Axenic mass cultivation of the free-living soil amoeba, Acanthamoeba castellanii in a laboratory fermentor

Axenic mass cultivation of Acanthamoeba castellanii in laboratory fermentors (14 l) yielded after 20 days approximately 3 g cells (wet weight). After a short lag phase amoebal cell numbers increased exponentially to a maximum of 3.5×10⁵ cells per ml until cell death occurred after 20 days. Optical density and protein concentrations revealed identical patterns. During amoebal growth only 12–19% of the initially added glucose (100 mM) as sole carbon source was used. Large amounts of ammonia (1 g in 10.5 l culture volume) were excreted into the medium which subsequently raised the pH from 6.6 to 7.7, and from 6.6 to 6.8 in 2 and 20 mM buffered media, respectively. Growth inhibition and cell death could not be explained by a depletion of glucose or oxygen limitations during growth. The production of ammonia had a growth inhibitory effect, however, the sudden termination of the exponential growth phase and cell death could not be explained by the toxic influence of ammonia only.     

Characterization of the genome of the small free-living amoeba Acanthamoeba castellanii.

The cellular DNAs of Acanthamoeba castellanii have been characterized by their behaviour in CsC1 density gradients, by their thermal denaturation and by their renaturation kinetics. Whole-cell DNA exhibits, on CsC1 density gradients, a major peak with a density of 1.717 g/cm3 (major component) and a minor peak with a density of 1.692 g/cm3 (minor component). The major component is nuclear and the minor component is of cytoplasmic origin. The latter contains mitochondrial DNA as well as an extramitochondrial DNA fraction. Reiterated sequences make up approximately 14% of the total and are mainly cytoplasmic. They are characterized by three families of nucleotide sequences. The mitochondrial DNA exhibits a complex renaturation pattern. The fast renaturing component has a calculated complexity of 4.107 daltons. The slower renaturing component has a kinetic complexity tentatively estimated as 1.1010 daltons. The melting profile of mitochondrial DNA suggests heterogeneity in base composition.     

Bacteriolytic activities of the free-living soil amoebae,Acanthamoeba castellanii,Acanthamoeba polyphaga andHartmannella vermiformis

Bacteriolytic activities of axenically grown free-living soil amoebaeAcanthamoeba castellanii, Acanthamoeba polyphaga andHartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometric assay revealed that the specific bacteriolytic activities of bothAcanthamoeba species were higher as those of the threeHartmannella strains.Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus andPseudomonas fluorescens were more easily lysed than the other bacteria tested.Agrobacterium tumefaciens, Klebsiella aerogenes andSerratia marcescens were hardly affected at all by the amoebal bacteriolytic activities. Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3–10 was performed to separate the bacteriolytic isoenzymes of amoebae. Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay. The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pI points of bacteriolytic activities inAcanthamoeba andHartmannella were shown. Bacteriolytic activities ofHartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0–9.3 while forAcanthamoeba the range was pI 4.5–8.9.Abbreviations IEF isoelectric focusing - PAA-IEF polyacrylamide-isoelectric focusing - CCAP culture collection of algae and protozoa - AS amoeba saline medium - pI isoelectric points     

Temperature-dependent parasitic relationship between Legionella pneumophila and a free-living amoeba (Acanthamoeba castellanii)

We analyzed the effects of temperature on the interaction of Legionella pneumophila with Acanthamoeba castellanii. At <20 degrees C, overexpression of type 1 metacaspase, a stimulator of A. castellanii encystation, was associated with a reduced number of bacteria within amoeba. At low temperatures, A. castellanii seems to eliminate L. pneumophila by encystation and digestion.     

Pseudomonas aeruginosa utilises its type III secretion system to kill the free-living amoeba Acanthamoeba castellanii

Pseudomonas aeruginosa is a free-living and common environmental bacterium. It is an opportunistic and nosocomial pathogen causing serious human health problems. To overcome its predators, such as macrophages and environmental phagocytes, it utilises different survival strategies, such as the formation of microcolonies and the production of toxins mediated by a type III secretion system (TTSS). The aim of this study was to examine interaction of TTSS effector proteins of P. aeruginosa PA103 with Acanthamoeba castellanii by co-cultivation, viable count, eosin staining, electron microscopy, apoptosis assay, and statistical analysis. The results showed that P. aeruginosa PA103 induced necrosis and apoptosis to kill A. castellanii by the effects of TTSS effector proteins ExoU, ExoS, ExoT, and ExoY. In comparison, Acanthamoeba cultured alone and co-cultured with P. aeruginosa PA103 lacking the known four TTSS effector proteins were not killed. The results are consistent with P. aeruginosa being a strict extracellular bacterium that needs TTSS to survive in the environment, because the TTSS effector proteins are able to kill its eukaryotic predators, such as Acanthamoeba.     

