A novel Sucrose/H+ Symport System and an Intracellular Sucrase in Leishmania Donovani.

The flagellated form of pathogenic parasitic protozoa Leishmania, resides in the alimentary tract of its sandfly vector, where sucrose serves as a major nutrient source. In this study we report the presence of a sucrose transport system in Leishmania donovani promastigotes. The kinetics of sucrose uptake in promastigotes are biphasic in nature with both high affinity Km (Km of �75 lM) and low affinity Km (Km � 1.38 mM) components. By contrast the virulent amastigotes take up sucrose via a low affinity process with a Km of 2.5 mM. The transport of sucrose into promastigotes leads to rapid intracellular acidification, as indicated by changes in the fluorescence of the pH indicator 20,70-bis-(2-carboxyethyl)-5-(6) Carboxyfluorescein (BCECF). In experiments with right side-out plasma membrane vesicles derived from L. donovani promastigotes, an artificial pH gradient was able to drive the active accumulation of sucrose. These data are consistent with the operation of a H+-sucrose symporter. The symporter was shown to be independent of Na+ and to be insensitive to cytochalasin B, to the flavonoid phloretin and to the Na+/K+ ATPase inhibitor ouabain. However, the protonophore carbonylcyanide P- (trifluromethoxy) phenylhydrazone (FCCP) and a number of thiol reagents caused significant inhibition of sucrose uptake. Evidence was also obtained for the presence of a stable intracellular pool of the sucrose splitting enzyme, sucrase, in promastigote stage parasites. The results are consistent with the hypothesis that L. donovani promastigotes take up sucrose via a novel H+–sucrose symport system and that, on entering the cell, the sucrose is hydrolysed to its component monosaccharides by an intracellular sucrase, thereby providing an energy source for the parasite