Pathogenic Mechanism Of Invasion Associated Genes In Avian Pathogenic E. Coli Duck Isolate DE205B

Avian colibacillosis caused by avian pathogenic Escherichia coli (APEC) manifested as pericarditis, parahepatitis, peritonitis, septicemia and so on, meningitis are continuously increasing in present. For the various serotypes of APEC,176 O antigens,103 K antigens, 83 H antigens, and antibotic resistance of bacteria, the difference of areas and species, it is difficult to eradicated for animal husbandry to serious harm, and is also a zoonotic pathogens. Recent studies show that poultry may be a vehicle or even a reservoir for human ExPEC strains,and APEC potentially serve as a reservoir of virulence-associated genes for human ExPEC strains such as UPEC and NMEC, The mortality and morbidity associated with neonatal gram-negative meningitis have remained significant despite advances in antimicrobial chemotherapy. Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis. Our incomplete knowledge of the pathogenesis of this disease is one of the main reasons for this high mortality and morbidity. In the study of E. coli meningitis, multiple genes involved in the bacterial penetration of the blood-brain barrier.have been identified, such as ibeA, ibeB, yijP, aslA, ypdP and ompA and so on, which were found widespread among APEC Isolates. Further study of their mechanisms involved in the pathogenesis is is necessary.Two pairs of primers were designed and synthesized to detected the prevalence of invasion associated virulence gene ibeB and yijP in 100 E.coli isolates from ducks and humen.The detection result shows that:Distribution Rate of both gene ibeB and yijP is 100% among the 100 strains. The full open Reading frame of gene ibeB and yijP were cloned from APEC strain DE205B,and then these two fragments were connected to pMDT18 vector and delivered for sequencing.Then the corresponding amino acid sequence homology and phylogenetic analysis were taken among the APEC O1, BL21(DE3), CFT073, RS218, UT189, O157:H7 strains and DE205B, respectively on the gene ibeB and yijP.The result shows that both of ibeB and yijP have high sequence homology among the strains analyzed above. In the evolutionary tree of both ibeB and yijP, APEC O1,RS218,UT189 and DE205B are in the same branch, possiblely have the common origin.The DNA fragment ibeB and yijP were cut by restriction endonuclease respectively from vectors pMD-T-ibeB and pMD-T-yijP, and then cloned into plasmid vector pET-32a(+) in the proper orientation.The positive recombinants were identified by endonuclease digestion. The recombinant plasmids pET-32a-ibeB and pET-32a-yijP were transformed into E. coli BL21(DE3). The expression of the histidine-tagged fusion proteins were induced by the addition of 1 mM isopropylβ-D-1-thiogalactopyranoside (IPTG) to recombinant E. coli BL21(DE3) for 4 h. The molecular weight of the IbeB, YijP fusion proteins was 69.3kDa and 83.4kDa, respectively. The fusion proteins were verified by Western blot analysis using the convalescent sera against APEC strain DE205B from rabbits. It suggested that the recombinant proteins have the same immunity epitope as the nature one in DE205B. After purification, the fusion proteins mixed with adjuvant were respetively vaccinated to rabbits.After third vaccination,two kind of high titer rabbit anti-protein serum were obtained.these anti-protein serum were used to the inhibition of invasion assay of DE205B.The result indicats that the serums respectively anti IbeB and YijP fusion proteins both has the ability to inhibit the invasion process of DE205B to the DF-1 cells.The mutant with deletion in the invasion associated gene ibeB was generated using the lambda red recombinase method. For genetic complementation, the ORF and putative promoter of ibeB were cloned into plasmid pUC18 and transformed into the mutant strain. The chracteration of wild type strain, mutant strain and complementation strain were compared and analyzed, which contributed to study the role of IbeB in the pathogenesis of APEC. The mutant strain shows decreased Growth capacity. The inactivation of ibeB in APEC DE205B led to the reduced invasion capacity in vivo and in vitro, whereas the invasion ability was fully restored in the complementation strain. The inactivation of ibeB resulted in the decreased expression of ibeA, which might be a reason for the decreased invasion capacity and defective virulence.