The Effect Of The Irp2、Fyua Deletion In HPI On The Pathogenicity Of APEC

Avian Pathogenic Escherichia coli (APEC) causes avian colibacillosis, the most significant infectious bacterial disease of poultry worldwide, and has brought a serious impact for the development of poultry industry. The Yersinia high pathogenicity island (high pathogenicity island, HPI) is a larger chromosome segment, it determines the virulence and pathogenicity of a variety of intestinal pathogens including APEC. The studies have shown that, irp2 and jyua genes are important structural genes of HPI, and their main functions are the regulation and absorption of iron, but the relationship between irp2|fyua and the virulence of APEC has no detailed report.The structural gene irp2 and fyua of HPI were choosed for this study, the gene deletion of irp2 and irp2|fyua strains were constructed using APEC. In order to study the relation between the deletion of APEC and the virulence of HPI, the mRNA levels of virulence genes、the ability of adherence、median lethal dose and the effect of pathological changes in infected chickens were studied. It reveals the relationship between irp2,fyua genes and the virulence of APEC, and it lay the foundation for the further research of the HPI in APEC. The specific contents and results are as follows: 1. Construction of irp2 and irp2|fyua mutant strain of Avian pathogenic E. coli strainIn this study, Red homologous recombination technology was used for the deletion of irp2 and fyua step by step. First, the homology arms of irp2 was cloned in zero-background cloning vector, then the chloramphenicol resistance fragment of pKD3 was inserted into the zero-background cloning vector, and then the targeting sequence was formed for the Red homologous recombination. Under the inducible protein of Red homologous recombination was expressed at the principle of 30℃, the PCR product of the targeting sequence was transformed electrically to the objective bacteria for the interchange of gene. The mark of chloramphenicol resistance was used for the screening of positive strains. And the vector of pCP20 was transformed electrically to the positive strains for the elimination of chloramphenicol resistance, then the irp2 mutant strain was constructed successfully. The irp2 mutant strain was choosed as starting strain, the irp2|fyua mutant strain was constructed by the same method. 2. The affect of irp2 deletion in the virulence of Avian pathogenic E. coli.In order to study the relation between the irp2 deletion and the virulence of APEC, the mRNA levels of virulence genes、the ability of adherence、median lethal dose and the effect of pathological changes in infected chickens were studied in the experiment. The Real-time PCR results showed that the mRNA levels of the virulence-related genes luxS, iss, ompa and fumc were significantly decreased in the irp2 mutant strain, when compared with the wild strain (p< 0.01). The determination of growth curve showed that the deletion of irp2 in APEC has no effect to its growth in LB medium. The irp2 mutant strain reduced significantly the abilities of adherence by 66.0% compared with the wild strain. Further studies showed that deletion of the irp2 gene in APEC reduced the bacterial virulence by 34-fold in Roman Chickens, based on the measurement of the 50% lethal dose. The histopathology of heart and liver showed that the pericardium of chicks has the infiltration of inflammatory cell and the surrounding of liver has the accumulation of inflammatory cells. And the pathological changes in chickens was significantly reduced by the infection irp2 mutant strain.3. The affect of irp2|fyua deletions in the virulence of Avian pathogenic E. coli.In order to study the relation between the irp2|fyua deletions and the virulence of APEC, the mRNA levels of virulence genes、the ability of adherence、median lethal dose and the effect of pathological changes in infected chickens were studied in the experiment. The Real-time PCR results showed that the mRNA levels of the virulence-related genes luxS,pfs, tsh, iss, ompa and fimc were significantly decreased in the irp2|fyua mutant strain, when compared with the wild strain (p< 0.01). The determination of growth curve showed that the deletion of irp2|fyua in APEC has no effect to its growth in LB medium. The irp2|fyua mutant strain reduced significantly the abilities of adherence by 53.5% compared with the wild strain. The irp2|fyua gene in APEC reduced the abilities of adherence by 14.7-fold compared with the irp2 mutant strain.Further studies showed that deletion of the irp2|fyua gene in APEC reduced the bacterial virulence by 500-fold in Roman Chickens, based on the measurement of the 50% lethal dose. The histopathology of heart and liver in Roman chicken showed there were no obvious changes after the infection of irp2|fyua mutant strain.The irp2 and irp2|fyua mutant strains were constructed successfully by the Red homologous recombination method. And the studies of virulence in irp2 and irp2|fyua mutant strains showed that the deletion irp2 and irp2|fyua in APEC will decrease the mRNA levels of some virulence-related genes, the abilities of adherence, the LD50,and the pathological changes of chicks. These results suggest that, irp2 and fyua has an important regulatory role in the virulence of APEC. This study provides some reference for the further research in HPI of APEC.