Delineation of functional roles of parasite- specific inserts in the malarial S-adenosylmethionine decarboxylase / ornithine decarboxylase By Marni Williams 25026519 Submitted in partial fulfilment of the requirements for the degree MSc. Biochemistry in the Faculty of Natural and Agricultural Science Department of Biochemistry University of Pretoria Pretoria South Africa February 2008 © University of Pretoria I declare that the thesis/dissertation, which I hereby submit for the degree MSc. Biochemistry at the University of Pretoria, is my own work and has not previously been submitted by me for a degree at this or any other tertiary institution. SIGNATURE: ………………………………. DATE: ………………………………. Acknowledgements • Prof A.I. Louw, University of Pretoria, South Africa (Promoter) • Dr L. Birkholtz, University of Pretoria, South Africa (Co-promoter) • Prof R.D. Walter, Dr C. Wrenger, Dr I.B. Müller, M. Eschbach, B. Bergmann, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany (Collaborative research visit in 2006) • Dr P.F.G. Sims, Manchester Interdisciplinary Biocentre, Manchester, United Kingdom (Mass spectrometric analysis) • Dr M. Rautenbach, University of Stellenbosch, South Africa (Design of synthetic peptides) • G.A. Wells, University of Pretoria, South Africa (Secondary structure predictions) • National Research Foundation (Grand holder’s bursary) • Family and friends Contents List of Figures ........................................................................................................... IV List of Tables ............................................................................................................ VI List of Abbreviations ............................................................................................... VII Chapter 1: Literature review ...................................................................................... 1 1.1 Malaria ........................................................................................................................ 1 1.2 The P. falciparum life cycle ......................................................................................... 2 1.3 Treating malaria .......................................................................................................... 3 1.3.1 Vector control ........................................................................................................ 3 1.3.2 Vaccine development ............................................................................................ 6 1.3.3 Current antimalarials ............................................................................................. 8 1.3.3.1 Quinolines ....................................................................................................... 9 1.3.3.2 Antifolates ..................................................................................................... 10 1.3.3.3 Artemisinins ................................................................................................... 11 1.3.3.4 Antibiotics ...................................................................................................... 13 1.3.4 Novel antimalarial targets ................................................................................... 13 1.3.4.1 Type II fatty acid biosynthesis ....................................................................... 13 1.3.4.2 DNA topoisomerases .................................................................................... 14 1.3.4.3 Polyamine biosynthesis ................................................................................. 14 1.4 Polyamines ............................................................................................................... 15 1.4.1 Polyamine metabolism in P. falciparum .............................................................. 16 1.4.2 Polyamine transporters ....................................................................................... 18 1.4.3 Polyamine levels during malarial infection .......................................................... 19 1.4.4 Polyamine biosynthetic enzymes as drug targets ............................................... 20 1.4.4.1 The bifunctional PfAdoMetDC/ODC protein .................................................. 23 1.4.4.2 Ornithine decarboxylase ............................................................................... 24 1.4.4.3 S-adenosylmethionine decarboxylase .......................................................... 25 1.4.4.4 Parasite-specific inserts ................................................................................ 26 1.5 Research aims .......................................................................................................... 29 Chapter 2: An efficient method for the deletion of parasite-specific inserts in malarial genes ....................................................................................... 30 2.1 Introduction ............................................................................................................... 30 2.2 Methods .................................................................................................................... 33 2.2.1 Deletion mutagenesis PCR ................................................................................. 33 2.2.1.1 Cloning of genes into pASK-IBA3 ................................................................. 33 2.2.1.2 QuickChange™ site-directed mutagenesis (QCM) ....................................... 33 2.2.1.3 ExSite™ PCR-based site-directed mutagenesis ........................................... 34 2.2.1.4 Overlapping primer method ........................................................................... 34 2.2.1.5 Inverse PCR method ..................................................................................... 34 2.2.1.6 RE-mediated inverse PCR method ............................................................... 34 2.2.1.7 Analysis of mutagenesis products ................................................................. 35 2.2.1.8 Electroporation into DH5α cells ..................................................................... 35 2.2.1.9 Plasmid isolation ........................................................................................... 36 2.2.1.10 Restriction enzyme mapping ......................................................................... 37 2.2.1.11 Nucleotide sequencing .................................................................................. 37 2.3 Results and Discussion ............................................................................................. 38 2.3.1 Analysis of mutagenesis products....................................................................... 38 2.3.2 Verification of the RE-mediated inverse PCR method ........................................ 42 Chapter 3: Delineation of the roles of structural features in the PfAdoMetDC parasite-specific inserts on bifunctional activity .............................. 45 3.1 Bifunctional enzymes in P. falciparum ...................................................................... 45 3.2 Parasite-specific inserts in PfAdoMetDC/ODC ......................................................... 46 3.3 Identification of secondary structures ....................................................................... 47 3.3.1 Hydrophobic cluster analysis .............................................................................. 48 I 3.3.2 The roles of secondary structures in protein-protein interactions and motif formation .......................................................................................................... 49 3.3.2.1 α-Helices and protein-protein interactions ..................................................... 49 3.3.2.2 β-Sheets, barrels and turns ........................................................................... 51 3.4 Methods .................................................................................................................... 52 3.4.1 Delineation of structural features within the PfAdoMetDC parasite-specific inserts ............................................................................................................... 52 3.4.2 Nucleotide deletion of the PfAdoMetDC parasite-specific inserts and mutagenesis of delineated areas ..................................................................... 55 3.4.2.1 Cloning of PfAdoMetDC/ODC into pASK-IBA3 ............................................. 55 3.4.2.2 Mutagenesis PCR ......................................................................................... 55 3.4.2.3 Post-PCR manipulations ............................................................................... 56 3.4.2.4 Plasmid isolation and nucleotide sequencing ............................................... 57 3.4.3 Recombinant protein expression and isolation of the mutated PfAdoMetDC/ODC proteins ............................................................................. 57 3.4.3.1 Heat shock transformation ............................................................................ 57 3.4.3.2 Protein expression induction and expression ................................................ 58 3.4.3.3 Isolation of the Strep-tagged recombinant proteins ...................................... 59 3.4.3.4 Protein concentration determination ............................................................. 59 3.4.3.5 SDS-PAGE analysis .....................................................................................