Studies on the Roles of mtr Efflux System and Pathogenicity Island of Neisseria Gonorrhoeae and Inhibition of the Two Gene Systems by External Guide Sequences
|School||Huazhong University of Science and Technology|
|Course||Dermatology and Venereology|
|Keywords||Neisseria gonorrhoeae Gonorrhea Drug tolerance Prokaryotic expression mtrC Hybridoma Monoclonal antibodies Multi- pass drug resistance Ciprofloxacin Exogenous guide sequence|
Part I The Relationship between the Multi-drug Resistant of Neisseria Gonorrhoeae and the Point Mutation of mtrR GeneObjective In order to investigate the relationship between the multi-drug resistant Neisseria gonorrhoeae and the point mutation of mtrR gene of mtrRCDE efflux system.Methods A total of 55 multi-drug resistant strains were obtained by the K-B and agar dilution methods. The chromosome DNA was extracted using little dose bacterial genome DNA extracting kit. The mtrR gene of Neisseria gonorrhoeae was examined by polymerase chain reaction （PCR）, and then their sequences were compared by softwares to find the difference between sensitive strains and resistant strains.Results The percentage of multi-drug resistant strain was 76. 36%（42/55）. There was no mtrR mutation in 4 sensitive and 2 penicillin-resistant strains. 10 multi-drug resistant strains mutated in three positions of mtrR gene, 6 strains mutated in the position 45 gly（GGC→GAC）asp, 3 strains mutated in the position 14 his（CAC→CGC）arg, 1 strain mutated in the position 51 phe（TTC→GTC） val . Conclusion The results indicated that the mutations in the position 45 gly（GGC→GAC）asp, 14 his（CAC→CGC）arg, 51 phe （TTC→GTC） val have a close relation with the multi-drug resistance of Neisseria gonorrhoeae. Part II Construction of Prokaryotic Expression Plasmid of MtrC Protein of Neisseria gonorrhoeae and ItsExpression in E.coliObjective To construct plasmid pET-28a （ + ） encoding MtrC gene of Neisseriagonorrhoeae and express protein in E. coli DE3. Provide a rational researchbasis for clinical detection and analysis of resistance mechanisms toantimicrobial hydrophobic agents.Methods Fragment of mtrC gene of Neisseria gonorrhoeae were amplifyied fromthe standard strains. Cloned the fragment into prokaryotic expressionplasmid pET-28a（+） with restriction endonuclease cut to construct recombineant pET-mtrC. Verified the recombinant with restriction endonuclease and DNAsequencing. The recombinant plasmid was transformed it into E. coli DE3 toexpress the protein mtrC after induced by IPTG.Results mtrC DNA fragment was proved correct through restrictionendonuclease and DNA sequencing . Its sequence was 99.5% the same as thesequence which was published on GeneBank （U14993） . A 48.5 kD fusion proteinwhich was induced by IPTG has been detected by SDS-PAGE.Conclusions Construction of prokaryotic expression plasmid of mtrCprotein of Neisseria gonorrhoeae was correct and the fusion protein wassuccessfully expressed in E. coli. The study provided a rational researchbasis for producing its monoclonal antibody and analysis of mechanisms ofmultiple transferable resistance efflux system. Part III Establishment and characterization of mice hybridoma cell line secreting anti mtrCmonoclonal antibody of Neisseria gonorrhoeaeObjective To develop and identify the monoclonal antibody against recombinant mtrC antigen, so as to provide Provide a rational research basis for analysis of quinolone resistance mechanisms and clinical detection. Methods Recombinant mtrC ptotein was used as antigen to immune Balb/c mice. Monoclonal antibodies against mtrC were prepared by normal hybridoma technology. The specificity and sensitivity of McAbs were studied using the sandwich method of double antibodies of ELISA and western blot. Results Two cell lines of hybridoma secreting constantly McAbs were obtained and named 2B3 and 4F7, which produce monoclonal antibodies specificity against mtrC. Chromosomes numbers 95—103. The McAbs were IgGl subclass. The titers of ascetic fluid were 1: 105 and 1: 106 respectively. Conclusions the method of western blot using our McAbs to detect mtrC protein of N. gonorrhoeae possessed high specificity and sensitivity. Part IV Effects of MtrC Monoclonal Antibody