Drug Targets of the Heartworm, Dirofilaria immitis

161 Pages·2012·3.45 MB·English

Drug Targets of the Heartworm,
Dirofilaria immitis

Inauguraldissertation
zur
Erlangung des Würde eines Doktors der Philosophie
vorgelegt der
Philosophisch-Naturwissenschaftlichen Fakultät
der Universität Basel

von

Christelle Godel
aus La Sagne (NE) und Domdidier (FR)
Schweiz

Avenches, 2012
Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät
auf Antrag von

Prof. Dr. Jürg Utzinger
Prof. Dr. Pascal Mäser
P.D. Dr. Ronald Kaminsky
Prof. Dr. Georg von Samson-Himmelstjerna

Basel, den 26th of June 2012

Prof. Dr. M. Spiess
Dekan
To my husband and my daughter
With all my love.
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Table of Content
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Acknowledgements ............................................................................................................... 5
Summary ............................................................................................................................... 8
Introduction ..........................................................................................................................11
Dirofilaria immitis ..............................................................................................................12
Phylogeny and morphology ...........................................................................................12
Repartition and ecology .................................................................................................14
Life cycle .......................................................................................................................16
Diagnosis, treatment and resistance ..............................................................................18
Old and new sequencing strategies ..................................................................................21
DNA sequencing ...........................................................................................................21
Premises - Sanger, Maxam and Gilbert .....................................................................21
Dye-labeling terminator sequencing ...........................................................................23
Automated sequencing and high-throughput sequencing development .....................23
454-Pyrosequencing ..................................................................................................24
Illumina sequencing ...................................................................................................25
Third generation of high-throughput sequencing ........................................................27
Summary and further analysis ...................................................................................27
Assembly methods ........................................................................................................29
Example of de novo genome sequencing ......................................................................30
Anthelmintics and targets ..............................................................................................31
Nicotinic acetylcholine receptors and cys-loop ligand-gated ion channels .........................33
Aims and objectives .............................................................................................................36
Chapter 1. Understanding the magic bullet: molecular opportunities for antiparasitic drug
selectivity ..............................................................................................................................38
Chapter 2. Efficacy testing of AADs against D. immitis (unpublished) ...................................61
Chapter 3. The genome of the heartworm, Dirofilaria immitis ...............................................66
Genome-wide survey for ligand gated ion channels in D. immitis .................................... 104
Chapter 4. Loss of DEG-3-subfamily acetylcholine receptors and lack of stereoselective
monepantel sensitivity in Dirofilaria immitis ......................................................................... 106
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Deeper investigations through the DEG-3-subfamily ....................................................... 117
In silico novel potential drug targets of D. immitis ............................................................ 118
Chapter 5. Drug sensitivity tests (unpublished) ................................................................... 121
RNA-dependant RNA polymerase .................................................................................. 122
Chitin synthase ............................................................................................................... 123
Potential novel receptors of Wolbachia, endosymbiont of D. immitis ............................... 124
Discussion .......................................................................................................................... 126
1. Nucleic acid synthesis and repair receptors ............................................................. 127
RNA-dependant RNA polymerase ............................................................................... 127
2. Glycosylation and sugar metabolism ........................................................................ 128
Beta-1,4-mannosyltransferase ..................................................................................... 128
UDP-galactopyranose mutase ..................................................................................... 130
Chitin synthase ............................................................................................................ 131
3. Parallel study performed on Wolbachia of D. immitis ............................................... 132
Conclusions ........................................................................................................................ 134
References ......................................................................................................................... 136
Appendices ........................................................................................................................ 146
List of abbreviations ........................................................................................................ 147
List of figures .................................................................................................................. 152
List of tables ................................................................................................................... 153
Curriculum vitae ................................................................................................................. 155
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Acknowledgements
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I would li ke to thank all the people who contributed to the success of this doctoral
thesis:

Pascal Mäser for his precious support and supervision throughout my PhD duration;
for his help in innovation and for bringing relevant new input. And I would like to
specially thank him for being present to answer important questions and to bring
judicious advices.

Christian Epe for the internal support and supervision within Novartis Animal Health
and good advices, trust and friendship. The CAP, companion animal parasiticides,
team for their help in laboratory work and parasites maintenance.

Ronald Kaminsky to lead the project in house and help me with the internal
questions, to guide me through the meanders of Novartis guidelines; to believe in me
through my entire PhD and in my results.

Georg von Samson-Himmelstjerna for his invaluable advice and comments as my
co-referee.

Jacques Bouvier for his support and help concerning screening tests and precious
advices and information about chemical compounds as well as for his valuable new
scientific input and ideas; for our open-minded interesting discussions.

Lucien Rufener for his precious advices in molecular biology; especially to help to
come across difficulties in the laboratory work; for brainstorming situations and
interesting scientific discussions as well as for friendship and sharing the same office.

Sandra Schorderet-Weber for her help and precious advices concerning the in vivo
tests with the rodents. The Rodents Model team for the laboratory work and the
parasites maintenance.

Daniel Nilsson from the Karolinska Institutet in Sweden for his great help and
knowledge in informatics, computer science and bioinformatic studies.
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Sujai Kumar, Georgios Koutsovoulos and Mark Blaxter from the University of
Edinburgh for their help to share their knowledge and success in bioinformatics as
well as their huge education concerning nematode parasites to set a good
publication.

Frederik Bringaud to share his educative information concerning transposons and
apply it to the heartworm.

Philipp Ludin and Christoph Schmid from the Swiss Tropical and Public Health
Institute for their collaboration and work in the genome publication.

Novartis Animal Health St-Aubin FR and the Swiss Tropical and Public Health
Institute to allow me to perform my PhD and share the results and interesting data
with relevant persons and for the fellowship grant and the internal support.

Special thanks go to Timothy Geary and Roger Prichard for their adequate and
quick advices as well as for their interesting scientific opinions and for their support to
my studies.

Finally, I would like to thank my husband, Sylvain, and my daughter, Mélie, who
supported me during the whole PhD and were always behind me as well as my family
and my friends.
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Summary