Denton, Helen (1996) Studies on the energy metabolism of Eimeria tenella and other coccidia. PhD thesis http://theses.gla.ac.uk/7233/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Studies on the energy metabolism of Eimeria tenella and other coccidia Helen Denton University of Glasgow This thesis is presented in submission for the degree of Doctor of Philosophy June 1996 ACKNOWLEDGEMENTS First and foremost, I would like to thank my academic supervisor, Professor Graham Coombs, for his positive guidance and encouragement throughout my time in his laboratory. I am also grateful to my industrial supervisor, Dr Kam-Wah Thong (Bob), in particular for making me welcome during my stay at Pfizer Central Research in Kent, and also for contributing Section 1.2.3 of this thesis. For specific practical assistance I would like to thank the following: Pete Giles and Phil Wood of Pfizer Central Research for the supply of Eimeria oocysts; Dr Michael Newman (also of Pfizer) for assistance with the NMR analyses; Dr Craig Roberts of Strathclyde University for supplying the tachyzoites and bradyzoites of Toxoplasma gondii; The laboratory of Dr Vincent McDonald at the London School of Hygiene and Tropical Medicine for supplying oocysts of Cryptosporidium parvum to Samantha Brown (and thus to me); And Samantha Brown, who let me have some of her oocysts, and also carried out the biotinylated inhibitor studies of Chapter 6. I am very grateful to Mr David Laughland and Mrs Dorothy Armstrong for their patient and capable technical assistance throughout my time in Biochemical Parasitology, and to many other members of the department who helped me along the way. I would particularly like to thank some of my fellow-postgraduates (Amanda McKie, Kathryn Brown, Paul Appleton, Mhairi Frame and Kathleen Milligan) who were generous, not only with their help and advice, but also with their friendship. This project was supported by a SERC/BBSRC-CASE award in conjunction with Pfizer Central Research, Kent. £0 c.Q This thesis is dedicated to Geoff Ballinger, for support and understanding over many years. 2 DECLARATION I hereby declare that this thesis has been composed by myself, that the work it describes is my own, except where stated in the text, and that it has not been accepted in any previous application for a degree. Helen Denton, 27th June 1996. 3 LIST OF CONTENTS ACKNOWLEDGMENTS 2 DECLARATION 3 LIST OF CONTENTS 4 LIST OF TABLES 10 LIST OF FIGURES 11 ABBREVIATIONS 13 SUMMARY 14 1. INTRODUCTION 19 1.1 THE COCCIDIA: 19 1.1.1 Taxonomy and phylogeny 19 1.1.2 Life cycles :- 23 1.1.3 Ultrastructure 26 1.1.4 Pathology and Pathogenesis 29 1.2 BIOCHEMICAL ASPECTS OF THE COCCIDIA ;.. 31 1.2.1 Energy metabolism 31 1.2.1.1 Energy Substrates 32 1.2.1.1.1 Endogenous polysaccharide energy reserves 32 1.2.1.1.2 Use of exogenous carbohydrates 35 1.2.1.2 Pathways of carbohydrate metabolism 36 1.2.1.2.1 Glycolysis 37 1.2.1.2.2 Mannitol metabolism 43 1.2.1.2.3 Tricarboxylic acid (TCA) cycle 46 1.2.1.2.4 Respiratory chain 48 1.2.1.2.5 Pentose phosphate pathway 50 1.2.1.2.6 Gluconeogenesis 51 '1.2.1.2.7 Glyoxylate cycle 51 1.2.1.3 Other catabolic pathways 51 1.2.1.4 Variation with phase of development 52 1.2.1.4.1 Oocysts and Sporulation 52 1.2.1.4.2 Sporozoites and excystation 54 4 1.2.1.4.3 Intracellular stages 56 1.2.1.5 Toxoplasma 57 1.2.1.6 Cryptosporidium 59 1.2.2 Protein and amino acid metabolism 60 1.2.2.1 Protein synthesis 60 1.2.2.2 Amino acid interconversions 60 1.2.2.3 Protein catabolism 60 1.2.3 Biochemical Action of Anticoccidial Agents 64 1.3 AIMS OF THE PROJECT 70 2. PARASITE METHODOLOGY 72 2.1 INTRODUCTION 72 2.2 CHEMICALS AND REAGENTS 74 2.3 EIMERIA: 74 2.3.1 Oocyst supply " 74 2.3.2 Bleach treatment of oocysts 75 2.3.3 Oocyst sporulation 75 2.3.4 In vitro excystation and purification of sporozoites ,.. 76 2.3.4.1 Efficiency of excystation 77 2.3.5 Motility and viability of sporozoites 79 2.3.6 Production ofmerozoites and schizonts 80 2. 4 TOXOPLASMA 80 2.4.1 Production and purification oftachyzoites 81 2.4.2 Production and purification ofbradyzoites 81 2.4.2.1 Efficiency ofbradyzoite production 82 2.5 CRYPTOSPORIDIUM 83 2.6 PREPARATION OF CELL HOMOGENATES 83 3. ENZYMES OF ENERGY METABOLISM IN THE COCCIDIA. 