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PubMed1992

Alterations in protein expression and complement resistance of pathogenic Naegleria amoebae.

Highly pathogenic strains of Naegleria fowleri activate the alternative complement pathway but are resistant to lysis. In contrast, weakly pathogenic and nonpathogenic Naegleria spp. activate the complement pathway and are readily lysed. The present study was undertaken to determine whether surface components on amoebae accounted for resistance to complement lysis. Enzymatic removal of surface components from highly pathogenic N. fowleri with phosphatidylinositol-specific phospholipase C or with endoglycosidase H increased the susceptibility of these amoebae to complement-mediated lysis. Similar treatment of nonpathogenic amoebae had no effect on susceptibility to complement. Tunicamycin treatment of highly and weakly pathogenic N. fowleri increased susceptibility to lysis by complement in a dose-related manner. Tunicamycin treatment did not alter the susceptibility of nonpathogenic amoebae to complement. Proteins of 234 and 47 kDa were detected in supernatant fluid from phosphatidylinositol-specific phospholipase C-treated highly pathogenic amoebae but not in supernatant fluid from phosphatidylinositol-specific phospholipase C-treated weakly pathogenic amoebae. Electrophoretic analysis of iodinated surface proteins of highly pathogenic N. fowleri revealed species of 89, 60, 44, and 28 kDa. Western immunoblots of lysates from surface-iodinated amoebae were stained with biotinylated concanavalin A or biotinylated Ulex europaeus agglutinin I. Surface proteins, identified in highly pathogenic amoebae by iodination, were shown to be glycoproteins by lectin analysis specific for the detection of mannose and fucose residues.

Animals↗

PubMed2006

[Free-living amoebae as vehicles of pathogenic bacteria].

The free-living amoebae are ubiquitous organisms. They are found in humid soil and all water reservoirs, i.e. fresh, sea, freezing and hot water. They mainly feed on bacteria. Pathogenic properties of amoebae and the mechanisms underlying pathological changes induced during human infection have not yet been fully elucidated. They are the causative agents of primary amoebic meningo-encephalitis (PAM), granulomatous amebic encephalitis (GAE), a chronic progressive disease of the central nervous system, amebic keratitis (AK), a chronic eye infection; amebic pneumitis (AP), a chronic lung infection, and skin infection. Only a few isolates are strongly and permanently pathogenic to humans. Some isolates lose their pathogenic properties after one passage. It has been assumed that such "temporary", unstable pathogenic properties of the amoebae may be caused by internal factors carried by them. It is generally known that the free-living amoebae may be naturally infected with pathogenic bacteria, which have the ability to survive for a long time and to proliferate in the amoebae cells. The role of the amoeba in the process of maintaining, propagating and transmitting human pathogens has not been well recognized. It has been suggested that some infections can be acquired by inhaling aerosols containing amoebae cells filled with bacteria. The presence of bacteria inside the free-living amoebae possess a great challenge to organisations responsible for testing and inspecting the quality and cleanliness of surface waters, swimming pools and drinking water intakes.

Acanthamoeba↗

PubMed1994

Membrane vesiculation of Naegleria fowleri amoebae as a mechanism for resisting complement damage.

