Single Amoeba Research Articles

Biological Movements as Objects of Natural Science (In memory of Michael Turvey, February 14, 1942 - August 12, 2023)

Michael Turvey was a great scientist, encyclopedist, fine lecturer, and, together with his wife and colleague Claudia Carello a wonderful friend of many. Mike Turvey was able to educate the next generation of students and influence the thinking of many outstanding researchers by focusing on a multidisciplinary approach to ecological psychology in which organisms directly perceive sensory stimuli and evaluate them depending on actions they can accomplish in the environment (Gibson 1968). This, “affordance principle” is applied to all biological kingdoms from a single cell amoeba to multi-celled organisms. Some other essential ideas in behavioral neuroscience advanced by Turvey are considered.

What Is the Weight of a Single Amoeba and Why Does It Matter?

Cell size is an important variable in the study of cellular growth, metabolism, and the cell cycle. The large size of Amoeba proteus and the ease with which it can be collected and cultured have made it a star in biology education—and it was a model for research on cell biology before the introduction of molecular genetic methods. Measuring the cytoplasmic density of a single amoeba without modern instrumentation seems like a difficult task, but this was done with supreme accuracy in the 1940s. The solution was based on the familiar Cartesian diver that is used to demonstrate Archimedes’s principle. It required the fabrication of a tiny diver that would respond to the additional mass of a cell. Experiments using this method allowed investigators to study changes in size and density associated with feeding, starvation, and cell division. This research is an illustration of the ingenuity of cell biologists in the pre-molecular genetic era of their field, which is often overlooked by contemporary scientists. The consideration of the mass, density, and buoyancy of free-living amoebas encourages a new hypothesis about the evolution of testate amoebas.

New data on the fine structure of Deuteramoeba mycophaga CCAP 1586/1 (Amoebozoa, Tubulinea)

The genus Deuteramoeba is one of the six amoebae genera belonging to the best-known amoeba family – Amoebidae (Amoebozoa, Tubulinea), containing such a popular species as Amoeba proteus. However, members of other genera of the family Amoebidae are much less known, and most of the studies of their morphology and ultrastructure date back to the 1970s and 1980s. Since these “classical” species are believed to be “well studied”, their morphology and fine structure rarely become a subject of re-investigation. The absence of modern morphological data may be critical when molecular data of the type strain are not available, and the only way to identify a species is by morphological comparison. For this paper, we performed an ultrastructural study of the strain CCAP 1586/1 – the type strain of the species Deuteramoeba mycophaga. Our study revealed new details of the nuclear structure, including a peripheral layer of filaments and a heterogeneous nucleolus, and provided new data on the cytoplasmic inclusions of this species. We performed a whole-genome amplification of the DNA from a single amoeba cell followed by NGS sequencing and searched for genetic evidence for the presence of a putative nuclear parasite detected in 2017, but found no evidence for the presence of Opisthosporidia.

Distribution of amoebal endosymbiotic environmental chlamydia Neochlamydia S13 via amoebal cytokinesis.

We previously isolated a symbiotic environmental amoeba, harboring an environmental chlamydia, Neochlamydia S13. Interestingly, this bacterium failed to survive outside of host cells and was immediately digested inside other amoebae, indicating bacterial distribution via cytokinesis. This may provide a model for understanding organelle development and chlamydial pathogenesis and evolution; therefore, we assessed our hypothesis of Neochlamydia S13 distribution via cytokinesis by comparative analysis with other environmental Chlamydiae (Protochlamydia R18 and Parachlamydia Bn9 ). Dual staining with 4',6-diamidino-2-phenylindole and phalloidin revealed that the progeny of Neochlamydia S13 and Protochlamydia R18 existed in both daughter cells with a contractile ring on the verge of separation. However, in contrast to other environmental Chlamydiae, little Neochlamydia S13 16S ribosomal DNA was amplified from the culture supernatant. Interestingly, Neochlamydia S13 failed to infect aposymbiotic amoebae, indicating an intimate interaction with the host cells. Furthermore, its infectious rates in cultures expanded from a single amoeba were always maintained at 100%, indicating distribution via cytokinesis. We concluded that unlike other environmental Chlamydiae, Neochlamydia S13 has a unique ability to divide its progeny only via host amoebal cytokinesis. This may be a suitable model to elucidate the mechanism of cell organelle distribution and of chlamydial pathogenesis and evolution.

