Table 1.
EC50 values for methotrexate and antimony in Leishmania promastigotes and amastigotes calculated from concentration-response curves.
Fig 1.
Evaluation of ROS accumulation and parasite survival in the absence and the presence of Sb and MTX.
Measurement of drug-induced (Sb and MTX) ROS accumulation (DCFDA fluorescence; Cytation 5; ex/em 485/535 nm) in L. infantum WT and LiFS-A and LiFS-B clinical isolates. Graphs represents the number of viable promastigotes normalized to 106 cells/mL (dotted line) and DCFDA fluorescence normalized to 106 promastigotes (bars). Each data point represents the average ± SEM. Differences were statistically evaluated using an unpaired two-tailed t-test (*,+ p <0.05; **,++ p <0.01; +++ p < 0.001; ****,++++ p < 0.0001).
Fig 2.
Normalized mRNA expression levels of mrpA, ptr1 and dhfr in non-exposed parasites.
mRNA expression levels of drug-resistance genes mrpA (A), ptr1 (B) and dhfr (C) were determined by quantitative real-time RT-PCR in LiWT, LiFS-A and LiFS-B strains and normalized using gapdh as housekeeping gene. Results are derived from three biological replicates. Each data point represents the average ± SEM. Differences were statistically evaluated using an unpaired two-tailed t-test *p<0.05; ** p<0.01.
Fig 3.
Phenotypic characterization of L. infantum WT reference strain and LiFS-A and LiFS-B clinical isolates after ‘pre-exposure’ to Sb and MTX.
Five days after ‘pre-exposing’ LiWT (A-B), LiFS-A (C-D), LiFS-B (E-F) promastigotes to the EC50 and EC90 (previously calculated; Table 1) of Sb or MTX, parasites were submitted to increasing concentrations of MTX and Sb to evaluate potential changes in their phenotype against these drugs. EC50 values were calculated from concentration-response curves performed with biological triplicates after nonlinear fitting with GraphPad Prism 10 software.
Table 2.
Summary of proteins identified in Sb-treated and MTX-treated LiFS-A, demonstrating a positive temperature shift.
Proteins that are common between the strains LiFS-A and LiFS-B are highlighted in bold for easy identification.
Fig 4.
Heat map representation (row Z-score) of the general thermal stability of LiFS-A soluble protein cell extracts.
Normalized protein abundance of LiFS-A proteins for which full melting curves were acquired in the absence (A) or in the presence (B) of 100 μM Sb (118 proteins) and in the absence (C) or in the presence (D) of 100 μM MTX (84 proteins). Color range depicts the relative protein abundance of the soluble fractions at different temperatures. Heat maps were generated through the Heat mapper webserver (www.heatmapper.ca/expression) using its protein expression plugin with average linkage as clustering method applied to rows and Euclidean as distance measurement method.
Fig 5.
Heat map representation (row Z-score) of the general thermal stability of LiFS-B soluble protein cell extracts.
Normalized protein abundance of LiFS-B proteins for which full melting curves were acquired in the absence (A) or in the presence (B) of 100 μM Sb (42 proteins) and in the absence (C) or in the presence (D) of 100 μM MTX (55 proteins). Color range depicts the relative protein abundance of the soluble fractions at different temperatures. Heat maps were generated through the Heat mapper webserver (www.heatmapper.ca/expression) using its protein expression plugin with average linkage as clustering method applied to rows and Euclidean as distance measurement method. Of note, within the two field strains, 20 proteins were identified as shared following exposure to Sb. These shared proteins encompass ribosomal proteins, elongation factors, and heat shock proteins. Additionally, 14 proteins were recognized as common to both strains subsequent to their interaction with MTX, highlighting a prevalence of ribosomal proteins and those associated with the parasite’s cellular respiration.
Table 3.
Summary of proteins identified in Sb-treated and MTX-treated LiFS-B, demonstrating a positive temperature shift.
Proteins that are common between the strains LiFS-B and LiFS-A are highlighted in bold for easy identification.