Molecular Design Of The Frog Antimicrobial Peptide Palustrin-OGL And Biological Activity,Antimicrobial Mechanism And Recombinant Expression Of Derivatives

Palustrin-OG1(OG1) is a31-amino acid cationic antimicrobial peptide derived from the skin secrection of Odorrana grahami. Our previous study demonstrated that the antimicrobial activity of OG1was higher than that of milk-derived antimicrobial peptides. However, OG1showed high hemolytic activity. Therefore, molecular design of OG1was carried out, and antimicrobial activities and cytotoxicities of OG1and its derivatives including OG2,OG2N, OG2A and OG2W were determined. Besides, the antimicrobial mechanism of OG1, OG2and OG2N were studied. Furthermore, Escherichia coli and Pichia pastoris expression systems were used to express the derivative OG2. The main results are as follows:1. Molecular design of OG1and screen of the modified peptidesThe protein blast and amino acid conservation analysis revealed that OG1belonged to the Brevinin superfamily which has conserved C-terminal Cys residues and the typical "Rana box" Circular dichroism spectroscopy showed that OG1was α-helical. On the basis of physichemical property analysis of OG1, the molecular design of OG1was carried out through amino acid deletions and subtitutions, deletion of a disulfide bridge, increase of amphipathicity and net positive charge and decrease of hydrophocitiy. Four derivatives named OG2,OG2N,OG2A and OG2W were obtained where the former three peptides were also α-helical and OG2W was β-sheet. Compared with OG1, the amphipathicities and net positive charges of four derivatives increased whereas the hydrophocitiies decreased according to the analysis result using CCS model. The antimicrobial activity test showed that all derivatives showed improved activity against harmful bacteria. The derivatives OG2and OG2N showed1～16times antimicrobial activity of OG1. Furhtermore, compared with OG1, the antimicrobial activity of OG2and OG2N against Bacillus subtilis showed a32-or8-fold decrease, respectively. The cytotoxicity assay showed that the derivatives OG2and OG2N exerted much lower cytotoxicities against porcine peripheral blood mononuclear cell than OG1(p<0.05) while OG2A and OG2W still had high cytotoxicities. The analysis of phsichemical properties and biological activities suggested that the low hydrophobicity of OG2and OG2N was responsible for their low cytotoxicities while the increase of net positive charge and improvement of amphipathicity contributed to the enhancement of antimicrobial activity.2. Antimicrobial mechanism of OG1and its modified peptide OG2and OG2NTransmission electron microscopy was used to observe the micrographs of S. aureus ATCC25923treated with OGl, OG2, and OG2N. OG1and OG2N at1－MIC damaged cell wall and cytoplasmic membrane with the leakage of cell contents while cells treated with1xMIC OG2showed some clear zones. No cells were observed when treated with3×MIC peptides, indicating that all peptides exerted damage on cell membranes. As to micrographs of E. coli ATCC25922, cells treated with1xMIC peptides showed large clear zones, cell wall roughening and significant decrease of electron density in cytoplasmic region.SYTOX uptake assay was used to evaluate the permeabilization activity of OG1, OG2and G2N. Compared with OG1, the modified peptides OG2and G2N showed lower permeabilization activity on E.coli and S. sureus cell membranes. However, there were still considerable permeabilizations. The cytotoxicity assay and membrane mechanism study implicated that the modified peptides OG2and G2N had highly selective permeabilization whereas OG1showed no selective activity.DNA binding assay and cell-free protein synthesis assay were used to evaluate whether OG1, OG2and OG2N had potential intracellular targets. The binding ability of OG2and OG2N with pGEM-3Z was higher than that of OG1, but all peptides had no effect on inhibition of protein synthesis and protein activity, indicating that OG2and OG2N may inhibit DNA replication rather than inhibit transcript, translation and biological activity of protein. Therefore, the modified peptides OG2and OG2N killed bacteria mainly by damage on cell membranes.3. Expression of OG2in E.coli and its purification and antimicrobial activityE. coli was the main pathogens in livestock and poultry production, and the derivative OG2showed not only twice antimicrobial activity against E. coli K88but also much lower antimicrobial activity against Bacillus subtilis than OG2N. Furthermore, there was no significant difference in cytotoxicitiy between OG2and