Metabolic Alteration of Trypanosoma cruzi during Differentiation of Epimastigote to Trypomastigote Forms

Abstract

Intracellular parasites such as Trypanosoma cruzi need to acquire valuable carbon sources from the host cell to replicate. Here, we investigated the energetic metabolism of T. cruzi during metacyclogenesis through the determination of enzymatic activities and quantification by HPLC of glycolytic and Krebs cycle short-chain carboxylic acids. Altered concentrations in pyruvate, acetate, succinate, and glycerate were measured during the growth of epimastigote in the complex medium BHI and their differentiation to trypomastigotes in the chemically defined medium, TAU3AAG. These alterations should represent significant differential metabolic modifications utilized by either form to generate energy. This paper is the first work dealing with the intracellular organic acid concentration measurement in T. cruzi parasites. Although it confirms the previous assumption of the importance of carbohydrate metabolism, it yields an essential improvement in T. cruzi metabolism knowledge.

Keywords: Trypanosoma cruzi; carbohydrate metabolism; carboxylic acid; enzyme activity; high-performance liquid chromatography; organic acid.

Figure 1

Concentration of parasites’ organic acids after different growth periods as a function of growth time in BHI media. The concentrations of succinate (−O−), malate (−∇−), and pyruvate (−Δ−) were nearly equivalent and appeared superimposed, while citrate (−X−) and acetate (−◊−) show similar profiles during cultivation. However, the glycerate 2p (−□−) concentration was noticeably higher than the others were. Values correspond to the mean ± SD (three biological replicates) of organic acid concentration measured in three independent cultures from a sample size of 3 × 10⁸ parasites. *: p ≤ 0.05, statistically significant from previous time point.

Figure 2

Concentration of the organic acid from parasites in different times of the cultivation in TAU3AAG medium: glycerate 2p (−□−), citrate (−X−), pyruvate (−Δ−), malate (−∇−), acetate (−◊−) and succinate (−O−). The values correspond to the mean ± S.D (three biological replicates) of the concentration in nmoles of organic acid of three experiments determined in a population of 3 × 10⁸ parasites. *: p ≤ 0.05, statistically significant from previous time point.

Figure 3

Specific enzymatic activity of pyruvate kinase (Pk), hexokinase (Hk), and aldolase (Ald) in T. cruzi during its differentiation to trypomastigotes in TAU3AAG medium. The percentage of trypomastigotes forms were < 1%, 25%, 50%, and 70% at 0, 24, 48, and 72 h, respectively. *: p ≤ 0.05, statistically significant from previous time point.

Figure 4

Activity of pyruvate kinase (Pk), hexokinase (Hk), and aldolase (Ald) in T. cruzi cultured in BHI medium for different times. Extracts from 2 × 10⁸ parasites were assayed in three independent experiments, and the values plotted correspond to the average. The unit for Pk and Hk was µmoles subst/min/mg protein, and for the aldolase nmoles subst/min/mg protein and means an S.D. are presented. *: p ≤ 0.05, statistically significant from previous time point.