Small Molecules From Traditional Chinese Medicine Inhibit Peb1 Protein For Campylobacter Jejuni Adhension

Campylobacter jejuni is one of common pathogens causing bacterial diarrhea worldwide.The main effective control method for Campylobacter jejuni is still by using antibiotics.However,WHO guidance of development of new antibiotics issued the priority medicine-resistant pathogens in 2017.The fluoroquinolone antibiotic-resistant Campylobacter was defined as a second-class highly important pathogen,indicating the urgency to establish new methods to control Campylobacter infectionThis study used a conserved adhesion protein Pebl in C.jejuni as a target to search for small molecules from traditional Chinese medicines through virtual screening.To search for the small molecules,the recombinant protein rPebl was expressed and applied to prepare monoclonal antibodies against Peb1,which were used to develop the adherence-ELISA to screen the small molecules inhibiting the adhesion of Pebl1.Virtual screening of small molecules from traditional Chinese medicines by Campylobacter jejuni adhesion protein PeblVirulence factors of C.jejuni with reliable three-dimensional structure were screened by searching protein database and homology modeling.The adhesion protein Pebl with crystal structure analyzed were determined as the target protein.Due to the extensive biological activity of traditional Chinese medicines,4918 medicine-like small molecules picked from TCM Database were used as ligands.After predicting the active pockets,the spatial positions of the predicted Cavities and natural ligand aspartic acid were compared,and Cavity₄ was chosen as the docking center for this study.Structure-based virtual screening was performed by Autodock Vina software,the screened result showed that 4903 small molecules could spontaneously react with Peb 1 protein,including 3125 high-scoring compounds with an absolute affinity of 6 kcal/mol or more,which accounted for 63.54%of all small molecules by virtual screening,134 small molecules with an absolute affinity of 8 kcal/mol or more,accounting for 2.72%,which is much more efficient than conventional medicine screening.Our results demostrate that small molecules of traditional Chinese medicines are indeed worthy of screening and further study2.Preparation and identifiaction of monoclonal antibodies against Campylobacter jejuni adhesion protein PeblTo develop the in vitro adhesion model of Peb1,monoclonal antibodies against Peb1 were prepared.Firstly,the peb1 gene was ligated into the expression vectors pColdl and pGEX-6p-1,respectively,to construct recombinant bacteria BL21?DE3??pColdI-pebI?and BL21?DE3??pGEX-6p-1-peb1?.The recombinant bacteria were induced by IPTG,and the expression products were obtained after purified by affinity chromatography column.The mixed natural proteins containing Peb1 were extracted from C.jejuni by glycine-hydrochloric acid buffer?pH 2.2?.The purified protein rHis-Peb1 was used as the immunogen,rGST-Peb1 and crude extract of Campylobacter jejuni containing natural Pebl were used as the detecting antigens.The monoclonal antibodies against Pebl was prepared by lymphocyte hybridoma technique.Six hybridoma cell lines secreting mAbs stably against Pebl were screened by indirect ELISA,which were named as 1D6,2A5,3B5,4E1.4F8 and 5B9.Their ascites titers were all over 1:2000000,and their immunoglobulin subclass all belonged to IgG1.Western blot analysis confirmed that the six monoclonal antibodies prepared in this study could specifically recognize the native Pebl protein,with a good positive reaction with C.jejuni.The monoclonal antibodies against Pebl were successfully prepared in this study,which laid the foundation for further study of the detection and function of Pebl protein.3.Verification and evaluation of Pebl adhesion inhibitorIn order to verify the adhesion inhibitors of C.jejuni Pebl protein,rPebl bound to Hela-MEF was established for the adherence ELISA.The receptor Hela-MEF was coated with 3?g/mL at 4? overnight,and 1 ?g/mL adhesin rPebl was incubated with small molecules in advance,then the mixtures were added to adherence ELISA system to determine the inhibitors.Amentoflavone inhibited the adhesion of Pebl to Hela-MEF and showed a dose-dependent fashion.Therefore,amentoflavone was selected for further study.The SIC of amentoflavone to C jejuni is 256 ?g/mL detected by micro-broth dilution method;the chemotaxis experiment showed that C.jejuni couldn't escape from 0.1 mM amentoflavone.MTT assay indicated 0.1 mM amentoflavone doesn't affect Hela cell activity.There was no significant effect of the amentoflavone on the adherence of C.jejuni to Hela cell,but it inhibites the invasion of C.jejuni significantly?p<0.05?.The effect of amentoflavone on the biofilm of C.jejuni was also studied,and it is found that amentoflavone showed significantly inhibition ability to the formation of C.jejuni biofilm with a dose-dependent fashion.All the above results demostrate that amentoflavone has certain effects on the prevention and control of C.jejuni,and further studies are necessary to explore for more effective anti-adhesion or anti-virulence small molecules for the control of C.jejuni.