A STUDY OF THE CELL BIOLOGY OF MOTILITY IN Eimeria tenella SPOROZOITES by David Robert Bruce Department of Biology University College London A thesis presented for the degree of Doctor of Philosophy in the University of London 2000 ProQuest Number: U643145 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U643145 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT A study on the cell biology of motility inEimeria tenella sporozoites Eimeria tenella is an obligate intracellular parasite within the phylum Apicomplexa. It is the causative agent of coccidiosis in domesticated chickens and under modem farming conditions can have a considerable economic impact. Motility is employed by the sporozoite to effect release from the sporocyst and enable invasion of appropriate host cells and occurs at an average speed of 16.7 ± 6 pms'\ Frame by frame video analysis of gliding motility shows it to be an erratic non­ substrate specific process and this observation was confirmed by studies of bead translocation across the cell surface occurring at an average speed of 16.9 ± 7.6 pms'^ Incubation with cytochalasin D, 2,3-butanedione monoxime and colchicine, known inhibitors of the motility associated proteins actin, myosin and tubulin respectively, indicated that it is an actomyosin complex which generates the force to power sporozoite motility. Western blotting analysis confirmed the presence of actin with an apparent molecular weight of 43kDa and an unconventional myosin with an apparent molecular weight of 93kDa. Antibodies against the actin binding proteins spectrin, vinculin, filamin, a-actinin, cofilin and tropomyosin failed to recognise any polypeptides in whole cell extracts. Immunofiuoresence studies showed actin was found predominantly in the anterior third of the sporozoite. Myosin appeared to have a more widespread distribution, with a strong signal found at the margins of the cell. Genomic DNA samples were prepared and two degenerate primers against highly conserved regions of the myosin head were used in a polymerase chain reaction (PCR) to probe for the presence of myosin genes. These PCR products were inserted into suitable plasmids followed by amplification in bacteria. Selection of appropriate bacterial colonies and subsequent DNA sequence analysis identified a clone with significant homology to a Homo sapiens myosin II gene previously described. CONTENTS Page Number Title 1 Abstract 2 Contents 4 List of tables and figures 9 Acknowledgements 13 List of publications 14 Chapter 1 - Introduction 16 Chapter 2 - Materials and Methods 79 2.1 Preparation of sporozoites and sporocysts 79 2.2 Preparation of cell cultures 81 2.2.a Resuscitation of cells jfrom liquid nitrogen 82 2.2.b Passaging adherent cells 83 2.2.C Preparation of cell monolayers 83 2.3 Microscopy 84 2.3.a General microscopy 84 2.3.b Observation of motility using videomicroscopy 84 2.3.b.I Prior chamber and microslides 85 2.3.b.II Potential inhibitory compounds 87 2.3.b.III Motility studies 88 2.3.b.IV Sperm tracker analysis 90 2.3.b.VExcystation studies 91 2.3.b.VI Invasion studies 92 2.3.b, VII Calculation of speeds 94 2.3.c Immunofiuoresence microscopy 95 2.3.C.I Preparation of coated coverslips 95 2.3.c.II Staining protocol 96 2.3.d Electron microscopy 97 2.3.d.I Fixation and embedding of material 97 2.3.d.II Sectioning 99 2.3.d.III Staining thin sections 99 2.3.d.IV Immuno-electron microscopy 99 2.4 Identification of cell proteins 100 2.4.a Preparation of polyacrylamide gels 100 2.4.b Preparation of protein samples for SDS electrophoresis 101 2.4.C Transfer of proteins to nitrocellulose membranes 104 2.4.d Western blotting 106 2.4.d.I Antibody treatment of nitrocellulose sheets 106 2.4.d.II Detection protocols 109 2.4.d.III F-actin overlay 111 2.4.e Chromatography 112 2.5.Identifîcation of myosin by molecular biology 113 2.5.a Preparation of genomic DNA and agarose gel electrophoresis 113 2.5.b Polymerase chain reaction using degenerate primers 114 2.5.C Ligation of PCR products into vectors 116 2.5.d Transformation of competent cells and colony selection 119 2.5.e Mini-prep of plasmid DNA 120 2.5.f Restriction enzyme digestion of plasmids 121 2.5.g Sequencing of products 122 Chapter 3 - Results 123 3.1 Motility speed calculations 123 3.1 .a Sporozoite gliding speeds 123 3.1 .b Sporozoite excystation 130 3.1.C Bead translocation speeds 139 3.2 Effect of inhibitors 142 3.2.a Gliding motility 145 3.2.b Sperm tracker analysis 148 3.2.C Excystation 159 3.2.d Invasion 159 3.3 