Identification and functional characterization of an ABC transporter of Haemonchus contortus, the P

317 Pages·2017·7.8 MB·English

by  Marion David

Identification and functional characterization of an ABC
transporter of Haemonchus contortus, the
P-glycoprotein 13
Marion A. David
To cite this version:
MarionA.David. IdentificationandfunctionalcharacterizationofanABCtransporterofHaemonchus
contortus, the P-glycoprotein 13. Toxicology. Université Paul Sabatier - Toulouse III, 2016. English.
￿NNT: 2016TOU30206￿. ￿tel-01665210￿
HAL Id: tel-01665210
https://theses.hal.science/tel-01665210
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Acknowledgements

First, I would like to thank the various fundings that made this work possible: the Natural Sciences
and Engineering Research Council of Canada, the FRQNT Centre for Host-Parasite Interactions,
Quebec, and EMIDA ERA-NET project CARES n° 11-EMID-003-02. I also thank the Center for host-
pathogens interactions (CHPI) that allowed me to extend my financial support for six months, the
Université Paul Sabatier (UPS) in Toulouse, and the Frontenac Program of Quebec government that
covered most of my travel fees between France and Canada.

I would like to acknowledge Pr. Elias Georges, Dr. Jean-Michel Jault and Pr. Alexis Valentin for
accepting to evaluate my PhD work.

Many thanks go to my supervisors Dr. Anne Lespine and Dr. Roger K. Prichard for giving me the
opportunity to do this PhD in co-direction between each of their laboratories in France and Canada.
This was a very rewarding experience in many ways, both scientific and personal. Thanks for believing
in me since the beginning and for always encouraging me to persist while Hco-Pgp-13 was giving us
hard times to learn to know it. Thanks for all your advice and for watching over my work so that it
followed the right track during all these years. Thanks for helping me with the extension of my PhD,
thus allowing it to finish in the best possible conditions.

I also would like to acknowledge my adivsory committee, Pr. Robin Beech and Dr. Cecile Menez,
who gave much advice and helped many times with results analysis during the three years of my PhD.

I would like to warmly thank the scientists who have highly contributed to the success of my PhD:
Thanks to Dr. François André for welcoming me to its laboratory at the CEA and
teaching me with bio/chemo-informatics. Thanks for teaching me patience and the art of perfectionism
that work together. Thanks for the extensive help with results analysis, and for revising each part of the
manuscripts as precisely as possible.
Thanks to Dr. Stéphane Orlowski for the incredible amount of work on ABC transporter
homologies studies. Thanks also for the help with writing and correcting articles. Thank you for all our
scientific talks, about my thesis project or other subjects of interest, and for being someone as passionate
about research as you are. I hope I learned everything I could from your philosophy of science.
Thanks to Dr. Chantal Lebrun, for being a skilled biochemist with a passion for
transmitting her knowledge. Thanks for having always been happy to help with experiments, to advice
with strategies and troubleshooting, and to participate in my manuscript correction. Thanks for your
contagious love of science and your great humanity.
Thanks to the students who critically helped me during these 4 years. Thanks to Shaima
Hashem for helping with my learning of in silico docking and homology modelling. Thanks for your
availability and sense of humor. Thanks to Thomas Duguet for helping in many ways with immuno-
fluorescence assays. Thanks for always being happy to collaborate and for being the best moaner in the
Parasitology building after me, thus contributing to the French reputation. Thanks to Clément Frainay
for his availability and the high amount of technical help provided for docking calculations run in
Toulouse. Thanks for being a ninja of bioinformatics.

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I also want to thank all other members of my both teams:
in Montreal: Thanks to Kathy for teaching me most of techniques in McGill,and for
various and extensive technical help during all my PhD, even from the other side of the ocean. Thanks
to Hua for her precious advice with the nested PCR and for her sweetness. Thanks to Catherine for
many scientific advice, long talks and valuable support. Thanks also to all the people who made this lab
such a nice cosmopolitan place: Ludmel, Virginie, Aissatou, Nour, Pablo, Shoaib, Durai and Phillip.
à ToxAlim, INRA : Merci à Jean-François, pour sa présence et pour tout le chocolat
salvateur de mon estomac. Merci à Elise, pour « la tristitude » et sa joyeuse folie contagieuse. Merci à
Léa, pour ne trouver aucune blague drôle mais pour avoir bien rit depuis son arrivée, et pour m’avoir
aidé précieusement sur la dernière ligne droite ! Merci à Mélanie et Alice pour leur aide au tout début
de ma thèse et à mon retour de Montréal. Enfin, merci aux étudiantes de licence pour avoir participé à
mon projet avec joie : Christina, Elsa et Mélanie.