An intracellular replication niche for Vibrio cholerae in the amoeba Acanthamoeba castellanii

Vibrio cholerae is a human pathogen and the causative agent of cholera. The persistence of this bacterium in aquatic environments is a key epidemiological concern, as cholera is transmitted through contaminated water. Predatory protists, such as amoebae, are major regulators of bacterial populations in such environments. Therefore, we investigated the interaction between V. cholerae and the amoeba Acanthamoeba castellanii at the single-cell level. We observed that V. cholerae can resist intracellular killing. The non-digested bacteria were either released or, alternatively, established a replication niche within the contractile vacuole of A. castellanii. V. cholerae was maintained within this compartment even upon encystment. The pathogen ultimately returned to its aquatic habitat through lysis of A. castellanii, a process that was dependent on the production of extracellular polysaccharide by the pathogen. This study reinforces the concept that V. cholerae is a facultative intracellular bacterium and describes a new host–pathogen interaction.     

Axenic cultivation of the anaerobic free-living ciliate Trimyema compressum

The strain N of Trimyema compressum, an anaerobic free-living ciliate, was cultivated axenically in a medium containing a buffered salt solution, yeast extract, trypticase, and glutathione. Dead bacteria were indispensable as food; a culture of the ciliate together with heat-killed Klebsiella pneumoniae has been established for more than one year. In the medium described, the ciliates grow to a higher cell density than in cultures with living bacteria as food. During the process of axenization, a nonmethanogenic bacterial endosymbiont was lost. In the microbodies of T. compressum, hydrogenase could be localized by the technique of indirect immunofluorescence.     

Conserved mechanisms of Mycobacterium marinum pathogenesis within the environmental amoeba Acanthamoeba castellanii

Mycobacterium marinum is a waterborne mycobacterial pathogen. Due to their common niche, protozoa likely represent natural hosts for M. marinum. We demonstrate that the ESX-1 secretion system is required for M. marinum pathogenesis and that M. marinum utilizes actin-based motility in amoebae. Therefore, at least two virulence pathways used by M. marinum in macrophages are conserved during M. marinum infection of amoebae.     

Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living amoeba Acanthamoeba castellanii

Vibrio cholerae species are extracellular, waterborne, gram-negative bacteria that are overwhelmed by predators in aquatic environments. The unencapsulated serogroup V. cholerae O1 and encapsulated V. cholerae O139 cause epidemic and pandemic outbreaks of cholera. It has recently been shown that the aquatic and free-living amoeba Acanthamoeba castellanii is not a predator to V. cholerae O139; rather, V. cholerae O139 has shown an intracellular compatibility with this host. The aim of this study was to examine the ability of V. cholerae O1 classical and El Tor strains to grow and survive in A. castellanii. The interaction between A. castellanii and V. cholerae O1 strains was studied by means of amoeba cell counts and viable counts of the bacteria in the absence or presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Confocal microscopy and electron microscopy were used to determine the intracellular localization of V. cholerae in A. castellanii. The results showed that V. cholerae O1 classical and El Tor strains grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were also found in the cysts of A. castellanii. The interaction showed a facultative intracellular behaviour of V. cholerae O1 classical and El Tor strains and a possible role of A. castellanii as an environmental host of V. cholerae species.     

Cellular response of the amoeba Acanthamoeba castellanii to chlorine, chlorine dioxide, and monochloramine treatments

Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested on A. castellanii trophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action on A. castellanii.     

Carbon monoxide- and oxygen-reacting haemoproteins in the mitochondrial fraction from the soil amoeba Acanthamoeba castellanii. Studies at subzero temperatures.

1. Mitochondria-enriched fractions of the soil amoeba Acanthamoeba castellanii contained four haemoproteins that in their reduced forms reacted with CO to give photodissociable CO complexes; these were cytochromes a 3, a 614, b- and c-type cytochromes. 2. Non-photodissociable oxygen-containing compounds were formed at temperatures between -130 and -150 degrees C after photodissociation of CO in the presence of 200 microM-O2, 3. Electron transport, indicated by the oxidation of cytochromes a + a3 and cytochrome c, did not occur until the temperature was raised to -80 degrees C.     

Interactions between the environmental pathogen Listeria monocytogenes and a free-living protozoan (Acanthamoeba castellanii)

Listeria monocytogenes can cause severe disease in animal hosts, but it has no recognized animal host reservoir. We tested the hypothesis that L. monocytogenes retains virulence traits to survive predation by amoebae and that listeriolysin O plays a crucial role in this process. Co-culturing of L. monocytogenes and Acanthamoeba castellanii demonstrated that L. monocytogenes does not actively kill amoebae, but in the presence of amoebae, high bacterial population densities can be maintained over a period of at least 96 h. A gentamicin protection assay demonstrated that there is no significant difference in the ability to survive predation between serovars (4b versus 1/2a and 1/2c; P  = 0.08) and between five species of Listeria ( P  = 0.14). Three of these species do not harbour the hly gene responsible for listeriolysin O production. A hly knockout strain had poorer survival compared with the parental strain ( P  = 0.04 at 24 h; P  = 0.04 at 48 h; P  = 0.02 at 72 h) and electron microscopy was consistent with a wild-type strain being able to escape the phagosome whereas the hly knockout strain did not appear to have this ability. Thus, while there is weak evidence that listeriolysin O can contribute to improved survival after ingestion by amoebae, listeriolysin O does not appear to provide a significant selective advantage under the conditions of this study.     

