Acanthamoeba polyphaga Mimivirus Prevents Amoebal Encystment-Mediating Serine Proteinase Expression and Circumvents Cell Encystment

Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.     

Naegleria-Like Cysts In Cretaceous Amber From Central Kansas

ABSTRACT. Fossil protist cysts are reported from the mid-Cretaceous amber of Ellsworth County, Kansas, which is rich in terrestrial microfossils but contains no known macrofossils. On the basis of their distinctive morphology, the cysts can be referred to the genus Naegleria (Schizopyrenida); they most closely resemble cysts of the living species Naegleria gruberi. This is the first known fossil record for this group of amoebas. the current phylogenetic position and paleoecological role of Naegleria are discussed in relation to this find; it provides direct confirmation of morphological stasis in this group, which had previously been inferred from rRNA sequence divergence data.,     

Testate amoebas of pine forests in Mexico

The population of testate amoebas in the soils of pine forests in Mexico has been studied. In total, 68 species, varieties, and types of testate amoebas with cosmopolite distribution were found. The species diversity of the testate population includes hygrophilous species that differ from hygrophilous species with Luvisols in higher Andosols. Comparative analysis using the results of one available study of soil testate amoebas from Mexico has been carried out (Bonnet, 1977).     

Soil amoebas as biological markers to estimate the quality of swimming pool waters

The legal biological survey of swimming pool waters is based on both the level of bacteriological contamination and the amount of material of fecal origin. The great number of soil amoebas and the occasional epidemiological risk involved led us to consider using these organisms as possible biological markers to estimate the quality of pool water and the extent of disinfection. During a 1-year survey of 54 public swimming pools, 765 superficial pool and tap water samples were collected. One portion (50 ml) drawn from 1-liter samples was filtered and cultured for amoebas. In specimens considered contaminated we detected at least 20 amoebas per liter, whereas uncontaminated samples contained fewer than 20 amoebas per liter. By keeping the threshold value voluntarily low, we were able to compare tap water with pool water and to monitor the quality of various disinfection procedures (i.e., chlorine, bromine, and Cu-Ag). The data suggest that the filters were not always protective against a high concentration of amoebas. Furthermore, these disinfection procedures were not equally efficient according to estimates based on biological criteria. In addition, the quality of swimming pool water also depends on the quality of its source tap water. Thus, the numeration of soil amoebas can be used as an additional biological marker to estimate the quality of swimming pool water.     

Soil amoebas as biological markers to estimate the quality of swimming pool waters.

The legal biological survey of swimming pool waters is based on both the level of bacteriological contamination and the amount of material of fecal origin. The great number of soil amoebas and the occasional epidemiological risk involved led us to consider using these organisms as possible biological markers to estimate the quality of pool water and the extent of disinfection. During a 1-year survey of 54 public swimming pools, 765 superficial pool and tap water samples were collected. One portion (50 ml) drawn from 1-liter samples was filtered and cultured for amoebas. In specimens considered contaminated we detected at least 20 amoebas per liter, whereas uncontaminated samples contained fewer than 20 amoebas per liter. By keeping the threshold value voluntarily low, we were able to compare tap water with pool water and to monitor the quality of various disinfection procedures (i.e., chlorine, bromine, and Cu-Ag). The data suggest that the filters were not always protective against a high concentration of amoebas. Furthermore, these disinfection procedures were not equally efficient according to estimates based on biological criteria. In addition, the quality of swimming pool water also depends on the quality of its source tap water. Thus, the numeration of soil amoebas can be used as an additional biological marker to estimate the quality of swimming pool water.     

srRNA evolution and phylogenetic relationships of the genus Naegleria (Protista: Rhizopoda)

A rapid RNA sequencing technique was used to partially sequence the small-subunit ribosomal RNA (srRNA) of four species of the amoeboid genus Naegleria. The extent of nucleotide sequence divergence between the two most divergent species was roughly similar to that found between mammals and frogs. However, the pattern of variation among the Naegleria species was quite different from that found for those species of tetrapods characterized to date. A phylogenetic analysis of the consensus Naegleria sequence showed that Naegleria was not monophyletic with either Acanthamoeba castellanii or Dictyostelium discoideum, two other amoebas for which sequences were available. It was shown that the semiconserved regions of the srRNA molecule evolve in a clocklike fashion and that the clock is time dependent rather than generation dependent.     

From drought sensing to developmental control: evolution of cyclic AMP signaling in social amoebas

Amoebas and other protists commonly encyst when faced with environmental stress. Although little is known of the signaling pathways that mediate encystation, the analogous process of spore formation in dictyostelid social amoebas is better understood. In Dictyostelium discoideum, secreted cyclic AMP (cAMP) mediates the aggregation of starving amoebas and induces the differentiation of prespore cells. Intracellular cAMP acting on cAMP-dependent protein kinase (PKA) triggers the maturation of spores and prevents their germination under the prevalent conditions of high osmolality in the spore head. The osmolyte-activated adenylate cyclase, ACG, produces cAMP for prespore differentiation and inhibition of spore germination. To retrace the origin of ACG function, we investigated ACG gene conservation and function in species that span the dictyostelid phylogeny. ACG genes, osmolyte-activated ACG activity, and osmoregulation of spore germination were detected in species that represent the 4 major groups of Dictyostelia. Unlike the derived species D. discoideum, many basal Dictyostelia have retained the ancestral mechanism of encystation from solitary amoebas. In these species and in solitary amoebas, encystation is independently triggered by starvation or by high osmolality. Osmolyte-induced encystation was accompanied by an increase in cAMP and prevented by inhibition of PKA, indicating that ACG and PKA activation mediate this response. We propose that high osmolality signals drought in soil amoebas and that developmental cAMP signaling in the Dictyostelia has evolved from this stress response.     

Sterols of Soil Amoebas and Ochromonas danica: Phylogenetic Approach

SYNOPSIS. Sterols of Ochromonas danica, Mayorella palestinensis, Acanthamoeba sp. and Hartmannella rhysodes were examined by thin-layer and gas-liquid chromatography. The sterol patterns of O. danica and the amoebas were strikingly similar. Two of the sterols present in both groups, were identified as ergosterol and stigmasterol. This similarity is construed as evidence that "soil" amoebas evolved from phytoflagellates having sterol patterns similar to that of O. danica. An explanation is suggested for the role of ergosterol in phytoflagellates, as originally an ultra-violet-protective agent, then a membrane component.     

Evidence for the existence of a monovalent cation-stimulated, magnesium-dependent adenosine triphosphatase activity in the isolated plasma membranes of amoebas of the slime mold dictyostelium discoideum

Evidence for a magnesium-dependent ATPase activity that can be stimulated by Na+ and K+, or equally by Na+ or K+ alone, has been found in the plasma membranes isolated from amoebas of the slime mold Dictyostelium discoideum when the membranes are isolated from cultures grown up to the stationary phase. This ATPase activity is scarcely inhibitable by ouabain or phlorizin, but is very sensitive to low concentrations of azide or thimerosal. When the plasma membranes are isolated from amoebas growing in logarithmic phase, this monovalent cation-stimulated Mg2+-dependent activity is barely detectable.     

阿米巴-细菌互作：进化、生态与环境效应

阿米巴是原生动物的主要类群,是陆地和水生生态系统的关键组成部分。阿米巴与细菌之间有着密切而复杂的相互作用。一方面,阿米巴通过捕食直接影响细菌群落与多样性,增强细菌活性。另一方面,细菌也进化出抵抗捕食的机制来抵抗甚至感染阿米巴,反向影响阿米巴的生长和多样性。近年来,阿米巴-细菌互作的研究开始受到了广泛关注。本文总结了阿米巴-细菌互作的进化历史、生态关系(捕食、偏利共生、寄生和互利共生)以及其对环境的潜在影响,旨在更好地理解阿米巴-细菌互作这一研究领域,为其他原生动物-微生物之间的研究提供新的思路,也为探究宿主-微生物互作的机制提供参考。     

Naked Amoebas and Bacteria in an Oil-Impacted Salt Marsh Community

Populations of soil amoebas were monitored in two salt marshes in Staten Island, NY for 2 years. One site, Gulfport Reach on the Arthur Kill, has been highly impacted by numerous oil spills. In particular, in 1990 a massive no. 2 fuel oil spill from a ruptured pipe flooded the area; its sediments had total petroleum hydrocarbon (TPH) concentrations in the range 800-46,000 ppm. A reference site 11 km away, on the Atlantic coast, had low TPH levels. Amoeba population densities were in general higher in the impacted sediments. In laboratory microcosm experiments, sediment samples from unimpacted sites were treated with added fresh (unweathered) hydrocarbons (no. 2 fuel oil) and cultured; these also yielded higher amoeba numbers than untreated controls. Four distinct amoeba morphotypes were monitored. Changes in population levels of total amoebas were correlated in the two sites, particularly for morphotype 2 (r = 0.83). The ratios of total amoebas to total bacterial numbers were also correlated (r = 0.85) between the sites. This suggests the amoebas may function as generalists, and that their trophic relation to bacterial prey is not much affected by the presence of petroleum hydrocarbons, but rather may reflect regional parameters such as ambient temperature or other physical factors.     

Evolution of developmental cyclic adenosine monophosphate signaling in the Dictyostelia from an amoebozoan stress response

The Dictyostelid social amoebas represent one of nature's several inventions of multicellularity. Though normally feeding as single cells, nutrient stress triggers the collection of amoebas into colonies that form delicately shaped fruiting structures in which the cells differentiate into spores and up to three cell types to support the spore mass. Cyclic adenosine monophosphate (cAMP) plays a very dominant role in controlling morphogenesis and cell differentiation in the model species Dictyostelium discoideum. As a secreted chemoattractant cAMP coordinates cell movement during aggregation and fruiting body morphogenesis. Secreted cAMP also controls gene expression at different developmental stages, while intracellular cAMP is extensively used to transduce the effect of other stimuli that control the developmental program. In this review, I present an overview of the different roles of cAMP in the model D. discoideum and I summarize studies aimed to resolve how these roles emerged during Dictyostelid evolution.     

Small-subunit ribosomal RNA sequence from Naegleria gruberi supports the polyphyletic origin of amoebas

We have sequenced the small-subunit ribosomal RNA gene of the amoebo- flagellate protozoan Naegleria gruberi. Comparison of this sequence with the rRNA sequences of other eukaryotes resulted in a phylogenetic tree that supports the suggested polyphyletic origin of amoebas and suggests a flagellate ancestry for Naegleria.      

Regulation of Predation by Prey Density: the Protozoan-Rhizobium Relationship

Tetramitus rostratus and strains of Hartmanella, Naegleria, and Vahlkampfia consumed large numbers of Rhizobium meliloti cells in a salt solution, but protozoan multiplication and the bacterial decline stopped when the prey density fell to about 10-6 to 10-7 cells/ml. At higher prey densities, the maximum numbers of Hartmanella sp. and Naegleria sp. were proportional to the quantity of R. meliloti initially provided to the amoebas. When supplemental rhizobia were supplied to Hartmanella sp. or Naegleria sp. after their active feeding had terminated, presumably because the remaining 10-6 or 10-7 bacteria/ml could not be captured, replication of the protozoa was initiated. The rate of elimination of rhizobia present in large populations was proportional to the initial abundance of Naegleria sp., but the final numbers of amoebas and surviving R. meliloti cells were independent of initial numbers of predators. The surviving bacteria were not intrinsically resistant to attack because 98% of the survivors, when concentrated, were consumed. It is suggested that large populations of bacteria in nature may be reduced in size by predatory protozoa, but many of the prey cells will not be eliminated.     

Germination and Amplification of Anthrax Spores by Soil-Dwelling Amoebas

While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.     

Amoebacidal effects of serum from the American alligator (Alligator mississippiensis)

Treatment of axenic Naegleria gruberi cultures with alligator serum resulted in time-dependent amoebacidal activity, with measurable activity at 5 min and maximal activity occurring at 20 min. The amoebacidal activity was concentration dependent, with measurable activity at 25% serum, whereas treatment of amoebas with undiluted serum resulted in only 16% survival. The efficacy was dependent on the concentration of amoebas, with higher survival rates at high amoeba densities and lower survival rates at low amoeba densities. The amoeba-killing effects of alligator serum were broad in spectrum because the serum was effective against 3 strains of Naegleria species tested and 4 Acanthamoeba species, which have been reported to be resistant to human serum complement-mediated lysis. The amoebacidal effects of alligator serum were temperature dependent, with optimal activity at 15-30 C and a decrease in activity below 15 C and above 30 C. The amoebacidal activity of alligator serum was heat labile and protease sensitive, indicating the proteinaceous nature of the activity, and was also inhibited by ethylenediaminetetraacetic acid, which indicated a requirement for divalent metal ions. These characteristics strongly suggest that the amoebacidal properties of alligator serum are because of complement activity.     