A special thanks to the students I met in neighbor teams and who helped me in many ways:
at the Institute of Parasitology, McGill: Adeline, thanks for coming back to the airport
to take me back to your home on the 13th of November 2015, and for everything else. Krittika, so many
thanks for running with me by -13°C, it was so “cool”! Thanks to all of the students of your team, for
making me discover both Canadian and US American customs. Thanks to the Fauchers: Nilmini and
Hana for making me travel to Sri Lanka and Tunisia just by talking with you. Finally, thanks to the best
roommates ever: Sarah and Oscar, for being the kindest Quebecer and cat I could ever meet.
à ToxAlim, INRA : Merci à Alix, Eli et Sarah d’avoir fait leur thèse la porte en face ou
au bout du couloir, et d’avoir toujours été prêtes à s’entraider de diverses manières, et en particulier
les derniers jours d’écriture, vous avez été géniales. Merci pour tous les précieux souvenirs accumulés
pendant ces dernières années, j’espère que de nombreuses occasions d’en rajouter suivront. Merci à
Isaura pour ton sourire permanent et les très bons moments partagés et merci aux étudiantes de l’équipe
05 qui y ont également participé.
Merci à tous les autres, famille ou amis, loin de la science, qui m’ont soutenue de façon toute aussi
importante :
Au Canada: Merci à Marie-Jo et Dominik, Suzanne, Cynthia and Djé, pour être des
exemples de sagesse, de force et de gaieté. Merci à Renata Heidersdorf pour ses merveilleux cours d’art
qui m’ont aidé plus qu’il n’est imaginable dans mon projet scientifique.

En France : Merci à mes parents pour avoir toujours accepté de me soutenir
financièrement tout au long de ces quatre ans. Merci de m’avoir fait pousser à la campagne et d’avoir
arrosé mes racines de votre amour pour les rendre plus solides que je n’aurais cru. Les fruits de mes
années de travail sont aussi pour vous. Merci à ma grande famille, auprès de laquelle j’ai aimé me
ressourcer. Merci aux précieux amis qui ont toujours été tout près quelquesoit la distance. Merci à Patty
pour les trop rares moments partagés ces dernières années et à Léti pour n’avoir jamais hésité à
m’accueillir à Paris dès qu’un séjour était nécessaire au CEA. Vous êtes les meilleures. Merci à Nicolas
pour le soutien et les encouragements essentiels à ma fin de thèse, merci d’avoir été au bon endroit au
bon moment ! Merci infiniment à Marie-Jo, pour les mots simples et percutants, qui reboostent en
quelques phrases pour au moins des mois. Enfin, un immense merci à la Team AGL, qui s’agrandit
toujours plus, merci à tous ces gens au grand cœur d’être entré dans ma vie ! Un merci spécial à
Geneviève, sans qui l’aventure n’aurait jamais eu lieu, et qui veille comme une maman poule sur tous
ses petits, et Valérie, inspirante de motivation et de bienveillance, une grande sœur inestimable. Enfin,
merci à Ludo pour ton grand cœur fort si complémentaire à mon petit cœur sensible.

