Many virulence factors are involed in the pathogenicity, besides enterococci’s extensive drug resistance. During quantitive of proved virulence factors, a serise of surface proteins were speculated to participate to the adhesion and colonization to host cells. Surface protein gene EF3183 was chosen by a previous study. A gene mutant was constructed with Enterococcus faecalis XJ11 as a parent strain which can cause encephalitis in lambs. The role of surface protein EF3183 during adhsion to He La cell, infection to raw264.7, infection to murine and capacity to form biofilm were investigated. Results showed that the capacity of E. faecalis XJ11 to adhere He La and infect raw264.7 were both stronger that its mutant. And the bacterial loads of E. faecalis XJ11 in liver, heart,kidney and spleen were outnumber than that of E. faecalis XJ11-Δ3183. The OD595 of biofilm formed by E. faecalis XJ11 were much higher and its structure were more complicated than biofilm formed by its mutant.The main research contents and results are as follows:1. Detection of surface protein genes among E. faecalis which can cause encephalitis in lambs: Specific primers were designed based on sequenced genome of E. faecalis XJ05 which can cause encephalitis in lambs. Distribution of surface protein genes EF1092, EF1269, EF2224, EF2505 and EF3183 among 11 E.faecalis which can cause encephalitis in lambs were tested by PCR. Results showed that the five surface protein were commonly detected among the 11 E. faecalis. EF2224 wasn’t detected in XJ09, but all the five surface proteins were detected in the other 10 E. faecalis.2. Cloning and bioinformatics analysis of EF3183 gene of E. faecalis which can cause encephalitis in lambs:The EF3183 gene of E. faecalis XJ11 was amplified by PCR, and sequencing and bioinformatics analysis were then conducted to investigate the structure and function of EF3183 protein. The function of this protein was predicted by bioinformatics software and the phylogenetic tree of EF3183 gene was obtained.Bioinformatics analysis showed that the sequence contained the complete coding region, which was 1056 bp, encoding 351 aa. The protein had no signal peptide, 2 transmembrane regions and 15 antigenic determinants were predicted by online analysis. The phylogenetic tree showed that the evolution distance of EF3183 gene was homogeneous to E. faecalis v583.3. The construction of E. faecalis-Δ3183: Amplifying the fragment which was used to disrupt the EF3183 gene with PCR, digested the amplified fragment and then reclaim and connected it to the shuttle vector p TX4577. Transform E. faecalis XJ11 competent cells with the recombination plasmid by electroporation after enzyme identification. Screening recombination with kanamycin and identify it by PCR. The genetic stability was proved by serially subculture. The results showed that the mutation was construted.4. The role of EF3183 in the adherence to HeLa cell, raw264.7 survival test and bacteria loaded in murine organs: Joining bacteria to He La cells and coincubated in 1 h and 2 h. Lysing cells with 0.1 % Triton-100.Viable bacteria were quantified by plating aliquots of serial dilutions on BHI agar; Joining bacteria into RAW264.7 cells, Mycillin was added and incubated in 4 h and 24 h, cells was lysed. Viable bacteria were quantified by plating aliquots of serial dilutions on BHI agar. Cultured bacteria were inoculated into mice,mice were dissected at different times and liver, kidney, spleen, and heart were harvested. The organs were lysed in a Tissuelyser. Viable bacteria were quantified by plating aliquots of serial dilutions on BHI agar.During the adhesion of E. faecalis XJ11 to He La, anti-EF3183 protein was added comparing to E. faecalis XJ11 without anti-EF3183 protein. Results showed a decreased adherence to He La cells, with a significant difference(p < 0.001). When anti-EF3183 protein was added, bacteria adhered to He La decreased with a significant difference(p < 0.05). Less mutant were survived in RAW264.7 cells compared to the wild type with no significant difference(p > 0.05). And the detected bacteria were reduced in liver, spleen and kidney(p < 0.001) and heart(p < 0.05), when compared to the wild type.5. The effect on biofilm formation of E. faecalis XJ11-Δ3183: Incubating E.faecalis XJ11-Δ3183 and E.faecalis XJ11 in 96-well plates under the same conditions at 37 ℃ for 8 h, 18 h,24 h,36 h and 48 h.Using the microplate reader determined the OD595 after the biofilms were stained with 1 % crystal violet.Incubating mutant and wild strain on coupons in 6-well plate at 37 ℃. Plates were washed with PBS and stained with FITC-con A and PI respectively. Observe the biofilms with confocal laser scanning microscope(CLSM) in each time point. Results showed that the OD595 of wild type biofilms were higher than the mutant biofilms. The biofilms formed by E. faecalis XJ11 was much excessive and intensive, and the distribution of embedded bacteria presented a regional property compared to E. faecalis XJ11-Δ3183viewed by CLSM. And E. faecalis XJ11-Δ3183 lost the ability to form biofilm at gas-liquid interface... |
PDF Full Text Request