The Role Of Omp T In Pathogenesis Of Uropathogenic Escherichia Coli

1. Background and objectiveUrinary tract infection(UTIs) is one of the most common bacterial infection in the world, lead to serious financial and medical burden. For the unique anatomic feature, female is more susceptible to UTIs. About 60% women suffer UTIs in their lifetime, and 27% primary infection victims will experience recurrence in half year, 48% in one year. Antibiotics is the main therapy of UTIs.The pathogenic agents of UTIs including Klebsiella spp, Enterobacter spp, Staphylococcus aureus, coagulase-negative Staphylococcus, Enterococci, and strains of uropathogenic Escherichia coli. Most of them originate from intestinal tract, go through urinary arrive at bladder. Among them, Uropathogenic Escherichia Coli (UPEC) account for about 90% in community infections and 50% in hospital acquired infections. The pathogens bind to and invade urothelial cells, followed by the formation of intracellular bacterial community (IBC) which are related to the pathogenic processes of UPEC in UTIs. Analysis of epidemiology, cell culture and animal experiment indicate that the virulence factors of UPEC including a-haemolysin, cytotoxic necrotizing factor-1 (CNF1), siderophores and adhesive organelles like typeⅠ, P, S, and F1C pilus allow it colonizes host cell, obtains nutrition they needed and escape from host defense mechanism. In these virulence factors, typeⅠpilus is critical that it conducts UPEC adherence and invasion to bladder epithelia cells which is the principal pathogenesis of UPEC. In this study, ompT gene deletion (COTD) was constructed and in vitro adhesion and invasion assays, in vivo murine experiment were performed to investigate the role of ompT in pathogenesis of UPEC. We demonstrates that ompT is necessary for the pathogenesis of UPEC. OmpT and its point mutant were also expressed, refolded and charactered to observe the ability of the purified protein to hydrolyze protamine, and the methods that expressed, refolded OmpT and its point mutant also lay the basis on further study on the role of OmpT in pathogenesis of UPEC.2. Methods(1) Red homologous recombinationAccording to corresponding sequence in NCBI, using software primer 5.0 to design the primers of linear DNA fragment including 50 bp extensions that homologous to regions adjacent to ompT and kanamycin resistance gene that use to replace the location of gene ompT. The linear DNA fragment was amplified from pET28a by PCR with OmpT_Kan_F2 and OmpT_Kan_R2(1.2.11). Then the linear DNA fragment was transformed to CFT073/pKD46 which expressedλRed homologous recombination system by electrotransformation. The mutant was screen by LB agar plate supplemented with Kanamycin(50μg/mL). To verified whether the colony was the mutant, Using wild-type CFT073 and the colony as templates, PCR with primers OmpT-2F, OmpT-2R(1.2.16) which designed at 5'and 3'Homology extensions and OmpT_F1, OmpT_R1 (1.2.16) which designed inner ORF of gene ompT respectively. If the colony was the mutant,2066 bp length PCR products would be obtained with primers OmpT-2F, OmpT-2R and there would be no 567 bp PCR products with OmpT_F1, OmpT_R1.(2) The growth curves and colonies formation of CFT073与COTDFor the growth curves, single colonies were picked and cultured overnight statically with fresh LB medium, After the first overnight growth in LB, subcultured the bacteria into fresh LB medium culturing at 160 r/min in 37℃. The samples were obtained at certain time to detect with OD600. For colony formation, the bacteria were subcultured into fresh LB medium of the ratio 1:100 and cultured overnight at 37℃, and carried out a tenfold serial dilution.plated the 10-6 and 10-7 dilution on LB agars to cuture overnight.(3) Adhesion and invasion assaysFor adhesion assays, cultured 5637 human bladder epithelial cells confluent monolayers in 24-well plates were incubated with CFT073, COTD and COTD(pST) (approximately 105 cells/wel) respectively for 2 h at 37℃. The cells were washed five times with PBS.and to lyse cells by 0.5% triton X-100. The bacteria were obtained and plating them on LB agar to enumerated the bacteria number as the amount of bacteria adhere to 5637 and the percentage of total number bateria added in each well was the adhesion frequencies.For invasion assays, CFT073, COTD and COTD(pST) were incubated with 5637 cell monolayers at the ration of 100:1 for 2 h at 37℃. The cells were washed three times with PBS. Each well was treated with gentamicin (final concentration was 200 ug/mL) for 1 h to kill the extracellular bacteria. Wash the cells three times with PBS then lyse cells and obtain the amount of invasion bacteria as adhesion assay described. Invasion frequencies were calculated as the number of invasion bacteria divided by the total number bateria added in.