Metabolic Profiling of the Protozoan Parasite Entamoeba invadens Revealed Activation of Unpredicted Pathway during Encystation

Abstract

Encystation, which is cellular differentiation from the motile, proliferative, labile trophozoite form to the dormant, resistant cyst form, is a crucial process found in parasitic and free-living protozoa such as Entamoeba, Giardia, Acanthamoeba, and Balamuthia. Since encystation is an essential process to deal with the adverse external environmental changes during the life cycle, and often integral to the transmission of the diseases, biochemical understanding of the process potentially provides useful measures against the infections caused by this group of protozoa. In this study, we investigated metabolic and transcriptomic changes that occur during encystation in Entamoeba invadens, the reptilian sibling of mammal-infecting E. histolytica, using capillary electrophoresis-tandem mass spectrometry-based metabolite profiling and DNA microarray-based expression profiling. As the encystation progressed, the levels of majority of metabolites involved in glycolysis and nucleotides drastically decreased, indicating energy generation is ceased. Furthermore, the flux of glycolysis was redirected toward chitin wall biosynthesis. We found remarkable temporal increases in biogenic amines such as isoamylamine, isobutylamine, and cadaverine, during the early period of encystation, when the trophozoites form large multicellular aggregates (precyst). We also found remarkable induction of γ-aminobutyric acid (GABA) during encystation. This study has unveiled for the first time the dynamics of the transcriptional and metabolic regulatory networks during encystation, and should help in better understanding of the process in pathogenic eukaryotes, and further development of measures controlling infections they cause.

The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation

“…This finding is consistent with recent work [56] in which the metabolome of encysting E. invadens was determined. In this study it was observed that during encystation and in mature cysts (48 h and 120 h post-encystation), basic metabolic processes such as glycolysis were drastically decreased, and glucose metabolism redirected to cyst wall synthesis.…”

Transcriptome Analysis of Encystation in Entamoeba invadens

“…However, despite its dormant nature, genes encoding several metabolic enzymes involved in nucleotide metabolism, energy, lipids, and sphingolipids metabolism were still transcribed during the late phase of encystation (Tables S6 and S7). In our previous study we discussed detailed analysis of metabolisms of glycolysis, amino acid, cyst wall biosynthesis [46]. Briefly, among genes involved in chitin biosynthesis, the transcript level of a gene encoding for glucosamine-fructose-6-phosphate aminotransferase (GFAT, EIN_136750), which is the first and rate limiting enzyme of the chitin biosynthetic pathway, was increased at 24 h of encystation (Table S3).…”

Entamoeba mitosomes play an important role in encystation by association with cholesteryl sulfate synthesis

“…Trophozoites of the E. invadens IP-1 strain were cultivated axenically as described previously in BI-S-33 medium at 26°C (17,29). To induce encystation, 1-wk-old trophozoite cultures were harvested and transferred to encystation medium at a final concentration of 6 × 10 5 /mL as described (17,29), with slight modifications. Cells were sampled at the indicated times after exposure to the encystation medium.…”

“…Cells were sampled at the indicated times after exposure to the encystation medium. The number and the percentage of cysts formed were determined as described previously (29). CS was dissolved in DMSO at 50 mM, and chlorate was dissolved in BI-S-33 or encystation medium at 500 mM, as stock solutions.…”