Construction And Characterization Of IbeB Gene Deletion Mutant Of Escherichia Coli Causing Calf Meningitis In Xinjiang

Extraintestinal pathogenic Escherichia coli(Ex PEC)are a new group of pathogenic E.coli which can colonize in extraintestinal tissues and result in extraintestinal infection.Because of Ex PEC’s special virulence factors,they can penetration across the intestinal barrier and blood-brain barrier of human and animals,They can cause diseases in clinic,including neonatal meningitis and urinary tract infections(UTIs),diarrhea,meningitis and septicemia for calves.Persistent infection of Ex PEC is an important cause of high mortality and morbidity.Ex PEC can cause central nervous system diseases,and many factors are involved in the pathogenic process.In particular,proteins Ibe family(ibe A,Ibe B,Ibe C and other virulence factors)play an important role in the adhesion and invasion of neonatal meningitis E.coli K1 strain to host cells.In the early stage,a strain of O161 serotype named E.coli-SN was isolate from the brain tissue of the calves with nervous symptoms.Whether the Ibe family proteins present in the E.coli-SN and participated in the pathogenicity of E.coli-SN strain.What is the relationship between the E.coli-SN strain and the neonatal meningitis K1 strain.They are not clear now.In this study,E.coli-SN of clinical isolated from calves encephalitis is as parent strain.The ibe B gene was detected using PCR method,then E.coli-SN-Δibe B strain was constructed by Red/ET homologoud recombination system,and compared differences in biological characteristics in vitro and pathogenicity in mice with parental strain.This study lay a foundation the pathogenesis mechanism of Ex PEC.The main research contents and results are as follows:1.Sequence analysis of ibe B gene of meningitic Escherichia coli isolates in calves: In order to analysis the ibe B gene of meningitic Escherichia coli isolates in calves.The ibe B gene was amplified by PCR from the isolated strain and connected to p MD19-T vector(p MD19-T-ibe B).The full length sequence of ibe B gene was obtained by sequencing,splicing and comparison,and analyzed by DNAStar software.These results showed that the nucleotide and amino acid sequence of ibe B gene of E.coli-SN strain shared 90.5% and 96.9% identity with Escherichia coli K1 RS218 ibe B gene,respectively,and they were clustered in a clade.While,E.coli-SG strain shared 99.4 % and 100.0 % identity with Escherichia coli K12 respectively,and they were clustered in a clade.The closely correlation and relationship of ibe B gene between meningitic Escherichia coli isolates E.coli-SN in calves and neonatal meningitis E.coli K1 RS218 strain has been preliminarily revealed.2.Construction and identification of E.coli-SN ibe B gene deletion strain(E.coli-SN-Δibe B): For further research of the function of ibe B gene in the pathogenicity of E.coli-SN,in this study,Red/ET recombination was used to knock out the target gene ibe B.According to the upstream and downstream sequence of ibe B gene and FRT-PGK-gb2-kan/neo-FRT gene,a paris of primers was desigened to constructe the mutant strain.First,the compenent cells of E.coli-SN was electrophoretic-transformed with the plasmid p Red ET,incubated at 30℃;the expression of gene mediating p Red ET was induced by L-arabinose.Second,the compenent cells of E.coli-SN-p Red E were prepared for electroporation with the FPF carrying ibe B homology arms.In the end,the compenent cells of E.coli-SN-Δibe B-FPF was electrophoretic-transformed with the plasmid 707-FLPe,marker gene was lose by 707-FLPe was induced at 37℃.All of the recombinant strains were identified by PCR.The deletion strain had no change by PCR testeing after cultured for 20 generations without antibiotic at 37℃,and indicated it had good genetic stability.So the E.coli-SN gene deleted strain was successful constructed and named as E.coli-SN-Δibe B strain.3.Partial biological characteristics of E.coli-SN-Δibe B: In order to understand the function of ibe B gene of E.coli-SN,through analyzed hemolysis test,acid and alkali resistance test in vitro,virulence and the amount of bacterium in the liver,brain,spleen of mouse infected E.coli-SN-Δibe B strain.The results showed that the haemolytic characteristics and acid and alkali resistance test in vitro of E.coli-SN-Δibe B strains was the same as the parent strain.The LD50 of the E.coli-SN-Δibe B strain was 101.16 higher than the E.coli-SN strain,but the difference was not significant.The amount of bacterium of E.coli-SN-Δibe B strain reduced in the liver(t=24h,p<0.01;t=48h,p<0.05)and spleen tissue(t=24h,p<0.05;t=48h,p<0.01),but no significant difference in the brain tissue.The survival time of mice infected E.coli-SN-Δibe B strain(average of 3.38 days)than the E.coli-SN strain(average of 2.25 days)was significantly prolonged(p<0.05).The results showed that the deletion of ibe B virulence gene could weaken the virulence of E.coli-SN strain.