Functional And Structural Characterization Of Apidaecin And Its N-terminal And C-terminal Fragments And Extracellular Production Of Apidaecin In Lactococcus Lactis

Apidaecins(apidaecin-type peptides)refer to a series of small,proline-rich (Pro-rich),18- to 20-residue peptides produced by insects.They are the largest group of Pro-rich AMP known to date.Apidaecins are active against gram-negative bacteria and show potential to be used as new candidates of peptide antibiotics.The present study elucidated the structure information of apidaecin through CD spectrum,the function target of apidaecin on E.coli cells and the function domain of its N-terminal and C-terminal.Apidaecin was also successfully extracellular produced in L.lactis. The main contents and results are as follows:1.Analysis of the function target of apidaecin on E.coli cell and the function domain of its N-terminal and C-terminal fragmentsTwo aspects were studied to elucidate the functional and structural characterization of apidaecin and its N-terminal and C-terminal fragments:(1) Functions of the N-terminal and C-terminal fragments of apidaecin were firstly studied by measuring their antibacterial activity,their ability to enter E.coli cells and their effects on the activities ofβ-galactosidase and alkaline phosphatase.The results indicated that neither the N-terminal nor the C-terminal of apidaecin contains intracellular delivery unit or active segment.(2)The effect of apidaecin on the ATPase activity of DnaK,and the interactions of apidaecin with E.coli lidless DnaK and DnaK D-E helix were studied by CD spectrum.Results showed that apidaecin could interact with the E.coli lidless DnaK protein and stimulate its ATPase activity,but not with E.coli DnaK D-E helix.This indicated that the antimicrobial activity of apidaecin may be shown by stimulating the ATPase activity of DnaK by binding to its conventional substrate-binding site,to decrease its cellular concentration of DnaK by competing with natural substrates and inhibit the enzymes' activities ofE.coli cells.It is the first study to suggest that the apidaecin-binding site of DnaK is the conventional substrate binging site.The total proteins of control and apidaecin-treated E.coli cells were separated with 2-DE.Gel images were analyzed by PDQuest soft and results showed that 9 differentially expressed protein spots were detected,and among them,4 were up-regulated,4 were down-regulated,and 1 unmatched.2.Investigation of the cytotoxicity of apidaecin on Caco-2 cells and intestinal epithelial cells of tilapia(Oreochromis niloticus)in vitroThe intestinal epithelial cells of tilapias,Oreochromis nilotica,and were cultured primarily and used as models,with the Caco-2 cell,to study the effect of different concentration apidaecin(Trial 1:10μg/ml,Trial 2:20μg/ml,Trial 3:30μg/ml)on the cells viability,livability,permeability,cytosolic free calcium and membranous PLA2 activity.Result showed that apidaecin within the concentration of 30μg/ml showed no significant effect on both Caco-2 and epithelial cells of tilapias comparing with the control.Furthermore,the effect of apidaecin on growth morphological variety of Caco-2 and intestinal epithelial cells of tilapias was also observed and the result did not show any morphological abnormity.This indicated that apidaecin within the concentration of 40μg/ml was harmless to the growth of Caco-2 and epithelial cells of tilapias for a period of determinate time.3.Extracellular Production of apidaeein in L.lactisHeterologous production of antibacterial peptide apidaecin was studied in the food-grade bacterium Lactococcus lactis.Expression of the apidaecin gene driven by the Lactococcal nisA promoter and the Usp45 signal peptide gene resulted in efficient extracellular production of apidaecin in L.lactis NZ9000.After 4 h induction with 10 ng/ml nisin,the secretory expression yield of recombinant apidaecin reached about 10μg/ml while no apidaecin was detected in the un-induced control.Recombinant apidaecin was purified by gel filtration on a Sephacryl S-100 HR column and semi-preparative RP-HPLC on a C18 Vydac column and about 10 mg pure active apidaecin was obtained from 1 L fermentation culture.15%/6M urea Tricine-SDS-PAGE indicated that recombinant apidaecin protein moleculer weight is about 2.0 kDa.ESI-MS/MS of purified apidaecin demonstrated a single large signal of the molecular ion[M+2H⁺]²⁺at 979.5.0 m/z,means a molecular weight of 1957.0 Da which is identical to that of the putative protein(1957.3 Da).The recombinant apidaecin showed antimicrobial activity against E.coli K88.This is the first report on the extracellular production of apidaecin in L.lacits.Expression and delivery of apidaecin in the food-grade L.lactis would facilitate the widespread application of apidaecin in the control and prevention of Gram-negative bacteria infection.