Mode of Action of Daptomycin, a Lipopeptide Antibiotic PDF
Preview Mode of Action of Daptomycin, a Lipopeptide Antibiotic
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 iii 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. v 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. vii Dedication Tomyparents,brothersandsisters Tomywifeandchildren ix
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