Studies On Molecular Design, Biological Activities, And Mechanisms Of β-Sheet Antimicrobial Peptides

In this current study, two research ideas were adopted to study the interactions between structure and function of β-sheet antimicrobial peptides and explore the biological activities and antibacterial mechanisms of antimicrobial peptides. The one idea designing antimicrobial peptides is as follows. On the one hand, according to the amino acid composition and structural characteristics of the antimicrobial peptides, a series of cyclic P-hairpin-like antimicrobial peptides were designed by positioning hydrophobic (V) or positively charged residues (R) in the following sequence template:Ac-C(VR)nDPG (RV)nC-NH2(n=1,2,3,4, or5). A short two-residue loop segment (DPG) was used to link the two-strand antiparallel β-sheet. The β-hairpin was further stabilized by a disulfide bridge. On the other hand, studies were further performed to test the effect of strand length on the biological activity. The four representative hydrophobic amino acids (I, F, W, and P) and R were chosen to design the XR antimicrobial peptide (XR)nH(RX)n-NH2,(n=1,2; X is I, F, W and P; H represents CRRRFC). The other idea is the transformation of natural antimicrobial peptides. Linear AvBD4(RL38) without disulfide bonds was used as a model peptide, and a series of derivatives with different length were obtained by truncation. The hydrophobicity scale of truncated peptides were increased by replacing cysteines (C) with isoleucine (I) and the biological functions of these derivatives were investigated. At the basis of preliminary studies, short peptides of the AvBD4carboxyl terminal were further transformed by replacing different location of amino acids with arginine (R). The interaction of increasingly net charge and biological functions were also investigated. High cell selectivity of de novo peptide and derivatives of AvBD4were screened by in vitro experiment. To elucidate the peptide-membrane interaction, synthetic lipid vesicles and whole bacteria were employed. In addition, the interactions of the peptides with DNA were studied. At last, screened peptide was expressed by Pichia pastoris.The relationship between the length of five β-hairpin-like antimicrobial peptides and the antibacterial activity was described by a quadratic function. Longer-chain peptides are likely to stimulate hemolytic activity. The VR3peptide with low cytotoxicity to mammalian cells was a potent killer to both gram-negative and gram-positive bacteria. With the increasing of β-strand length, the antimicrobial activity and cytotoxicity were increasing. IR2including aliphatic amino acid I displayed the highest cell selectivity.The linear AvBD4derivatives (RL38) has great antimicrobial activity, and the truncation resulted in the reduction of antibacterial activity against gram-negative and gram-positive bacteria, especially pathogenic Salmonella Pullorum C79-13. GLI23, GLI18and GLI13were derived by the replacement of cysteines with isoleucines of GL23, GL18and GL13which are the C-terminal truncated sequence of RL38. Results showed that antibacterial activity of GLI23, GLI18and GLI13against the observed bacteria including Salmonella Pullorum C79-13recovered to the original level of RL38. To increase the number of net positive charge of GLI13, proline (P), tyrosine (Y), serine (S), and glycine (G) of GLI13were substituted with arginine (R). The increase of net charge by substituting P with R improved antimicrobial activity of GLI13-5. However, antimicrobial activities of GLI13-6, GLI13-7, and GLI13-8were not further increased and still weak against S. Pullorum C79-13. The minimum hemolytic concentration of RL38and a series of derivatives of C-terminus terminal are16uM and over128uM, respectively. Therapeutic index (TI) is the ratio of the MHC to the geometric mean of LCs (GM). Larger values indicate greater cell specificity. Collectively, GLI23and GLI13-5have higher cells selectivity than other derivatives.The peptides kept different degrees of antimicrobial activity after the treatment with salt, protease and heat. CD spectra indicated that β-hairpin-like antimicrobial peptides appeared to be β-hairpin and β-sheet structure in10mM sodium phosphate buffer, pH7.4(mimicking an aqueous environment),50%TFE (mimicking the hydrophobic environment of the microbial membrane), and30mM SDS micelles (negatively charged prokaryotic membrane comparable environment,). XR antimicrobial peptides displayed random structure in aqueous and showed different secondary structure in TFE and SDS buffer, respectively. The derivatives of AvBD4displayed a typical spectra of an unordered conformation in aqueous. Only RL38and GLI13-5showed some β-sheet structure, and other derivatives indicated the a-helical-rich structure in bacteria-mimicking membranes.An intraperitoneal model of sepsis caused by Salmonella typhimurium was established and the peptide VR3was administered intraperitoneally1h afterwards or prior to bacterial infection. The mortality and peritoneal bacterial counts were significantly decreased for the groups administered VR3with2.5mg/kg and5mg/kg of body weight, compared with the PBS-treated group. The peptide cured bacterial sepsis in a dose-dependent manner. The group injected2.5mg/kg VR3of bodyweight in advance, showing protection to the mice from Salmonella typhimurium infection as well.Two kinds of vesicles (either PE/PG, a phospholipid composition typical of bacteria, or PC/cholesterol, a phospholipid composition used to mimic the outer leaflet of human erythrocytes) were prepared to compare membrane-binding affinities of the peptides. The peptide VRW3(AC-C(VR)3DPG(RV)3CW-NH2) was derived from the peptide VR3by adding W to its C-terminus to monitor lipid-peptide interactions using tryptophan fluorescence. A larger blue shift (17and18) of VR3or melittin was observed in the presence of PE/PG phospholipid vesicles. However,4and16blue shifts were observed for VR3or melittin in the presence of PC/cholesterol phospholipid vesicles. The blue shift (15,16and15) of AvBD4derivatives were observed in the presence of PE/PG phospholipid vesicles.5,1and1blue shifts were observed for AvBD4derivatives in the presence of PC/cholesterol phospholipid vesicles. Peptide-membrane interaction was examined by using the membrane potential-sensitive dye NPN and DiSC3-5. Results showed that the peptides disrupted the integrity of cell membranes and enhanced the potential to depolarize the cytoplasmic membranes. Electron microscopy results showed that the VR3, IR2, GLI23, and GLI13-5can damage cell membrane, and led to leak out of cytoplasm. Gel retardation experiment displayed that AvBD4derivatives had DNA binding activity at low concentrations, suggesting that peptide may interact with intracellular molecular.The nucleic acid sequence corresponding to the GLI23peptide was synthesized according to the codon bias of Pichia pastoris, and then was cloned into the expression vector pGAPHaM. The constructed recombinant expression vector pGAPHaM-GLI23was successfully transformed into Pichia pastoris GS115by electroporation. Antimicrobial activity assay showed that the product of expression inhibited the growth of bacteria, yet the yield of antimicrobial peptide in Pichia pastoris was not very high.In conclusion, a series of different length of β-hairpin-like antimicrobial peptides were designed, and VR3with16amino acid residues was screened for its high cell selectivity. XR antimicrobial peptides were designed by combining different hydrophobic amino acid and hydrophilic amino acid R, and IR2with highest cell selectivity was selected. In addition, natural antimicrobial peptide AvBD4was transformed by truncation and amino acid substitutions. Results showed that GLI23and GLI13-5have the highest cell selectivity. With structure-fuctions relationships, peptide-membrane interactions, and peptide expression in Pichia pastoris examined are to be explored. And the work will provide the guide for antimicrobial peptides research in clinical and livestock application.