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A Grégory Lemarchal,
Pour m’avoir inspiré ma quête :

« Tenter, sans force et sans armure
D’atteindre l’inaccessible étoile
[…]
Je ne sais si je serai ce héros
Mais mon cœur serait tranquille »
La Quête – Jacques Brel

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Table of contents
TABLE OF CONTENTS ............................................................................................................ 1
LIST OF FIGURES AND TABLES OF THE LITERATURE REVIEW ............................... 5
ABBREVIATIONS ..................................................................................................................... 6
CONTRIBUTION OF AUTHORS ............................................................................................ 7
SCIENTIFIC VALORIZATION ................................................................................................ 8
GENERAL INTRODUCTION ................................................................................................... 9

LITERATURE REVIEW ................................................................................................. 15
I. HAEMONCHUS CONTORTUS AND CAENORHABDITIS ELEGANS .................................................... 15
1. Haemonchus contortus, a parasitic gastro-intestinal nematode .................................................. 15
2. Caenorhabditis elegans, a model nematode ................................................................................ 21
II. ANTHELMINTICS AND MECHANISMS OF RESISTANCE ................................................................ 25
1. Main classes of anthelmintic drugs ............................................................................................. 26
2. Mechanisms of resistance to anthelmintics ................................................................................. 31
III. ABC MDR TRANSPORTERS AND ANTHELMINTICS RESISTANCE .............................................. 34
1. ABC transporters: structures and functions ................................................................................ 34
2. ABC MDR transporters and resistance to anthelmintics............................................................. 53
3. Overcoming ML resistance in parasitic nematodes .................................................................... 59
HYPOTHESIS AND OBJECTIVES ....................................................................................... 64

EXPERIMENTAL WORK .............................................................................................. 67

PART I - IN SILICO FUNCTIONAL CHARACTERIZATION OF CEL-PGP-1 .............. 69

A. ........ MANUSCRIPT N°1 IN PREPARATION: MODELING MULTISPECIFIC DRUG
RECOGNITION BY CAENORHABDITIS ELEGANS P-GLYCOPROTEIN 1 ............................ 70
I. INTRODUCTION .............................................................................................................................. 72
II. COMPUTATIONAL METHODS ....................................................................................................... 75
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1. Structure of Cel-Pgp-1 ................................................................................................................ 75
2. Preparation and conformational analysis of ligands ................................................................... 76
3. Docking calculations ................................................................................................................... 76
4. Data analysis ............................................................................................................................... 77
5. Determination of the residues constituting the "hotspots for drug binding" ............................... 79
III. RESULTS ......................................................................................................................................... 80
1. Docking experiments on compounds stimulating or not the ATPase activity of Cel-Pgp-1 ....... 80
2. Binding mode of other substrates of mammalian Pgp previously tested on the ATPase activity of
Cel-Pgp-1 …………………………………………………………………………………………………...85
3. Binding mode of other molecules of interest .............................................................................. 87
IV. DISCUSSION .................................................................................................................................... 89
1. Validation of in silico docking approach on Cel-Pgp-1 ............................................................... 89
2. Further analysis of the in silico/in vitro correlation .................................................................... 91
3. Molecular properties of the multispecific binding domain of Cel-Pgp-1 .................................... 92

B. .......... MANUSCRIPT N°2 PUBLISHED IN THE INTERNATIONAL JOURNAL FOR
PARASITOLOGY: DRUGS AND DRUG RESISTANCE: IN SILICO ANALYSIS OF
ANTHELMINTICS BINDING TO CAENORHABDITIS ELEGANS P-GLYCOPROTEIN 1 .. 136

PART II - IDENTIFICATION, LOCALIZATION AND FUNCTIONAL
CHARACTERIZATION OF H. CONTORTUS P-GLYCOPROTEIN 13 ................................... 161

