Farnesyl diphosphate synthase is a cytosolic enzyme in Leishmania major promastigotes and its overexpression confers resistance to risedronate

Abstract

Farnesyl diphosphate synthase is the most likely molecular target of aminobisphosphonates (e.g., risedronate), a set of compounds that have been shown to have antiprotozoal activity both in vitro and in vivo. This protein, together with other enzymes involved in isoprenoid biosynthesis, is an attractive drug target, yet little is known about the compartmentalization of the biosynthetic pathway. Here we show the intracellular localization of the enzyme in wild-type Leishmania major promastigote cells and in transfectants overexpressing farnesyl diphosphate synthase by using purified antibodies generated towards a homogenous recombinant Leishmania major farnesyl diphosphate synthase protein. Indirect immunofluorescence, together with immunoelectron microscopy, indicated that the enzyme is mainly located in the cytoplasm of both wild-type cells and transfectants. Digitonin titration experiments also confirmed this observation. Hence, while the initial step of isoprenoid biosynthesis catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase is located in the mitochondrion, synthesis of farnesyl diphosphate by farnesyl diphosphate synthase is a cytosolic process. Leishmania major promastigote transfectants overexpressing farnesyl diphosphate synthase were highly resistant to risedronate, and the degree of resistance correlated with the increase in enzyme activity. Likewise, when resistance was induced by stepwise selection with the drug, the resulting resistant promastigotes exhibited increased levels of farnesyl diphosphate synthase. The overproduction of protein under different conditions of exposure to risedronate further supports the hypothesis that this enzyme is the main target of aminobisphosphonates in Leishmania cells.

FIG. 1.

LmFPPS purification. Lane 1, crude extract (21 μg protein); lane 2, 0 to 40% saturated (NH₄)₂SO₄ fraction (21 μg of protein); lane 3, protein from hydroxyapatite chromatography (21 μg of protein).

FIG. 2.

Determination of LmFPPS expression by Western blot analysis of wild-type and transfectant cells. Lane 1, wild-type promastigotes; lane 2, pX63NEO control parasites; lane 3, pX63NEO-FPPS parasites overexpressing FPPS protein. The membrane was incubated with a rabbit anti-LmFPPS polyclonal antibody. Nine micrograms of protein was loaded into each lane. The net intensities of the individual LmFPPS bands were determined with 1D-manager analysis software.

FIG. 3.

Analysis of FPPS levels in wild-type and risedronate-resistant cells. (A) Western blotting was performed with extracts prepared from 10⁶ wild-type cells (lane 1) and 10⁶ resistant cells (lane 2). The membrane was incubated with a rabbit anti-LmFPPS polyclonal antibody. The blot was normalized with an anti-LmdUTPase polyclonal antibody. (B) Northern blotting was performed with 5 and 10 μg of total RNA from wild-type (lanes 1 and 3) and resistant (lanes 2 and 4) parasites. The blot was hybridized with an [α-³²P]dCTP-labeled LmFPPS probe and normalized with an [α-³²P]dCTP-labeled LmdUTPase probe.

FIG. 4.

Digitonin titration of LmFPPS. Activities of pyruvate kinase (open triangles), hexokinase (open squares), citrate synthase (open circles), and farnesyl diphosphate synthase (closed triangles) were assayed in supernatants of whole L. major suspensions in solutions with increasing digitonin concentrations. Total enzyme activity is expressed as a percentage, taking as 100% the total activity obtained in supernatants after permeabilization with 1 mg/ml of digitonin. (Left) Activities recovered from L. major wild-type supernatants; (right) activities recovered from supernatants of L. major parasites transfected with pX63NEO-LmFPPS and overexpressing FPPS.

FIG. 5.

Indirect immunofluorescence microscopy. (A, B, C, D, I, J, K, and L) Wild-type cells; (E, F, G, and H) FPPS-overexpressing cells. (A, E, and I) Phase-contrast images; (B and F) DAPI and Mitotracker staining, indicating the localization of DNA in the nucleus and kinetoplast in blue and the mitochondrion in red, respectively; (C and G) FITC staining (green) using the LmFPPS antibody diluted 1:1,200 for wild-type cells and 1:20,000 for transfected cells; (D and H) overlays of FITC, MitoTracker, and DAPI staining; (J) FITC staining (green) using the anti-ALD antibody diluted 1:1,200 and DAPI; (K) staining with the LmFPPS antibody labeled directly with Alexa Fluor 594 (red); (L) overlay of FITC (anti-ALD), Alexa Fluor 594 (anti-LmFPPS), and DAPI staining.

FIG. 6.

Immunoelectron microscopy localization of FPPS in control cells and transfectant cells overexpressing the enzyme. Leishmania major promastigotes were fixed in 4% paraformaldehyde and 0.4% glutaraldehyde and embedded in LRWhite resin. Thin sections were immunolabeled with purified anti-LmFPPS (diluted 1:3,000 for transfectant cells and 1:1,000 for wild-type control cells) polyclonal antibody followed by goat anti-rabbit immunoglobulin conjugated to gold (10 nm). Most of the gold-labeled particles were localized in the cytosol in wild-type cells (A) as well as in overexpressing FPPS transfectant cells (B and C). Magnification: ×16,000 for panel A, ×25,000 for panel B, and ×40,000 for panel C. N, nucleus; M, mitochondrion; G, glycosome; K, kinetoplast; AC, acidocalcisome.