Immunofiuoresence 161 3.3.a Actin 161 3.3.b Myosin 161 3.4 Cell ultrastructure 164 3.5 Western blotting 181 3.5.a Actin 181 3.5.b Myosin 183 3.5.C Actin associated proteins (AAPs) 186 3.6 Fast Protein Liquid Chromatography (FPLC) 188 3.7 Identification of myosin sequence 189 3.7.a Extraction of genomic DNA 189 3.7.b PCR products 189 3.7.C Selection of inserts 191 3.7.d Sequence data analysis 191 Chapter 4 Discussion 198 4.1 Speed of movement 198 4.2 Erratic nature of movement 199 4.3 Cell ultrastructure 201 4.4 Cell-substratum interactions 202 4.5 Bead-cell surface interactions 230 4.6 Excystation 231 4.7 Nature of the motor 234 4.8 Invasion 241 References 245 Appendices 288 1 Chemicals 288 2 Standard proteins 291 3 Antibodies 292 (a) Primary antibodies 292 (b) Secondary antibodies 293 4 Molecular biology 294 5 Miscellaneous 295 LIST OF FIGURES AND TABLES Figures: Page Number Figure 1.1: A cartoon of a typical apicomplexan zoite 17 Figure 1.2: The classical coccidian life 19 Figure 1.3: The life cycle of Eimeria tenella in the caecum of the chicken 22 Figure 1.4: Model of the moving junction of invasion 29 Figure 1.5: Cartoon of a Gregarina blaberae fold 34 Figure 1.6: The cellular functions of the 15 myosin classes 53 Figure 1.7: Hydrolysis of ATP by the myosin head 55 Figure 1.8: Similarities in the basic structure of kinesin myosin 58 Figure 1.9: Myosin neck domains 58 Figure 1.10: A phylogenetic tree of myosin motor domains 76 Figure 2.1 : Diagram of the Prior perfusion chamber apparatus 86 Figure 2.2: Diagram of the protein transfer ‘sandwich’ apparatus 105 Figure 2.3: Diagram of the slot blot apparatus 107 Figure 2.4: Schematic diagram of the HRP conjugate detection system 110 Figure 2.5: Generation of new DNA strands during PCR 117 Figure 3.1(a) & (b): Graph of cumulative & individual distances moved by zoites 127 Figure 3.2: Cartoons of RIM studies on gliding sporozoites 129 Figure 3.3: Excystation of two sporozoites from a single sporocyst 132 Figure 3.4: High magnification observation of an excystation 133 Figure 3.5: Graph of individual and cumulative during excystation 137 Figure 3.6(a) & (b): Graph of individual & cumulative distances moved by a 2/xm bead 141 Figure 3.7: Sequential photographs of a bead translocation 143 Figure 3.8: Cartoon of the bead translocation shown in figure 3.7 144 Figure 3.9(a) & (b): Inhibition of sporozoite motility in CD & BDM 146 Figure 3.10: Graph of Hobson Sperm Tracker analysis 149 Figure 3.11(a) & (b): Sporozoite excystation control conditions & in DMSO 153 Figure 3.12(a) & (b): Sporozoite excystation in 0.1 &1 /igml ‘ Cytochalasin D 154 Figure 3.13(a) & (b): Sporozoite excystation in 75 &100mM BDM 155 Figure 3.14(a) & (b): Sporozoite excystation in ImM Colchicine & l/xgml'* Salinomycin 156 Figure 3.15: Mean % invasion of MDBK cells by sporozoites 158 Figure 3.16: Photograph of actin immunofiuoresence 161 Figure 3.17: Photograph of myosin immunofiuoresence 162 Figure 3.18: TEM section through an uninvaded CaC02 cell 164 Figure 3.19: TEM of a flexing sporozoite 166 Figure 3.20: Cartoon of the flexing sporozoite in figure 3.19 167 Figure 3.21 : CaC02 cells 20 minutes post invasion 169 Figure 3.22: TEM of a CaC02 cell 20 minutes post invasion 170 Figure 3.23: TEMs of a CaC02 cell 20 minutes post invasion 171 Figure 3.24: TEM of a possible sporozoite Golgi complex 172 Figure 3.25: TEM of a CaC02 60 minutes post invasion 173 Figure 3.26: High magnification TEM of in intracellular 174 Figure 3.27: TEM of the anterior region of a sporozoite 175 Figure 3.28: TEM of a sporozoite at the base of an INT 407 cell 176 10 Figure 3.29: A high resolution TEM of the apical tip of a sporozoite 177 Figure 3.30: A high magnification TEM of a horizontal sporozoite section 178 Figure 3.31 : A high magnification TEM of an intracellular sporozoite 179 Figure 3.32: Anti-actin Western blot and corresponding Coomassie stained gel 181 Figure 3.33: Actin dilution series 183 Figure 3.34: Anti-myosin Western blot 184 Figure 3.35: Anti-myosin Western blot using two anti-myosin antibodies 186 Figure 3.36: UV illuminated photograph of Eimeria total genomic DNA 189 Figure 3.37: UV illuminated photograph of PCR products 190 Figure 3.38: UV illuminated photograph of restriction enzyme digests and inserts 192 Figure 3.39: Sequences for the Eimeria tenella cloned insert DB-07 193 Figure 3.40: Alignment of the Eimeria tenella insert protein 195 Figure 3.41: Alignment with Plasmodium falciparum & Toxoplasma gondii 196 Figure 4.1 : Interaction