(4) In vitro hemagglutination assaysThe erythrocyte were obtain from heart of guinea pig. The cell pellet was wash with PBS and resuspend with PBS to 2% concentration. Doubling dilution the bacteria in 96 V-type pore plate and and the 2% erythrocyte were added to each pore, incubated in room temperature. To observe the result after 2 h.(5) Murine-model of urinary tract infectionThe bacterial internalization and formation of IBCs of the mutant COTD were examed by murine model of urinary tract infection.7 weeks old female C57B/L6 mice which susceptibility to UPEC were used in our study. After anesthesia by 0.6% pentobarbital sodium, Intravenous needle was use as a urinary catheter to through urethra arrive at bladder, the syringe was insert to the catheter, push the syringe plunger to inject a 50μL inoculum of-107 bacteria to the bladder in 40 s.After 6 h,bladders were harvested aseptically and wash with PBS, incubated with PBS containing gentamicin(final concentration was 200μg/mL) for 1 h to kill the extracellular bacteria, wash three times with PBS and homogenized in 0.8 mL PBS, dilution and plating them onto LB agar to recover the intracellular bacteria.At the same time, to exam the imformation of IBCs, the bladders were harvested and fixed with 10% neutral buffered formalin solution(NBF) at least 8 h at room temperature. Bisect the bladder longitudinally, proceed paraffin embeding and hematoxylin-eosin(H&E) staining.(6) Expression, refolding and characterization of Escherichia coli outer membrane T and its mutantFirst, we amplified gene ompT from E. coli geneome by PCR and inserted it into expression vector pET28a to construct the recombinant plasmid pET-ompT. And the OmpT mutant was constructed from pET-ompT by site-directed mutagenesis. To express the protein, the two recombinant plasmid was transformed into BL21(DE3) and induced with IPTG, large scale inclusion bodies of OmpT and the mutant were obtained and purified, then refolded them by N-Dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate.The addition of Lipopolysaccharide(LPS) was critical to recover OmpT enzymatic activity. According the ability to hydrolyze protamine and RMCK, we identify the function of OmpT and the mutant by microscope and SDS-PAGE.(7) Statistical analysisThe data of colonies formation were analyzed by Independent-Sample T Test. The data of adhesion, invasion assays, HA and murine model of urinary tract infection were heterogeneity ofvariance, all analyzed by K Independent Samples Test.3. Results (1) Isogenic deletion of ompT gene in CFT073The linear DNA fragment was constructed from pET28a by PCR with OmpT_Kan_F2 and OmpT_Kan_R2, the fragment of 1602 bp was consistent with that expected. After transformed the DNA fragment into E. coli CFT073/pKD46, twelve colonies were screen on LB agar with kanamycin. Pick 14 colonies to verify and No.l,2,3,4,12,14 were consistent with the positive colony we expected. At last we performed BLAST and comfirm that we have constructed the isogenic ompT deletion in CFT073 successfully.And the CFT073 and COTD have no difference in their growth curves, the colonies formation also have no notable difference.(2) Isogenic deletion of ompT gene reduced the adhesion and invasion of CFT073 to 5637The mean rank of CFT073 adhesion rate was 38.00, that of COTD was 8.00, and that of COTD(pST) was 23.00. The mean rank of CFT073 invasion rate was 35.93, that of COTD was 8.00, and that of COTD(pST) was 25.07. All data analyse with SPSS 13.0, K Independent Samples Test and statistical significance(P<0.001).The mean antigen titer of CFT073, COTD and COTD(pST) in hemagglutination was 14.00. All hemagglutination can be suppressed by D-mannose. According to K Independent Samples Test, no significant differences in the data of these group, indicating that ompT gene couldn't influence the expression of type I pilus.(3) Isogenic deletion of ompT gene decreased in vivo invasion of CFT073 and suppressed the IBC formationThe mean rank of bacteria in bladder of mice was 19.89, that of COTD was 5.00, and that of COTD(pST) was 17.11. According to K Independent Samples Test, statistical significance (P<0.001). The data indicates ompT involve in the in vivo invasion of CFT073. The bladder slices with H & E staining showed that IBCs of COTD were suppressed when compared with that of CFT073 and COTD(pST). (4) Purified OmpT hydrolyzed protamine strongly and the mutant have no enzymatic activityOmpT and the mutant were expressed and refolded sucessfully. RMCK that incubated with OmpT were hydrolyzed to several fragment. The protamine that didn't incubate with OmpT had killed COTD and suppress its growth in 6 h, that incubated with OmpT didn't harm COTD. And the protamine incubated with OmpT point mutant also harm COTD.4. Conclusion(1) ompT gene might involve in adhesion, invasion and IBC process of UPEC, which couldn't by mean of affecting the expression of UPEC type I pilus.(2) The purified OmpT hydrolyzed protamine strongly and protected COTD from protamine.