Cyclic AMP inhibits dedifferentiation in the cellular slime mold Dictyostelium discoideum

When aggregating amoebas of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, the amoebas will reaggregate in less than $\text{1}\text{10}\text{th}$ the original time. When aggregating amoebas are disaggregated and resuspended either in full nutrient medium or in buffered salts solution containing dextrose, they retain this developmentally acquired capacity to rapidly reaggregate for approximately 1 hr and then lose it completely in a synchronous and discrete step which we have referred to as the “erasure event.” In this report, it is demonstrated that micromolar concentrations of cAMP completely block this transition from the developmental to vegetative state, and that other cyclic nucleotides also inhibit it, but they do so at 20-fold higher concentrations. Neither the hydrolysis products of cAMP nor the vegetative chemoattractant folic acid inhibit dedifferentiation at concentrations as high as 10^?3M, demonstrating a specificity for cyclic nucleotides and cAMP in particular. The addition of cAMP at any time during the lag period preceding the erasure event inhibits it and addition immediately after the erasure event reverses it. Since cAMP may inhibit the transition from the developmental to vegetative state intracellularly or extracellularly, we have also examined the intracellular concentration of cAMP and the levels of cAMP binding sites on the cell surface during the erasure process. Evidence is presented that the majority of cAMP binding sites on the cell surface are not necessary for the inhibition of erasure by cAMP. The results of these latter studies are discussed in terms of alternative models for the involvement of cAMP in the transition from the developing to vegetative state.     

Effects of amino acids and glucose on adenylate cyclase and cell differentiation of Dictyostelium discoideum.

Starvation triggers the differentiation of Dictyostelium discoideum amoebas to aggregation competence. To determine more precisely the nature of the starvation signal, the ability of various components of the growth medium to inhibit differentiation was examined. Changes in adenylate cyclase (the enzyme which generates the cAMP pulses basic to the differentiation process), various physiological and biochemical markers of developing cells, and the ability of amoebas to form specific intercellular contacts were monitored. We show that amino acid mixtures inhibit cell differentiation by preventing the increase of adenylate cyclase activity which normally occurs during the early hours of starvation. High concentrations of glucose also inhibit the differentiation process but at a later stage: The rise in adenylate cyclase still occurs when cells are starved in the presence of sugar, but the enzyme does not appear to function in vivo. Exogenously generated cAMP pulses are not able to bypass the block exerted by amino acids but can bypass the block exerted by glucose. Results support the hypothesis that the presence of amino acids inhibits adenylate cyclase synthesis, while the presence of 3% glucose blocks endogenous activation of adenylate cyclase, perhaps as a consequence of high osmotic pressure.     

Development of the simple cellular slime mold Dictyostelium minutum

An ultrastructural study has been made of the life cycle of the cellular slime mold Dictyostelium minutum. The development of D. minutum is rather simple if compared with Dictyostelium discoideum. After 2 hr of starvation, amoebas move in a nonpulsatile manner towards an acrasin-secreting founder cell. The chemotactic signal is not relayed by the amoebas and stream formation toward primary aggregation centers does not occur. Usually, more than one fruiting body arises from one pseudoplasmodium. No migration of the pseudoplasmodium takes place. The first signs of spore differentiation are found in late aggregates, where prespore cells can be distinguished from the surrounding undifferentiated cells by the increased electron density of their cytoplasm. Vacuoles comparable with the prespore vacuole of D. discoideum appear in both cell types; they fuse with the plasma membrane during sporulation of electron-dense cells and are lysed in electron-light cells, which eventually form the stalk. In contrast with D. discoideum no spatial separation between prespore and prestalk cells is found until very late in fruiting body development.     

Analysis of phenotypic evolution in Dictyostelia highlights developmental plasticity as a likely consequence of colonial multicellularity

Colony formation was the first step towards evolution of multicellularity in many macroscopic organisms. Dictyostelid social amoebas have used this strategy for over 600 Myr to form fruiting structures of increasing complexity. To understand in which order multicellular complexity evolved, we measured 24 phenotypic characters over 99 dictyostelid species. Using phylogenetic comparative methods, we show that the last common ancestor (LCA) of Dictyostelia probably erected small fruiting structures directly from aggregates. It secreted cAMP to coordinate fruiting body morphogenesis, and another compound to mediate aggregation. This phenotype persisted up to the LCAs of three of the four major groups of Dictyostelia. The group 4 LCA co-opted cAMP for aggregation and evolved much larger fruiting structures. However, it lost encystation, the survival strategy of solitary amoebas that is retained by many species in groups 1–3. Large structures, phototropism and a migrating intermediate ‘slug’ stage coevolved as evolutionary novelties within most groups. Overall, dictyostelids show considerable plasticity in the size and shape of multicellular structures, both within and between species. This probably reflects constraints placed by colonial life on developmental control mechanisms, which, depending on local cell density, need to direct from 10 to a million cells into forming a functional fructification.