Benzodiazepine binding to mitochondrial membranes of the amoeba Acanthamoeba castellanii and the yeast Saccharomyces cerevisiae

Benzodiazepine binding sites were studied in mitochondria of unicellular eukaryotes, the amoeba Acathamoeba castellanii and the yeast Saccharomyces cerevisiae, and also in rat liver mitochondria as a control. For that purpose we applied Ro5-4864, a well-known ligand of the mitochondrial benzodiazepine receptor (MBR) present in mammalian mitochondria. The levels of specific [(3)H]Ro5-4864 binding, the dissociation constant (K(D)) and the number of [(3)H]Ro5-4864 binding sites (B(max)) determined for fractions of the studied mitochondria indicate the presence of specific [(3)H]Ro5-4864 binding sites in the outer membrane of yeast and amoeba mitochondria as well as in yeast mitoplasts. Thus, A. castellanii and S. cerevisiae mitochondria, like rat liver mitochondria, contain proteins able to bind specifically [(3)H]Ro5-4864. Labeling of amoeba, yeast and rat liver mitochondria with [(3)H]Ro5-4864 revealed proteins identified as the voltage dependent anion selective channel (VDAC) in the outer membrane and adenine nucleotide translocase (ANT) in the inner membrane. Therefore, the specific MBR ligand binding is not confined only to mammalian mitochondria and is more widespread within the eukaryotic world. However, it can not be excluded that MBR ligand binding sites are exploited efficiently only by higher multicellular eukaryotes. Nevertheless, the MBR ligand binding sites in mitochondria of lower eukaryotes can be applied as useful models in studies on mammalian MBR.     

Gene discovery in the Acanthamoeba castellanii genome

Acanthamoeba castellanii is a free-living amoeba found in soil, freshwater, and marine environments and an important predator of bacteria. Acanthamoeba castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct diseases in humans. In order to provide a genomic platform for the study of this ubiquitous and important protist, we generated a sequence survey of approximately 0.5 x coverage of the genome. The data predict that A. castellanii exhibits a greater biosynthetic capacity than the free-living Dictyostelium discoideum and the parasite Entamoeba histolytica, providing an explanation for the ability of A. castellanii to inhabit a diversity of environments. Alginate lyase may provide access to bacteria within biofilms by breaking down the biofilm matrix, and polyhydroxybutyrate depolymerase may facilitate utilization of the bacterial storage compound polyhydroxybutyrate as a food source. Enzymes for the synthesis and breakdown of cellulose were identified, and they likely participate in encystation and excystation as in D. discoideum. Trehalose-6-phosphate synthase is present, suggesting that trehalose plays a role in stress adaptation. Detection and response to a number of stress conditions is likely accomplished with a large set of signal transduction histidine kinases and a set of putative receptor serine/threonine kinases similar to those found in E. histolytica. Serine, cysteine and metalloproteases were identified, some of which are likely involved in pathogenicity.     

Vertical distribution of the free-living amoeba population in soil under desert shrubs in the Negev desert, Israel

A field study was designed to examine the effect of desert shrubs on the dynamics of free-living amoebae in arid soil. Soil samples from 0- to 50-cm depths were collected at 10-cm intervals in each of the four seasons. The vertical distributions of the four main morphological types of amoebae, grouped according to their mobility, and of small flagellate populations were measured under the canopies of Hammada scoparia and Atriplex halimus, shrubs belonging to the chloride-absorbing xerohalophytes. The result obtained from the field study demonstrated that the total number of protozoa was significantly higher during the wet seasons (winter and spring) than during the dry seasons. The protozoan population was more diverse under the canopy of H. scoparia during the wet seasons, reaching 8,000 individuals per 1 g of dry soil, whereas during the dry seasons, the populations were higher under the canopy of A. halimus, with a mean of 250 individuals. The protozoan population in the deeper layers (40 to 50 cm) was found to be as active as that in the upper layers, demonstrating that, in the desert, soil columns below 20 cm are fertile and worth studying. The type 1 amoebae (e.g., Acanthamoeba and Filamoeba spp.) were the most abundant throughout the study period, and their numbers were significantly higher than those of the other amoeba types.     

A phylogenetic study of the free-living amoeba

Immunological distances were determined for four strains of the free-living amoeba classified as Amoeba proteus, two strains classified as Polychaos dubia and a single strain classified as Chaos carolinensis. The data show that the _ShP strain does not belong to the proteus group; that A. proteus is more closely related to C. carolinensis and is derived from Chaos as is the _ShP strain; that P. dubia and C. carolinensis are the more distantly related species and appear to be the first of the above to have diverged from a common ancestor; and that the amoebae have a long evolutionary history. The accuracy of the phylogenetic tree and the distance Wagner network was discussed. Since the amoebae may be polyphyletic in origin, the latter was assumed to be a more accurate representation of the immunological distance data.