Roles Of Uropathogenic Escherichia Coli Outer Membrane Protein T In Pathogenesis Of Urinary Tract Infection

Background and objectiveThe urinary tract is one of the most common sites of bacterial infection in humans. Lower urinary tract infections (UTIs), such as cystitis, are typically characterized by symptoms including frequency, urgency, and dysuria. If left untreated, these infections can progress to an upper UTI, known as acute pyelonephritis or kidney infection, which can be associated with additional symptoms such as fever, nausea, vomiting, and flank pain. These infections also carry the risk of possible progression to bacteremia. UTIs can be classified as uncomplicated or complicated. UTIs can occur at any age, the incidence rate of male is about0.3%,but after the age of65, because of prostate disease and urology surgery, the infection increased rapidly to13%to14%. Due to women’s special physiological structure, women are more susceptible than men, according to statistics, about81%of UTIs occur in women which aged between16-35years old,27%of women relapse within six months after the initial urinary tract infection, approximately48%of women recurrence UTIs within a year. UTIs are the most common clinical bacterial infections and antibiotics are the mainly treatment for clinical pathogens becoming more resistant, which makes it easy to repeate.Most urinary tract pathogens originated in the intestinal tract, there are about95%of UTIs, up to the bladder and urethra,which the initial site of infection is bladder. UTI is caused by a range of pathogens, with Uropathogenic Escherichia coli (UPEC) being the most common etiological agent.caused80%～90%of UTIs. Recently the research work is mainly focus on the UPEC. The vast majority of UPEC carries ompT gene,which encoding the outer membrane protein T (OmpT). OmpT has a strong activity in a denaturing solution of proteolytic enzymes,causing the expression of exogenous protein in severe degradation caused concern. The OmpT is a enzyme which is homologous with murine Pla of Yersinia Bacillus.The. Pla was confirmed that it could adhere to the extracellular matrix, activate plasminogen converting to plasmin, and promote the degradation of the extracellular matrix. Whether the OmpT have the same function? In this study,we compared the ablities of ompT wild strains CFT073and knockout strains COTD adhesion to ECM.Found that the wild-type strain CFT073has a strong ablity adhere to de ECM compared with knockout strains COTD.The results suggested that OmpT may reduce the colonization ability of pathogenic bacteria to the bladder tissue. Finally,we examine the expression of the adhesion and virulence factors among three types bacterial,by real-time PCR analysis.The urinary tract pathogenic E. coli CFT073ompT knockout strains COTD and compensation strains COTD(pST) were used in this study. Murine model of urinary tract infection was build. The Histological analysis of the bladder and kidney tissues of infected mice with Escherichia coli strains was taken to compare the change among three types of OmpT. ELISA method was used to detect the expression of IL-6, IL-8in urinary system organs and plasma.The purpose of this topic is to make clear what the role of ompT in the pathogenesis of urinary tract pathogenic E. coli.Methods1. The OmpT and ECM interact with each other on the bacteria adhesion.HBEC5637subcultured covered the bottom of the bottle use of antibiotic-free medium,then washed with sterile PBS three times,incubated with PBS contain0.4%Triton X-100and20mM NH4OH5min.Then incubated with100ug/mL DNase I and100ug/mL RNase A1in37℃, fixed with0.1%neutral buffered formalin solution(NBF)6h at room temperature, stored at4℃. Then we culture the HBEC to the dish for ahhesion test. Adhesion rate=number of bacterial adhesion to cells/total number of bacteria×100%. The basement membrane preparation was diluted1/25in ice-cold PBS, and250μL of the dilution was pipetted on membranes in Transwell3415culture chamber inserts. The plates were initially kept for2.5h at37℃. Then quenchined with0.1%BSA1h at room temperature. Bacteria (1×107～108cells in300μL) were added to the plates kept for3h in room temperature. The control group were250μL BSA(200μg/mL). After incubated3h, washed with0.1%BSA-PBS. Results were observed under a microscope.2. The OmpT affect the expression of Iha and IroN mRNA expression levels of the bacteria.Overnight-cultured E. coli strains were collected. The total RNA extraction with TRIzol(?).was carried out according to the vendor’s instruction manual. The purified total RNA was reverse transcripted with the ExScript RT reagent kit, according to the manufacturers instructions. The16s-rRNA was used as an internal control. Primers were synthesized and used for cDNA amplification of iha and iron.3. The role of OmpT in proinflammatory of urinary tract infection in mice.7weeks old female C57B/L6mice which susceptibility to UPEC were used in this study.After12h, bladders and kidneys were harvested aseptically and wash with PBS,4%neutral buffered formalin solution(NBF). After