OG2N. Therefore, the derivative OG2was taken as the target peptide for recombinant expression.Firstly, fusion partners including TrxA, Synechocystis sp DnaB (inteinl), Mycobacterium xenopi GyrA (intein2) and small ubiquitin-related modifier (SUMO) were used to express OG2in E.coli. Four recombinant proteins were successfully expressed. Trx, inteinl and intein2improved solubility of fusion proteins, with the yield of soluble recombinant proteins at50,44and24.5mg/L, respectively.Secondly, both enterokinase (EK) and tobacco etch virus (TEV) protease were used to cleave corresponding fusion proteins to obtain an efficient cleavage. Fusion proteins were purified by Ni-NTA affinity chromatography, desalted on Sephadex G25and concentrated by ultrafiltration, achieving85%purity. When the cleavage site was along with N-terminus of OG2, Trx-EK-OG2was less amenable to EK. In contrast, Trx-TEV-OG2, where TEV protease cleavage site was introduced between EK site and OG2, was completely cleaved by EK after4h cleavage or by TEV after16h cleavage. The released OG2from Trx-TEV-OG2after4h cleavage showed antimicrobial activity against S. aureus. However, the released OG2from TEV protease cleavage was not stable while that from EK cleavage had10additional amino acids at N-terminus.Finally, the effect of intein on expression and purification of OG2in E. coli was studied. Two proteins including OG2-intein2-CBD (C-terminal fusion expression) and CBD-inteinl-OG2(N-terminal fusion expression) were successfully expressed. The soluble OG2-intein2-CBD in total target protein increased from30%to70%, when IPTG concentration decreased from0.5mM to0.1mM and induction temperature decreased from37℃to30℃. The yield of soluble OG2-intein2-CBD and the total yield of OG2-intein2-CBD were up to75mg/L and107.1mg/L, respectively. The yield of target peptide OG2was8.9mg/L. About42%CBD-intein-OG2was soluble after6-h induction by0.1mM IPTG at20℃. Therefore, the decrease of IPTG concentration and induction temperature improved the solubility. OG2was purified by chitin affinity chromatography, cleaved on-column by DTT and desalted on Sephadex G10. More than85%OG2-intein2-CBD was cleaved, and the released OG2showed high activity against E. coli. The CBD-intein-OG2was less amenable due to the inhibition of cleavage by the N-terminal Lys. Therefore, intein2was the best fusion partner to express and purify OG2in E. coli. 4. Expression of OG2in yeast and its purification and antimicrobial activityFirstly, OG2and PS-OG2were extracellularly expressed in P. pastoris. Three genes including OG2(38%GC), OG2O(44%GC) and PS-OG(50%GC) were optimized and obtained by SOE-PCR. OG2O and PS-OG2were expressed in YPM using pPICZaA and GS115, and the supernatant showed antimicrobial activity against S. aureus. However, no expression and activity were detected when OG2O was expressed in BMMY, indicating that GC content and induction medium affected expression. Meanwhile, PS-OG2was also successfully expressed and secreted into YPM medium and showed antimicrobial activity.Secondly, OG2was expressed intracellularly as tandem expression cassettes in P. pastoris. Tandem expression plasmids (pAO815-OG2,1-,2,4-and8-copy) were constructed using Bgl Ⅱ and Bam Ⅰ and transformed into GS115. The agar diffusion test was used to test cell lysis supernatant of recombinant strains against S. aureus. The activity was enhanced when the copy number of gene increased. The result of expression of OG2in Saccharomyces cerevisiae was similar to that in P. pastoris. The supernatant of cell lysis from recombinant strain showed higher antimicrobial activity than that from host strain. However, the host strain without target gene also showed activity.Finally, HIS-tagged OG2was expressed in P. pastoris intracellularly. GS115-PICZ-OG2-HIS was induced in MMH medium and purified using Ni-NTA. Both SDS-PAGE and Western blot showed the visible expression of OG2about3.2kDa, indicating that OG2gene can successfully transcript and translate in P. pastoris.In summary, after molecular design of OG1which has very high cytotoxicty, two modified peptides, OG2and OG2N, showed higher antimicrobial acidity and lower cytotoxicity than OG1. Compared with OG1, OG2and OG2N had high seclective activity on cell membrance, and OG2showed a higher selectivity of antimicrobial activity. Furthermore, OG2was successfully expressed in both E. coli and yeast and showed antimicrobial activity, which provides a cost-effective way to obtain adequate OG2for structure-function relationship study with a goal of developing a new antimicrobial agent.