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The polyphemusin family of antimicrobial peptides : activity through structure and membrane interactions Powers, Jon-Paul Steven

Abstract

Cationic antimicrobial peptides are a class of small, positively charged peptides known for their broad-spectrum antimicrobial'activity. These peptides have also been shown to possess anti-viral activity and, most recently, the ability to modulate the innate immune response. Peptides from the horseshoe crab, the polyphemusins and tachyplesins, are some of the most active peptides isolated from nature, possessing high antimicrobial activity against both Gram negative and Gram positive bacteria. Despite their excellent antimicrobial activity, the mechanism of action is not well defined. The goal of this thesis was to investigate structure-activity relationships of two representative polyphemusins, polyphemusin I (PM1) and PV5. The solution structures of PM1 and PV5 were determined using ¹H-NMR spectroscopy. Both peptides were found to form amphipathic, β-hairpin conformations stabilized by disulfide bond formation. A linear analogue, PM1-S, with all cysteines simultaneously replaced with serine, was found to be dynamic in nature with no favoured conformation. The antimicrobial activity of PM1-S was found to be 4 to 16-fold less than that of PM1 and corresponded with a 4-fold reduction in ability to depolarize bacterial cytoplasmic membranes. Although both PM1-S and PM1 were able to associate with lipid bilayers in a similar fashion,only PM1-S lost its ability to translocate model membranes.. It was concluded that the disulfide constrained, β-sheet structure of PM1 is required for maximum antimicrobial activity. Model membrane interactions of PM1 and PV5 were investigated using fluorescence spectroscopy and differential scanning calorimetry (DSC). Both peptides were found to have limited affinities for neutral vesicles containing phosphatidylcholine (PC), and/or cholesterol or phosphatidylethanolamine (PE); however partitioning was increased through the inclusion of anionic phosphatidylglycerol (PG) into PC vesicles. DSC studies supported the partitioning data and demonstrated that neither peptide interacted readily with neutral PC vesicles while both peptides showed affinity for negatively charged membranes incorporating PG, causing significant perturbation of these membranes. The affinity of PV5 was much greater than that of PM1, as the pretransition peak was absent at low peptide to lipid ratios and the reduction in enthalpy of the main, gel to liquid crystalline transition was greater than that produced by PM1. Both peptides decreased the lamellar to inverted hexagonal phase transition temperature of PE, indicating the induction of negative curvature strain. Using a biotin-labeled PM1 analogue and fluorescence microscopy, it was demonstrated that the peptide accumulates in the cytoplasm of wild type E. coli within 30 min after addition without corresponding membrane damage. Comparison between peptide treated and untreated cells revealed that DNA in peptide treated cells appeared less condensed than untreated cells and was found at the periphery of cells. Collectively, the results presented here, combined with previous findings that PM1 promotes lipid flip-flop but does not induce significant vesicle leakage, ruled out a membrane disruption mechanism of antimicrobial action for PM1. In addition, the localization of a biotin-labelled PM1 within the cytoplasm of E. coli following peptide treatment indicated that the polyphemusins are capable of translocating biological membranes and may act on cytoplasmic components as their target of antimicrobial action.

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