Coupling chemical mutagenesis to next generation sequencing for the identification of drug resistance mutations in Leishmania

Arijit Bhattacharya, Philippe Leprohon, Sophia Bigot, Prasad Kottayil Padmanabhan, Angana Mukherjee, Gaétan Roy, Hélène Gingras, Anais Mestdagh, Barbara Papadopoulou and Marc Ouellette ()
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Arijit Bhattacharya: Division of Infectious Disease and Immunity, CHU de Québec Research Center
Philippe Leprohon: Division of Infectious Disease and Immunity, CHU de Québec Research Center
Sophia Bigot: Division of Infectious Disease and Immunity, CHU de Québec Research Center
Prasad Kottayil Padmanabhan: Division of Infectious Disease and Immunity, CHU de Québec Research Center
Angana Mukherjee: Division of Infectious Disease and Immunity, CHU de Québec Research Center
Gaétan Roy: Division of Infectious Disease and Immunity, CHU de Québec Research Center
Hélène Gingras: Division of Infectious Disease and Immunity, CHU de Québec Research Center
Anais Mestdagh: Division of Infectious Disease and Immunity, CHU de Québec Research Center
Barbara Papadopoulou: Division of Infectious Disease and Immunity, CHU de Québec Research Center
Marc Ouellette: Division of Infectious Disease and Immunity, CHU de Québec Research Center

Nature Communications, 2019, vol. 10, issue 1, 1-14

Abstract: Abstract Current genome-wide screens allow system-wide study of drug resistance but detecting small nucleotide variants (SNVs) is challenging. Here, we use chemical mutagenesis, drug selection and next generation sequencing to characterize miltefosine and paromomycin resistant clones of the parasite Leishmania. We highlight several genes involved in drug resistance by sequencing the genomes of 41 resistant clones and by concentrating on recurrent SNVs. We associate genes linked to lipid metabolism or to ribosome/translation functions with miltefosine or paromomycin resistance, respectively. We prove by allelic replacement and CRISPR-Cas9 gene-editing that the essential protein kinase CDPK1 is crucial for paromomycin resistance. We have linked CDPK1 in translation by functional interactome analysis, and provide evidence that CDPK1 contributes to antimonial resistance in the parasite. This screen is powerful in exploring networks of drug resistance in an organism with diploid to mosaic aneuploid genome, hence widening the scope of its applicability.

Date: 2019
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DOI: 10.1038/s41467-019-13344-6

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