New insights in the mode of action of anti-leishmanial drugs by using chemical mutagenesis screens coupled to next-generation sequencing

Abstract

Leishmania parasites are responsible for a range of clinical manifestations ranging from self-resolving cutaneous sores to life-threatening diseases. The management of leishmaniasis is complicated in part by the scarcity of treatment options but also by the emerging or established resistance to available drugs. A major driver of resistance in Leishmania is the amplification of resistance genes taking advantage of the highly repetitive genomic landscape of the parasite. The recent advent of whole genome gain of function screens gave new momentum to the study of such resistance mechanisms, leading to the identification of novel resistance factors and drug targets against approved drugs, which include antimony (SbIII), miltefosine (MIL), paromomycin (PMM), and amphotericin B. However, these screens do not pinpoint single nucleotide variations (SNVs), an important contributor of drug resistance. To fill the gap, our recent study describes the optimization of chemical mutagenesis coupled to next generation sequencing, an approach called Mut-seq, as a way to explore networks of drug resistance genes in organisms with a diploid to mosaic aneuploid genome like Leishmania. Our Mut-seq screen revealed associations between genes linked with lipid metabolism and resistance to MIL, and highlighted the role of a protein kinase in translation leading to resistance to PMM.

Keywords: Leishmania; Mut-seq; chemical mutagenesis; miltefosine; paromomycin; resistance.

Figure 1. FIGURE 1: A Mut-seq genomic screen highlights the role of the CDPK1 kinase in drug resistance.

(A) Mut-seq strategy. Optimisation in mutagens concentrations, recovery time, and plating leads to drug resistant mutant cells. Clones are individually expanded and their DNA sequenced by next-generation sequencing. A bioinformatics pipeline allows the detection of SNVs and recurrent ones are tested experimentally (by transfection and/or gene editing). New genes were isolated using Mut-Seq after selection for miltefosine (MIL), paromomycin (PMM), or antimony (SbIII) resistance. (B) CDPK1. Interactome experiments indicated that many partners are potentially interacting with CDPK1. Reciprocal immunoprecipitations confirmed that the ribosomal proteins L23a and L28, and ARM56 a protein associated with SbIII resistance are interacting with CDPK1. L23a is phosphorylated by CDPK1. The Mut-seq screen after selection with either PMM or SbIII revealed that the CDPK1 gene is frequently mutated. Diverse mutations in CDPK1 were proven to lead to PMM resistance and to modulation in translation. It remains to be seen whether the phosphorylation, or lack of, of L23a is important for those phenotypes. CDPK1 mutations are closely associated with SbIII selection and future work will determine whether its association with ARM56 is necessary for this phenotype. Some of the graphical items in the figure were taken from the Servier Medical Art website and are used under the terms of the Creative Commons Attribution 3.0 Unported License.