Function Of The Pathopoiesia About Outer Membrane Protein T Of Uropathogenic Escherichia Coli

BackgroundInfectious disease caused by Bacteria, viruses, fungi and parasitic pathogens is still human death and disability, new cases caused more than17million around the world each year.Urinary tract infection is the most common clinical bacterial infections. Susceptibility to women than men,about81%of UTIs happened in women,aged from16to35,30years old of women who have a third-a quarter have acute urinary tract infection of the history, and27%of patients in the six months again,48%recurrence in a year.Second,the elderly and children is also suffer UTIs.Urinary infection according to anatomical different divided into the lower urinary tract infection and upstream, the former includes urethritis and cystitis, the latter refers to the pyelonephritis.Pyelonephritis is the most serious urinary infection of the form, when more than six months or a year history have turned to the possibility of chronic pyelonephritis, and chronic pyelonephritis chronic renal failure is one of the most common cause.Urinary pathogenic Escherichia coli cause UTIs accounted for80%-90%.Most of the urinary tract pathogen originated in the intestine, and follow runs to the urinary bladder, about95%of the infected site is starting UTIs bladder.At present the main point of research is around the relationship between UPEC with host.UPEC same with other pathogenicity of escherichia coli,chromosome is poisonous force factor information. UPEC poisonous force factors include:alpha hemolysin, CNF1; On one hand, the bacteria through the poisonous force factor in the host to survive in the breed of purpose; On the other hand, the host using the body’s immune system, defense against bacteria, produce inflammation response; In constant interaction process, bacteria produce some enzymes from the defense system against the host. For example, the experiment that UPEC virulence factors can be hydrolysis ompT inherent immune components people defense element, thus bacteria in the host body live provide conditions.In the urinary tract infection, UPEC urothelial cells of the host of adhesion ability is the most important factor of pathogenic, capsule, lipopolysaccharide adhesion, and some other organs and adhesion, expression is often the key to disease, the help UPEC integrated with urinary and avoid being urine scour cleared. The experimental research shows that:ompT gene knockout, can lead to FimH expression;FimH is a proven adhesion element, and the results demonstrate that the body and in vitro, ompT knock out can make UPEC adhesion rate, and the experimental results are consistent. In the same way, compared to other poisonous force ompT regulation, also in this paper is discussed. These virulence genes function and adhesion, invade, serum resistance and metabolic and related, and confirmed by experiment. About ompT regulate these poisonous force of the factors related mechanism is not confirmed by experimentThis study used RED the homologous recombination of urinary system construction of CFT073pathogenic Escherichia coli ompT knockout strains, investigation ompT missing the genes of plant disease resistance, the influence of people defense hydrolysis. And through the reference and predict get, ompT through the effect some poisonous force of precursor protein components in production and urinary tract infection in action. And through the fluorescence analysis ompT genes on quantitative PCR main poisonous force of protein expression whether have influence. Confirm ompT missing the genes of bacteria in the body is the survivability effect. The purpose of this topic is preliminary studies gene ompT urinary pathogenic escherichia coli in the pathogenesis as role.Methods and Results1. Expression of HDB-4For the further study in OmpT urinary infection pathogenesis the role of expression of3cloning people defense HBD-4simulation model in vitro infection. This experiment with e. coli bladder cancer for total RNA template, PCR amplification HBD-four genes, links to pET28a, constructing the expression vector pET28a-HBD-4. Will be restructured plasmid transform BL21(DE3), the IPTG induction HBD-4and mutants inclusion body of expression in form. purified protein the gradient method of dialysis resilience, and join the crude lipopolysaccharide (lipopolysaccharide, LPS) resume protein activity. Use OmpT can hydrolysis substrate continuous basic amino acids characteristics, through sds-page, count and the growth curve method colonies observed OmpT success hydrolysis HBD-4and beneficial to bacteria live.2. Will logarithm growth period of escherichia coli cft073, cft073enables delta ompT and ompT/pUCT (CFU:1×106) vaccination in40LB edged medium, add10μl defense grain protein HBD-4incubation, make each tube protein concentration of the end for200μg/ml.37℃things will train incubation8h, removed a hour in the600nm determine the pipe in the absorbency. Will the tube diluted106times evenly coated in LB solid culture medium,37℃after incubation overnight colony count. Each concentration of vice tube. 3people defense element HBD-4the antibacterial activity detection Because the bacterium hair type I FimH to prove the adhesion of element, so using fluorescence quantitative PCR CFT073observation and CFT073enables delta ompT FimH in expression. CFT073and CFT073enables delta ompT training for the night, centrifugal and TRIzol cracking, organic solvent extraction) total RNA, reverse transcriptase; Fluorescent dye quantitative calculation method of PCR two bacteria in fimH gene expression of the volume.4CFT073and CFT073enables delta ompT training for the night, centrifugal and TRIzol cracking, organic solvent extraction) total RNA, reverse transcriptase; Ordinary PCR, gel electrophoresis, the calculation of two groups of bacteria grey value, the calculation of two kinds of bacteria ompA,hly, CNF1respectively of the expression.Result1.Expression of HDB-4We have successfully constructed the expression vector pET28a-of HBD-4, after transforming it into engineering BL21,we successfully expression, purification and refolding of HBD-4.2.he inhibitory effect of Defensin to CFT073、CFT073ΔompT and ompT/pUCTWe observede colony counts after diluted the bacteria1×106times, the CFT073group get an average of79colony forming units, the CFT073Δ ompT group get an average of5and ompT/pUCT group get an average of232;Colony forming unit of ompt knockout strains is Statistically significant less than wild strains and covering strains.Observed the growth curve,we can see that OD value is smaller at wavelength of600,this phenomenon shows that defensin can strong inhibit the CFT073ΔompT; Compared with CFT073ΔompT, the growth of CFT073is not affected, ompT/pUCT although growth was affected, but overall still showed proliferation.3.resulte of time fluorescence quantitative PCRThe relative fluorescence quantitative shows that compared with knockout strains the wild strains is256times in expression of fimH gene,then we analysis the optical density value after gel electrophoresis respectively, after statistical analysis,there are significant differences between the wild strains and knockout strains, t=7.998,P<0.05.In addition,the relative fluorescence quantitative shows no differerce between wild strains and knockout strains in reference gene copy number,this phenomenon indicates that wild strainsof fimH gene expression maybe stronger than the knockout strains, knockout gene ompt make the expression of fimH gene drop down.Ompt may affect the fimH gene expression.Discuss1After successfully expression purification and refolding of HBD-4,knock out genes ompt is more sensitive than wild strains and covering strains;OmpT can hydrolysis defensin,it may make the UPEC survival in the urinary tract.2The result of time fluorescence quantitative PCR shows that knockout gene ompt make the expression of fimH gene drop down.Ompt gene may through by affect the expression of FimH working in the adhesion.3Grayscale scanning analysis shows that ompT may not affect to the expression of the gene ompA,but to the Hly gene and CNF1gene.