onCiprofloxacin Resistance Level of Neisseria GonorrhoeaeObjective To study the effects of of MtrC Monoclonal Antibody on Ciprofloxa cin Resistance Level of Neisseria Gonorrhoeae.Methods MtrC Ab、 NaN3 were used as inhibitors to mtr efflux pump protei ns , and the quantity of ciprofloxacin up take and accumulation in Neisseria gonorrhoeae were determined under three kinds of situation , with MtrC Ab、 with NaN3 and without inhibitors respectively. Results The quantity of ciprofloxacin uptake in Neisseria gonorrhoeae was limited , and the limitation of ciprofloxacin-susceptible and ciprofloxacin-resistant Neisseria gonorrhoeae were 70-120ng and 20-80ng , respectively. Without inhibitors, the quantity of ciprofloxacin accumulation in ciprofloxa cin-susceptible Neisseria gonorrhoeae was 93.7ng, which was significantly more than 37.6ng in ciprofloxacin-resistant Neisseria gonorrhoeae （P< 0.01）. After MtrC Ab was Added , the former was 92.3ng , no different from that without inhibitors（P>0.05）, but the latter increased to 75.3ng, obviously more than that without inhibitors（P<0.05）, and it was no different from that in ciprofloxain -susceptible Neisseria gonorrhoeae without inhibitors（P >0.05）. After NaN3 was added , the former was 94.8ng, no different from that without inhibitors （P>0.05）, but the latter increased to 77.6ng, obviously more than that without inhibitors（P<0.05）, and it was no different from that in ciprofloxacin-susceptible Neisseria gonorrhoeae without inhibitors（P >0.05）. There were no active efflux pump proteins among 6 strains ciprofloxa cin-susceptible Neisseria gonorrhoeae and among 2 strains low level cipro floxacin-resistant Neisseria gonorrhoeae. Mtr Efflux pump proteins were active among 4 strains high level ciprofloxacin-resistant Neisseria gonorr hoeae.Conclusions The efflux pump could actively tranfer ciprofloxacin out of Neisseria gonorrhoeae cytoplasm , the quantity of ciprofloxacin accumulation in cytoplasm reduced , which was an important factor of Neisseria gonorrhoeae high level resistance to ciprofloxacin. MtrC Ab、 NaN3 could recover the susceptiveness to ciprofloxacin by inhibiting the function of mtr efflux pump of Neisseria gonorrhoeae.Part V Conversion of Clinical Multiple Drug-resistant Isolates of Neisseria gonorrhoeae into Drug-sensitive Ones by Using EGS TechniqueObjective To explore the possibility of converting the multiple drug-resistant isolates of Neisseria gonorrhoeae into drug-sensitive ones by employing external guide sequence （EGS） technique in vitro. Methods Recombinant plasmids with EGS-MtrC （an important protein of multi ple transferable resistance efflux system）and kanamycin drug resistant gene, named PET-28a（+）-EGS-MtrC, were constructed. Routine CaCl2 method was used to introduce the plasmids into multiple drug-resistant clinical isolates Neisseria gonorrhoeae. PCR was used to identify the bacteria and A600 was used to detect the growth rates in liquid and solid cultures containing ampicillin. The MIC of Neisseria gonorrhoeae was detected before and after conversion respectively.Results The multiple drug-resistant clinical isolates Neisseria gonorr hoeae grew well in Ampicillin containing （50μg/mL, 100μg/mL） culture whe reas the transformants with PET-28a（+）-EGS-MtrC failed to grow in the concen trations of 100μg/mL and could not grow well in the concentrations of 50 μ g/mL;Conclusions The plasmid PET-28a（+）-EGS-MtrC converted the Neisseria gonorr hoeae successfully. The sensitivity to the Ampicillin、 Azithromycin、 Crystal viole、 Eryrhromycin、 TritonX-100 and Tetracycline of transformants with PET-28a（+）-EGS-MtrC was partly reversed.Part VI Bioinformatical Analysis of Mtr Efflux SystemObjective To analysis the physical and chemical parameters、 hydrophobici ty、 transmembrane helices、 three-dimensional structure and function of multiple transferable resistance efflux system（protein MtrR、 MtrC、 MtrD、 MtrE） by employing methods of bioinformatics.Methods The sequences of genes were Z25796 （mtrR）、 AF037041（mtrC） 、 U60099（mtrD）、 X95635（mtrE） and The sequences of proteins were CAA81045 （MtrR）, AAA80193 （MtrC）、 AAC45560 （MtrD）, CAA64891 （MtrE） . The parameters include the molecular weight, theoretical