Polychaos centronucleolus n. sp. – a new terrestrial species of the genus Polychaos (Amoebozoa, Tubulinea) with nontypical nuclear structure

A new species of the “proteus-type” naked amoebae (large cells with discrete tubular pseudopodia) was isolated from tree bark sample of a birch tree in the surrounding of Kislovodsk town, Russia and named Polychaos centronucleolus n. sp. (Amoebozoa, Tubulinea). Amoebae of this species have a filamentous cell coat and a nucleus with a central compact nucleolus. This type of nucleolar organization has not been previously known for the genus Polychaos. A sequence of the 18S rRNA gene of this strain was obtained using whole genome amplification of DNA from the single amoeba cell, followed by NGS sequencing. The analysis of molecular data robustly groups this species with Polychaos annulatum within the family Hartmannellidae. Our results, together with the results of our previous studies, show that the taxonomic assignment of “proteus-type” amoebae species is becoming increasingly complex, and the taxonomic characters that can be used to classify these organisms are becoming more shadowed.

Dynamic Analysis of Quorum-sensing Genetic Oscillators with the Influence of External Medium

Living organisms have the capacity to exhibit elaborate rhythmic behaviors. These complex behaviors are controlled by biochemical networks themselves as well as environmental clues. Quorum sensing refers to the typical intercellular communication mechanism executed by certain small molecule. This paper undertakes a dynamic analysis of single amoeba Dictyostelium cell, which is a representative genetic unicellular oscillator, communicating with others through quorum sensing. Apply the theory of vibration rather than center manifold method, our focal attention is dedicated to the kinetic influence of external signaling medium cAMP. The equilibrium existence condition is discussed, and near Hopf bifurcation point, the approximate solution is derived by Lindstedt’s method. Several numerical simulations are conducted to demonstrate our results.

Ag2S atomic switch-based 'tug of war' for decision making.

For a computing process such as making a decision, a software controlled chip of several transistors is necessary. Inspired by how a single cell amoeba decides its movements, the theoretical 'tug of war' computing model was proposed but not yet implemented in an analogue device suitable for integrated circuits. Based on this model, we now developed a new electronic element for decision making processes, which will have no need for prior programming. The devices are based on the growth and shrinkage of Ag filaments in α-Ag2+δS gap-type atomic switches. Here we present the adapted device design and the new materials. We demonstrate the basic 'tug of war' operation by IV-measurements and Scanning Electron Microscopy (SEM) observation. These devices could be the base for a CMOS-free new computer architecture.

Raman spectroscopic study on the excystation process in a single unicellular organism amoeba (Acanthamoeba polyphaga).

An in vivo Raman spectroscopic study of amoeba (Acanthamoeba polyphaga) is presented. The changes of the spectra during the amoeba cyst activation and excystation are analyzed. The spectra show the changes of the relative intensities of bands corresponding to protein, lipid, and carotenoid components during cyst activation. The presence of carotenoids in the amoeba is observed via characteristic Raman bands. These signals in the Raman spectra are intense in cysts but decrease in intensity with cyst activation and exhibit a correlation with the life cycle of amoeba. This work demonstrates the feasibility of using Raman spectroscopy for the detection of single amoeba microorganisms in vivo and for the analysis of the amoeba life activity. The information obtained may have implications for the estimation of epidemiological situations and for the diagnostics and prognosis of the development of amoebic inflammations.

