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The Cytochrome P450 Family in the Parasitic Nematode Haemonchus Contortus

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The Cytochrome P450 Family in the Parasitic Nematode Haemonchus Contortus Laing, Roz (2010) The cytochrome P450 family in the parasitic nematode Haemonchus contortus. PhD thesis. http://theses.gla.ac.uk/2355/ 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] The cytochrome P450 family in the parasitic nematode Haemonchus contortus Roz Laing BSc (Hons) BVMS Institute of Infection and Immunity Faculty of Veterinary Medicine University of Glasgow Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy at the University of Glasgow September 2010 ii Abstract Haemonchus contortus, a parasitic nematode of sheep , is unsurpassed in its ability to develop resistance to the anthelmintic drugs used as the mainstay of its control. A reduction in drug efficacy leads to prophylactic and therapeutic failure, resulting in loss of productivity and poor animal welfare. This situation has reached crisis point in the sheep industry, with farms forced to close their sheep enterprises due to an inability to control resistant nematodes. The mechanisms of anthelmintic resistance are poorly understood for many commonly used drugs. Altered or increased drug metabolism is a possible mechanism, yet has received little attention despite the clear role of xenobiotic metabolism in pesticide resistance in insects. The cytochrome P450s (CYPs) are a large family of drug-metabolising enzymes present in all species. Their expression is induced on exposure to their substrate and over-expression of a single CYP has been shown to confer multi-drug resistance in insects. The H. contortus genome is currently being sequenced and assembled at the Wellcome Trust Sanger Institute, Cambridge. Despite the lack of a completed genome, the public provision of read, contig and supercontig databases has facilitated the identification of 73 partial gene sequences representing a large family of H. contortus CYPs. Their constitutive expression is highest in larval stages although adult expression was also detected. The majority of CYPs are most highly expressed in the worm intestine, which is thought to be the main organ of detoxification in nematodes and is consistent with a role in xenobiotic metabolism. A small number of CYPs were more highly expressed in anthelmintic resistant isolates than in an anthelmintic-susceptible isolate and may represent candidate genes for further research. The identification of putative H. contortus orthologues of the Caenorhabditis elegans nuclear hormone receptors controlling CYP transcription and the cytochrome P450 reductase gene catalysing electron transfer to CYPs suggests that regulatory and functional pathways may be conserved between the species. Transcriptome analysis using next generation sequencing was undertaken to guide a pilot annotation of 590 Kb genomic sequence. A high degree of iii conservation was observed between the conceptual translations of H. contortus and C. elegans genes, although at a genomic level, H. contortus consistently had a larger number and size of introns, which may reflect a larger genome than previously predicted. Gene order was not conserved, although regions of microsynteny were present and a bias for intra-chromosomal rearrangements resulted in putative orthologues frequently residing on the corresponding chromosome in both species. Partial conservation of a number of C. elegans operons in H. contortus was identified. These findings have important implications for the H. contortus genome project and the transcriptome databases provide a valuable resource for future global comparisons of gene expression. iv Contents Abstract ..................................................................................... ii Contents.....................................................................................iv List of Tables ..............................................................................vii List of Figures ............................................................................ viii Acknowledgement......................................................................... x Declaration .................................................................................xi Definitions/ Abbreviations ..............................................................xii 1 General Introduction ................................................................ 1 1.1 Drug resistance in parasites.................................................... 1 1.1.1 Measuring resistance....................................................... 3 1.2 Anthelmintics..................................................................... 4 1.2.1 Benzimidazoles ............................................................. 4 1.2.2 Imidazothiazoles ........................................................... 5 1.2.3 Macrocyclic lactones....................................................... 5 1.2.4 New anthelmintics ......................................................... 6 1.3 Alternative methods of parasite control ..................................... 7 1.4 The parasitic nematode H. contortus ........................................ 7 1.4.1 Genetic diversity of H. contortus ....................................... 10 1.4.2 H. contortus isolates...................................................... 11 1.4.3 The H. contortus genome project ...................................... 12 1.4.4 C. elegans as a model nematode ....................................... 12 1.5 Drug transfer and metabolism in nematodes ............................... 14 1.6 Cytochrome P450s (CYPs) ..................................................... 16 1.6.1 Location .................................................................... 17 1.6.2 Structure ................................................................... 17 1.6.3 Nomenclature.............................................................. 19 1.6.4 CYP family structure and evolution .................................... 19 1.6.5 Transcriptional regulation of CYPs...................................... 21 1.6.6 Factors affecting CYP activity........................................... 22 1.6.7 Induction and inhibition of CYPs by xenobiotics...................... 23 1.6.8 CYPs and insecticide resistance......................................... 25 1.6.9 CYPs and anthelmintic resistance ...................................... 26 1.7 Aims of this project..............................................................28 2 Materials and Methods .............................................................29 2.1 Standard reagents .............................................................. 29 2.2 Parasite .......................................................................... 29 2.2.1 H. contortus maintenance and culturing .............................. 29 2.2.2 Sexing adult H. contortus ................................................ 31 2.2.3 Xenobiotic exposure experiments ...................................... 31 v 2.3 Template preparation.......................................................... 32 2.3.1 DNA lysates for microsatellite analysis ................................ 32 2.3.2 RNA extraction............................................................. 33 2.3.3 First strand cDNA Synthesis.............................................. 33 2.4 Reverse transcriptase PCR..................................................... 34 2.5 Agarose gel electrophoresis ................................................... 34 2.6 Real-time PCR screen .......................................................... 34 2.6.1 Normalising and control genes .......................................... 34 2.6.2 Primer design .............................................................. 36 2.6.3 RT-QPCR reaction parameters........................................... 36 2.6.4 Analysis ..................................................................... 36 2.7 Illumina transcriptome analysis .............................................. 37 2.7.1 RNA and cDNA preparation............................................... 37 2.7.2 Sequencing and analysis.................................................. 38 2.8 Bioinformatic identification of CYP sequence.............................. 39 2.9 Bioinformatics software ....................................................... 40 3 Cytochrome P450 gene expression ..............................................41 3.1 Introduction ..................................................................... 41 3.2 Results............................................................................ 43 3.2.1 Identifying CYP gene sequences in an incomplete genome ......... 43 3.2.2 Development of a PCR assay of CYP gene expression................ 45 3.2.3 CYP gene expression in different life stages, sexes and tissues.... 50 3.2.4 CYP gene expression in xenobiotic-exposed H. contortus ........... 54 3.2.5 CYP gene expression in resistant isolates of H. contortus .......... 57 3.3 Tables and Figures.............................................................. 64 3.4 Discussion
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