MAPK1 of Leishmania donovani modulates antimony susceptibility by downregulating P-glycoprotein efflux pumps

Abstract

Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Mitogen-activated protein kinases (MAPKs) are well-known mediators of signal transduction of eukaryotes, regulating important processes, like proliferation, differentiation, stress response, and apoptosis. In Leishmania, MAPK1 has been shown to be consistently downregulated in antimony-resistant field isolates, suggesting that it has a role in antimony resistance. The present work investigates the molecular mechanism of MAPK1 in antimony resistance in Leishmania donovani. The L. donovani MAPK1 (LdMAPK1) single-allele replacement mutants exhibited increased resistance to Sb(III) (5.57-fold) compared to wild-type promastigotes, while overexpressing parasites became much more susceptible to antimony. The LdMAPK1-mediated drug sensitivity was directly related to antimony-induced apoptotic death of the parasite, as was evidenced by a 4- to 5-fold decrease in cell death parameters in deletion mutants and a 2- to 3-fold increase in MAPK1-overexpressing cells. LdMAPK1-underexpressing parasites also exhibited increased P-glycoprotein (P-gp)-mediated efflux pump activity, while a significant decrease in pump activity was observed in overexpressing cells. This change in efflux pump activity was directly related to expression levels of P-gp in all cell lines. However, episomal complementation of the gene restored normal growth, drug sensitivity, P-gp expression, and efflux pump activity. The data indicate that LdMAPK1 negatively regulates the expression of P-glycoprotein-type efflux pumps in the parasite. The decrease in efflux pump activity with an increase in LdMAPK1 expression may result in increased antimony accumulation in the parasite, making it more vulnerable to the drug.

FIG 1

Modulation of LdMAPK1 expression in Leishmania parasites. (a) Agarose gel stained with ethidium bromide showing PCR products of MAPK1, NEO, and HYG genes with Dd8^+/+ (wild-type parasites), Dd8^+/− (parasites with MAPK1 single-allele replacement), and Dd8^−/− (parasites with MAPK1 double-allele replacement). (From left) Lanes 1 and 8, 1 kb plus DNA ladders; lanes 2 to 4, MAPK1 PCR products from Dd8^+/+, Dd8^+/−, and Dd8^−/− parasites; lanes 5 to 7, NEO PCR products from Dd8^+/+, Dd8^+/−, and Dd8^−/− parasites; lanes 9 to 11, HYG PCR products from Dd8^+/+, Dd8^+/−, and Dd8^−/− parasites. (b) Effect of modulation of LdMAPK1 expression on promastigote growth. (c) Changes in expression of LdMAPK1 in wild-type (Dd8^+/+) parasites, vector control (Dd8Vc) parasites, overexpressing transfectants (Dd8^++/++), single-allele replacement mutants (Dd8^+/−), and add-back mutants (Dd8^+/−/+) by Western blot analysis using anti-LdMAPK1 antibodies. α-Tubulin was used as a control. (d) Quantitative estimation of expression of LdMAPK1 in cells by measuring the relative density of each band. The data are representative of the results of 3 independent experiments. The error bars represent SD. ***, P < 0.001; ns, not significant.

FIG 2

Effect of modulation of expression of LdMAPK1 on percent infectivity (number of infected macrophages × 100 cell nuclei) (a), number of intracellular amastigotes per cell nucleus after 24 and 72 h of infection (b), and antimony [Sb(III)] susceptibility of the parasite (c). The data represent means ± SD of the results of 3 independent experiments. **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 3

Effect of modulation of LdMAPK1 expression on antimony-induced apoptosis. (a) Cell cycle analysis by flow cytometry. The dark-gray peaks represent the G₀/G₁ and G₂/M phases, and the shaded region represents S phase, while the light-gray peaks represent the sub-G₀/G₁ (apoptotic) phase. (b) Histogram depicting DNA fragmentation by TUNEL assay. M1 is marked according to unstained cells, while M2 represents fluorescence of dUTP-FITC bound to nicked DNA ends. (c) Dot plot of annexin V and PI staining. The lower left quadrant represents unstained (healthy) cells, the upper left represents PI-positive (necrotic) cells, the lower right represents annexin V-positive (early apoptotic) cells, and the upper right represents annexin- and PI-positive (late apoptotic) cells. (d) ROS generation by H₂CDCF-DA staining. (e) Dot plot representing loss of mitochondrial transmembrane potential upon antimony treatment. The regions were marked on the basis of untreated cells. The dots outside region R1 represent red-fluorescent J aggregates (for healthy cells), and the dots within region R1 represent green-fluorescent monomers (for cells with depolarizing mitochondria). The data are representative of the results of three independent experiments.

FIG 4

Graphical representation of a rhodamine assay. (a) Mean fluorescence intensities of accumulated Rhodamine123 after 1 h of loading in the absence or presence of verapamil or trifluoperazine. (b) Mean fluorescence intensities representing rhodamine123 retention at 0 and 1 h of efflux in the absence or presence of verapamil or trifluoperazine. The data represent means ± SD of the results of 3 independent experiments. ***, P < 0.001; **, P < 0.05; ns, not significant.

FIG 5

P-glycoprotein expression and localization. (a) Histogram depicting cells stained with anti-P-glycoprotein antibody conjugated to phycoerythrin or its isotype antibody. (b) Ratio of mean fluorescence intensity of P-gp versus the isotype control. The data represent means ± SD of the results of 3 independent experiments. (c) Subcellular localization of P-gp on the plasma membrane of the cell by immunofluorescence microscopy, using anti-P-gp antibody conjugated to phycoerythrin. ***, P < 0.001; ns, not significant. DIC, differential interference contrast.