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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 interlibrary 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 sitedirected 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 intermolecular 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 ......................................................................................................................XVI 1. LITERATURE REVIEW.......................................................................................................................1

1.1 1.2
Background .............................................................................................................................1 Malaria Vectors.......................................................................................................................2
Anopheles funestus .........................................................................................................3
Malaria Control .......................................................................................................................5
Treatment of Infection: Chemotherapy and Chemoprophylaxis....................................6 Malaria Vaccines .............................................................................................................6 Vector control .................................................................................................................7
Insecticides............................................................................................................................11
Pyrethroids....................................................................................................................11 Organophosphates (OPs) ..............................................................................................18 Carbamate.....................................................................................................................18 Organochlorines (OCs) ..................................................................................................19 Other insecticides .........................................................................................................19
Insecticide Resistance ...........................................................................................................20
Introduction ..................................................................................................................20 Types of Insecticides Resistance Mechanisms..............................................................20 Interaction of Resistance Mechanisms.........................................................................28
Cytochrome P450-Dependent Monooxygenases (P450s) ....................................................29

1.2.1

1.3
1.3.1 1.3.2 1.3.3
1.4
1.4.1 1.4.2 1.4.3 1.4.4 1.4.5
1.5
1.5.1 1.5.2 1.5.3
1.6
V
1.6.1 1.6.2 1.6.3 1.6.4 1.6.5 1.6.6
Introduction ..................................................................................................................29 Classes and Structural Features of P450s .....................................................................30 Insects Cytochrome P450s............................................................................................32 Genomic Variation of Insects P450s .............................................................................34 Mechanism of Action of CYP450s .................................................................................34 Redox Partners of Cytochrome P450s ..........................................................................37
Molecular Basis of Monooxygenase-mediated Insecticide Resistance ................................44
Introduction ..................................................................................................................44 Distribution of Insecticide Resistance in An. funestus across Africa.............................45 Role of Cytochrome P450s in Insecticide Resistance in An. funestus...........................50
Hypothesis.............................................................................................................................53 Aim and Objectives ...............................................................................................................54
1.7
1.7.1 1.7.2 1.7.3
1.8 1.9
2. POLYMORPHISM SURVEY AND PREDICTION OF IMPACT BY MODELLING AND DOCKING

SIMULATIONS........................................................................................................................................55

2.1 2.2 2.3
Background ...........................................................................................................................55 Objectives..............................................................................................................................56 Methods................................................................................................................................57
Mosquito Strains...........................................................................................................57 Amplification of full length CYP6P9a and CYP6P9b Alleles...........................................58 Sequence Analysis and Mapping of Polymorphisms ....................................................61 Modelling of CYP6P9a and CYP6P9b alleles by Satisfaction of Spatial Restraints........61 Models Assessment (Validation)...................................................................................63 Ligand Structures ..........................................................................................................64 Preparation of Receptors (Models) and Ligands for Molecular Docking......................64 Molecular Docking with GOLD......................................................................................64 In silico Analysis of Protein Pockets and Cavities..........................................................66
Results...................................................................................................................................67
Sequence Characterisation of CYP6P9a and CYP6P9b Alleles ......................................67 Modelling and Molecular Docking Simulation Analyses...............................................74 Active Site Residues and Enzymes-Substrates Interactions..........................................87 Identification of Substrate Access Channel pw2a.........................................................92
Discussion and Conclusion....................................................................................................94
2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 2.3.7 2.3.8 2.3.9
2.4
2.4.1 2.4.2 2.4.3 2.4.4
2.5

VI
3. ASSESSMENT OF METABOLIC EFFICIENCY OF ALLELIC VARIANTS USING FLUORESCENT PROBES, METABOLISM ASSAY AND TRANSGENIC EXPRESSION OF CYP6P9a and CYP6P9b ALLELES .................99

3.1 3.2 3.3
Background ...........................................................................................................................99 Aim and Objectives .............................................................................................................101 Methods..............................................................................................................................101

gambiae Cytochrome P450 Reductase (CPR) .............................................................................101

3.3.1.2 Construction of pACYC-184:: Cytochrome P450 Reductase Plasmid...........................106 3.3.1.3 Construction and Purification of pB13 ::( His) 4-Cytochrome b5 ..................................107 3.3.1.4 Co-transformation of pB13:: ompA+2CYPP9a and pB13::ompA+2CYP6P9b with pACYC-

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  • A Substrate-Specific Cytochrome P450 Monooxygenase

    A Substrate-Specific Cytochrome P450 Monooxygenase

    Insect Biochemistry and Molecular Biology 41 (2011) 244e253 Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella) Guodong Niu a, Sanjeewa G. Rupasinghe b, Arthur R. Zangerl a, Joel P. Siegel c, Mary A. Schuler b, May R. Berenbaum a,* a Department of Entomology, University of Illinois, Urbana, IL 61801, USA b Department of Cell and Developmental Biology, University of Illinois, Urbana, IL 61801, USA c USDA-ARS, S. Riverbend, Parlier, CA 93648, USA article info abstract Article history: The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree Received 13 August 2010 crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular Received in revised form mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, 30 December 2010 full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this Accepted 31 December 2010 insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selec- tively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl Keywords: compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed Amyelois transitella Cytochrome P450 monooxygenase P450 protein, P450 reductase and cytochrome b5 on 16 compounds, including phytochemicals, myco- fi Imperatorin toxins, and synthetic pesticides, indicated that CYP6AB11 ef ciently metabolizes imperatorin (0.88 pmol/ Piperonyl butoxide min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis Insecteplant interactions indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain.