The Effect Of The PhoP/Q Mutant On The Pathogenicity Of Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC) is an important pathogen of serious harm poultry health and hinder the development of poultry farming industry. Pho P/Q two-component system is a signal transduction regulation system which regulating bacterial virulence and many cell activity in Gram-negative bacteria. Pho P/Q two-component system plays an important role in the pathogenicity of bacteria. The specific regulation mechanism of Pho P/Q two-component system has no detailed report in APEC.The gene mutant and complementation strains of phoP and phoQ will be constructed. In order to study the relation between Pho P/Q and pathogenicity of APEC, the m RNA levels of virulence genes, bacterial motility assay, the ability of adhesion and invasion to CEF and median lethal dose were studied. It lay the foundation for the further research of the Pho P/Q two-component system in APEC. The specific contents and results are as follows:1.Construction of phoP and phoQ mutant strains and complementation strains of Avian pathogenic E. coli.In this study, 29 strains from clinical isolates of avian pathogenic Escherichia coli had been detected whether or not containing phoP gene and phoQ gene. Then bacteria were detected whether sensitivity to chloramphenicol and ampicillin, and ultimately selected the object strain AE17. Red homologous recombination technology was used for the mutant of functional areas on the phoP gene and phoQ gene. The single gene mutant strains(AE18 and AE19) and double mutant strains(AE20) were successfully constructed. Three complementation strains were constructed by use of low copy plasmid PSTV-28 based on mutant strains. The same gene complement fragment of phoP gene and phoQ gene were successfully constructed according to the bioinformatics software analysis and related literature. Containing the promoter of the gene fragment digested reply after connected to low copy plasmid vector PSTV-28. The complementation strains of CAE18, CAE19 and CAE20 were successfully constructed.2. The effect on the biological characteristics and virulence of gene mutant in APEC.Comparison of changes in mutant strains, complementation strains and wild strain between virulence gene transcription level and biochemical characteristics. Growth curve determination, biochemical identification, motility assay and detection of virulence genes transcription levels were used to explore of Pho P/Q two-component system of gene mutant effects on biochemical characteristics and virulence of APEC. The determination of growth curve showed three mutant strains and three complementation strains growth in LB medium have no obvious change. Biochemical characterization test results show: three mutant strains and three complementation strains with wild-type gene have no different of eleven enterobacteriaceae biochemical characteristics. The results show that the mutant of phoP and phoQ have no effect on biochemical characteristics. Motility assay results showed that phoP and phoQ gene mutant can significantly reduce the athletic ability of AE17. The Real-time PCR results showed that the m RNA levels of the virulence-related genes Mar A, met J, ibe A, sod A, iss, omp A and so on had been detected in APEC. The virulence genes transcription levels of three mutant strains showed varying degrees of upward or downward than wild strains.3. The effect of the phoP and phoQ mutant to pathogenicity on cell and organism.Real-time PCR results showed that Pho P/Q two-component system can regulate the transcription and expression of virulence factors in APEC, so as to effect of bacterial pathogenicity. To explore the effect of Pho P/Q two-component system of APEC pathogenicity, the ability of adhesion and invasion, median lethal dose and pathological observation of animal tissue were studied in the experiment. Adhesion and invasion of chicken embryo fibroblast cells showed phoP mutant strain, phoQ mutant strain and double genes mutant strains reduced significantly of the abilities of adherence by 63.11%,65.42% and 30.26% compared with the wild strain. Three mutant strains reduced significantly of the abilities of invasion by 59.83%, 57.82% and 37.90% compared with the wild strain. Further studies showed that mutant of the phoP and phoQ genes in APEC reduced the bacterial virulence by 31.57 �fold, 10-fold and 13.31-fold in Roman Chickens. Acquisition of pathological tissue of Luo Mandan cock after infection and made into slice showed that AE17 can cause the typical symptoms of Escherichia coli.The phoP and phoQ mutant strains were constructed successfully through the above experiments. This study has identified that Pho P/Q two-component system can regulate the transcription of virulence genes, effect of gene mutation in APEC in CEF cells and change the pathological of chicks. The research results show that phoP and phoQ gene play an important role in regulation of APEC pathogenicity, provide some experimental basis for further study of the regulatory mechanism of Pho P/Q two-component system in APEC.