Recruitment of erythrocyte membrane components by apicomplexan parasites Babesia divergens and Plasmodium falciparum DISSERTATION zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) Dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Preetish Gangopadhyay aus Kalkutta, Indien Marburg am der Lahn 2015 Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation angenommen am: Erstgutachter: Prof. Dr. Klaus Lingelbach Zweitgutachter: Prof. Dr. Ralf Jacob Tag der Disputation am: “I slept and dreamt that life was joy. I awoke and saw that life was service. I acted and behold, service was joy” Rabindranath Tagore To my Ma, Baba, Didi, Sonai, Mom-mom and Ayan A major part of the results presented in the thesis have been published in: Repnik, U., Gangopadhyay P., S. Bietz, J. M. Przyborski, G. Griffiths and K. Lingelbach (2015). "The apicomplexan parasite Babesia divergens internalizes band 3, glycophorin A and spectrin during invasion of human red blood cells." Cell Microbiol. And has been prepared to be published in a review as Novel insights into red blood cell physiology using parasites as tools (manuscript in preparation) Stefan Baumeister, Preetish Gangopadhyay, Urska Repnik, Klaus Lingelbach Other publications Thavayogarajah, T., P. Gangopadhyay, S. Rahlfs, K. Becker, K. Lingelbach, J. M. Przyborski and A. A. Holder (2015). "Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum." PLoS One 10(4): e0125191 Table of Contents Abbreviations Summary Zusammenfassung 1 Introduction ........................................................................................................................... 1 1.1 Intracellular parasitism.................................................................................................... 1 1.1.1 A brief introduction to Apicomplexa .......................................................................... 2 1.1.1.1 Morphology of Apicomplexans ......................................................................... 3 1.1.1.2 Intracellular life of Apicompexans .................................................................... 6 1.2 Human erythrocyte: an unusual cell and an uncommon host ......................................... 7 1.2.1 Erythrocyte membrane components: Lipids ............................................................... 8 1.2.2 Erythrocyte membrane components: Proteins ............................................................ 9 1.2.2.1 Integral membrane proteins: .............................................................................. 9 1.2.2.1.1 Band 3 ........................................................................................................... 9 1.2.2.1.2 Glycophorins ................................................................................................. 9 1.2.2.1.3 Aquaporins .................................................................................................. 10 1.2.2.2 Cytoskeletal protein: ........................................................................................ 10 1.2.2.2.1 Spectrin........................................................................................................ 10 1.2.2.2.2 Actin ............................................................................................................ 12 1.2.2.2.3 Protein 4.1 ................................................................................................... 12 1.2.2.2.4 Adducin ....................................................................................................... 12 1.2.2.2.5 Ankyrin........................................................................................................ 12 1.3 Plasmodium................................................................................................................... 13 1.3.1 Intra-erythrocytic development of Plasmodium falciparum ..................................... 14 1.3.2 Infection induced alterations of erythrocytes: Plasmodium falciparum ................... 15 1.3.2.1 Morphological alterations: Knobs and cytoadherence .................................... 16 1.3.2.2 Physiological alterations: Novel permeation pathways ................................... 17 1.3.3 Invasion induced compartmentation in erythrocytes ................................................ 18 1.3.3.1 Maurer’s cleft and Tubovesicular membrane network .................................... 18 1.3.3.2 The parasitophorous vacuole: an unusual compartment .................................. 20 1.3.3.2.1 Formation of the parasitophorus vacuole .................................................... 21 1.3.3.2.2 Components of the plasmodial PVM .......................................................... 22 1.3.3.2.2.1 Lipids of the PVM ................................................................................ 23 1.3.3.2.2.2 Proteins of the PVM ............................................................................. 24 1.3.3.2.3 Functions of the PV ..................................................................................... 27 1.3.3.2.3.1 Transitional compartmentation ............................................................ 27 1.3.3.2.3.2 Nutrition acquisition and maintenance of the iconic environment ...... 29 1.3.3.2.3.3 The PV and merozoite egress ............................................................... 30 1.4 Babesia .......................................................................................................................... 31 1.4.1 Intra-erythrocytic development of Babesia .............................................................. 32 1.4.2 Infection induced alterations of erythrocyte: Babesia .............................................. 33 1.4.2.1 Morphological alterations: Ridges and cytoadherence .................................... 34 1.4.2.2 Physiological alterations: Novel permeation pathways ................................... 36 1.4.3 Invasion induced compartmentation: Babesia .......................................................... 38 1.5 Two parasites one host cell: .......................................................................................... 40 1.6 Objective ....................................................................................................................... 42 2 Materials and methods ........................................................................................................ 44 2.1 Materials ....................................................................................................................... 44 2.1.1 Instrumentation ......................................................................................................... 44 2.1.2 Utilities ...................................................................................................................... 44 2.1.3 Chemicals /reagents .................................................................................................. 45 2.1.4 Cell culture materials ................................................................................................ 46 2.1.5 Antibodies ................................................................................................................. 47 2.1.6 Cells and organisms .................................................................................................. 47 2.1.7 Buffers and solutions for protein biochemistry ........................................................ 47 2.1.8 Cell culture solutions ................................................................................................ 49 2.2 Methods......................................................................................................................... 49 2.2.1 Cell culture: Plasmodium falciparum 3D7 and Babesia divergens .......................... 49 2.3 Parasitemia and subculture of Plasmodium .................................................................. 50 2.3.1 Synchronisation of Plasmodium falciparum ............................................................. 51 2.3.1.1 Synchronisation of Plasmodium falciparum using a high gradient magnetic field (Paul et al., 1981) ...................................................................................................... 52 2.3.1.2 Synchronisation of Plasmodium falciparum via Gelafundin floatation (Pasvol et al., 1978) ....................................................................................................................... 53 2.3.1.3 Synchronisation of Plasmodium falciparum with Sorbitol treatment (Lambros and Vanderberg, 1979)...................................................................................................... 53 2.3.2 Parasitemia and subculture of Babesia divergens ..................................................... 54 2.3.3 Synchronisation of Babesia divergens ...................................................................... 54 2.3.3.1 Synchronisation of Babesia divergens: Transient enrichment of differentiated merozoites ......................................................................................................................... 55 2.3.4 Cryopreservation of Plasmodium falciparum and Babesia divergens ...................... 56 2.3.5 Thawing of cryo-preserved parasites: Plasmodium falciparum and Babesia divergens ................................................................................................................................ 56 2.3.6 Methods with non-infected erythrocytes .................................................................