Slime mould logic gates based on frequency changes of electrical potential oscillation

Physarum polycephalum is a large single amoeba cell, which in its plasmodial phase, forages and connects nearby food sources with protoplasmic tubes. The organism forages for food by growing these tubes towards detected foodstuff, this foraging behaviour is governed by simple rules of photoavoidance and chemotaxis. The electrical activity of the tubes oscillates, creating a peristaltic like action within the tubes, forcing cytoplasm along the lumen; the frequency of this oscillation controls the speed and direction of growth. External stimuli such as light and food cause changes in the oscillation frequency. We demonstrate that using these stimuli as logical inputs we can approximate logic gates using these tubes and derive combinational logic circuits by cascading the gates, with software analysis providing the output of each gate and determining the input of the following gate. Basic gates OR, AND and NOT were correct 90%, 77.8% and 91.7% of the time respectively. Derived logic circuits XOR, half adder and full adder were 70.8%, 65% and 58.8% accurate respectively. Accuracy of the combinational logic decreases as the number of gates is increased, however they are at least as accurate as previous logic approximations using spatial growth of P. polycephalum and up to 30 times as fast at computing the logical output. The results shown here demonstrate a significant advancement in organism-based computing, providing a solid basis for hybrid computers of the future.

Counting Legionella Cells within Single Amoeba Host Cells

Here we present the first attempt to quantify Legionella pneumophila cell numbers within individual amoeba hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 1,348 (mean, 329) and 385 (mean, 44) CFU trophozoite(-1), respectively.

Zigzag Turning Preference of Freely Crawling Cells

The coordinated motion of a cell is fundamental to many important biological processes such as development, wound healing, and phagocytosis. For eukaryotic cells, such as amoebae or animal cells, the cell motility is based on crawling and involves a complex set of internal biochemical events. A recent study reported very interesting crawling behavior of single cell amoeba: in the absence of an external cue, free amoebae move randomly with a noisy, yet, discernible sequence of ‘run-and-turns’ analogous to the ‘run-and-tumbles’ of swimming bacteria. Interestingly, amoeboid trajectories favor zigzag turns. In other words, the cells bias their crawling by making a turn in the opposite direction to a previous turn. This property enhances the long range directional persistence of the moving trajectories. This study proposes that such a zigzag crawling behavior can be a general property of any crawling cells by demonstrating that 1) microglia, which are the immune cells of the brain, and 2) a simple rule-based model cell, which incorporates the actual biochemistry and mechanics behind cell crawling, both exhibit similar type of crawling behavior. Almost all legged animals walk by alternating their feet. Similarly, all crawling cells appear to move forward by alternating the direction of their movement, even though the regularity and degree of zigzag preference vary from one type to the other.

Inside the Lifestyle of the Virophage

Objective(s): We sought to better characterize Sputnik, the first isolated virophage, and to analyze its parasitic lifestyle during co-infection with Marseillevirus (a new giant virus) in Acanthamoeba castellanii. Methods: A combination of electron microscopy, immunofluorescence microscopy, and real-time PCR was used to characterize the kinetics of the viral replication cycle. RT-PCR was performed to detect RNAs inside the Sputnik virions. Results: Sputnik is a new viral entity carrying an almost complete ready-to-use set of viral RNAs (20 out of 21). Sputnik does not replicate with Marseillevirus but delays its replication cycle. While Marseillevirus is successfully internalized by A. castellanii following co-infections with Mamavirus and Sputnik, it does not initiate a replication cycle. In contrast, both Marseillevirus and Mamavirus can replicate in the amoeba in case of co-infection, but the development of one is exclusive from the other inside a single amoeba cell. Conclusions: This work provides new insight into the Sputnik replication cycle with another giant virus and confirms that Sputnik is a virophage. It shows new dimensions of the interactions existing among giant viruses.

Foraging Strategies for Starving and Feeding Amoeba

Do individual cells have a search strategy when their target is outside their range of detection? Does this strategy change when a high density of targets is encountered?To answer these questions, we observed single, well-isolated cells of the social amoeba Dictyostelium as they forage for bacteria on a flat surface. Time-lapse movies of this predator-prey system were recorded and analyzed. By varying the concentration of the food source over several orders of magnitude the dynamics of the amoebae as they responded to their environment could be studied.At low bacterial density the amoebae scooped up the bacteria as they passed over them without any detectable change in motion. At higher bacterial density the amoebae slowed down, picking up essentially all bacteria encountered, and were found to change their long-timescale behavior from an unbiased random walk to a self-avoiding random walk. This way the amoebae avoids searching for food in already depleted regions. A single amoeba typically ingested 250 bacteria before pausing for 15 min to divide.