A. ......................... MANUSCRIPT N°3 IN PREPARATION: CHARACTERIZATION OF
HAEMONCHUS CONTORTUS P-GLYCOPROTEIN 13 ............................................................ 162
I. INTRODUCTION ............................................................................................................................ 164
II. MATERIAL AND METHODS .......................................................................................................... 166
1. Parasites .................................................................................................................................... 166
2. RNA extraction and reverse transcription. ................................................................................ 166
3. Amplification of Hco-pgp-13 cDNA sequence ......................................................................... 167
4. Prediction of Hco-Pgp-13 protein sequence and phylogenic analysis ...................................... 168
5. Construction of 3D models of Hco-Pgp-13 based on Cel-Pgp-1 4F4C PDB structure as template
and in silico docking calculations ................................................................................................................... 169
6. Cloning and transfection of Hco-pgp-13 gene in Pichia pastoris ............................................. 170
7. ATPase activity measurement of Hco-Pgp-13 stimulated by actinomycin D ........................... 171
8. Design of specific antibodies against Hco-Pgp-13 ................................................................... 172
9. Polyacrylamide gel electrophoresis and Western-blot .............................................................. 172
10. Immunohistochemistry on larvae and adult H. contortus sections............................................ 173
III. RESULTS ....................................................................................................................................... 173
1. Amplification and sequencing of Hco-pgp-13 cDNA ............................................................... 173
2. Translation product amino-acid sequence and topology of amplified Hco-pgp-13 .................. 174
3. Phylogenetic analysis of Hco-Pgp-13 ....................................................................................... 175
4. Homology modelling of Hco-Pgp-13 on Cel-Pgp-1 ................................................................. 176
5. In silico docking of actinomycin D on Hco-Pgp-13 ................................................................. 177
6. Expression of Hco-Pgp-13 in Pichia pastoris cells and stimulation of its ATPase activity by
actinomycin D ................................................................................................................................................. 178

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7. Immunolocalization of Hco-Pgp-13 protein in larvae and adult parasites ................................ 179
III. DISCUSSION .................................................................................................................................. 180
1. The Hco-pgp-13 corrected cDNA sequence encodes a protein matching the topology of an ABC
transporter ……………………………………………………….. .…………………….. ………………….181
2. Hco-Pgp-13 can interact with actinomycin D ........................................................................... 182
3. Hco-Pgp-13 sequence and localization are very close to those of Cel-Pgp-12, Cel-Pgp-13 and
Cel-Pgp-14 …………………………………………………………………………………………………...184

B. SUPPLEMENTARY EXPERIMENTS FOR THE LOCALIZATION OF HCO-PGP-13
MRNA IN THE PARASITE ........................................................................................................... 216
I. MATERIAL AND METHODS .......................................................................................................... 216
1. DNA Probes amplification ........................................................................................................ 216
2. RNA probes amplification and validation................................................................................. 217
3. Fluorescence in situ Hybridization ........................................................................................... 219
II. RESULTS ....................................................................................................................................... 219
1. Probes amplification and validation .......................................................................................... 219
2. Fluorescence in situ hybridization ............................................................................................ 221
III. DISCUSSION .................................................................................................................................. 221

C. SUPPLEMENTARY EXPERIMENTS FOR THE CHARACTERIZATION OF HCO-
PGP-13 FUNCTION ....................................................................................................................... 225
I. MATERIAL AND METHODS .......................................................................................................... 225
1. Codon optimization and transfection of Hco-pgp-13 in LLCPK1 cells .................................... 225
2. Characterization of mRNA expression ..................................................................................... 226
3. Characterization of protein expression...................................................................................... 227
4. Transport assays ........................................................................................................................ 228
5. Cytotoxicity assays ................................................................................................................... 228
6. ATPase activity assays .............................................................................................................. 229
7. In silico docking of various molecules on the two Hco-Pgp-13 3D structural models ............. 229
II. RESULTS AND DISCUSSION.......................................................................................................... 229
1. Expression of Hco-Pgp-13 in LLCPK1 .................................................................................... 229
2. Functional characterization of Hco-Pgp-13 expressed in LLCPK1 cells .................................. 230
3. Passage-dependent expression and function of Hco-Pgp-13 in LLCPK1 cells ......................... 231
4. Functional characterization of Hco-Pgp-13 expressed in Pichia pastoris ................................ 231
5. In silico docking calculations on Hco-Pgp-13 3D structural models ........................................ 232

GENERAL DISCUSSION .............................................................................................. 269
GENERAL DISCUSSION AND PROSPECTS ................................................................... 271
1. Cel-Pgp-1 is a multidrug transporter with some homologies with mammalian Pgp ................. 272
2. Cel-Pgp-1 interacts with ML with high affinity ........................................................................ 272
3. Hco-Pgp-13 presents many homologies with Cel-Pgp-1, which makes it a putative ABC
multidrug transporter ...................................................................................................................................... 273

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