Interactions of a lytic peptide with supported lipid bilayers investigated using surface-selective techniques

© 2015 Dr. Andrew Cyrus RapsonThe development of antimicrobial resistance, which is the ability of microorganisms to prevent cell death by antimicrobial agents (i.e. antibiotics, antivirals), is an increasing problem. Traditional antimicrobial drugs target aspects of microorganisms responsible for infectious diseases cell structure than can be altered, greatly reducing their cell-killing efficiency. To combat this, research has been performed into the application of naturally-occurring antimicrobial peptides, sourced from venoms or host-defence systems of plants and animals. These peptides display broad-spectrum cell-killing activity by directly attacking the lipid matrix of cell membranes, thereby bypassing the means through which cells develop resistance. In this study, a proline-substituted, fluorescently-labelled analogue of the antimicrobial peptide melittin (a major component of venom from the European honey bee) was designed and synthesised. The, analogue, named melittin P14K-Alexa 430 (MK14-A430), displayed twice the membrane-lytic activity of native melittin through means of a lipid vesicle dye-release assay. The interactions of MK14-A430 with lipid membranes consisting of 1,2-dipalmitoyl-sn-glycero-3-phoshatidylcholine (DPPC) were investigated using circular dichroism and surface-selective analytical techniques. The structure and orientation of MK14-A430 were studied by circular dichroism and oriented circular dichroism. In contrast to native melittin, MK14-A430 displayed high helicity under solution conditions mimicking physiological conditions. This helicity was retained in small unilamellar vesicles, except at low peptide density in ordered DPPC vesicles. In oriented lipid multibilayers, MK14-A430 inserted completely into a transmembrane orientation in fluid-phase DPPC, while only a fraction inserted in ordered-phase bilayers (up to an observed maximum of 25%). The interaction mechanism of MK14-A430 with supported lipid bilayers (SLBs) was studied using quartz crystal microbalance with dissipation monitoring. MK14-A430 initially associated with all SLBs in a surface-aligned state, after which different behaviour was observed depending on the lipid phase. Similar to the oriented circular dichroism experiments, MK14-A430 inserted completely into a transmembrane orientation in fluid-phase DPPC, while a significant fraction in a membrane surface-aligned state was observed for both ordered-phase SLBs. The predominating orientation of MK14-A430 could be reversibly changed with the lipid phase: transmembrane above the phase transition temperature and surface-aligned below. No mass removal indicative of detergent-like membrane solubilisation was observed. The location and mobility of MK14-A430 was assessed through fluorescence of the AlexaFluor 430 label by time resolved, evanescent wave-induced fluorescence spectroscopy and anisotropy. MK14-A430 penetrated the lipid headgroup structure in pure, ordered-phase DPPC membranes but was located near the headgroup-water when cholesterol was included. MK14-A430 formed lytic pores in DPPC SLBs, and an increase in pore formation with incubation time was observed through an increase in polarity and mobility of the probe. When associated with the DPPC-Chol SLB, the probe displayed polarity and mobility that indicated a population distributed near the lipid headgroup-water interface with MK14-A430 arranged predominantly in a surface-aligned state. The information from this study showed that the lytic activity MK14-A430 occurred through a pore-forming mechanism. The lipid headgroup environment experienced by the fluorescent label where MK14-A430 displayed pore information indicated that pore formation was best described by the toroidal pore model