Regulation of Phosphatidic Acid Levels in Trypanosoma cruzi

Abstract

Lipid kinases and phosphatases play essential roles in signal transduction processes involved in cytoskeletal rearrangement, membrane trafficking, and cellular differentiation. Phosphatidic acid (PtdOH) is an important mediator lipid in eukaryotic cells, but little is known regarding its regulation in the parasite Trypanosoma cruzi, an agent of Chagas disease. In order to clarify the relationship between PtdOH metabolism and developmental stages of T. cruzi, epimastigotes in culture were subjected to hyperosmotic stress (~1,000 mOsm/L), mimicking the environment in the rectum of vector triatomine bugs. These experimental conditions resulted in differentiation to an intermediate form between epimastigotes and trypomastigotes. Morphological changes of epimastigotes were correlated with an increase in PtdOH mass accomplished by increased enzyme activity of diacylglycerol kinase (DAGK, E.C. 2.7.1.107) and concomitant decreased activity of phosphatidate phosphatases type 1 and type 2 (PAP1, PAP2, E.C. 3.1.3.4). Our results indicate progressive increases of PtdOH levels during the differentiation process, and suggest that the regulation of PtdOH metabolism is an important mechanism in the transition from T. cruzi epimastigote to intermediate form.

Trypanosoma cruzi: Morphological and Microtubular Changes Related to Epimastigote Differentiation under Hyperosmotic Stress

“…As previously reported [8], in the hyperosmotic media the proportion of IF increased steadily, and reaching 85-90% of parasite forms at day 13, whereas in the control culture epimastigotes remained the predominant form (65%). The IF generated in hyperosmotic media showed normal viability, a delay in the lag period of the growth curve, increased generation time and a reduction in the maximal growth to ~ 50% of the control value ( Figure 1D).…”

“…Cells in logarithmic growth phase (day 6) were harvested by centrifugation at 1,000 g for 10 min, and washed twice with 25 mM Tris-HCl, pH 7.35, 1.2 mM MgSO 4 , 2.6 mM CaCl 2 , 4.8 mM KCl, 120 mM NaCl, and 100 mM glucose [Krebs-Ringer-Tris (KRT) buffer]. Intermediate Forms (IF) were obtained as described previously [8]. Briefly, epimastigote forms were grown in the same culture medium but in the presence of 0.5 M NaCl or 1 M mannitol, and the cells were harvested at day 13 by centrifugation at 1,000 g for 10 min and washed twice with KRT buffer.…”

“…Parasites were fixed with absolute methanol, stained with 10% Giemsa in 0.1 M sodium phosphate buffer, pH 7.2, and observed in a Zeiss Axiolab Microscope with a 100x objective. Parasites were classified as epimastigotes, trypomastigotes, or intermediate forms based on cell shape, size, and Giemsa staining, as described previously [8].…”

“…cruzi Intermediate Forms (IF) were obtained by growing of epimastigote forms in hyperosmolar media, as described previously [8]. The morphological study of these parasites was first carried out by observation (100x objectives) of preparations of Giemsa-stained samples.…”

Transcript Abundance of Putative Lipid Phosphate Phosphatases During Development of Trypanosoma brucei in the Tsetse Fly

“…The genome of Trypanosoma cruzi codes for at least three LPPs, which might be involved in cell differentiation. 7 In this study, four T. brucei genes encoding putative LPP proteins (TbLPP) were identified, and their tsetse tissue-specific transcription were profiled. In addition, we evaluated the transcriptional regulation of these genes both during parasite development in vivo and in vitro.…”