Ligand Screening And Functional Study Of Chemoreceptor Tlp1 In Campylobacter Jejuni

Campylobacter jejuni,a food-borne pathogen existing in the gastrointestinal tract of animals especially poultry,infects humans through contaminated food and causes acute gastroenteritis and other diseases,posing a serious threat to human health.In order to invade the host’s gastrointestinal cells to grow and proliferate,pathogenic bacteria have evolved specific sensing mechanisms to detect signals in the environment.One of the behaviors that enables bacteria to recognize and sense stimulus signals is chemotaxis.Chemotaxis plays an important role in the host infection and pathogenicity of gastrointestinal pathogens,including C jejuni.The key to reveal the link between chemotaxis and pathogenicity of pathogenic bacteria lies in the discovery of ligand signaling molecules that chemoreceptors can sense.However,the research on the chemoreceptors and ligand recognition of C.jejuni is still in infancy,and only the ligand specificity of several chemoreceptors has been clarified.The ligand molecule that the chemoreceptor Tlp1 of C.jejuni can directly bind to and sense is not clear.In addition,there are many difficulties in the screening of ligand molecules.Therefore,this study takes C jejuni Tlpl as the model receptor,aiming to develop a novel ligand screening strategy,discover the ligand compound it recognizes,and explore the chemotactic response and physiological function through the Tlp1.The main results are described below:1.A novel strategy for ligand screening based on hybrid receptors was developed,and a functional hybrid receptor connecting C.jejuni and Escherichia coli chemoreceptor was construsted for Tlp1 ligand screening.Six hybrid receptors with different fusion sites were constructed by connecting the periplasmic ligand-binding domain(ligand-binding domain,LBD)of C.jejuni Tlp1 to the cytoplasmic signaling domain of the E.coli chemoreceptor Tar.The activities of the constructed hybrid receptors were measured by soft agar plate and microfluidic,and the hybrid receptor with the highest chemotactic activity,Tlp344Tar207,was finally obtained for the subsequent ligand screening.2.The potential ligand of Tlpl was screened by E.coli expressing the hybrid receptor Tlp344Tar207 and formate was found to be the probable ligand.The structure type of Tlp1LBD is a dCache(double Calcium channels and chemotaxis receptor)domain.Based on the analysis of its ligand-binding pocket,a compound library containing 54 small molecules was constructed for ligand screening.We measured the chemotactic response of E.coli expressing Tlp344Tar207 to these 54 compounds using microfluidics and found that Tlp344Tar207 exhibited a significant chemoattractant response to formate,indicating that formate may be the ligand signaling molecule of Tlp1-LBD.3.The binding ability of Tlp1-LBD to formate was verified by isothermal titration calorimetry-(ITC).Based on the results of molecular docking between Tlp1-LBD and formate.the key amino acids in Tlpl-LBD,H251.Y287.and S290.were predicted to bind to formate.These key residues were verified by constructing mutant proteins and the ITC experiments.When the amino acids at these three sites were mutated to alanine,the binding affinity of Tlp1-LBD to formate weakened significantly.4.The chemotactic responses of C.jejuni NCTC 11168 wild type(WT).Tlpl knockout strain ΔTlp1,complemented strain ΔTlp1C and chemotactic gene cheY knockout strainΔCheY to formate were determined by microfluidics,respectively.It was found that formate is a chemoattractant of C.jejuni,and its chemoattractant response to formate is mediated by chemotactic system and chemoreceptor Tlp1.5.The growth of C.jejuni WT and its mutant strains in an environment containing formate gradient was measured through growth competition experiments.The results showed that the presence of formate gradient could significantly promote the growth of C.jejuni WT.When WT competed with ΔTlp1 or ΔCheY strains for growth,the Tlp1-mediated chemoattractant effect of C.jejuni on formate gave WT strains a growth advantage,indicating that formate promotion of C.jejuni growth is related to the chemoattractant effect of C.jejuni on formate.In summary,through a novel ligand screening strategy,this study found that the ligand signaling molecule directly and specifically binds to C.jejuni Tlp1 is formate.As far as we know,this is the first report on the specific binding of dCache-type chemoreceptors to formate and the physiological function of formate chemotaxis.This study reveals the molecular mechanism and physiological significance of formate chemotaxis and will contribute to further research on the function of microbial chemotaxis in pathogen-host interactions.