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Actomyosin Motors and Malaria Parasite Invasion of the Host Cell

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Actomyosin Motors and Malaria Parasite Invasion of the Host Cell Actomyosin motors and malaria parasite invasion of the host cell Noor Azian Binti Md Yusuf Division of Parasitology MRC National Institute for Medical Research The Ridgeway Mill Hill, London NW7 1AA Department of Structural and Molecular Biology University College London This thesis is submitted in part fulfilment of the requirements of University College London for the degree of Doctor of Philosophy June 2013 1 Declaration I, Noor Azian Binti Md Yusuf, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis Noor Azian Binti Md Yusuf June 2013 2 Abstract In the blood stream the merozoite is the invasive form of the malaria parasite that must enter a new erythrocyte. Merozoites lack structure for locomotion such as cilia and flagella, but use an actomyosin motor complex that provides the force to drive them into the cell. This molecular motor is located at the pellicle membrane of the parasite. The motor protein complex or glideosome includes a class XIV myosin heavy chain (MyoA), Myosin Tail Interacting Protein (MTIP) and two anchoring proteins known as Glideosome Associated Protein 45 (GAP45) and GAP50. A ternary complex of MyoA- MTIP-GAP45 is first formed and this later associates with GAP50. Using a GFP- tagged MyoA transgenic parasite line the expression of MyoA was examined in both Plasmodium falciparum and P. knowlesi. The protein was expressed at the periphery of the parasite and associates with MTIP, GAP45 and GAP50. In time course studies it was shown that recruitment of GAP50 to the complex occurs late relative to the onset of GAP50 synthesis, likely reflecting a role for GAP50 in cellular processes prior to host cell invasion. A second class XIV myosin, Myosin B (MyoB) was also tagged with GFP in a transgenic parasite line. Whilst the timing of MyoB expression followed that of MyoA, with clearly detectable protein in segmenting schizonts at 40 hours post invasion, MyoB did not associates with MyoA or any of the other invasion complex proteins. The localisation of MyoB-GFP in P. falciparum was at the apical end of the merozoite, in front of other apical organelle markers such as Rhoptry neck protein 4 (RON4). During erythrocyte invasion, MyoB -GFP remained in the same location, whilst RON4 moved out onto the parasite surface as part of the moving junction and migrated to the rear of the parasite. Although the exact role of MyoB is unknown, it is suggested that it may be involved in the invasion process but within its own protein complex that is distinct from the glideosome. 3 Dedication To my late father Allahyarham Md Yusuf B. Awang and my late mother Allahyarhamah Rubiah Binti Ismail who always encouraged me to follow my dreams and to my loving and caring stepmother Rokiah Binti Md Noor who took all the responsibilities to let me lift my dreams……. 4 Acknowledgement Firstly, I would like to thank my supervisor Dr. Anthony A. Holder for providing me with the opportunity to work in his lab and for the continuous guidance and support throughout my project. I would also like to thank Dr. Robert W. Moon, Dr. Ellen Knuepfer, Dr. Justin Molloy, Dr. Judith L. Green, Dr Claire H. Hastings and Dr. David K. Moss for their generous help and guidance as well as their continuous support and interest in this field of malaria invasion. I also would like to thank my thesis committee panel, Dr. Michael J. Blackman and Dr Roger Buxton for their time and helpful advice. I also would like to express my gratitude to my home institution, The Institute for Medical Research (IMR), and the Ministry of Health, Malaysia for granting me a full time study leave and The Islamic Development Bank (IDB), Saudi Arabia for awarding me the PhD Merit–scholarship to pursue my study concerning the most intriguing ancient disease in the world in this wonderful country. A special thank you to Dr. Malik Shah B. Mohd Yusoff, Head of Scholarship Programme and Mr. Shaharuqul Huq who spent a lot of their precious time to answer all my emails and who flew from Saudi Arabia to London just to make sure that all concerns of the scholars were taken care of appropriately in order for us to finish this programme, which will benefit our respective home countries. My appreciation is extended to Ms. Sola Ogun, Dr. Rachel E. Farrow, Dr. Steven Howell, Dr. Madhusudan Kadekopala, Mrs. Munira Grainger, Ms. Oniz Suleyman, Dr. Liz McMinn, Mrs. Rachel Chung, Ms. Suraya Diaz, Ms. Lesley Calder, Ms. Kate Sullivan, Dr. Donna Brown, Ms Jirina Markova and all members of Tony Holder’s, Blackman’s and Delmiro’s Labs who helped