Dehydrated, embedded, sliced, analysis the histological of bladders and kidneys proceeding paraffin embed and hematoxylin-eosin(HE) stain. We aslo detected the bacteremia in blood and detected the burden of bacterium of the bladder and kidney tissue.4. The role of OmpT on the urinary tract infection in mice in vivo cytokine expression.After12h, bladders and kidneys of mice were harvested aseptically and washed with PBS,4%neutral buffered formalin solution(NBF), bladders and kidneys homogenized in1mL PBS, after centrifugation, get the supernate for the ELISA assays. Bladder and kidney tissue were formalin-fixed, paraffin-embedded sections, SP method was used for detection and comparison of urinary tract infections in mouse tissue IL-6and IL-8protein expression. Results1. The OmpT and ECM interact with each other on the bacteria adhesion.The difference of adhesion rate among different strains is significant (χ2=21.316, P<0.05). The4.62%of ompT mutant COTD adhesion to ECM was significantly lower than E. coli CFT073. Were observed compared with the control group, the adhesion to the pre-decking of the the Matrigel bacteria significantly more; knockout strains of bacterial adhesion significantly less than the wild-type strain and the revertant strains.2. OmpT involved in regulating the expression of iha and iron.In order to determine whether OmpT modulated expression of the the virulence factors Iha and IroN in CFT073, real-time RT-PCR was used in this part. After normalization with the copy number of the16s-rRNA gene, the relative expression level of iha in COTD(1.02±0.04) was significantly less than that in CFT073(2.02±0.07), while the relative expression level of the COTD(pST) strain increased to1.56±0.05). The COTD strain also significantly reduced the relative expression level of iroN (from3.81±0.07to1.01±0.10).3. OmpT impact model of acute urinary tract infection in mice induced inflammatory response.To determine if OmpT has a role in uropathogenesis, bladder bacterial loads of the ompT mutant relative to wild-type CFT073and complemented COTD(pST) were evaluated by an murine cystitis model.All groups have varying degrees of inflammatory response compared with the control groups.Bacterial colony counts due to the degree of bacteremia, one-way ANOVA analysis showed that the strains between the number of colonies was.P<0.05, according to statistical significance. Log10conversion after the wild-type strain model of the blood in the average colony number of2.52±0.05, knockout strains was2.01±0.02. The wild strain bacteremia higher than the knockout strains.Mouse bladder tissue after logarithmic transformation of wild strains, the number of carrier volume as6.36±0.06, while the knock except strain was6.01±0.07,6.29±0.06, of the covering strain one-way analysis of variance (P<0.05) differencestatistically significant. Bacterial load in kidney tissue, wild-type strain to6.25±0.05to5.87±0.06knockout strains, one-way analysis of variance, P<0.05, statistically significant difference between the groups after the knockout gene ompT bacteria in the bladder and kidney tissue load is significantly reduced.4. OmpT enhance the expression of cytokine in urinary tract infection of mice.After statistical analysis can be seen in plasma IL-6expression levels the CFT073, COTD and contrast COTD(pST), a statistically significant difference among the three groups (F=767.43, P=0.00), the strains COTD groups of mice blood IL-6were significantly lower than the wild-type strain and the revertant strains of group. Plasma IL-8expression among the three groups were significantly different (F=187.52, P=0.00). Compare kidney tissue IL-6and IL-8expression found the COTD group expression were less than CFT073group. As can be seen from the results of immunohistochemistry, after the ompT gene knockout, IL-6and IL-8expression in the bladder tissue and kidney tissue decreased. Analysis of interaction, found that the treated and cytokines has the interaction(F=18.42, P=0.00). It prompted that OmpT effect the expression of IL-6and IL-8in bladder and kidney.Statistical analysisThe statistical content covered in this article using SPSS13.0statistical software for processing. All values are expressed as mean±standard deviation. Using two independent samples t-test comparison of the measurement data of the two samples, when the homogeneity of variance.Statistical analysis was performed with One-Way ANOVA.Nonparametric test was used in the ranked data.Differences with P<0.05were considered to be statistically significant.Conclusions1. The OmpT and ECM interaction with each other enhanced urinary tract pathogenic E. coli adhesion to extracellular matrix, which affects the pathogenic bacteria invade into host cells.2. OmpT may regulate the expression of the adhesion gene iha and the transferrin gene iron, which play the role of network regulation, which will affect the adhesion of the bacteria and the host pathogenic. 3. OmpT may be provided play a role in the pathogenic which influence of urinary tract pathogenic E. coli in vivo inflammatory responses.