Multiple mutations in heterogeneous miltefosine-resistant Leishmania major population as determined by whole genome sequencing

Abstract

Background: Miltefosine (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 findings: Analysis 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/significance: Whole 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.

Figure 1. Miltefosine susceptibilities of Leishmania major mutants and wild-type parasites.

The EC₅₀ values were determined by culturing promastigote parasites in the presence of increasing concentrations of MF. The average of three independent experiments is shown. EC₅₀ values for MF in L. major wild-type parasites (1); MF80.1 (2); MF80.2 (3);MF80.3 (4); MF80.5 (5) MF-resistant mutants. The MF80.3 and MF80.5 mutants were grown in the absence of MF for 30 passages (6 and 7 respectively). * p<0.05; ** p<0.01.

Figure 2. Mutations in the miltefosine transporter (MT) and pyridoxal kinase (PK) in the MF80.3 lineage.

The top and bottom half circle represents the MT and PK genes respectively. White represents wild-type sequences; semi-colored an heterozygous mutation and fully colored an homozygous mutation. The type of mutation is also indicated.

Figure 3. Miltefosine susceptibilities of Leishmania major wild-type and MF-resistant transfectants.

The EC₅₀ values were determined by culturing promastigote parasites in the presence of increasing concentrations of MF. The average of three independent experiments is shown. LmjF WT (A), MF80.3 (B) and MF80.5 (C) parasites were transfected with pSP72αHYGα (1); pSP72αHYGα-MT (2); pSP72αHYGα-MT* (G852D) (3); pSP72αHYGα-MT* (M547del) (4); pSP72αNEOα-PK (5); and pSP72αHYGα-adaptin like protein (6). * p<0.01; ** p<0.001.

Figure 4. Miltefosine EC₅₀ values of L. major parasites.

Drug susceptibility of wild-type cells (white bars) and MF-resistant mutant population (P) or clones (gray shaded bars) were measured. The clones were derived from MF5.3, MF10.3 and MF15.3 population and clone numbers are relate to Fig. 2. The average of three independent measures is shown. * p<0.05; ** p<0.01; *** p<0.001.

Figure 5. Miltefosine susceptibilities of L. major parasites transfected with the PK gene.

The EC₅₀ values were determined by culturing promastigotes parasites in the presence of increasing concentrations of MF. The average of three independent experiments is shown. L. major MF15.3 transfected with the empty vector pSP72αNEOα (1); pSP72αNEOα-PK (2); or pSP72αNEOα-PK* (T216M) (3). * p<0.05.

Figure 6. Inactivation attempts of the L. major PK gene.

(A) A schematic drawing of the PK locus with PstI sites. (B) Southern blot analysis of L. major genomic DNA digested with PstI and hybridized with a 3′ UTR probe of PK gene (a 600 bp fragment just upstream the stop codon of PK gene). Lane 1, L. major wild-type; lane 2, L. major with one PK allele disrupted with the NEO marker; lane 3, L. major with two alleles disrupted (HYG and NEO). Molecular weight is indicated on the right and the alleles are indicated on the left. (C) Miltefosine susceptibilities of Leishmania major wild-type (1), SKO-PK (2) and SKO-PK complemented with a wild-type PK gene (3) parasites. The EC₅₀ values were determined by culturing promastigotes parasites in the presence of increasing concentrations of MF. The average of three independent experiments is shown. * p<0.01.