The Role Campylobacter Jejuni Mcp1/2/3 Genes Play In Chemotactic Behavior In Vitro And Colonization In Animal Hosts Of Campylobacter Jejuni

Background and Objective Campylobacter jejuni is a major foodborne causative agent of human gastroenteritis throughout the world.Sequelae of the campylobacteriosis can be severe and include arthropathies and Guillain-Barre syndrome(GBS),an autoimmune disease resulting in acute motor nerve paralysis.In becteria,two-component signiling system(TCS),which usually composed of sensor protein and response regulator protein,has the functions to sense and response to enviromental signals.The TCS responsible for bacterial movement towards favorable locations in hosts,i.e.chemotaxis,is termed as Che-TCS.In a Che-TCS there are three major groups of functional proteins:MCPs to receive signals of attractants,histidine kinase to start intracellular signal transmission though phosphoryl group transfer and flagellar motor switch proteins to decide the rotation orientation of flagella.Up to now the Che-TCS of Campylobacter jejuni has not been well studied.In our study,we constructed prokaryotic expression systems of mcp1,mcp2 and mcp3 genes,and made antiserum and IgG F(ab')₂ of rMCP1-MCP3,established chemotactic model in vitro of the microbe for determing chemotaxis-inducing substances.We constructed mcp1^—,mcp2^—,mcp3^—and mcp1^—/mcp2^—mutants.We detected the chemotactic ability of mcp mutants toward DOC.At last we determined the coloniozation of the mutants on jejunal mucosa of mice.All the work are expected to increas our understanding of the relationship among MCPs and chemotactic inducers.Methods The segments of mcp1,mcp2 and mcp3 genes were amplified by PCR and then sequenced after T-A cloning.Prokaryotic expression systems of the genes were subsequently constructed.SDS-PAGE plus BioRad Gel Image Analyzer were used to examine the expression of target recombinant proteins rMCP1,rMCP2 and rMCP3,and Ni-NTA affinity chromatography was performed to purify the rMCPs.Rabbits were immunized with each the three rMCPs to obtain antisera.Immunodiffusion assay was performed to measure the titers of antisera.IgG in each of the antisera were extracted by ammonium sulfate precipitation and DEAE-32 ion exchange chromatography,and IgG F(ab')₂s were then prepared by pepsin enzymolysis and Sephadex G-100 chromatography.We constructed mcp1^—,mcp2^—,mcp3^—and mcp1^—/mcp2^—mutants with homologous recombination.Chemotactic model in vitro of C.jejuni based on HAP(hard-agar plus) method was established to determine the chemotaxis-inducing effect of eight candidate substances.Chemotaxis inhibition test based on IgG F(ab')₂ blocking was applied to deterimne the function and diversity of MCPs.Colonization model in mouse was established to determine the role MCPs play in coloning mice.Results The segments with expected sizes amplified from mcp1,mcp2 and mcp3 genes were obtained by PCRs,and their nucleotide and putative amino acid sequences were completely same as the reported.The constructed prokaryotic systems could efficiently express rMCPs with the yields about 10%of the total bacterial proteins. Immunization with rMCP1,MCP2 and rMCP3 enables the rabbits to produce specific antibody.All the antisera had 1:4 immunodiffusion titers.Both bovine bile and sodium deoxycholate(DOC) were able to induce chemotactic movement of C.jejuni in a dosage-dependent manner(P＜0.05).When MCP1 and MCP2 were blocken with their IgG F(ab')₂s,the chemotactic ability of C.jejuni were remarkably decreased(P＜0.05). However,MCP3 blocking did not affect the chemotaxis(P＞0.05).Furthermore,mcp2, mcp3 and both the two gene knock-out mutants(mcp1^—,mcp2^—and mcp1^—/mcp2^—) presented 16%,22%and 51%in diameter of the DOC-induced chemotactic ring of wild-type strain(P＜0.05).mcp1^—,mcp2^—and mcp1^—/mcp2^—mutants also displayed significantly attenuated colonization on jejunal mucosa of mice compared to wild-type strain(P＜0.05).And the CFU counting number of mcp1^—/mcp2^—mutant in the murine jejunal contents was much lower than that of mcp1^—and mcp2^—mutants(P＜0.05).Conclusion All the results of this study lead a conclusion that DOC is an efficient chemical chemotactic attractant of C.jejuni in vitro,and Mcp1 and Mcp2 are the DOC-specific sensor proteins to evoke the chemotactic movement of C.jejuni tawards DOC.