Studies On The Comparative Proteomics Of Uropathogenic Escherichia Coli 132

Objective:To study proteome differences between domestic strain uropathogenic E.coli (UPEC) 132,model strain abroad UPEC J96 and non-uropathogenic E.coli K-12 MG1655 whose genomic sequencing has been completed.Methods:1.Identificated UPEC 132,UPEC J96 and E.coli K-12 MG1655 by Gram stain,test of biochemical reaction and papC PCR.2.Counted the bacteria number of three strains and described their growth curves with turbidity method.According the result above,cells were harvested respectively at the early logarithmic growth phase(6h) or late log-to-stationary phase(12h) after centrifugation.Prepared protein samples for two-dimensional gel electrophoresis(2-DE) after cells disrupted by ultrasound and measure concentration of these samples by Bradford method.3.Optimized experiments conditions of bacterial culture and two-dimensional gel electrophoresis as follows:(1)Compared and analyzed the 2-DE patterns of UPEC 132 with different growth times and pH gradients;(2)Counted isoelectric points and molecular weights with pI/Mr Prediction Tool(http://www.expasy.org/tools/ pi_tool.html) according to E.coli K-12 MG1655 GenBank notes,constructed its theoretic 2-DE pattern and estimated the distribution of whole-cell proteins according its theoretic 2-DE pattern.(3)Compared the 2-DE pattern of E.coli K-12 MG1655 of pH 4-7 with its theoretic 2-DE pattern,calculated the resolution.4.The 2-DE patterns acquired under the better conditions above were matched and analyzed with the software of PD Quest 7.0 after Coomassie brilliant blue staining. The differential expression protein spots(up-regulation expressing more than twofold compared with UPEC 132) were digested in gel by enzyme and measured by MALDI-TOF-MS.Results: 1.Identification of UPEC 132,UPEC J96 and E.coli K-12 MG16551) They were recognized as Gram negative bacilli and their biochemical reaction conformed to the biochemical pattern of Escherichia coli.2) Tth results of papC PCR showed the strains of UPEC 132 and J96 appear electrophoresis strip of 328bp prospectively while E.coli K-12 MG1655 did not.So we recognized UPEC 132 and J96 are UPEC strains.2.Three strains appeared similar growth curves:got in logarithmic phase(2-12h) after short lag phase.3.Protein concentration of three strains harvested in 6h was:10.63mg/ml,11.93 mg/ml and 11.55 mg/ml;harvested in 12h was:11.06mg/ml,12.84 mg/ml and 12.52mg/ml.The results were up to our expectations.4.Optimization of conditions of two dimensional gel electrophoresis1) The 2D patterns of UPEC 132 with different growth times(6h and 12h) appeared similar distribution of proteins.The protein spots were calculated by PD Quest software that the fromer pattern has 472 spots while the other one has 513 spots,the latter has more than 8.69%sopts than the fromer.2) The protein spots separated on the pH 3-10 IPG strip of UPEC 132 were confined to pH 4-7 that basic protein spots were rare while the spots showed uniform distribution on the pH 4-7 IPG strip,and the latter had better resolving power.Their spots number was 268 and 513 calculated by software, the resolving power of latter better than fromer.3) The theoretic 2-DE pattern of whole proteins of E.coli K-12 MG1655 showed that 62.9%theoretic protein spots were cencenrated in pH 4 to 7, 27.1%theoretic isoelectric points were situated in pH 8 to 10,only 5.3% points situated in pH 7 to 8.4) There were 356(13.2%) spots showed from the 2-DE pattern of intra-cellular proteins of E.coli K-12 MG1655 on pH 4-7 IPG strip.The resolving power was ideal compared with general condition of 2-DE technique now(only fewer than 10%proteins separated).5.The results of two-dimensional gel electrophoresis showed that mean number of protein spots recognized from UPEC 132 was 466±11,382±12 from J96 and 338±15 from E.coli K-12 MG1655.there were 298(59.7%) protein spots shared by the three strains,33(6.6%) spots shared by UPEC 132 and E.coli K-12 MG1655,56(11.2%) spots shared by UPEC 132 and J96;89(17.8%) protein spots characterized by UPEC 132,22(4.4%) characterized by J96 and only 1 (0.2%)characterized by E.coli K-12 MG1655.6.22 differential protein spots involved in virulence factors,metabolism and transportation,regulation of protein syntheses,biological oxidation and unknown functions,were identified by MALDI-TOF-MS,15 spots were differential expressed or significantly increased expressed in UPEC 132 while 7 spots were increased expressed both in UPEC 132 and J96 compared with E.coli K-12 MG1655,Conclusion:In this study,we got the 2-DE patterns of intra-cellular proteins of UPEC 132,UPEC J96 and E.coli K-12 MG1655.Through research of differential proteins expressed in three strains,we acquired that the proteome from UPEC strains and non-uropathogenic Escherichia coli,or UPEC 132 and J96 were differentially expressed.It will provide important fundament on the pathogenesis of UPEC.