Study On Molecular Modification Of The PMAP-36 And Its Mechanism Of Action

Pig myeloid antimicrobial peptide-36(PMAP-36), which was identified by cDNA cloning from RNA pools of pig bone marrow, is a highly positively charged 36-residue antimicrobial peptide(AMP). Based on the amino acid composition and structural characteristics of PMAP-36, a series of short AMPs with imperfect amphipathicity or low α-helical propensity were designed and characterized for their biological activities and mechanisms of action. In addition, a Pseudomonas aeruginosa-targeted AMP was designed and evaluated for its bactericidal activity. Further, a series of short peptides containing heptad repeats to mimic naturally occurring AMP sequences were designed and determined for their anti-pseudomonal activities. The main results are shown as follows:(1) Design of imperfectly amphipathic a-helical AMPs. Amphipathicity is traditionally believed to be crucial to the de novo design or systematic optimization of AMPs. In this study, a series of short a-helical AMPs with imperfect amphipathicity were designed to augment the arsenal of strategies and to gain further insights into their antimicrobial and hemolytic activity. These imperfectly amphipathic a-helical AMPs were designed by replacing the paired charged amino acid residues on the polar face of an amphipathic peptide with Trp residues on the basis of a-helical protein folding principles. PRW4, an imperfectly amphipathic a-helical AMP with hydrogen bonds formed by paired Trp residues, was observed to be more selective towards bacterial cells than toward human red blood cells. PRW4 was also effective against Gram-negative and Gram-positive bacteria, and fluorescence spectroscopy, flow cytometry, scanning electronic microscopy(SEM)and transmission electronic microscopy(TEM) indicated that PRW4 killed microbial cells by permeabilizing the cell membrane and damaging their membrane integrity. Therefore, disruptive amphipathicity had excellent potential for the rational design and optimization of AMPs with promising antimicrobial activities.(2) Design of low amphipathic AMPs with different a-helical propensity. Previous studies indicated that disruptive amphipathicity formed by replacing the paired charged amino acid residues on the polar face of an amphipathic helix with Trp residues linked with hydrogen bonds on the basis of a-helical protein folding principles endowed the AMPs with increased cell selectivity. In an attempt to augment and hone this strategy further, a series of imperfect amphipathic peptides by placing different types of amino acid residues at the hydrogen bond linked positions of a-helix structures were designed to characterize their antimicrobial properties and mechanism of action. The D-Trp-substituted sequence(PRW4-d) showed greater antimicrobialpotency than Cys-(C4), Asp-(D4), Ile-(I4), and Pro-(P4) substituted sequences, comparable to the L-Trp-substituted parent sequence(PRW4). Furthermore, the total replacement of Lys residues with Arg residues along the peptide sequence(PRW4-R) exhibited enhanced antimicrobial activity and cell selectivity. In addition, no cytotoxicity was observed among these synthetic peptides.PRW4-d and PRW4-R maintained their activities in the presence of physiological salts and human serum. The fluorescence spectroscopy, flow cytometry, and electron microscopy observations indicated that the optimized sequences exhibited excellent antimicrobial potency by inducing cytoplasmic membrane potential loss, membrane permeabilization and disruption. Therefore, the results could be useful for designing short AMPs with great antimicrobial activity and cell selectivity.(3) Importance of Trp in an amphipathic peptide. Here, it was found that simple substitution of amino acids in the middle position of the hydrophobic face of an amphipathic peptide RI16, the N-turminal of PMAP-36, with Trp(T9W) considerably transformed into an antimicrobial peptide specifically targeting P. aeruginosa. Minimal inhibitory concentration(MIC) results demonstrated that T9 W had a strong and specifically antimicrobial activity against P. aeruginosa, including antibiotic-resistant strains, but was not active against E. coli, S. typhimurium, S. aureus and S.epidermidis.T9W also displayed high activity with lethal concentration(LC) of 1 to 4 μM against P.aeruginosa, including ciprofloxacin-, gentamicin-, and ceftazidime-resistant strains, even in the presence of 50 to 300 m M of Na Cl, 1 to 5 m M of Ca2+, or 0.5 to 2 mM of Mg2+. The time-kill curves(TKC) analysis demonstrated concentration-dependent activity, with T9 W achieving complete killing in less than 30 min at 1 × LC and in less than 5 min at 4 × LC. Combination TKC analysis additionally demonstrated a synergistic effect with ciprofloxacin and gentamicin. The selectivity of T9 W was further supported by its ability to specifically eliminate P. aeruginosa in a coculture with macrophages without toxicity to the mammalian cells. The results from fluorescent measurement indicated that T9 W bound to LPS and induced P. aeruginosa membrane depolarization, and microscopic observations and flow cytometry further indicated that T9 W targeted the P. aeruginosa cell membrane and disrupted the cytoplasmic membrane integrity,thereby causing cellular contents release and leading to cell death. Furthermore, the strong antibiofilm activity was also observed with the peptide T9 W, which decreased the percentage of biomass formation in a dose-dependent manner. This study revealed the potential usefulness of T9 W as a novel antimicrobial agent against P. aeruginosa.(4) De novo design of heptadic peptides as antimicrobial agents. A series of short peptides containing heptad repeat sequence were designed to mimic naturally occurring AMP sequences.The designed peptides were active against a panel of P. aeruginosa strains, including antibiotic-resistant strains, as well as gram-positive S. aureus and gram-negative E. coli. Peptides with Leu-containing heptad repeat sequence exhibited more antimicrobial activity than peptides with Val-, Phe- or Trp-containing HRS. Optimized synthetic sequence LW3 also exhibited excellentselectivity for the microbial membrane, reduced sessile biofilm biomass, neutralized endotoxins,and remained activity under physiological salt concentrations. Moreover, these synthetic peptides permeabilized the bacterial membrane and disrupt membrane integrity. Therefore, these findings clearly demonstrated the potential of heptad repeat sequence templates as therapeutics in a wide range of localized, systemic, or external therapeutic applications.Collectively, in this paper, an alternative approach was proposed to the design and/or optimization of AMPs based on the principles of α-helical peptide/protein folding theory. A series of engineered peptides with imperfect amphipathicity or low helical propensity were synthesized and evaluated for their biological activities. PRW4 and PRW4-R shown broad-spectrum activities against Gram-negative and Gram-positive bacteria. While T9 W was demonstrated to be specifically targeting P. aeruginosa, including clinical and antibiotic-resistance strains, indicating that design of single-pathogen antimicrobial agents can be achieved by simple amino acid mutation in naturally occurring peptide sequences and suggesting a model of optimization/design of anti-pseudomonas drugs in which the Trp residue was a conserved element. In addition, the de novo designed heatadic peptides were highly selective, having great potential for use in infection-related applications. The results from peptide-membrane interactions presented that the optimized AMPs were membrane-lytic, permeabilizing the bacterial membrane and disrupting membrane integrity, thereby leading to cell death. These findings will pave the way for AMPs research in clinical and livestock application.