Year: 2013
Type: dissertation
Abstract: Daptomycin is a lipopeptide antibiotic that contains 13 amino acids and an N-terminally attached \nfatty acyl residue. The antibiotic kills Gram-positive bacteria by membrane depolarization. \nIt has long been assumed that the mode of action of daptomycin involves the formation \nof oligomers on the bacterial cell membrane; however, at the outset of my studies, this had not \nbeen experimentally demonstrated. \nIn the work described in this thesis, I have used fluorescence energy transfer (FRET) between \nnative daptomycin and an NBD-labeled daptomycin derivative to demonstrate that the \nantibiotic indeed forms oligomers on bacterial cell membranes. In a liposome model, oligomer \nformation depends on calcium and on phosphatidylglycerol (PG). The oligomer forms rapidly \nand is stable for a length of time longer than required for the bactericidal effect. Through variation \nof the ratio of FRET donor (native daptomycin) and acceptor (NBD-daptomycin), I have \ndetermined that the oligomer consists of approximately 6–7 molecules, or, depending on the \nstructure of the oligomer, possibly up to twice that number. \nOligomer formation on liposomes and on bacterial membranes was confirmed using excimer \nfluorescence of a perylene-labeled daptomycin derivative. Excimer fluorescence was \nalso used to demonstrate a stoichiometric interaction between daptomycin and PG. \nIt has previously been shown that the bactericidal activity of daptomycin requires calcium \nand correlates with the concentration of PG in the bacterial cell membrane; these requirements \nmirror those observed here for oligomer formation. Furthermore, membrane permeabilization \nis selective, and electron microscopy of bacterial membranes exposed to daptomycin has \nrevealed no discontinuities or accretions of electron density. Both of these findings suggest \nformation of a small membrane lesion, which is compatible with the small size of the oligomer \nthat was determined here. In conjunction with these previous findings, the experiments contained \nin my thesis strongly suggest that the oligomer is the bactericidal form of daptomycin.
Source: UWSpace (University of Waterloo)
Author: Jawad K. Muraih
Language: English
Subfield: Microbiology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
Funder: University of Waterloo
OpenAlex ID: W769980331