Infection of Mayorella penardi (Gymnamoebia) in oak-hornbeam forest soil by the ectoparasitic fungus Amoebophilus simplex (Zygomycota)

During research on naked amoebae in terrestrial habitats in the oak-hornbeam forests of Malé Karpaty Mts. (Western Slovakia), a mass fungal infection in Mayorella penardi Page, 1972 (Euamoebida: Paramoebidae) was observed. In one of the 243 soil and moss samples examined a remarkably high percentage (80%) of amoebae were infected by the ectoparasitic soil fungus Amoebophilus simplex Barron, 1983 (Zygomycota: Zoopagales), as revealed by detailed light microscopical observations of infected specimens. The parasitic fungus displayed characteristic cylindrical conidia (length 10 μm, breadth 3 μm) projecting out from the posterior part of the amoeba cell. Infecting conidium frequently produced a chain of two to three conidia and, rarely, secondary chains from the distal end. Two to four infections in a single amoeba were frequently observed. From the present study, and from earlier records, it can be deduced that Amoebophilus simplex is specialized in parasitizing mayorellid amoebae, maybe even Mayorella penardi exclusively. Two other species ( Korotnevella diskophora and Korotnevella stella) from the family Paramoebidae were recorded in the same sample, but neither of them showed signs of infection.

Decreasing prevalence of rhizosphere IAA producing and seedling root growth promoting bacteria with barley development irrespective of protozoan grazing regime

Barley was grown in soil with either bacteria and a mixed protozoan community (Mixed protozoa) or bacteria and a single vahlkampfiid amoebal species (Single amoeba). We assessed the influence of plant age (day 29, 43 and 57 after sowing) on two aspects of rhizosphere bacterial functioning: (1) the proportion of indole-3-acetic acid (IAA) producing bacteria and (2) the effect of mixed rhizosphere bacterial assemblages on barley seedling root growth in an agar based assay. The proportion of IAA producers was significantly lower at day 57 than at day 29 and 43, and mixed bacterial assemblages extracted from rhizospheres of 29 days old plants were significantly less harmful to seedling growth than bacterial assemblages from older plants. Hence both assays indicated that bacterial communities from rhizospheres of older plants were less beneficial for root growth than bacterial communities from younger plants. Genetic fingerprinting of rhizosphere bacterial communities was compared by use of length heterogeneity polymerase chain reaction (LH-PCR). This analysis showed a clear succession from the inoculum bacterial community with a rather low diversity to a community with much higher diversity at day 29. However, diversity did not change after day 29, and no relationship between protozoan treatment nor plant age and genetic fingerprinting was found.

Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge.

This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.

Endogenous Autoinhibitors Regulate Changes in Actin Tyrosine Phosphorylation During Dictyostelium Spore Germination

Phosphorylation of proteins on tyrosine residues has been shown to govern many cellular processes, but little work has focused on the role of tyrosine phosphorylation during germination. Under optimal conditions, D. discoideum spores synchronously germinate each liberating a single amoeba. The total amount of phosphotyrosine containing proteins observed in spores was greatest during quiescence with a gradual decline during spore activation and emergence of nascent amoebae. During dormancy, tyrosine residues of actin were heavily phsophorylated, but they gradually underwent dephosphorylation upon spore activation and this process continued through emergence. Interestingly, an endogenous autoinhibitor(s), which blocks germination, induces tyrosine phosphorylation of actin. Conversely, the removal of the autoinhibitor(s) was followed by a decrease in phosphorylation. Thus, during germination of Dictyostelium spores, actin is dephosphorylated, with the level of phosphorylation regulated by the autoinhibitor(s) and/or the autoactivator. This change in actin phosphorylation appears to play a direct role since actin dephosphorylation and reorganization is a necessary prelude to germination.

