Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Nitrogen Metabolism in Haemonchus contortus and Teladorsagia circumcincta A thesis presented In partial fulfilment of the requirements For the degree of DOCTOR OF PHILOSOPHY at Massey University Palmerston North New Zealand Sallah Umair 2012 Table of Contents Table of Contents ...................................................................................................................... i Abstract ...................................................................................................................................... vi Acknowledgements............................................................................................................. viii List of Figures ............................................................................................................................ x List of Tables ........................................................................................................................... xv List of Abbreviations ......................................................................................................... xvii Introduction ............................................................................................................................... 1 CHAPTER 1 (Literature Review) ................................................................................... 3 1. NITROGEN METABOLISM IN NEMATODES .......................................................... 4 1.1. NEMATODE NEUROTRANSMITTERS .................................................................. 4 1.2. GLUTATHIONE METOBOLISM ................................................................................ 5 1.3. CHITIN METABOLISM ................................................................................................. 6 1.4. CHORISMATE MUTASE .............................................................................................. 6 1.5. AMINO ACID METABOLISM IN NEMATODES .................................................. 7 1.5.1. GLUTAMATE METABOLISM ................................................................................ 8 1.5.1.1. Aminotransferases .................................................................................................. 8 1.5.1.2. Glutaminase ................................................................................................................ 9 1.5.1.3. Glutamine synthetase-Glutamate synthase (GS-GOGAT) pathway ...... 9 1.5.1.4. Glutamate dehydrogenase ................................................................................. 10 i 1.5.1.5. Gamma-amino butyric acid (GABA)............................................................... 12 1.5.2. ARGININE METABOLISM ....................................................................................... 1 1.5.2.1. Ornithine urea cycle (OUC) ............................................................................... 14 1.5.2.2. Ornithine synthesis .............................................................................................. 15 1.5.2.2.1. Arginase ................................................................................................................ 15 1.5.2.2.2. Arginine deiminase pathway ........................................................................ 16 1.5.2.2.3. Arginine dihydrolase pathway ..................................................................... 16 1.5.2.3. Nitric oxide synthesis .......................................................................................... 17 1.5.2.4. Polyamine synthesis ............................................................................................ 18 1.5.2.4.1. Polyamine synthesis by ornithine decarboxylase ................................ 18 1.5.2.4.2. Polyamine synthesis by arginine decarboxylase .................................. 19 1.5.2.5. Phosphagen synthesis ......................................................................................... 19 1.5.3. PROLINE METABOLISM ...................................................................................... 21 1.5.4. LYSINE METABOLISM .......................................................................................... 22 1.5.4.1. The α-amino adipic acid pathway .................................................................. 22 1.5.4.2. The pipecolate pathway ..................................................................................... 23 1.5.4.3. Non-mammalian pathways ............................................................................... 24 1.6. Conclusions .................................................................................................................. 24 Chapter II ................................................................................................................................ 26 Glutamate synthase, but not GABA shunt enzymes, contributes to nitrogen metabolism of the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta ................................................................... 27 Chapter III .............................................................................................................................. 32 Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase ................................................................................................. 33 ii Chapter IV .............................................................................................................................. 43 Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus ........................................ 44 Chapter V ................................................................................................................................ 52 Enzymes of the ornithine-proline-glutamate pathway in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta 53 Chapter VI .............................................................................................................................. 59 Arginine metabolism in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta ................................................................... 60 Chapter VII ............................................................................................................................. 72 Lysine catabolism in Haemonchus contortus and Teladorsagia circumcincta ..................................................................................................................................................... 73 Chapter VIII ........................................................................................................................... 79 GENERAL DISCUSSION ..................................................................................................... 80 8.1. LYSINE CATABOLISM ............................................................................................... 81 8.2. PROLINE METABOLISM .......................................................................................... 83 8.3. ARGININE METABOLISM........................................................................................ 84 8.3.1. Arginase ........................................................................................................................ 85 8.3.2. Polyamine Synthesis ................................................................................................ 86 8.4. GLUTAMATE METABOLISM ................................................................................. 88 8.4.1. Gaba Metabolism ...................................................................................................... 89 8.4.2. Glutamate Synthase (GOGAT) .............................................................................. 89 8.4.3. Glutamate Dehydrogenase (GDH) ...................................................................... 90 iii 8.4.3.1. H. contortus GDH ................................................................................................... 91 8.4.3.2. Nucleotide allosteric regulation ...................................................................... 92 8.4.3.3. Recombinant T. circumcincta GDH ................................................................. 92 8.5. METABOLIC DIFFERENCES IN ABOMASAL NEMATODES ...................... 93 8.5.1. Difference between parasite and host .............................................................. 93 8.5.1 Difference between life-cycle stages .................................................................. 95 8.5.2 Difference between parasite species ................................................................. 95 8.6. FUTURE WORK ........................................................................................................... 96 References ............................................................................................................................. 98 Appendix 1. Statements of contribution to doctoral thesis containing publications .......................................................................................................................... 116 Appendix 2. Parasitology ..............................................................................................