Role Of Notch Molecules In The Dynamic Interactions Of Neutrophil Clearance Of Vibrio Parahaemolyticus

The Vibrio parahaemolyticus（Vp）is an aquatic zoonotic pathogen that causes vibriosis in marine animals as well as sepsis,gastroenteritis and wound infection in human.Transmigration of neutrophilsis acritical defense mechanism of the innate immune systemagainst infection and injury.Zebrafish（Danio rerio）has many advantages that mammal do not have,especially it only has innate immune within three weeks after fertilization,so,it has been used to studyneutrophil function and host-pathogen interactions in several research fields,especially in innate immunity.To explore the interactions between Vp and neutrophils,and the role of Notch in innate immunity,we tagged a pathogenic Vp strain Vp57 by tri-parental mating method with red fluorescent protein（RFP）.After testing the stability of plasmid and comparing their differences between growth and virulence,we observed interactions of the labeled Vp57(Vp57^RFP)with neutrophils in 3 days post fertilization（dpf）larva zebrafish.In order to exploring the Notch signal mechanism in zebrafish innate immunity,the expressionlevels of inflammatory cytokine and complement relevant geneswere tested after infecting zebrafish larva with the Vp57^RFP.In order to study the role of immune genes such as cytokines in innate immunity,we use bioinformatics software to analyzecytokine tnfb、il11b and il13 5 ’upstream sequence of about 2000 bp and predict its related transcription factors.We cloned promoter and build dual luciferase reporter vector,and identified its sequence with the double enzyme digestion and sequencing method.Finally,we used the dual luciferase reporter assay system detecting the activity of reorganized vector.The results of this study were as followed:（1）We tagged Vp57 with RFP by tri-parental mating method,and the plasmid was stable,it was found to be maintained at 18.2% in the strain Vp57^RFPafter320 generations in M9 medium.The growth curve of Vp57 and Vp57^RFP were similar,both strains had a proliferation by exponential growth at 0.3 h,and then entered the stationary phase at about 2 h.The result of LD₅₀ showed that Vp57^RFP had similar values as Vp57.The LD₅₀ of Vp57 and Vp57^RFP was2.18x10⁷CFU/ml and 2.00 x10⁷CFU/ml respectively.All of these made a foundation for the following infection.Zebrafish neutrophil was round or circular,the nucleus was morphological and mostly the lobulated nuclear.（2）We microinjected 579 CFU Vp57^RFP into the zebrafish caudal vein,and observed that Vp57^RFP were mainly distributed in the tail,eyes,heart and otic vesicle within 3 hours post infection（hpi）.After 3 hpi,the fish died.Several Vp57^RFP were even detected in somite by fluorescence microscopy.When Vp57^RFPwas injected at 974 CFU,somite of larvae was covered with an abundance of the bacteria,and neutrophils were not numerous enough to engulf the Vp57^RFP.The larva died with serve spine bending at 2.5 hpi.（3）We used Q-PCR to test the expressionlevels of inflammatory cytokine and complement relevant genes after infecting zebrafish of wt and notch1a^-/-by microinjection and immersion,the results showed that the expression level of cytokines,such as il1 b,ifnphil,ccl-c5 a,cxcl8a and complement c3,c6,c9,bf and bf3 in the microinjectionwas higher than that in immersion,whether it was wt or notch1a^-/-.It indicated that the acute immune response is more intense caused by injection.Furthermore,in the injected group,the expression level of all cytokines was lower in wt than that in notch1a^-/-,but among the expression level of complement,only c6 and c9 werelowerin wt than that in notch1a^-/-,while other complement genes expressionswere higher than in notch1a^-/-,it illustrated that notch1 a had negative regulatoryeffect on neutrophils in the innate immune,moreover,different complement genes had different response patterns.（4）We found the transcription factor binding sites of tnfb including Oct-1,TBP,GATA-1,RSRFC4,GLO,C/EBPalpha,NF-kappaB,ER,Pit-1a,Oct-2,Oct-2.1,RAP1.The transcription factor binding sites of il11 b including Oct-1,SRF、GR,RAR-alpha1,COUP,NF-kappaB,MEB-1,NF-1,AP-1.The transcription factor binding sites of il13 including AP-1、GATA-1、NF-1、C/EBPalpha、TBP、Oct-1.The Cp G island was not found inpromoter region of tnfb,il11 b and il13.The 5 ’flanking sequences contain the transcription components including,TATA Box and CAAT Box.We cloned the promoter fragment oftnfb,il11 b and il13 into pGL3-enhancer to construct reorganized plasmid.Fragment size was consistent by enzyme digestion,and the sequence was correct.Relative luciferase activity of Raw264.7 cells transfected of recombined vectors was higher than that of control cells.Dual luciferase report gene detection system proved that recombined vectors had promoter activity.This can provide a powerful research tool to study tnfb,il11 b and il13 involved in regulation between immune related signaling pathways in zebrafish.In conclusion,our study provided a new way of thinking about exploring the effective way of transformation with Vp.Here we established an effective artificial infection disease model studying neutrophils function in innate immune response to Vp infection.At last,our research providedapreliminaryfoundationfor studying the mechanism of Notch participating in the innate immunity of zebrafish.