Investigation of the pathways related to intrinsic miltefosine tolerance in Leishmania (Viannia) braziliensis clinical isolates reveals differences in drug uptake

Abstract

In Brazil, cutaneous leishmaniasis is caused predominantly by L. (V.) braziliensis. The few therapeutic drugs available exhibit several limitations, mainly related to drug toxicity and reduced efficacy in some regions. Miltefosine (MF), the only oral drug available for leishmaniasis treatment, is not widely available and has not yet been approved for human use in Brazil. Our group previously reported the existence of differential susceptibility among L. (V.) braziliensis clinical isolates. In this work, we further characterized three of these isolates of L. (V.) braziliensis chosen because they exhibited the lowest and the highest MF half maximal inhibitory concentrations and were therefore considered less tolerant or more tolerant, respectively. Uptake of MF, and also of phosphocholine, were found to be significantly different in more tolerant parasites compared to the less sensitive isolate, which raised the hypothesis of differences in the MF transport complex Miltefosine Transporter (MT)-Ros3. Although some polymorphisms in those genes were found, they did not correlate with the drug susceptibility phenotype. Drug efflux and compartmentalization were similar in the isolates tested, and amphotericin B susceptibility was retained in MF tolerant parasites, suggesting that increased fitness was also not the basis of observed differences. Transcriptomic analysis revealed that Ros3 mRNA levels were upregulated in the sensitive strain compared to the tolerant ones. Increased mRNA abundance in more tolerant isolates was validated by quantitative PCR. Our results suggest that differential gene expression of the MT transporter complex is the basis of the differential susceptibility in these unselected, naturally occurring parasites.

Keywords: Isolates; Leishmania braziliensis; Miltefosine; RNAseq; Susceptibility; Uptake.

Graphical abstract

Fig. 1

Uptake of labeled phosphocholine MT-EtBDP (A) and PC-BODIPY (B) by L. (V.) braziliensis isolates and the reference strain M2903. Isolates were incubated with labeled molecules and fluorescence intensity inside parasite was measured by flow cytometry. Results are representative of three independent experiments and show the mean and standard deviation of fluorescence measured for three technical replicates.

Fig. 2

Labeled miltefosine (MT-EtBDP) uptake evaluated by confocal microscopy. Parasites were adhered to poly-lysine coated plates and MF was added. Uptake was monitored during 8 min and fluorescence intensity over time was calculated. Uptake of MT-EtBDP (green) by L. (V.) braziliensis isolates LTCP 16012 (A) and LTCP 19446 (B) at the time of MT-EtBDP addition and after 3:30 and 7:39 min. The complete video is available as a supplementary file. (C) Measured fluorescence of each parasite in the focal field was plotted for area under the curve (AUC) analysis. Black lines represent the mean and gray shade represents the standard deviation of fluorescence intensity measured for each isolate population (LTCP 16012 n = 17 and LTCP, 19446 n = 11). Small dotted line represents the mean and SD of fluorescence units measured for background in different regions of the plate.

Fig. 3

Residual fluorescence inside L. braziliensis isolates LTCP 16012 and LTCP 19446 was assessed by flow cytometry. After initial uptake determination, fluorescence associated with cell bodies was measured after 1 h, 2 h, 24 h and 48 h. Fluorescence intensity at each point time was normalized by the initial uptake of MT-EtBDP since these isolates showed differences in drug uptake. Each point represents mean and SD of three technical replicates and the graph show a representative experiment of three.

Fig. 4

Localization of MT-EtBDP in L. braziliensis promastigotes was analyzed by confocal microscopy. For both isolates, the same pattern of MT-EtBDP labeling (green) was observed (A and B) being mostly concentrated in the anterior portion of parasite, near the flagellar pocket and around nucleus. For co-localization assays, parasites were initially labeled with Hoechst (blue), (A) Mitotracker Deep Red FM (red) or (B) Lysonyr (red). After incubation with MT-EtBDP co-localization was determined by overlapping images obtained for each labeling.

Fig. 5

RNAseq revealed genes differentially expressed in both tolerant isolates compared to the sensitive strain, but absent in comparisons with M2903. Those genes were selected as possibly related to MF tolerance in these L. (V.) braziliensis clinical isolates. The table lists the description of these 36 DEG genes. Genes marked in green are upregulated in the sensitive isolate (LTCP 16012) compared to both tolerant strains (LTCP 16907 and LTCP, 19446). Genes in red are downregulated in the sensitive strain compared to tolerant strains. Genes shown in gray were found to be differentially expressed in these contrasts but not in the same direction, being upregulated in one comparison but down in the other. The detailed list of these 36 DEG containing logFC and p-value information is available as Supplementary Table 4.

Fig. 6

Relative abundance of Ros3 mRNA normalized by GAPDH mRNA expression for each L. (V.) braziliensis isolate compared to M2903. mRNA quantification was assessed by qRT-PCR and normalized using the 2^ΔΔCt method. Three biological replicates of each isolate were evaluated in three technical replicates of each sample.