An understanding of intracellular signaling pathways leading to resting cyst formation (encystment) is essential for the development of clinical drugs to prevent the life cycle of pathogenic unicellular eukaryotes. Recent studies imply that there are common signaling pathways among pathogenic and nonpathogenic unicellular eukaryotes. This paper describes molecular events, including signaling pathways, in the encystment of the nonpathogenic unicellular eukaryote Colpoda, based on results obtained mainly in our laboratory in the past 20 years.
Keywords: Colpoda; Resting Cysts; Encystment; Ca2+/Calmodulin; cAMP; Phosphorylation
Abbreviations: Ca2+/CaM: Ca2+/calmodulin; UV: Ultraviolet rays; PKA: Protein kinase A; Phos-tag/ECL: Phosphate-binding tag/enhanced chemiluminescence; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; 2-D PAGE: Two-dimensional polyacrylamide gel electrophoresis; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis: EF-1α: Elongation factor 1α; AMPK: AMP-activated protein kinase; eEF2K : Eukaryotic Elongation Factor-2 Kinase
One strategy of some pathogenic unicellular eukaryotes is to form resting cysts that are resistant to the environmental stresses of the host, such as stomach acid or immune system attack. Therefore, an understanding of intracellular signaling pathways leading to resting cyst formation (encystment) is essential for the development of clinical drugs to prevent cyst formation. It has been reported that the induction of the encystment of pathogenic Entamoeba histolytica is Ca2+/calmodulin (Ca2+/CaM)-dependent  and cAMP-dependent in the pathogenic unicellular eukaryote Giardia . Ca2+/CaM and cAMP are also essential components in intracellular signaling pathways of the nonpathogenic free-living soil ciliate Colpoda . These facts imply that common signaling pathways leading to encystment occur in some pathogenic and nonpathogenic unicellular eukaryotes. Recently, early molecular events during the encystment of Colpoda, including signaling pathways, were elucidated extensively. In this paper, we describe the encystment process of Colpoda based mainly on our research results. In hazardous environments, Colpoda promptly transforms into resting cysts that are resistant to desiccation, freezing, high temperature, acid, and UV light [4-7]. In the case of Colpoda cucullus Nag-1 , encystment can be induced by suspending vegetative Colpoda cells at a high cell density in the presence of Ca2+ .
Morphogenetic Events During Encystment of C. cucullus Nag-1
The morphogenetic changes in encystment-induced Colpoda cells are described as follows:
2~3 h after Encystment Induction: The cells stop swimming and begin to aggregate by an excretion of sticky mucus. The cells then round up, and then small sticky globules showing a fibrous or crystal-like fine structure  called lipidosomes  are extruded to be trapped by the mucus layer, followed by the formation of an ectocyst layer (a rigid single-cyst wall layer) . In this stage, the cellular structures characterizing vegetative cells such as cilia begin to disintegrate and mitochondrial activity is arrested .
3~5 h after Encystment Induction: At 3~5 h after encystment induction, the first-synthesized endocyst layer is formed by the excretion and gelation of an endocyst-precursor substance between an ectocyst layer and the plasma membrane . In order to digest vegetative structures, several autophagosomes are formed to digest the vegetative structures.
5 h~1 week after Encystment Induction: The formation of endocyst layers is repeated (presumably twice per day), and several layers of endocyst are formed in several days. Auto phagocytosis is nearly completed within 24h. In the 1-week-old mature cyst, the cytoplasm is filled with an amorphous electron-lucent material and many electron-lucent ellipsoidal granules (presumably reserve grains) accumulate .
Signaling Pathways Leading to Encystment and Early Molecular Events During Resting Cyst Formation
Using a pharmacological method, we have proposed that intracellular signaling pathways leading to the encystment of C. cucullus Nag-1 are activated by Ca2+-calmodulin, followed by an increase in intracellular cAMP concentrations . Thereafter, an encystment-dependent elevation of the intracellular Ca2+ concentration was demonstrated by using Fura 2 ratiometry . Intracellular Ca2+ may activate calmodulin, which is thought to activate adenylate cyclase to elevate cAMP, followed by activation of protein kinase A (PKA) . Actually, cAMP enzyme immunoassay (EIA) showed up to ten-fold elevation of cAMP levels in encystment-induced Colpoda cells [13,14]. cAMP-dependently phosphorylated proteins were detected by means of biotinylated Phos-tag/enhanced chemiluminescence (ECL) assay  and then identified by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis [16,17]. The proteins whose phosphorylation levels are up-regulated at 1h after encystment is induced by Ca2+/ overpopulation of Colpoda vegetative cells or by the addition of membrane-permeation-type cAMP acetoxymethyl ester (cAMPAM) are listed as follows (most phosphorylated proteins are common in Ca2+/overpopulation induction and cAMP induction):
a. Ribosomal P0 protein (localized in macronucleus) Presumed function: Regulation of gene expression and metabolism
b. Ribosomal S5 protein
Presumed function: Arrest of cell cycle
c. Rieske iron-sulfur protein (RISP)
Presumed function: Arrest of mitochondrial activity d. Histone H4 (hyperacetylated form)
Presumed function: Chromatin condensation of the macronucleus
Presumed function: Resorption of cilia
During Colpoda encystment, the expression of encystmentspecific proteins is expected to occur, and most proteins expressed in vegetative cells may be silenced. Actually, mRNA levels are extremely reduced within 5h after encystment induction . We analyzed the alteration of water-insoluble protein expression levels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) or two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and identified these proteins by LC-MS/MS analysis [11,18]. The proteins whose expression levels are modified within 12h after encystment induction are listed as follows:
a. Elongation factor 1α (EF-1α) [up-regulated 3h after encystment induction]
Presumed function: Acceleration of protein synthesis
b. ATP synthase β chain [downregulated 4h after encystment induction]
Presumed function: Disappearance of the mitochondrial membrane potential
c. Heat shock protein 60 (HSP 60) [temporarily up regulated 5h after encystment induction]
Presumed function: Molecular chaperon
d. Actin-related 49 kD protein [up-regulated 1.5h after encystment induction]
Presumed function: Chromatin remodeling
The 2-D PAGE of water-insoluble components (containing ciliary microtubules) of C. cucullus Nag-1 showed that the amount of tubulin is drastically reduced within several hours after the onset of encystment induction . In this case, tubulin gene expression may not be downregulated but instead probably resulted from the disassembly of microtubules of ciliary axoneme. Knowledge of the signaling pathways for encystment, including those proposed by our research group, has been extensively advanced by the Colpoda aspera transcriptome analysis performed by Jiang et al. . In the signaling pathways activated by Ca2+-calmodulin, the expression of genes for AMP-activated protein kinase (AMPK), eukaryotic elongation factor-2 kinase (eEF2K), AKT (protein kinase B) and several genes for autophagy is up-regulated.
Colpodais a nonpathogenic unicellular eukaryote, an understanding of molecular events in the early stages of
Colpodaencystment is expected to help elucidate the signaling pathways leading to the encystment of pathogenic unicellular eukaryotes.
This research was financially supported by a Sasagawa Scientific Research Grant (#24-407) from Japan Science Society, by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (#13J08784), and by Grant-in-Aid for Scientific Research (B) (#19H03447).
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