|dc.description.abstract||Daptomycin is a lipopeptide antibiotic that contains 13 amino acids and an N-terminally attached
fatty acyl residue. The antibiotic kills Gram-positive bacteria by membrane depolarization.
It has long been assumed that the mode of action of daptomycin involves the formation
of oligomers on the bacterial cell membrane; however, at the outset of my studies, this had not
been experimentally demonstrated.
In the work described in this thesis, I have used fluorescence energy transfer (FRET) between
native daptomycin and an NBD-labeled daptomycin derivative to demonstrate that the
antibiotic indeed forms oligomers on bacterial cell membranes. In a liposome model, oligomer
formation depends on calcium and on phosphatidylglycerol (PG). The oligomer forms rapidly
and is stable for a length of time longer than required for the bactericidal effect. Through variation
of the ratio of FRET donor (native daptomycin) and acceptor (NBD-daptomycin), I have
determined that the oligomer consists of approximately 6–7 molecules, or, depending on the
structure of the oligomer, possibly up to twice that number.
Oligomer formation on liposomes and on bacterial membranes was confirmed using excimer
fluorescence of a perylene-labeled daptomycin derivative. Excimer fluorescence was
also used to demonstrate a stoichiometric interaction between daptomycin and PG.
It has previously been shown that the bactericidal activity of daptomycin requires calcium
and correlates with the concentration of PG in the bacterial cell membrane; these requirements
mirror those observed here for oligomer formation. Furthermore, membrane permeabilization
is selective, and electron microscopy of bacterial membranes exposed to daptomycin has
revealed no discontinuities or accretions of electron density. Both of these findings suggest
formation of a small membrane lesion, which is compatible with the small size of the oligomer
that was determined here. In conjunction with these previous findings, the experiments contained
in my thesis strongly suggest that the oligomer is the bactericidal form of daptomycin.||en