pI、 amino acid composition、 atomic composition, extinction coefficient、 estimated half-life、 instability index、 aliphatic index and grand average of hydropathicity （GRAVY） were calculated by employing Protparam; hydrophobicity of the proteins were analyzed by employing ProtScale; transmembrane helices of the proteins were analyzed by employing TMpred; three-dimensional structure of the proteins were analyzed by employing SWISS-MODELand function of the proteins were predicted by employing AmiGO.Results Obtained the parameters include the molecular weight、 theoretical pI、 amino acid composition、 atomic composition、 extinction coefficient, estimated half-life、 instability index 、 aliphatic index 、 GRAVY and hydrophobicity; transmembrane helices and three-dimensional structure of the proteins were predicted. The GO number of MtrR protein was 0006355 , MtrC was 0015559, MtrD was 0006810, MtrE was 0015562. Conclusions The physical and chemical parameters 、 hydrophobicity 、 transmembrane helices, three-dimensional structure and function of protein of Neisseria gonorrhoeae can be analyzed quickly and effectively by employing methods of Bioinformatics.Part VII Bioinformatical Analysis of PathogenicityIsland of Neisseria GonorrhoeaeObjective To analysis the physical and chemical parameters, hydrophobi city, transmembrane helices and three-dimensional structure of TraH、 TraG、 AtlA by employing methods of bioinformatics. Analysis the sites of integration and GC content in GGI and genome of Neisseria gonorrhoeae. Methods The parameters include the molecular weight、 theoretical pI、 amino acid composition、 atomic composition、 extinction coefficient、 estimated half-life、 instability index、 aliphatic index and grand average of hydropa thicity （GRAVY） were calculated by employing Protparam; hydrophobicity of the proteins were analyzed by employing ProtScale; transmembrane helices of the proteins were analyzed by employing TMpred ; three-dimensional structure of the proteins were analyzed by employing SWISS-MODEL. The sites of integration and GC content in GGI and genome of Neisseria gonorrhoeae were analyzed by employing Islander and IslandPath respectively. Results Obtained the parameters include the molecular weight、 theoretical pI、 amino acid composition, atomic composition, extinction coefficient, estimated half-life、 instability index、aliphatic index, GRAVY and hydrophob icity; transmembrane helices and three-dimensional structure of the proteins were predicted. GC content of GGI （44%） was lower than that of genome of Neisseria gonorrhoeae（52%）. Gene of site specific intergrase XerCD was found at dif site which located at the end of GGI.Conclusions The physical and chemical parameters, hydrophobicity、 transmem brane helices, three-dimensional structure and problems of comparative genomics can be analyzed quickly and effectively by employing methods of Bioinformatics. Part VIII Effect of Inhibiting traH Gene of NeisseriaGonorrhoeae by External Guide Sequence to the Function of Type IV Secretion System Objective To study the effect of inhibiting traH gene of Neisseria gonorr hoeae by employing external guide sequence （EGS） technique in vitro to the function of type IV secretion system.Methods Recombinant plasmids with EGS-traH （an important gene which codes the type IV secretion system of Neisseria gonorrhoeae） and kanamycin drug resistant gene, named PET-28a（+）-EGS-traH, were constructed. Routine CaCl2 method was used to introduce the plasmids into Neisseria gonorrhoeae MS11. PCR was used to identify the bacteria and whether the bacteria contain the GGI. The function of type IV secretion system was detected by Live/Dead staining and DNA secretion assays before and after conversion respectively. Results The transformants with PET-28a（+）-EGS- traH were confirmed. GGI existed in Neisseria gonorrhoeae MS11.The viability and the function of secreting DNA were decreased after inhibiting traH gene by employing EGS technique.Conclusions The plasmid PET-28a（+）-EGS- traH converted the Neisseria gonorrhoeae successfully. GGI existed only in Neisseria gonorrhoeae MS11. The viability and the function of secreting DNA were decreased after inhibiting traH gene.