Unveiling drug-tolerant and persister-like cells in Leishmania braziliensis lines derived from patients with cutaneous leishmaniasis

Abstract

Introduction: Resistance against anti-Leishmania drugs (DR) has been studied for years, giving important insights into long-term adaptations of these parasites to drugs, through genetic modifications. However, microorganisms can also survive lethal drug exposure by entering into temporary quiescence, a phenomenon called drug tolerance (DT), which is rather unexplored in Leishmania.

Methods: We studied a panel of nine Leishmania braziliensis strains highly susceptible to potassium antimonyl tartrate (PAT), exposed promastigotes to lethal PAT pressure, and compared several cellular and molecular parameters distinguishing DT from DR.

Results and discussion: We demonstrated in vitro that a variable proportion of cells remained viable, showing all the criteria of DT and not of DR: i) signatures of quiescence, under drug pressure: reduced proliferation and significant decrease of rDNA transcription; ii) reversibility of the phenotype: return to low IC₅₀ after removal of drug pressure; and iii) absence of significant genetic differences between exposed and unexposed lineages of each strain and absence of reported markers of DR. We found different levels of quiescence and DT among the different L. braziliensis strains. We provide here a new in-vitro model of drug-induced quiescence and DT in Leishmania. Research should be extended in vivo, but the current model could be further exploited to support R&D, for instance, to guide the screening of compounds to overcome the quiescence resilience of the parasite, thereby improving the therapy of leishmaniasis.

Keywords: Leishmania; antimonials; drug tolerance; persisters; quiescence.

Figure 1

Drug susceptibility of Leishmania braziliensis promastigotes and their growth features under drug pressure. (A) Drug susceptibility to potassium antimonyl tartrate (PAT) in nine strains as estimated by the resazurin test after 24 h of drug pressure. For each strain, the IC₅₀ was calculated in a lineage without prior exposure to the drug (post-PBS) and after the exposure to 9 µg/mL of PAT (~ 4 -fold the median IC₅₀ considering all lineages, post-PAT). The results represent the mean ± SEM of three biological replicates. (B) Evaluation of the cytostatic effect of PAT at 9 µg/mL. The gray dashed line represents no change in the original resorufin signal compared with the day the cells were initially plated. A log2 FC above 1 indicates parasite duplication and a log2 FC below 0 indicates parasite arrest and/or cellular death. The asterisks represent statistically significant differences after Tukey’s post-hoc test (P < 0.001) between cells after 24 h of plating without drug compared with cells under drug pressure.

Figure 2

rEGFP expression in promastigotes (nine strains) and amastigotes (three strains) of Leishmania braziliensis under drug pressure; measures are made on viable cells. (A) rEGFP expression of L. braziliensis lineages after 24 and 48 h of exposure to 9 µg/mL of PAT. The results represent the mean ± SEM of three biological replicates. The asterisks represent statistically significant differences after Tukey’s post-hoc test; ** P < 0.01, *** P < 0.001. (B) Relative rEGFP reduction (in %) in relation to increasing concentrations of PAT after 24 and 48 h of PAT exposure.

Figure 3

Lethal doses (LD) on Leishmania braziliensis promastigotes and amastigotes. (A) Viability of individual promastigotes after 24 h of exposure to increasing PAT concentration. (B) Lethal doses 10 and 50 on promastigotes. The results represent the mean ± SEM of three biological replicates. (C) The relationship between viability of promastigotes and reduction of rEGFP expression at different PAT concentrations. The Pearson’s correlation coefficients and P-values are shown. (D) Viability of individual amastigotes after 24 and 48 h of exposure to increasing PAT concentration. (E) Lethal doses 10 and 25 on amastigotes, calculated after 48 h of PAT exposure. The results represent the mean ± SEM of three biological replicates.

Figure 4

Genomic differences between the nine strains of Leishmania braziliensis and each lineage before (post-PBS) and after (post-PAT) drug pressure; bulk whole genome sequencing. (A) Ploidy. (B) Maximum likelihood phylogenetic tree based on 163,823 high-quality SNPs.

Figure 5

Promastigote (PER191 EGFP Cl5) viability under conditions of longer and stronger environmental stress. (A) Experimental outline. Cell viability as measured by flow cytometry in i) long-term stationary phase on day 14 (Sta D14) or ii) after exposing proliferating cells to 14 days of PAT pressure (log PAT D14); we tested the resilience of the survivors on day 14 after treating them with a second exposure to PAT or PBS as control over 24 h. The concentration of the first and second exposure to PAT was 9 µg/mL. See Materials and methods for details on sequential gating. (B) The density plots showing the percentage of survivors on day 14 in one representative biological replicate per experimental condition. (C) Cell viability and their levels of relative rEGFP reduction in long-term cultures as described in panel (A). The bars represent the mean ± SEM of three biological replicates.