Antimicrobial Peptide Daptomycin and its Inhibition by Pulmonary Surfactant: Biophysical Studies using Model Membrane Systems

Abstract

Daptomycin is a lipopeptide antibiotic that is clinically used to treat severe infections caused by Gram-positive bacteria. It is highly potent against resistant strains of bacteria such as methicillin-resistant Staphylococcus aureus. However, in cases of community-acquired pneumonia (a leading cause of death worldwide), daptomycin is somehow inhibited by lung surfactant and therefore unable to exert its bactericidal activity against Streptococcus pneumoniae, the primary cause of this disease. This thesis presents the successful development of lipid model systems to mimic the lipid composition of S. pneumoniae bacterial membranes, human cell membranes, and both synthetic and natural lung surfactant. Experiments were performed that help to elucidate the basis for daptomycin’s inhibition by lung surfactant, culminating in a new, detailed model of daptomycin sequestration that summarizes the findings from these studies. Daptomycin is believed to be sequestered by lung surfactant and has been shown to insert into this surfactant. Fluorescence spectroscopy experiments were used to test the interaction of daptomycin with different lipid model membranes in the presence of calcium. The results provided strong evidence that daptomycin is sequestered by lung surfactant and that daptomycin has a similar affinity for both lung surfactant and bacterial membrane, suggesting these two entities play a competitive role in the binding of daptomycin. Increased emission spectra for daptomycin and bacterial membranes at higher concentrations of calcium suggest that calcium may remove an inhibited late step of daptomycin pore formation that has previously been shown. Using Langmuir-Blodgett monolayer techniques, studies were performed on how daptomycin affects monolayer properties. Compression isotherms provided data on monolayer compressibility, and it was found that daptomycin and calcium reduce the compressibility of lung surfactant monolayers, possibly improving its function. Constant-area insertion assays provided additional data that verified daptomycin’s avid binding to lung surfactant at low calcium concentrations. Scanning probe microscopy techniques were employed to obtain atomic force microscopy and Kelvin probe force microscopy images for monolayers in air. In the presence of daptomycin and calcium, the lung surfactant monolayers exhibited multilayer formation and increased electrical surface potential. Atomic force microscopy images taken of model lipid bilayers in liquid show multi-bilayer formation for the lung surfactant bilayers in the presence of daptomycin and calcium. This provides further evidence that daptomycin and calcium induce multilayer formation in lung surfactant. These findings allowed for the development of a novel model of daptomycin inhibition by lung surfactant. In the presence of physiological levels of calcium, daptomycin binds to lung surfactant and is sequestered. This binding causes a decrease in lung surfactant compressibility, allowing it to easily form multilayers that effectively reinforce the sequestration of daptomycin. The lipid models, methods, and experimental protocols developed in this thesis will help foster future studies in the field of membrane biophysics.