Potential selection of antimony and methotrexate cross-resistance in Leishmania infantum circulating strains

Abstract

Background: Visceral leishmaniasis (VL) resolution depends on a wide range of factors, including the instauration of an effective treatment coupled to a functional host immune system. Patients with a depressed immune system, like the ones receiving methotrexate (MTX), are at higher risk of developing VL and refusing antileishmanial drugs. Moreover, the alarmingly growing levels of antimicrobial resistance, especially in endemic areas, contribute to the increasing the burden of this complex zoonotic disease.

Principal findings: To understand the potential links between immunosuppressants and antileishmanial drugs, we have studied the interaction of antimony (Sb) and MTX in a Leishmania infantum reference strain (LiWT) and in two L. infantum clinical strains (LiFS-A and LiFS-B) naturally circulating in non-treated VL dogs in Spain. The LiFS-A strain was isolated before Sb treatment in a case that responded positively to the treatment, while the LiFS-B strain was recovered from a dog before Sb treatment, with the dog later relapsing after the treatment. Our results show that, exposure to Sb or MTX leads to an increase in the production of reactive oxygen species (ROS) in LiWT which correlates with a sensitive phenotype against both drugs in promastigotes and intracellular amastigotes. LiFS-A was sensitive against Sb but resistant against MTX, displaying high levels of protection against ROS when exposed to MTX. LiFS-B was resistant to both drugs. Evaluation of the melting proteomes of the two LiFS, in the presence and absence of Sb and MTX, showed a differential enrichment of direct and indirect targets for both drugs, including common and unique pathways.

Conclusion: Our results show the potential selection of Sb-MTX cross-resistant parasites in the field, pointing to the possibility to undermine antileishmanial treatment of those patients being treated with immunosuppressant drugs in Leishmania endemic areas.

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.

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.