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Allelic Variation and Multigenic Metabolic Activity of Cytochrome Allelic Variation and Multigenic Metabolic Activity of Cytochrome P450s Confer Insecticide Resistance in Field Populations of Anopheles funestus s.s., a Major Malaria Vector in Africa Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Sulaiman Sadi Ibrahim January 2015 I DECLARATION This work has not previously been accepted in substance for any degree and is not being currently submitted in candidature for any degree. Signed ........................................................................................(Candidate) Date ........................................................................................... Statement 1 This thesis is the result of my own investigation, except where otherwise stated. Other sources are acknowledged and bibliography appended. Signed ........................................................................................(Candidate) Date ........................................................................................... Statement 2 I hereby give my consent for this thesis, if accepted, to be available for photocopying and for inter- library loan, and for the title and summary to be made available to outside organisations. Signed ........................................................................................(Candidate) Date ........................................................................................... I DEDICATION This work is for all the individuals (teachers, parents, loved ones and friends) that have been there for me when I needed them most, and encourage me most when in doubt on the vicissitudes of life. II ABSTRACT Malaria control relies heavily on the use of insecticides, especially the pyrethroids, for control interventions such as Long Lasting Insecticide Nets (LLINs) and Indoor Residual Spraying (ITNs). However, widespread resistance to insecticides in major malaria vectors, such as An. funestus is threatening to derail these control tools. To design and implement suitable resistance management strategies which will ensure the continued effectiveness of these control tools it is necessary to elucidate the molecular basis of the resistance. In An. funestus, resistance is mainly metabolic with the duplicated P450s CYP6P9a and CYP6P9b implicated as the major pyrethroid resistance genes. Despite the detection of these key resistance genes the detailed molecular mechanisms through which they confer pyrethroid resistance remain uncharacterised. Because CYP6P9a and CYP6P9b were shown to exhibit significant allelic variation between resistant and susceptible mosquitoes, we hypothesised that this allelic variation is potentially a key mechanism conferring pyrethroid resistance. Here, I characterised the role of these genes in the resistance to pyrethroids and identified other candidate genes which confer cross-resistance to non-pyrethroid insecticides. The role of allelic variation in pyrethroid resistance was investigated using polymorphism survery and in silico prediction of activity. Metabolic activities and efficiencies of allelic variants of CYP6P9a and CYP6P9b were investigated using fluorescent probes, metabolism assays and transgenic expression in D. melanogaster system. Pyrethroid resistance causative mutations were detected using the site- directed mutagenesis. Other candidate P450s that confer cross-resistance to pyrethroids carbamates and organochlorines were identified and characterised. This study revealed that CYP6P9a and CYP6P9b from resistant populations of An. funestus are undergoing directional selection with reduced genetic diversity and beneficial mutations selected, compared to the alleles from susceptible strain (FANG), which exhibited high genetic variation. Modelling and docking simulations predicted the alleles of CYP6P9a and CYP6P9b from the resistant strains all across Africa to metabolise pyrethroids with high efficiency while the susceptible alleles FANGCYP6P9a and FANGCYP6P9b were predicted to have low activity toward pyrethroids. Validation of the docking predictions with probes and metabolism assays established that the resistant alleles of CYP6P9a and CYP6P9b possess high activities toward pyrethroids with kinetic profiles significantly different (high affinity and catalytic efficiency) from those obtained from the FANG, indicating that allelic variation is playing a major role in pyrethroid resistance. These findings were further strengthened by results from transgenic expression with GAL4/UAS technology showing that flies expressing the resistant alleles of both genes were significantly more resistant to pyrethroids than those expressing the susceptible alleles. Using mutagenesis, three key residues (Val109, Asp335 and Asn384) from the resistant allele of CYP6P9b were established as the important amino acid changes responsible for resistance with impact on substrate channelling, possible enhancement of interaction with redox partners and inter- molecular hydrogen bonding interactions, respectively conferring high metabolic efficiency. The finding of these resistance markers make it possible to design a diagnostic tool that can allow detection and tracking of the resistant alleles in the field population of An. funestus across Africa. Other up-regulated P450s in multiple resistant populations from southern Africa were also characterised, revealing that pyrethroid resistance is mediated by other P450s as well: CYP6M7, CYP6Z1, CYP9J11 and CYP6AA4 all of which metabolise pyrethroids. CYP6Z1 and CYP9J11 are cross- resistance genes which metabolise bendiocarb, while CYP6Z1 metabolise DDT in addition. In conclusion, allelic variation is a key mechanism conferring pyrethroid resistance in An. funestus s.s. from sub-Saharan Africa. Key amino acid changes control pyrethroid resistance factors and these molecular markers can be used to design DNA-based diagnostic tests which will allow tracking of the resistance alleles in the field. Pyrethroid resistance is multi-genic in the field populations of An. funestus with other P450s involved apart from CYP6P9a and CYP6P9b. The finding of cross-resistance P450s is of concern to resistance management and should be taken into account when designing resistance management strategies. III ACKNOWLEDGEMENTS The list is endless, especially since the three years spent created more questions in my mind than answers. However, I must acknowledge the risky investment my Supervisor (Dr. Charles Wondji) and Prof. Hilary Ranson made in choosing me out of the potential applicants back in 2011 and the rarest chances for training offered by my secondary Supervisor (Dr. Mark Paine). I have learnt a lot more than I could possibly comprehend and hope that I have managed to connect few dots, in here. Charles has always been a source of inspiration, encouragement and opportunities, while Mark provides me with excellent openings to learn new techniques, all the time whipping my mind to reflect deeply on ramifications of things I ignorantly claimed or deliberately avoided. In the beginning there was Jacob (Dr Riveron J.M.) and at the end there stood Jacob, guiding, explaining in Spang-lish even if frustrated, arguing and making sure things are ‘all-or-none’ perfect. Thanks a lot Jacob, for your time and patience. My deepest gratitude goes to Bibby Jaclyn who helped me with the modelling and molecular docking component of this research even at the eleventh hour and Michael Voice (along with the other staff of the Cypex BioDundee who helped me with some part of the P450 characterisation experiment), as well as my advisory panel supervisors (Dr. Alvaro Acosta-Serrano and Dr. Gareth Lycett). Other individuals who have helped me one way or another on this journey and on the course of this research include Drs. Abdulsalami Yayo Manu, Hanafy M. Ismail, Eva Maria Hodel, Cristina Yunta Yanes, Rodolphe Poupardin, Clare Strode, Craig Wilding, Lee Haines, Lisa Stone, Ms Helen Irving, Mr. Michael Kusimo, Ms Kayla G. Barnes and the rest of staff members and students from the Vector Biology Department. I am grateful for your patience and help. Finally, thanks to the management of Bayero University Kano, Nigeria, especially Prof. Hafiz Abubakar for making this possible. IV TABLE OF CONTENTS DECLARATION .......................................................................................................................................... I DEDICATION ............................................................................................................................................ II ABSTRACT ............................................................................................................................................... III ACKNOWLEDGEMENTS .......................................................................................................................... IV TABLE OF CONTENTS ............................................................................................................................... V LIST OF TABLES ....................................................................................................................................... XI LIST OF FIGURES .................................................................................................................................... XII LIST OF APPENDICES ............................................................................................................................. XV LIST OF ABBREVIATIONS .....................................................................................................................
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