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Entamoeba invadens: cell biology & development (Title)
Entamoebae are anaerobe eukaryotes living in host’s intestine and an interesting research object of the developmental cell biology. They are capable of asymmetric cell division, self renewal and stemness, mitotic cell cycle arrest and differentiation [1]. Key regulator of amoebic growth and differentiation is the ambient oxygen content. Host’s intestine and oxygen consuming cultures (OCB cultures) [2] are low oxygen micro-environments favouring Entamoeba’s asymmetric cell fate, stemness and cyclic differentiation (sequential cyst formation) [3,4]. In contrast, axenic cultures and extraintestinal oxygen pressure favour symmetric cell fate, identical progeny and logarithmic growth [5]. In OCB cultures one of both daughter cells is the self-renewing cell and the other enters a state of G1 arrest or G0 quiescence (vegetative/somatic cells) or differentiates terminally forming a tetranucleated cyst (reproductive cells). Quiescent G0 cells have dual potential for proliferation and differentiation similar to the cycling cells in a state of G1, prior RP commitment. Entry, maintenance and exit of quiescence are controlled by a network of intrinsic mechanisms including oxygen sensing and oxygen signalling pathways. Strong hypoxic conditions near anoxia abolish asymmetric cell division and asymmetric cell fate. Increased oxygen pressure as occurring in upper intestinal gradient zones (≤ 5.5% O2) favours cyst differentiation. Oxygen contents above 5.5% as occurring in axenic cultures replace asymmetric by symmetric cell division
1. The primitive cycle of life of E. invadens
The life cycle of Entamoebae starts by infectious cysts excysting in intestine. Entamoeba’s mature innercyst cell is a tetranucleated 8N polyploid cell. It hatched out as a non-proliferative metacyst segregating its whole genome copies by reductive nuclear division to eight subnuclei that cellularize forming eight daughter cells of stem cell capacity (amoebulae, microcells). Entamoeba’s life cycle finishes by the tetranucleated mature cyst that is the mother cell of the microcell progeny. In developmental opinion Entamoeba’s cyst is a reproductive cyst. A subsequent life cycle starts by the disseminating progeny either in subcultures or in other infected hosts. Sometimes, intracolonic formed cysts lead to reinfections and amoebulae dissemination in the same host. However, repetitive life cycles are rare in amoebiasis.
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2. The multilined stem and progenitor cell lineage
Protists such as Entamoeba and Giardia have primitive stem und progenitor cell lineages hidden in their life cycle [1,3,4,6,7]. Entamoeba’s primitive lineage consists of primitive stem cells, vegetative (somatic) cells and reproductive cells producing cysts in OCB cultures; the inner cyst cell is a primitive-terminal differentiated cell of reproductive function. Its progeny forms a undifferentiated stem cell line (primary cell line) [8,9] that converses to two more differentiated sublines: one is the reproductive ACD+ subline producing cysts by autonomous cyclic differentiation (secondary cell subline) [8,9] and the other is the vegetative/ somatic ACD- subline not producing cysts in OCB cultures (tertiary cell subline) [8,9]. In not optimal oxygenic conditions and not optimal OCB cultures the more oxygenic ACD+ subline converses to a somatic (invasive) ACD- subline. In oxygenic/low hypoxic conditions of about 5.0 % O2 content ATD+ sublines proliferate and differentiate ACD cysts. Oxygen consuming cell cultures (OCB cultures) contain usually both cell sublines: the ACD+ subline form a minor subpopulation of only few percent while ACD- subline form a dominant subpopulation of >95% cells. All cell lines and sublines proliferate by asymmetric cell division. Modern day protists such as Entamoeba, Giardia [10] and Colpoda [11] inherit the primitive multilined lineage from the common eukaryotic ancestor [12]. It is also conserved in the “dark genome” of metazoans and humans [13].
3. Differentiation potential
Both sublines of Entamoeba invadens have differentiation potential forming cysts in OCB cultures (reproductive cells) or encystment media (vegetative/ somatic cells).
3.1 Reproductive cells
Daughter cells produced in cultures by asymmetric cell division are non-identical. In the case of the reproductive ACD+ subline, one of two daughter cells is the self-renewing cell and the second is the committed precursor cell that exits cell cycle by the G1/G0 checkpoint; it enters polyploidisation and cyst differentiation (cyst formation).
3.2 Somatic cells
In contrast, the vegetative/somatic subline ACD- does not commit the second daughter cell to differentiation; the non-committed daughter cell arrests in a pre-differentiated state of G0/G1. These mitoticly arrested cells (pre-differentiated MAS cells) produced by the somatic ACD- subline are the counterpart of committed precursor cells produced by the ACD+ subline. MAS cells may reenter the mitotic cell cycle or differentiate to cysts under conditions of stress and nutrient depletion. Switching from one cell state into the other cell state occurs by epigenetic reprogramming. In the course of the amoebiasis, early somatic clones change to more invasive and virulent genotypes capable of invading the liver and other organs [14][15][16].
Somatic/vegetative cells of ATD- subline respond in hypo-osmotic nutrient free media to hypo-osmotic differentiation signals by induced encystment (ratio 1:1 for G1 cells, and 1:2 for G2/M cells). The differentiating signals can be received in all cell cycle phases, except the S-phase. In contrast to the cyclic formation of autonomously cysts (ACD cysts) from committed precursor cells (cells exiting mitotic cell cycle at the G1/G0 exit point) induced cyst formation (ICD cysts) is a mass differentiation process addressing to all G1/G0 and G2/M cells, indifferently if quiescent or proliferating. Changes in epigenetic configuration could be received also in G2/M, however, G2/M cells cannot be driven out of the cell cycle and finish cell division by symmetric cell division. Both identical daughter cells are capable of polyploidisation and ITD cysts formation in hypo-osmotic media; they enter differentiation from the post mitotically pre-G1 state (in nutrient free media cells do not start G1 phase) in a ratio 1:1.