me in one way or another. A special thanks to Mrs. Alana Sargent for always helping me with administrative works and dropping by at my house to pick up things early in the morning, Dr. Claire H. Hastings for the ‘Haggis Night’, Rob Moon for the unforgettable ‘ice skating experience’, David Moss for the introduction to British delicacies, Mr. Antonio Casal for the coffee indulgence, Mrs. Kush Meghji for the food during Ramadhan, Ms Hayley Wood for photographic advice, Ms. Harsha Seth and Mr. Omar Ayad for their caring 5 thoughts and last but not least, Dr. Mohd Ridzuan Mohd Abdul Razak for being my unpaid IT manager who always tried to resolved my IT problems. My most sincere thanks and appreciations goes to Mrs. Salini Halim and her family who were always at my side during both happy and difficult times throughout my stay in London. To my colleagues and friends at the IMR: Dr. Lokman Hakim Sulaiman, Dr. Noor Rain Abdullah, Dr. Shamilah Hisam, Hj.Mohd Yusri Yusof, Mrs Aishah Mahmod, Mrs. Maslawaty Md Noor, Mrs. Parina Ismail, Mrs. Rosilawaty Harun, Mrs Rozie Sarip, Mrs. Paridah Yusuf, Mrs. Rozana Rozali and to the never- ending lists of great and wonderful people in London, Saudi Arabia and Malaysia who in known or unknown ways contributed to the successes of my study, not only in the academic field but also in life experiences. Last but not least, my endless thanks and never-ending love go to my big family of 12. To my step mother Rokiah, to my elder sisters, Ummi Kalsom, Umi Sofian, Noriza, Noor Zila and Noor Zurawati, to my elder brother Md Amran, to my younger sisters Noor Ziana and Nurul Asyikin, to my younger brothers, Mohd Amirul Azdi, Azrul Hisyam and Mohd Fikry Affendy whom I always refer to by numbers and to all my in- laws, Suhaimi, Razif, Rohaida, Hashimah, Dunian, Amir, Rosli, Azmir, Nazif, and Mira, thank you for your boundless caring, love and support throughout my studies although we are thousands miles apart. To my nephews and nieces: Fahim, Azril, Amzal, Azra, Afza, Azhan, Alia, Aina, Akmal, Shafiq, Firdaus, Balqis, Kamil, Dayana, Hisyamudin, Fahada, Fariha, Firzan, Farisha, Danial, Adam, Hani, Luqman, Elena, Tolia, Fatina, Husna, Damia, Rania and Azfar…Facebook is amazing!!!..I love you all…. …..Thank You…. 6 Table of contents Pages Title Page 1 Declaration 2 Abstract 3 Dedication 4 Acknowledgement 5 Table of contents 7 List of figures 12 List of tables 14 Abbreviations 15 References 170 Appendixes 187 Chapter 1 – Introduction 1.1 Introduction to malaria 17 1.1.1 The burden 17 1.1.2 The disease 18 1.1.3 The parasite 18 1.2 The Plasmodium life cycle 19 1.2.1 The zoites 19 1.2.2 The life cycle 22 1.2.3 The mosquito stage 24 1.2.4 The liver stage 26 1.2.5 The asexual blood stage 27 1.3 The erythrocyte invasion by merozoites 30 1.3.1 Initial attachment 31 1.3.2 Apical orientation 31 1.3.3 Moving junction (MJ) formation 32 1.3.4 Sealing and completion of the parasitophorus vacuole (PV) 34 1.4 The myosin superfamily 35 7 1.5 The inner membrane complex (IMC) 42 1.6 Acto-myosin motor complex in Apicomplexa 42 1.6.1 Plasmodium falciparum myosin A (PfMyoA) 43 1.6.2 Interaction of MyoA with its myosin light chain, MTIP 47 1.6.3 The glideosome associated protein (GAPs) 48 1.6.4 MyoA-actin interrelationship 49 1.7 Apicomplexan Myosin B 54 1.8 Aims of this thesis 55 Chapter 2 – Material and Methods 2.1 DNA manipulation and transformation 57 2.1.1 Oligonucleotides 57 2.1.2 Polymerase Chain Reaction(PCR) 57 2.1.3 Agarose gel electrophoresis 57 2.1.4 Restriction endonuclease digestion 58 2.1.5 Ligation of DNA fragments 58 2.1.6 The propagation and transformation of E.coli by plasmid 58 DNA 2.1.7 Isolation of plasmid DNA 59 2.1.8 Sequencing of DNA 59 2.2 In vitro Expression and purification of recombinant MyoA, MTIP and 59 GAP45 protein 2.2.1 Construction of recombinant DNA 59 2.2.2 Transcription of DNA to mRNA 60 2.2.3 Translation of mRNA to protein 60 2.2.4 Co-translation of proteins 60 2.2.5 SDS-PAGE 61 2.2.6 Immunoblot 61 2.3 Pull down assay and Co-immunoprecipitation assay 62 2.3.1 GST pull down for cell free protein 62 2.3.2 His pull down for cell free protein 62 2.3.3 Co-immunoprecipitation with GFP-TRAP system for parasite 62 lysate 8 2.4 The functional analysis of MyoA 63 2.4.1 In vitro motility assay 63 2.4.2 Single molecules studies 64 2.5 In vitro parasite culture and maintenance 66 2.5.1 Maintenance of Plasmodium falciparum and purification of 66 the schizonts 2.5.2 Maintenance of Plasmodium knowlesi and purification of the 67 schizonts 2.5.3 Freezing and thawing of parasites 67 2.6 The parasite DNA manipulation and transfection 68 2.6.1 Transfection: The construction of single crossover integration 68 constructs for integration of P.
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