Mode of Action of Daptomycin, a Lipopeptide Antibiotic

148 Pages·2013·3.09 MB·English

by  Jawad Kadhum Muraih

Mode of Action of Daptomycin, a Lipopeptide Antibiotic
by
Jawad Kadhum Muraih
Athesis
presentedtotheUniversityofWaterloo
infulfillmentofthe
thesisrequirementforthedegreeof
DoctorofPhilosophy
in
Chemistry
(Biochemistry)
Waterloo,Ontario,Canada,2012
©JawadKadhumMuraih2012

Author’s Declaration
I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis,
includinganyrequiredfinalrevisions,asacceptedbymyexaminers.
Iunderstandthatmythesismaybemadeelectronicallyavailabletothepublic.
JawadK.Muraih
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Abstract
Daptomycin is a lipopeptide antibiotic that contains 13 amino acids and an N-terminally at-
tachedfattyacylresidue. TheantibiotickillsGram-positivebacteriabymembranedepolariza-
tion. It has long been assumed that the mode of action of daptomycin involves the formation
ofoligomersonthebacterialcellmembrane;however,attheoutsetofmystudies,thishadnot
beenexperimentallydemonstrated.
In the work described in this thesis, I have used fluorescence energy transfer (FRET) be-
tween native daptomycin and an NBD-labeled daptomycin derivative to demonstrate that the
antibioticindeedformsoligomersonbacterialcellmembranes. Inaliposomemodel,oligomer
formation depends on calcium and on phosphatidylglycerol (PG). The oligomer forms rapidly
andisstableforalengthoftimelongerthanrequiredforthebactericidaleffect. Throughvari-
ationoftheratioofFRETdonor(nativedaptomycin)andacceptor(NBD-daptomycin),I have
determined that the oligomer consists of approximately 6–7 molecules, or, depending on the
structureoftheoligomer,possiblyuptotwicethatnumber.
Oligomer formation on liposomes and on bacterial membranes was confirmed using ex-
cimer fluorescence of a perylene-labeled daptomycin derivative. Excimer fluorescence was
alsousedtodemonstrateastoichiometricinteractionbetweendaptomycinandPG.
It has previously been shown that the bactericidal activity of daptomycin requires calcium
andcorrelateswiththeconcentrationofPGinthebacterialcellmembrane;theserequirements
mirror those observed here for oligomer formation. Furthermore, membrane permeabiliza-
tion 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
formationofasmallmembranelesion,whichiscompatiblewiththesmallsizeoftheoligomer
that was determined here. In conjunction with these previous findings, the experiments con-
tainedinmythesisstronglysuggestthattheoligomeristhebactericidalformofdaptomycin.
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Acknowledgements
Eventhoughtherearemanypeoplewhohavehelpedmetoreachthispointofmylife,theone
towhomIowemydeepestthanksandgratitudeisMichaelPalmer. Iamnotexaggeratingwhen
I say that he is one of the best supervisors and advisors in the Department of Chemistry at the
University of Waterloo. I am highly indebted to Professor Palmer for his valuable and fruitful
guidance, suggestions and discussions; without his knowledge, perceptiveness, enthusiasm,
andencouragement,thisthesiswouldneverhaveseenthelight. Inparticular,IthankProfessor
Palmerforhisincrediblepatienceforalwayskeepingthedoortohisofficeopenforquestions,
andforhiscontinualsupport. ThankyouforeverythingProfessorPalmer.
I would like to extend my thanks to my advisory committee members, Professors John
Honek,ElizabethMeieringandShawnWettigfortheirusefulsuggestionsandcomments.
I would like to thank my lab members Eric Brefo-Mensah, Oscar Zhang, Muhamad Salah,
and former members Mohammad Khan, Dr. Waseem el Huneidi, Dr. Lisa Pokrajac for their
helpandusefuldiscussions;alltheundergraduatestudentsAna,Alex,Jesse,Kathy,Minyoung,
and Celine. All of you contribute to make our lab is a nice place to work. In addition, I would
liketothankmyfriendKamalMroueforthemomentswesharedtogetherandallofmyfriends.
In particular, I want to thank my parents, my brothers and my sisters for their support and
encouragement. Finally, I would like to express my gratitude to my wonderful wife Najah,
for her patience and support, also I would like to thank my son Mohammad for his help and
patience and my children Rand, Rahaf, Fatemah, Ali and Sarah whom have provided full joy
andpleasuretoourjointlife.
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Dedication
Tomyparents,brothersandsisters
Tomywifeandchildren
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