Molecular and biochemical characterization of Entamoeba histolytica fructokinase.

Julia Matt,Michael Duchêne

doi:10.1007/s00436-015-4383-5

Abstract

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess. The medium for its axenic culture contains glucose as energy source, and we addressed the question whether E. histolytica can also use fructose instead. As the amoebic hexokinases do not phosphorylate fructose, a separate fructokinase is essential. The genome project revealed a single candidate gene encoding an E. histolytica homolog of bacterial fructokinases. This gene was cloned, and the recombinant enzyme had a magnesium-dependent fructose 6-kinase activity (EC 2.7.1.4) with a K m for fructose of 0.156 mM and a V max of 131 U/mg protein. Recombinant fructokinase also showed a much weaker mannokinase activity, but no activity with glucose or galactose. The amoebae could be switched from glucose to fructose medium without any detectable consequence on doubling time. Fructokinase messenger RNA (mRNA) was modestly but significantly upregulated in amoebae switched to fructose medium as well as in fructose-adapted E. histolytica.Electronic supplementary materialThe online version of this article (doi:10.1007/s00436-015-4383-5) contains supplementary material, which is available to authorized users.

Highlights

The protozoan parasite Entamoeba histolytica is the cause of amoebic dysentery and liver abscess
We investigated whether E. histolytica is able to grow in a medium with fructose replacing glucose and if this medium switch would cause an upregulation of the putative fructokinase gene on the messenger RNA level and on the level of enzyme activity
E. histolytica trophozoites were switched from 10 g/l glucose to 10 g/l fructose medium

Summary

Introduction

The protozoan parasite Entamoeba histolytica is the cause of amoebic dysentery and liver abscess. The intestinal parasite exists in a microaerophilic environment and lacks a functional Krebs cycle, mitochondria and oxidative phosphorylation, so glycolysis is the major source of energy (Reeves 1984). Glucose is readily taken up (Serrano and Reeves 1974) and phosphorylated by one of the two hexokinases (Ortner et al 1995) as the first step of glycolysis. These two steps together with glycogen breakdown were found to have the largest influence on the glycolytic flux (Pineda et al 2014)

Methods

Results

Conclusion