| Pathogenic Escherichia coli can be divided into intestinal pathogenic Escherichia coli and extraintestinal pathogenic Escherichia coli(ExPEC).Avian pathogenic Escherichia coli(APEC)is one of the extraintestinal pathogenic Escherichia coli(ExPEC),which can infect through different routes including respiratory and genital tracts and causes multiple system injury syndrome diseases in chickens.The disease is responsible for significant economic losses in the chicken industry,which has become one of the important bacterial diseases of the poultry industry.Consequently,how to prevent and control the disease has become an arduous task in the poultry industry.In addition,APEC is greatly similar with uropathogenic Escherichia coli(uropathogenic E.coli,UPEC)causing human urinary tract infections,Therefore,APEC is considered to be a food borne pathogen,which has important public health significance.DNA microarray is a stable,practical and feasible genome-wide analysis method.It can explore the molecular pathogenesis for the pathogen,and it can also be used to analyze the whole gene expression pattern of bacterial pathogens when they interact with the host,and it devotes to identify virulence factors and their corresponding regulatory mechanisms.This study selected APEC 02 serotype representative strain E058 as a model strain to study its transcriptome of the infected chicken by DNA microarray,the differentially expressed genes were screened by data analysis,and study the potential virulence genes,The aim of this research is to find out the gene expression profiles of APEC in infected host and further explore the potential virulence genes,it provides theory knowledge to reveal the pathogenic mechanism of APEC.1.The transcriptional expression profile of APEC strain E058 in infected chickens based on DNA microarray analysisIn this study,1-day-old specificpathogen free(specificpathogen free,SPF)chickens were infected with a representative APEC E058 isolated strain.The bacteria were collected from the infected chicken blood,and the E058 strain under the condition of LB culture was used as the control in vitro.A commercial Affymetrix multigenome DNA microarray,which contains most of the genomic open reading frames of E.coli K-12 strain MG1655,was used to detect the expression level of APEC genome-wide in infected chickens and in vitro.So as to reveal the gene expression pattern of APEC in infected host.It shows that 245 genes were significantly upregulated and 369 genes were significantly downregulated during bacterial growth in vivo.The differentially expressed genes were involved in the physiological metabolism,iron acquisition,virulence,stress response and biological regulation and so on.According to the results of gene microarray analysis,12 differentially expressed genes(8 up-regulated and 4 down regulated genes)were selected to verify the microarray obtained data by fluorescence quantitative PCR analysis.The results showed that the expression level of selected genes consistent with the gene microarray results,which can determine the reliability of transcription data obtained by microarray analysis.At the same time,according to the microarray results,Several significantly upregulated genes cotained yjiY,sodA,phoB and spy of the expression of APEC in vivo were selected to construct deletion mutations using gene knockout technology,we can get preliminary understanding and speculation about the influence of selected genes on pathogenicity by comparing 1-day-old chickens,pathogenicity of parent and mutant strains.The results showed that the virulence of APEC E058 was not decreased due to the deletion of yjiy,phoB and spy gene,but the pathogenicity of APEC E058 was significantly decreased in chicken after deleting sodA gene,these results suggest that sodA may be a potential virulence gene involved in the pathogenesis of APEC,then the function and pathogenicity of sodA gene was evaluated.The study aimed to clarify the relationship between the expression level of target genes in vivo and its pathogenic effects,that is,whether the genes obviously up-regulated in vivo are the true virulence genes.2.Construction of the sodA gene mutant of avian pathogenic Escherichia coli and evaluation of their pathogenicitySuperoxide dismutase(sodA),an antioxidant stress factor,plays an important role in the antioxidant effect of bacteria.It has not been reported that the direct virulence effect to APEC.The aim of this study was to investigate the relationship between the sodA gene and the pathogenicity of APEC,lay the foundation for reveal the pathogenic mechanism of APEC and further construction of APEC vaccine strains.In this study,a mutant E058?sodA was constructed using the X Red recombination system,subsequently,the sodA gene complete reading frame with corresponding promoter were inserted into the low copy plasmid pACYC184 to generate revert strain by electroporation.The growth kinetics of mutant was similar with that of their wild-type strain E058 in the LB medium,it didn't show significantly differences,but the growth rate of E058?sodA was significantly lower than that of the wild strain in the LB medium containing H2O2.In vitro competition test results showed that the mutant was slightly attenuated in the LB medium.Serum bactericidal test showed that the mutant and wild strains is not sensitive to the serum bactericidal of SPF chicken.Strain biofilm assay showed that the biofilm formation ability of mutant is slightly higher than the wild strain.Under the selected environmental stress conditions in vitro in order to imitate the environment in vivo,the bacterial survival experimental results showed that the mutants are sensitive to H2O2,however,it is not sensitive to strong acid,strong alkali,high osmotic pressure and urea.The test results of phagocytosis and intracellular bacteria proliferation in HD-11 cells showed that,the ability to prohibit swallow and proliferation rate of the mutant E058?sodA to HD-11 cells were lower than that of the wild-type strain.LD50 test results showed that compared with wild-type,the virulence of mutant decreased about 100 times.The colonization and persistence ability of the mutant E058?sodA in different organs of 35-day-old SPF chickens was lower than the wild strain E058.Above results showed that sodA gene is an important virulence factor of avian pathogenic Escherichia coli,which plays a key role in its pathogenicity. |
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