During environmental stress, the vegetative cells of the facultative pathogenic amoeba Acanthamoeba castellanii reversibly differentiate into resistant dormant stages, namely, cysts or pseudocysts. The type of resistant stage depends on the nature and duration of the stressor. Cell differentiation is accompanied by changes in morphology and cellular metabolism. Moreover, cell differentiation is also expected to be closely linked to the regulation of the cell cycle and, thus, to cellular DNA content. While the existence of the resistant stages in A. castellanii is well known, there is no consensus regarding the relationship between differentiation and cell cycle progression. In the present work, we used flow cytometry analysis to explore the changes in the DNA content during Acanthamoeba encystation and pseudocyst formation. Our results strongly indicate that A. castellanii enters encystation from the G2 phase of the cell cycle. In contrast, differentiation into pseudocysts can begin in the G1 and G2 phases. In addition, we present a phylogenetic analysis and classification of the main cell cycle regulators, namely, cyclin-dependent kinases and cyclins that are found in the genome of A. castellanii.
- MeSH
- Acanthamoeba castellanii klasifikace genetika MeSH
- buněčná diferenciace genetika MeSH
- fylogeneze MeSH
- fyziologický stres genetika MeSH
- proteiny buněčného cyklu genetika MeSH
- protozoální DNA analýza MeSH
- průtoková cytometrie MeSH
- stadia vývoje genetika MeSH
- Publikační typ
- časopisecké články MeSH
The non-reducing disaccharide trehalose can serve as a protectant against a range of environmental stressors, such as heat, cold, or dehydration, in both prokaryotes and eukaryotes, with the exception of vertebrates. Here, we analyzed trehalose metabolism in the facultatively parasitic organism Acanthamoeba castellanii, known to respond to unfavorable external conditions by forming two resistant stages: a cyst, produced in the case of chronic stress, and a pseudocyst, formed in reaction to acute stress. The possible role of trehalose in the resistant stages was investigated using a combination of bioinformatic, molecular biological and biochemical approaches. Genes for enzymes from a widespread trehalose-6-synthase-trehalose-6-phosphate phosphatase (TPS-TPP) pathway and a prokaryotic trehalose synthase (TreS) pathway were identified. The expression patterns of the genes during encystation and pseudocyst formation were analyzed and correlated with the time course of cellular trehalose content determined mass spectrometrically. The data clearly demonstrate fundamental differences between encystation and pseudocyst formation at the level of cellular metabolism.
- MeSH
- Acanthamoeba castellanii genetika metabolismus MeSH
- fylogeneze MeSH
- genom protozoální * MeSH
- messenger RNA genetika metabolismus MeSH
- metabolické sítě a dráhy MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA protozoální genetika metabolismus MeSH
- trehalosa biosyntéza MeSH
- Publikační typ
- časopisecké články MeSH
Genes for mannitol-metabolizing enzymes, mannitol phosphate dehydrogenase (MPDH) and mannitol dehydrogenase (MDH), have been recently identified in the genome of Acanthamoeba castellanii and their potential role in stress tolerance was proposed. Using qRT-PCR, comparison has been made of mRNA levels of the enzymes for mannitol metabolism at various time intervals during the stress defence reactions of encystation and pseudocyst formation. Gradual decrease of both enzymes during encystation and slight increases at the beginning of pseudocyst formation were observed. Detailed analysis of mRNA sequences of the two genes revealed similarities with various alcohol dehydrogenases rather than mannitol dehydrogenases. Our results indicate there is probably no protective role for mannitol in Acanthamoeba as no mannitol was detected using HILIC ESI MS, in any Acanthamoeba life cycle stage. Possible misinterpretation of previously published sequences as encoding enzymes of the mannitol metabolic pathway is discussed.
- MeSH
- Acanthamoeba castellanii enzymologie metabolismus fyziologie MeSH
- anotace sekvence MeSH
- fyziologický stres MeSH
- genetická transkripce MeSH
- konzervovaná sekvence MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- mannitol metabolismus MeSH
- mannitoldehydrogenasy genetika metabolismus MeSH
- metabolismus sacharidů MeSH
- molekulární sekvence - údaje MeSH
- protozoální proteiny genetika metabolismus MeSH
- regulace genové exprese enzymů MeSH
- sekvence aminokyselin MeSH
- spory protozoální enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
