Initial Research Of Potential Activation Factors Of The Cpx Two Component System And Gene YccA Of Haemophilus Parasuis

Haemophilus parasuis is the causative agent of Gl?sser's disease,which characterized by pleurisy,peritonitis,arthritis and meningitis under stress conditions.Two-component system is a signal transduction systems of envelope stress response that responds to environmental signals and regulates genes expression.The Cpx is one of signal transduction systems in H.parasuis which is composed of histidine kinase sensor CpxA and response regulator CpxR.When Cpx system activated by stress environment induces,CpxR alleviates membrane pressure by changing the transcriptional level of genes.This study is based on initial exploring stimulus of Cpx activation and function of the regulating factor.Main research results are as follows:1.Cpx system may be activated by factors of high osmotic pressure,heavy metals and antibioticsAccording to a variety of challenges H.parasuis encountered from respiratory tract to host internal,some potential activated factors were analyzed : temperature,high pH,osmolality,reactive oxygen species and antibiotics etc.Whether Cpx was activated by above factors or not,growth environment around H.parasuis JS0135 changed and Cpx genes transcription information were detected.The results showed that the transcription of cpxA/cpxR was increased under high osmolality,heavy metal ion and antibiotics and predicted that Cpx may be activated by these stimulus.2.Transcription of H.parasuis yccA/secY enhanced under induction by copperRelative genes transcription of H.parasuis JS0135 were investigated.Real-time PCR was used to determinate various genes expression under induction.The results showed that drug resistance genes yccA/secY remarkable up regulated with copper induction,suspected CpxR protein may regulate yccA/secY in relation with drug resistance of H.parasuis.3.CpxR protein bound promoter region of yccAAccording to the fluorescence quantitative results,it was speculated that CpxR regulated the drug resistance genes under the stimulation of gentamicin.CpxR protein were prokaryotic expressed and purified,regulated genes' promoter was amplified.The results showed binding bands between CpxR protein and the promoter of yccA in vitro,suggested that CpxR protein regulated gene yccA,coded the inner member protein.Speculated that yccA may be regulated by CpxR protein response to copper.4.Exploring H.parasuis JS0135?yccA biological functionJS0135?yccA was constructed in vitro and biological function of JS0135?yccA was explored.The results showed that the growth and biofilm formation ability of JS0135?yccA under high pH condition was weaker than JS0135,meanwhile the transcription of yccA up regulated within copper stress condition.