4. Commitment of differentiation and differentiation switches
The decision of whether a cell commits for differentiation or not, or becomes a self-renewing pre-differentiated cell (such as MAS cells) depends probably on molecular switches by epigenetic changes [11]. A differentiation switch (DS) decides whether the second cell produced by asymmetric division becomes a MAS cell or commits for final cyst differentiation. The molecular DS switch regulates the cell fate to mitotic proliferation (DS/OFF) or differentiation (DS/ON). Differentiation commitment means DS/ON, proliferation DS/OFF. In the process of cyclic differentiation the regulatory switch is always open (DS/ON) while in the case of induced differentiation of MAS cells it must change from DS/OFF to DS/ON. Somatic MAS cells express their hidden differentiation potential by opening the regulatory DS switch.
5. Concluding remarks
In summary, cells of Entamoeba invadens show: (1) all cells have differentiation potential; ACD+ cells express the differentiation potential autonomously while ACD- cells express the hidden differentiation potential only by stress; (2) differentiation always occurs from a state of G1; (3) under conditions of stress a mitotic blocked cell (such as the MAS cell) escapes cell death bypassing to an amitotic reproductive solution; it forms multiple microcell progeny by polyploidisation and depolyploidisation; (4) most of the individual cell states may turn into each other; (5) switching from one amoebic cell state to the other – including the transition from the stressed, mitotic blocked cell (MAS) to the reproductive polyploidisation-depolyploidisation process is epigenetically accomplished.
References
1. Niculescu Vladimir F. (2017) Regulatory Mechanisms of Asymmetric/Symmetric Cell Division and Quiescence in the Primitive-/ Stem Progenitor Cell Lineage of Entamoeba. Stem Cells Regen Med. 1(2): 1-7
2. Niculescu VF (2013) Growth of Entamoeba invadens in sediments with metabolically repressed bacteria leads to multicellularity and redefinition of the amoebic cell system. Roumanian Archives of Microbiology and Immunology 72(1):25-48 PMID:239470
3. Niculescu VF (2015) The stem cell biology of the protist pathogen Entamoeba invadens in the context of eukaryotic stem cell evolution. Stem Cell Biol Res. 2015; 2:2. http://dx.doi.org/10.7243/2054-717X-2-2
4. Niculescu VF (2016) Low-oxygen environments slow down precursor cell development in the pathogen anaerobe protist Entamoeba invadens. J Stem Cell Res Med 1(1): 27-35 doi: 10.15761/JSCRM.1000104
5. Niculescu VF (2015) Axenic stress leads the minor stem cell line of Entamoeba histolytica to defective mitosis and aberrant reversible endopolyploidy. Conference: XVIII Seminar of Amebiasis, Campeche, Mexico, October 13-16, 2015 DOI: 10.13140/RG.2.1.1851.4648
6. Niculescu VF (2016) Developmental and Non Developmental Polyploidy in Xenic and Axenic Cultured Stem Cell Lines of Entamoeba invadens and E. histolytica. Insights Stem Cells. 2:1.
7. Niculescu VF (2017) Development, cell line differentiation and virulence in the primitive stem-/progenitor cell lineage of Entamoeba. J Stem Cell Res Med, 2(1): 1-8 doi: 10.15761/JSCRM.1000115
8. Niculescu VF (2015) The magna-minuta/precyst classification in the light of Entamoeba invadens’ Researchgate. doi: 10.13140/RG.2.1.3064.9762
9. Niculescu VF (2014) The three line PST stem cell system of Entamoeba invadens as one of the earliest stem cell systems today (pre-print). Researchgate. doi:10.13140/RG.2.1.4162.7043
10. Niculescu VF (2014) The cell system of Giardia lamblia in the light of the protist stem cell biology. Stem Cell Biol Res.1:3. http://dx.doi.org/10.7243/2054-717X-1-3
11. Niculescu VF (2014) Evidence for asymmetric cell fate and hypoxia induced differentiation in the facultative pathogen protist Colpoda cucullus.Microbiol Discov. 2:3. http://dx.doi.org/10.7243/2052-6180-2-3
12. Niculescu VF VF (2014) On the Origin of Stemness and Ancient Cell Lineages in Single-Celled Eukaryotes. SOJ Microbiol Infect Dis 2(2): 1-3. http://dx.doi.org/10.15226/sojmid.2014.00115
13. Niculescu VF (2018) Carcinogenesis: recent insights in protist stem cell biology lead to a better understanding of atavistic mechanisms implied in cancer development. MOJ Tumor Res 1(1): 00004. doi: 10.15406/mojtr.2018.01.00004
14. Bruchhaus I, Roeder T, Lotter H, Schwerdtfeger M, Tannich E (2002) Differential gene expression in Entamoeba histolytica isolated from amoebic liver abscess. Mol Microbiol 44: 1063-1072. https://doi.org/10.1046/j.1365-2958.2002.02941.x
15. Biller L, Davis PH, Tillack M, Matthiesen J, Lotter H et al. (2010) Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties. BMC Genomics 11:63-76. doi: 10.1186/1471-2164-11-63
16. Meyer M, Fehling H, Matthiesen J, Lorenzen S, Schuldt K et al. (2016) Overexpression of differentially expressed genes identified in non pathogenic and pathogenic Entamoeba histolytica clones allow identification of new pathogenicity factors involved in amoebic liver abscess formation. PLOS Pathog 12: e1005853. doi:10.1371/journal.ppat.1005853