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Leishmania mexicana amazonensis: plasma membrane adenine nucleotide translocator and chemotaxis - PubMed

Leishmania mexicana amazonensis: plasma membrane adenine nucleotide translocator and chemotaxis

S Detke et al. Exp Parasitol. 2008 Mar.

Abstract

Leishmania cannot synthesize purines de novo and rely on their host to furnish these compounds. To accomplish this, they possess multiple purine nucleoside and nucleobase transporters. Subcellular fractionation, immunohistochemical localization with anti-adenine nucleotide translocator (ANT) antibodies and surface biotinylation show that the mitochondrial ANT is also present in the plasma membrane of both promastigotes and amastigotes. Leishmania, however, do not appear to rely on this transporter to supplement their purine or energy requirements via preformed ATP from its host. Rather, Leishmania appear to use the plasma membrane ANT as part of a chemotaxis response. ATP is a chemorepellant for Leishmania and cells treated with atractyloside, an inhibitor of ANT, no longer exhibit negative chemotaxis for this compound.

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Figures

Figure 1
Figure 1. Localization of YFP tagged adenine nucleotide translocator in Leishmania

Leishmania transformed with YFP-ANT or ANT-YFP were counterstained with MitoTracker® Red 580 and observed by confocal microscopy. Panels A-C and G-M, Leishmania transformed with the ANT-YFP gene; panels D-F, Leishmania transformed with the YFP-ANT gene; panels N-P, wild type Leishmania. Panels A, D, G, K and N, differential interference contrast (DIC) microscopy; panels B, E, H, L and O, yellow fluorescence channel; panels I, M and P, red fluorescence channel; panels C and F, merged image of yellow and DIC images; panel J, merged image of yellow and red channels. The cells in panels G-J and N-P were counterstained with MitoTracker® Red 580.

Figure 2
Figure 2. Subcellular localization of adenine nucleotide translocator in Leishmania promastigotes determined by subcellular fractionation

Western blots of mitochondria, plasma membrane and cytoplasmic fractions were probed with the antibodies indicated adjacent to each panel. Equal amounts of protein (5μg/lane) from the three fractions were resolved by denaturing poly acrylamide gel electrophoresis and blotted onto PVDF membrane. The results are representative of at least six separate experiments. Lane1, mitochondria; lane 2, plasma membrane, lane 3, cytoplasm.

Figure 3
Figure 3. Localization of the adenine nucleotide translocator in the plasma membrane by indirect immunofluorescence

Promastigotes were fixed with either 0.5% (w/v) gluteraldehyde or 4% (w/v) paraformaldehyde as described in the methods section. The fixed cells were probed with the antibody indicated adjacent to each panel. Polyclonal antibodies used to analyze ANT, CPT and gp63 were visualized with FITC-labeled goat anti rabbit secondary antibody whereas anti LPG monoclonal antibody was visualized with rhodamine-labeled goat anti mouse secondary antibody. DIC, differential interference microscopy.

Figure 4
Figure 4. Biotinylation of plasma membrane adenine nucleotide translocator by a membrane impermeable biotinylation reagent

Late log phase Leishmania promastigotes were incubated with the membrane impermeable EZ-Link™ sulfo-NHS-LC-biotin for 1 hour and processed as described in the methods section. Biotinylated proteins were isolated by chromatography on immobilized avidin columns and analyzed by western blotting with the antibodies indicated adjacent to each panel. +, cells with biotin cross linker; -, cells without biotin cross linker.

Figure 5
Figure 5. Chemotactic response of Leishmania to ATP

The chemotactic response of Leishmania to ATP and glucose was assessed by the capillary method as described in the methods section. The relative ratio is the cell density in the capillary tube with the attractant relative to the density in the capillary tube with no attractant. The data are the mean ± standard deviation from two experiments each done in triplicate. The statistical significance was determined by Student’s t test: *, p < 0.01. Black bar, control cultures; stippled bar, Leishmania with 100 μg/ml sodium atractyloside, an inhibitor of ANT; grey bar, Leishmania with 10 μM ATP added to the main reservoir of cells.

Figure 6
Figure 6. Localization of the adenine nucleotide translocator on the plasma membrane of amastigotes by indirect immunofluorescence

Leishmania were fixed with 0.5% (w/v) gluteraldehyde as described in the methods section and probed with preimmune or anti ANT antibody. The distribution of the primary antibodies was visualized with FITC-labeled goat anti rabbit secondary antibody. Top three panels, amastigotes; bottom three panels, promastigotes. Panels B and E, preimmune serum; panels A, C, D and F, anti ANT antibody. Panels B, C, E and F, Leishmania co-stained with MitoTracker® Red 580. DIC, differential interference microscopy; Green, green fluorescence channel; Red, red fluorescence channel. The bar in panel A is 10 μm.

Figure 7
Figure 7. Biotinylation of plasma membrane adenine nucleotide translocator in amastigotes by a membrane impermeable biotinylation reagent

Leishmania amastigotes were purified from J774G8 cells, incubated with the membrane impermeable EZ-Link™ sulfo-NHS-LC-biotin for 1 hour and processed as described in the methods section. Biotinylated proteins were isolated by chromatography on immobilized avidin columns and analyzed by western blotting with anti ANT antibodies. +, cells with biotin cross linker; -, cells without biotin cross linker.

Figure 8
Figure 8. Comparison of the relative abundance of adenine nucleotide translocator in amastigotes and promastigotes

Western blots of whole cell lysates (i.e., 20 μg total protein per lane) from amastigotes and promastigotes were probed with anti ANT and anti tubulin antibodies. The data are the mean ± standard deviation (N = 3). The difference in ANT levels between amastigotes and promastigotes was not statistically significant as determined by Student’s t test (p > 0.05) but the difference for tubulin between these two stages was statistically significant (*, p < 0.01). The western on the right shows the relative intensity of the bands from amastigotes and promastigotes from one of the above experiments.

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