Characteristics Of Campylobacter Jejuni Cj0371 Mutant And The Role Of Cj0371 Gene In The Chemotaxis Pathway

Campylobacter jejuni is gram-negative and micro-aerobic bacteria,it is the leading cause of bacterial food-borne diarrhoeal disease throughout the world.Up to now,the factors and mechanisms of why C.jejuni can successfully colonize in poultry without symptomatic but cause human illness remain poorly understood.Despite the availability of both genomic information of different C.jejuni strains,the complete understanding of the virulence of C.jejuni is still an ongoing effort.In contrast to other diarrhea-causing bacteria,C.jejuni does not express a large number of classical virulence factors in human campylobacteriosis.Even so,many atypical virulence factors have been discovered.Using in vivo-induced antigen technology(??AT),an immunoscreening method,our group has identified virulence-associated genes expressed during human and chicken infection or invasion by C.jejuni.cj0371 is one novel gene of these virulence-associated genes.Based on the cj0371 mutant constructed by our laboratory,this paper analyses many biological characteristics and virulence of this mutant.In addition,the immunological characteristics of Cj0371 protein were also preliminarily evaluated in this research.At the same time this paper explores the chemotaxis pathway of the gene may play a role in,which adds new information for the study of Campylobacter jejuni.1.The analysis of biological characteristics of Campylobacter jejuni cj0371 mutant.This paper localizes the place where cj0371 plays a role in firstly.A shuttle plasmid which expressing fuse protein Cj0371-GFP was constructed and introduced into C.jejuni Acj0371,then observing its fluorescence by confocal microscopy,the result showed that Cj0371-GFP localized at the two poles of C.jejuni.In addition,the subcellular localization of Cj0371 was found that it existed in cytoplasm and cell membrane.So,Cj0371 protein may be a transmembrane protein.Using a soft-agar motility plate assay to compare the motility of C.jejuni ?cj0371,C.jejuni 11168 and C.jejuni ?cj0371+.C.jejuni Acj0371 exhibited hypermotility compare to C.jejuni 11168 and C.jejuni ?cj0371+ showed a motility defect.Using transmission electron microscopy to observe the flagella,there is no difference between the wild type and mutant strain.In this study,the growth curves of the mutant and the wild type strains were tested,the growth rate of the mutant was still greater than that of the wild type strain,and there was significant difference from the beginning of the growth of 6 h.The result of chemotaxis analysis also showed that when using DL-malic acid,a-ketoglutarate and succinate as the chemoattractant the chemotaxis ability of mutant was higher than that of wild type strain.The virulence analysis of mutant strain includes in vitro adhesion and invasion,in vivo colonization and quantitative analysis of inflammatory cytokines in infected cells.The result of adhesion and invasion showed that the invasion ability of mutant was higher than that of wild type strain,especially that C.jejuni Acj0371 showed significantly increased invasion ability(P<0.01)compared to C.jejuni 11168.Similarly,C.jejuni Acj0371 exhibited slightly increased colonization ability(though without statistically significant differences)in infant rabbits after infection 24 h,and at 48 h post-infection,the colonization level of C.jejuni 11168 was significantly(P<0.05)less than C.jejuni ?cj0371.The cell model for the quantitative analysis of the inflammatory cytokines after the mutant and wild type strains infected were HD 11 and mouse peritoneal macrophages cell.The inflammatory cytokines were analysis including IL-6,IL-1? and chemokines.The expression of inflammatory cytokines was significantly increased in both HD 11 and peritoneal macrophages cells after infection,but there was no significant difference between mutant and wild type strains.Expressing and purifying of Cj0371 protein,then using 10 ?g/mL Cj0371 protein to stimulate the HD11 and peritoneal macrophages cells.After the stimulation of HD 11 cells,the expression of chemokines and iNOS genes were significantly up-regulated expression,but there was no difference in the expression of IL-6,IL-1?.But after the stimulation of peritoneal macrophages cells,the IL-6,IL-1? were significantly up-regulated expression.2.The role of cj0371 gene in the chemotaxis pathway of Campylobacter jejuniTo research the function of cj0371 gene deeply,this study used co-immunoprecipitation coupled mass spectrometry to screen the known proteins that interacted with Cj0371 protein.Based on the information of known proteins to speculate the function of Cj0371 protein.About 40 suspected proteins were obtained in this mass spectrometry analysis,and these proteins main participate the pathway of chemotaxis and metabolize.This research chooses chemotactic pathways continue to pursue.Firstly,using qRT-PCR to detect the expression of chemotaxis pathway genes and flagellar genes affected by chemotaxis.The results showed that the genes cheV.cj 1110c?cj1564 and cj0262c in the chemotaxis of cj0371 mutant all up-regulated expressing no matter in the early and late stage of bacterial growth;Especially,compare to the wild type C.jejuni11168,cheV were significantly increased express in C.jejuni?cj0371(12h,P<0.05;24h,P<0.01);The gens in the base of flagellar also universal up-regulated expression in the early stage of bacterial growth.Secondly,this research expressed the chemotaxis genes cheV?cheA?cheY?cj1110c?cj1564?cj0262c of C.jejuni and GST pull-down technology was used to verify the interaction between these proteins and Cj0371 protein respectively.Only CheV protein has interaction with Cj0371 protein in this research.In addition,this study purified CheV?CheA?CheY?Cj0371?Cj6462 and used these protein to simulate the chemotaxis pathway of Campylobacter jejuni in vitro,and then tested the ATPase activity of CheA protein when the Cj0371protein in or does not in the pathway by Enzyme-coupled spectrophotometric assay.The result showed that when Cj0371 protein did not in the pathway,ATP was consumed faster.So it can be sure that cj0371 has a negative influence on the chemotaxis pathway.But,when CheV protein did not in the pathway,the consumed rate of ATP was slowest,the detection result can explain that CheV plays an important role in chemotaxis pathway,but can not explicate relationship between CheV and Cj0371 protein.At the end of this study,it tried to analyze the domain of the interaction between Cj0371 and CheV,the CheW-like domain and the regulatory domain of CheV were expressed in prokaryotic expressing system,the interaction between CheV domains and Cj0371 protein was confirmed by GST pull-down analysis.The result showed that Cj0371 protein interacted with the regulatory domain of CheV protein.So we infer that Cj0371 may though regulate the receiver(REC)domain of CheV to influence the chemotaxis pathway.