Characterization of npf mutants identifying developmental genes in Physarum

In Physarum polycephalum, uninucleate haploid amoebae develop into macroscopic multinucleate plasmodia. Wild-type, sexual development is triggered when two amoebae carrying different alleles of matA fuse to form a zygote which develops into a diploid plasmodium. Mutations in the matA genetic region give rise to apogamic strains in which a single haploid amoeba can develop into a haploid plasmodium. An essential stage in both sexual and apogamic plasmodium formation is an extended cell cycle in uninucleate cells, which ends with the formation of a binucleate cell by mitosis without cytokinesis. Using a 'brute force' screening method, we have isolated mutants blocked in apogamic plasmodium development. Genetic analysis showed that the mutations we have identified were unlinked to matA, unlike mutations previously identified following an enrichment step. Most of the loci revealed by our screen were represented by only one allele, indicating that further screening should lead to the identification of additional genes required for plasmodium development. Phenotypic analysis showed that different mutants were blocked at different stages of plasmodium formation. Some of the mutations blocking apogamic development at an early stage, close to the start of the long cell cycle, failed to block sexual development in zygotes homozygous for the mutation. Since the two modes of plasmodium formation differ only in the initiation of development, these mutations presumably interfere with the initiation process. In the remaining mutants, in which both sexual and apogamic development were blocked, development first became abnormal towards the end of the long cell cycle. This suggested that the wild-type gene products were required by this time and was consistent with previous evidence that many changes in cellular organization and gene expression occur during the long cell cycle. Each of these mutants showed a different terminal phenotype and some aspects of plasmodium development occurred normally although others were blocked, suggesting that development involves multiple pathways rather than a dependent sequence of events. Phenotypic analysis of double mutants supported this conclusion and also revealed epistatic interactions, presumably due to blocks in the same pathway. In several of the mutants, terminally differentiated cells died by an apoptosis-like mechanism; since this was never observed in vegetative cells, it was presumably triggered by the failure of development. Phenotypic analyses of additional mutants will extend our understanding of the pathways involved in plasmodium development.

Reticulamoeba gemmipara n. gen., n. sp., an “Amoebaflagellate” with Reticulopodia and Zoosporogenesis

The “amoeboflagellate” Reticulamoeba gemmipara n. gen., n. sp. was isolated from samples which were collected in the littoral zone of the Greek island Thera (Santorin). It could be cultivated with the pennate diatom Amphiprora sp. as food organism. On a “lawn” of the diatoms, the amoeboid stages create clusters. Every cluster contains a single amoeba which gathers the diatoms with its reticulopodia. When the density of the diatoms is low, the reticulepodia of different amoebae fuse with each other to form a “feeding community”. The multiplication of Reticulamoeba gemmipara entails the successive formation of buds which can transform into biflagellate swarmers or zoospores. The course of reticulopodial digestion, zoosporogenesis and the morphology of the zoospores exhibit a striking similarity to the meroplasmodial species Leucodictyon marinum (Grell 1991a), indicating a relationship.

Specificity of glycosphingolipid recognition by Entamoeba histolytica trophozoites

The ability of purified glycosphingolipids to enhance liposome-stimulated Entamoeba histolytica actin polymerization was assessed as a means of defining the specificity of mammalian cell membrane lipid glycan recognition by this parasite. Synthetic liposomes containing a variety of individual glycosphingolipids bearing neutral, straight-chain oligomeric glycans with galactose or N-acetylgalactosamine termini stimulated rapid (90-s) polymerization of amoeba actin. Glycans with terminal N-acetylglucosamine residues were not stimulatory at all or were only weakly stimulatory. Glycans with glucose, N-acetylglucosamine, galactose, and N-acetylgalactosamine as the penultimate residue were recognized. Attachment of N-acetylneuraminate to the terminal residue of a stimulatory glycosphingolipid eliminated activity; attachment of fucose to the penultimate sugar reduced activity. Glycans with a terminal beta 1-4 or 1-3 glycosidic bond were most effective; glycans with terminal alpha 1-4 or 1-3 glycosides were less effective. The activity of glycans with both beta- and alpha-linked terminal glycosides was inhibited by lactose, suggesting recognition of both configurations by a single amoeba protein. The ability of liposomes to stimulate actin polymerization reflected the extent of liposome phagocytosis.