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.
Highlights
Current genome-wide screens allow system-wide study of drug resistance but detecting small nucleotide variants (SNVs) is challenging
We used four different mutagens ENU, ethyl methanesulfonate (EMS), MMS, and HMPA against a freshly picked L. infantum clone while optimizing the mutagen concentrations, exposure (6–8 h) and recovery (24–36 h) times, and drug selection dose for both MIL and PMM
A similar deletion was detected in MIL5 (Supplementary Fig. 2a), which showed 1.55 ± 0.03 fold resistance to PMM compared to the wild-type source clone (p = 0.0004, unpaired two-tailed t-test)
Summary
Current genome-wide screens allow system-wide study of drug resistance but detecting small nucleotide variants (SNVs) is challenging. CRISPR-Cas system has been implemented effectively in Leishmania[10,11] which will eventually allow for genome-wide loss of function screens as demonstrated in mammalian cells[12,13] These techniques, whether based on gain- or loss of function usually will not pinpoint SNVs, which include single nucleotide polymorphisms (SNPs) and small insertions or deletions (indels), an important contributor of drug resistance in most organisms. To address this limitation we coupled chemical mutagenesis to NGS. We have performed extensive mechanistic studies on the role of a protein kinase involved in PMM resistance
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