86 3.1 INTRODUCTION 86 3.1.1. Why look at enzymes? 86 3.1.2 Pyrophosphate-dependent glycolytic enzymes 87 3.1.3 Aims of this study 91 3.2 MATERIALS AND METHODS 93 3.2.1 Preparation of extracts for use in enzyme assays 93 5 3.2.2 Concentration of extracts 93 3.2.3 Buffer exchange 93 3.2.4 Enzyme assays 94 3.2.4.1 Phosphofructokinase 95 3.2.4.2 Pyruvate kinase (and pyruvate phosphate dikinase) 95 3.2.4.3 Hexokinase 96 3.2.4.4 Lactate dehydrogenase 97 3.2.4.5 Isocitrate dehydrogenase (NAD+ and NADP+-dependent) 97 3.2.4.6 Succinate dehydrogenase 97 3.2.4.7 Pyrophosphatase 97 3.2.4.8 Other enzymes: 98 3.2.4.9 Microtitre plate protocols 98 3.2.5 MonoQ fractionation 98 3.2.6 SDS;..PAGE ; 99 3.2.7 Protein determinations 99 3.3 RESULTS 100 3.3.1 Enzyme activities in coccidial extracts 100 3.3.1.1 Phosphoryl substrate specificites of PK and PFK 104 3.3.1.2 Pyrophosphatase activity 105 3.3.1.3 Differences between developmental stages 105 3.3.1.4 Detection of other enzyme activities 105 3.3.2 Characterisation ofPPj-PFKs from E. tenella and C. parvum 107 3.3.2.1 Stability 107 3.3.2.2 Kinetic properties 107 3.3.2.3 Cation dependence 108 3.3.2.4 The reverse reaction 108 3.3.2.5 pH optima 108 3.3.3 Characterisation of the PKs of E. tenella, T gondii and C. parvum 112 3.3..3.1 Stability 113 3.3.3.2 Effect ofDTT 113 3.3.3.3 pH optima 113 3.3.3.4 Cation dependence 113 3.3.3.5 Kinetic properties ~ 114 6 3.3.4 Characterisation of hexokinase from Eimeria 123 3.3.4.1 Assays and MonoQ fractionation 123 3.3.4.2 Glucokinase versus fructokinase activities 124 3.3.4.3 Regulatory studies 124 3.4 RESULTS SUMMARY 131 4. SUBSTRATES AND END-PRODUCTS OF ENERGY METABOLISM IN EIMERIA TENELLA 133 4.1 INTRODUCTION: 133 4.1.1 Substrates and end-products of energy metabolism in parasitic protozoa 133 4.1.2 Techniques for analysing metabolites 136 4.1.3 Aims of this study 138 4.2 MATERIALS AND METHODS 140 4.2.1 Qualitative analyses: NMR 140 4.2.1.1 Incubation ofsporozoites 140 4.2.1.2 Preparation of samples for NMR 140 4.2.1.3 NMR spectroscopy 141 4.2.1.4 Identification of metabolites ,141 4.2.2 Quantitative analyses 142 4.2.2.1 Sporozoite incubations 142 4.2.2.2 Preparation of oocyst extracts for metabolite analyses 142 4.2.2.3 Enzymatic assays 143 4.2.2.4 Amino acid hplc 144 4.2.2.5 Organic acid hplc: 144 4.2.2.5.1 Extraction procedure 144 4.2.2.5.2 Separation procedure 144 4.3 RESULTS 147 4.3.1 Glucose catabolism by sporozoites: NMR analyses 147 4.3.1.1 Identification of soluble products of glucose catabolism 147 4.3,.1.2 Catabolism of selectively labeled 13C-glucose 149 4.3.1.3 Intracellular products 149 4.3.2 Glucose catabolism by sporozoites: quantitative analyses 152 4.3 .2.1 Kinetics of glucose catabolism 152 7 4.3.2.2 Quantitative analysis of products under aerobic and anaerobic conditions: fixed length incubations 154 4.3.2.3 Production of end-products from endogenous energy reserves 158 4.3.3 Investigation of alternative substrates 159 4.3.3.1 Amino-acids 159 4.3.3.2 Mannitol 163 4.3.4 Sporulation studies 163 4.3.4.1 Investigation of released products 164 4.3.4.2 Levels of organic acids and glycerol in sporulated and unsporulated oocysts 164 4.3.4.2.1 The 'X' peak! 166 4.3.4.3 Comparison of free amino acids in sporulated and unsporulated oocysts ....................................................................................................................... 168 4.3.4.3.1 Unknown amino compounds 171 4.4 RESULTS SUMMARY 172 5. PROTEINASE STUDIES 173 5.1 INTRODUCTION: PROTEINASES OF PARASITIC PROTOZOA 173 5.1.1 Aims of this study 174 5.2 MATERIALS AND METHODS 176 5.2.1 Parasites 176 5.2.2 Preparation of samples for SDS-PAGE 176 5.2.3 Substrate SDS-PAGE 176 5.2.4 Investigation of proteolysis in crude homogenates 177 5.2.5 Western blotting 177 5.2.6 Use ofbiotinylated inhibitors 177 5.3 RESULTS 179 5.3.1 Presence of trypsin in parasite homogenates 179 5.3.2 Proteolysis in parasite homogenates 181 5.3.3 Reaction with antibodies against leishmanial cysteine proteinases 183 5.3.4 Reaction with biotinylated inhibitors 184 5.4 RESULTS SUMMARY 187 6.