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Probing the interaction between daptomycin and model membranes Zhang, Jin


Daptomycin is the first approved antibiotic from the lipopeptide family, with antibacterial activity against a wide variety of Gram-positive bacteria. It is composed of 13 amino acids with a fatty acyl chain attached at the N-terminus. Although it has been approved for clinical usage for over two decades and a number of studies have worked on the mechanism, many details of the mode of action of daptomycin still remain to be understood. This thesis focuses on shedding light on: i) how daptomycin binds to membranes and whether it forms well-defined oligomers; and ii) whether once in the membrane, daptomycin causes leakage. We have used photon correlation spectroscopy (PSC) to determine the condition under which fusion among vesicles occurs in the presence of daptomycin. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPC/POPG) liposome is a better system as higher concentrations of Ca²⁺ is required to trigger membrane fusion. Based on these findings of membrane fusion caused by daptomycin, binding and kinetics experiments were conducted to study the interaction between daptomycin and the lipid membrane. The binding between daptomycin or NBD-labeled daptomycin and POPC/POPG or 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPC/DMPG) liposomes is strong, with micromolar dissociation constants. Förster resonance energy transfer (FRET) experiments were conducted under conditions where fusion is not present. The results show that the oligomerization number n is concentration-dependent. A two-stage equilibrium process is proposed when the daptomycin binds and oligomerizes in the membrane, in which daptomycin accumulates firstly in the membrane to form larger oligomers, facilitating further effects which lead to the cell death, such as the lipid extracting effect on the membrane. In addition, fluorescence experiments were conducted to investigate whether daptomycin causes leakage in POPC/PG and DMPC/PG liposomes, with or without the proton ionophore carbonyl cyanide m-chlorophenyl hydrazine (CCCP). Experiments using the ion-selective pore-forming aurein peptide were also done, as a control. For stable liposomes, daptomycin does not cause ion permeabilization of the membrane, suggesting that the membrane depolarization resulting from daptomycin is different from pore-formation by the aurein peptide. The work described in this thesis provides evidence leading to a clearer mechanism of action for daptomycin.

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