WHOLE GENOME IDENTIFICATION AND CHARACTERIZATION OF NOVEL MECHANISMS OF RESISTANCE TO TOPOISOMERASE-TARGETING DRUGS IN PROTOZOAN PARASITES

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Protozoan parasites represent one of the most relevant groups of pathogens for both animals and humans as they are, in many cases, highly zoonotic pathogens. These emergent parasites have an important impact on the economy of Québec due to their direct impact on livestock productions and on the health and well-being of companion animals.

 

As no vaccine or effective therapeutic treatment are currently available for any of these animal diseases, there is an urgent need to develop novel therapeutic strategies to eradicate these infections. In this way, during his Ph.D., Dr. Fernandez-Prada proved that DNA Topoisomerases (Topo) are suitable targets for treating protozoan infections in animals using Leishmania infantum, the causative agent of highly-zoonotic canine leishmaniasis, as a model parasite. In fact, Topo are key enzymes for many essential biological functions such as replication, transcription, recombination, repair and DNA segregation. These enzymes are responsible for the regulation of the DNA topological state by introducing or removing supercoiling, knots or catenations in DNA molecules. Dr. Fernandez-Prada’s discovery opened the door to the development of novel molecules for the treatment of zoonotic canine leishmaniasis. However, as seen for other drugs, Leishmania can rapidly bypass the effect of these promising inhibitors by deploying pleiotropic resistance mechanisms, which could include genome rearrangements, changes in genes dosage and single point mutation, among others. Consequently, in order to avoid drug-resistance emergence and be able to propose durable and efficient combinatory treatments, it is of vital importance to address the impact of leishmanial Topo (1 and 2) inhibition in a genome-wide context. To this end, we propose to explore all these potential mechanisms leading to resistance using two fast and robust, high-throughput whole-genome approaches.

 

We will use the Cos-seq gain-of-function approach to identify the genomic regions that bestow a selective advantage against Topo inhibitors. We will also proceed to directly select for Leishmania mutants and perform genomic comparative analyses. This second approach will help us to better identify copy number variations and single nucleotide polymorphisms linked to drug resistance and Leishmania survival.  The ensemble of these two genome-wide methods will lead us to identify unknown Topo-inhibitors targets as well as determinants of drug resistance. Moreover, for the first time, we will be able to highlight all the unknown auxiliary genes/pathways that are involved in buffering the cellular damages triggered by DNA topological stress in Leishmania parasites. Finally, this project will further innovation of animal infectious diseases and antimicrobial resistance research in the province of Québec, laying the foundations for subsequent grant proposals aiming at the validation of novel genes and pathways.

FRQNT New university researchers start up program 2019-NC-253631 awarded to CFP