Multiple Mutations in Heterogeneous Miltefosine-Resistant Leishmania major Population as Determined by Whole Genome Sequencing

Abstract

BackgroundMiltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance.Methods/Principal FindingsAnalysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins—pyridoxal kinase and α-adaptin like protein—were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes.Conclusions/SignificanceWhole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes.

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

“…Although overexpression of these proteins leads to a gain-offunction resistance phenotype, Cos-Seq does not lend itself to isolation of loss-of-function transporter mutations, such as for the MTF transporter MT (10). Additional technical limitations associated with Cos-Seq include the failure to detect nonprotein drug targets or selection against specific regions whose overexpression would be toxic.…”

“…Eight cosmids were individually isolated for characterization, seven of which imparted MTF resistance between 1.7-and 3.2-fold (Table 1). MTF resistance is an easily selected phenotype as a result of single-point mutations in the MTF transporter (MT) (10,21). The latter is a loss-of-function event that would not be selected in the current screen.…”

Genomic Appraisal of the Multifactorial Basis forIn VitroAcquisition of Miltefosine Resistance in Leishmania donovani

“…For example, whole-genome sequencing was used to study experimental HePC resistance in L. major (43), and distinct mutations were identified, with a potential role in the mechanism of resistance. NGS also encompasses the study of the entire transcriptome of an organism via RNA-seq, with the advantage of quantifying the most significant changes in gene expression.…”

Drug Resistance in Leishmania

“…Finally, the sequencing of the genome of miltefosine-resistant mutants pinpointed a mutation in the pyridoxal kinase (PLK) gene (Fig. 4e), and the role of this gene in MIL resistance was confirmed by gene transfection experiments (Coelho et al 2012). …”

“…Interestingly, the resistance phenotype seems stable in in vitro macrophages infected with MT-null mutant and MT-mutated parasites (Seifert et al 2007), suggesting that what is observed in MIL-resistant promastigotes might be applied to intracellular amastigotes. Moreover, several mutations in MT can lead to resistance, and within a resistant population, they may be several different MT genotypes, leading to resistance (Coelho et al 2012).…”