Identification And Characterization Of A Novel Gene R049 In Uropathogenic Escherichia Coli

ObjectiveTo find a novel pathogenicity-associated gene from uropathogenic Escherichiacoli(UPEC)132,and preliminarily analyze its characteristics and lay a foundation offurther study on the pathogenic mechanism of UPEC.Methods1.PCR was used to detect the distribution of a specific CDS fragment R049(789bp) in UPEC and fecal E.coli strains.R049 positive strains were analyzed byPFGE and Southern blot to show the distribution features of the R049 fragment inbacterial chromosome.2.Genome walking was used to identify the flanking regions of R049 fragmentin order to obtain a complete ORF (R049-ORF).3.The expression strain E.coli BL21(DE3)/pET32a-R049 was constructed,andthe recombinant protein was purified by nickel affinity chromatography.Antiserum tothe purified protein was generated,and then was used to detect the whole-cell lysatesand outer and inner membrane proteins of UPEC132 through Western blot.4.The adherence rate of EJ cells by UPEC132 was calculated,and the invasionof EJ cells by GFP-labeled UPEC132 was observed by a confocal microscope.AcDNA microarray was used to identify the gene expression differences of EJ cellsinfected with UPEC132 or transfected with R049-ORF.5.A recombinant E.coli K-12 p678-54/pACYC184-R049 was used to infectBALB/c mice through urinary tract in order to assess the pathogenicity of R049-ORFby bacteria amounts of urine and kidneys and renal pathological damages.OtherBALB/c mice were immunized with recombinant R049 protein,and then weretransurethrally inoculated with UPEC132.The immune protective effects wereevaluated by above criterion.Results1.The R049 fragment was found in 8 of the 20 (40%) UPEC strains and in 3 ofthe 40 (7.5%) fecal isolates,which had significant difference (P＜0.01).Southernblot showed the size of positive bands of six UPEC strains was 150kb,which wasquite different from the UPEC model strain J96,suggesting the R049 fiagmentpossessed the feature of clustering distribution in domestic isolated UPEC strains.2.A 1207 bp downstream and 1502bp upstream sequences of R049 fiagmentwere obtained though genome walking,and the presence of an R049-ORF (1311 bp)was predicted by bioinformatics software,which had no similarity with the submitted sequences in GenBank and was registered with No.EF488001.3.The recombinant R049 protein was expressed as inclusion body,and thepurity of the purified protein was up to 95%.The titer of antiserum to recombinantR049 protein was more than 1:102400,and were able to react with the whole-celllysates and the outer membrane protein of UPEC 132 in the size of approximately 47KD which was consistent with the predicated mass of R049 protein.These resultsshowed R049-ORF encoded an outer membrane protein.4.The adherence rate of EJ cells by UPEC 132 was (73.20±5.26) %,and theobservation through confocal microscope revealed UPEC 132 was an invasive strain.A total of 29 genes,including 28 genes up-regulated and 1 gene down-regulated,werefound in EJ cells infected by UPEC132 through cDNA microarray,which consisted ofregulators of growth and proliferation,cytokines,and modulators of apoptoticresponses and involved in three signal transduction pathways (MAPK,Toll-likereceptors,and TNF receptors pathways).EJ cells transfected with R049-ORF showedIL-6 gene was up-regulated more than 2-fold,which was related with inflammation.5.The recombinant E.coli K-12 p678-54 expressing R049 protein showed nopathogenicity to animals compared with E.coli K-12 p678-54.The bacteria amountsof urine and kidneys of immunization group were significantly lower than those ofunimmunization group (P＜0.01,P＜0.05).However,the renal pathological damagesof part mice in both groups did not show significant difference.These resultssuggested R049-ORF-encoding protein possessed a certain immune protective effecton animals challenged by homologous strain.Conclusion1.A novel gene R049 of UPEC132 was specifically associated with UPECstrains.2.R049-ORF(1311 bp)encoded an outer membrane protein(47KD)of UPEC 132,and showed no similarity with known DNA sequences through BLAST search.3.IL-6 gene was up-regulated in R049-ORF-transfected EJ cells,suggestingR049 gene was probably related with inflammation.4.Animal experiments showed R049 protein had a certain immune protectiveeffect on mice challenged by homologous strain.