Pathogenic and nonpathogenic Naegleria amoebae activate the alternative C pathway; however, only pathogenic amoebae are resistant to C-mediated damage. The present study was undertaken to determine the mechanism by which highly pathogenic N. fowleri amoebae resist C-mediated damage. Nomarski optics microscopy and electron microscopy of Naegleria amoebae revealed membrane blebbing on the surface of C-resistant N. fowleri, but not on C-sensitive N. gruberi, in response to incubation in normal human serum diluted 1:4 to 1:16. Immunofluorescent staining of pathogenic amoebae, by using antiserum to human C proteins comprising the membrane attack complex, C5b through C9, and FITC-labeled goat anti-rabbit IgG, confirmed that the membrane attack complex was concentrated on the membrane blebs. Binding studies with the use of radioiodinated C9 demonstrated a decrease in the 125I-labeled C9 cpm associated with N. fowleri amoebae and an increase in the 125I-labeled C9 cpm associated with the released membrane vesicles after increasing incubation periods in normal human serum. Treatment of pathogenic, C-resistant N. fowleri with cytochalasin D or cytochalasin B to inhibit actin-dependent exocytic processes increased the susceptibility of the amoebae to C damage. In contrast, incubation of nonpathogenic, C-sensitive amoebae with cytochalasins did not alter their susceptibility to C lysis. These data indicate that pathogenic N. fowleri use membrane vesiculation to remove membrane-deposited C proteins, specifically the membrane attack complex (C5b-C9). The ability to remove surface-associated membrane attack complexes serves as one mechanism by which pathogenic N. fowleri resist C lysis.

Animals↗

PubMed1976

Endosymbiosis in amoebae: recently established endosymbionts have become required cytoplasmic components.

A strain of large, free-living amoeba that became dependent on bacterial endosymbionts which had infected the amoebae initially as intracellular parasites, was studied by micrurgy and electron microscopy. The results show that the infected host cells require the presence of live endosymbionts for their survival.Thus, the nucleus of an infected amoeba can form a viable cell with the cytoplasm of a noninfected amoeba only when live endosymbionts are present. The endosymbiotic bacteria are not digested by the host amoebae and are not themselves used as nutritional supplement. While the host amoebae are dependent specifically on the endosymbionts, the latter can live inside amoebae of different strains, indicating that their dependence on the host cells is not yet strain specific.

Acid Phosphatase↗

PubMed2004

Rho/Rho-dependent kinase affects locomotion and actin-myosin II activity of Amoeba proteus.

The highly motile free-living unicellular organism Amoeba proteus has been widely used as a model to study cell motility. However, the molecular mechanisms underlying its unique locomotion are still scarcely known. Recently, we have shown that blocking the amoebae's endogenous Rac- and Rho-like proteins led to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. In order to elucidate the mechanism of the Rho pathway, we tested the effects of blocking the endogenous Rho-dependent kinase (ROCK) by anti-ROCK antibodies and Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, a specific inhibitor of ROCK, on migrating amoebae and the effect of the Rho and ROCK inhibition on the actin-activated Mg-ATPase of the cytosolic fraction of the amoebae. Amoebae microinjected with anti-ROCK inhibitors remained contracted and strongly attached to the glass surface and exhibited an atypical locomotion. Despite protruding many pseudopodia that were advancing in various directions, the amoebae could not effectively move. Immunofluorescence studies showed that ROCK-like protein was dispersed throughout the cytoplasm and was also found in the regions of actin-myosin II interaction during both isotonic and isometric contraction. The Mg-ATPase activity was about two- to threefold enhanced, indicating that blocking the Rho/Rho-dependent kinase activated myosin. It is possible then that in contrast to the vertebrate cells, the inactivation of Rho/Rho-dependent kinase in amoebae leads to the activation of myosin II and to the observed hypercontracted cells which cannot exert effective locomotion.

Acanthamoeba↗

PubMedDate not supplied

High numbers of naked amoebae in the planktonic waters of a mangrove stand in southern Florida, USA.

This is the first study to examine the abundance of naked amoebae in the water column of a mangrove stand. A total of 37 different morphotypes was noted and at least 13 of these are probably new species. Over a one-year sampling interval, amoebae averaged 35,400 cells liter(-1) (range 2,000-104,000) by an indirect enrichment cultivation method. Densities in the upper end of this range are the highest ever reported for any planktonic habitat. Variation between samples was related to the quantity of suspended aggregates (flocs) in the water column emphasizing that amoebae are usually floc-associated. The study also showed that it is essential to disrupt floc material prior to withdrawing sample aliquots for the indirect counting method since several amoebae can occupy the interstices of aggregates. There is concern that indirect enumeration methods that require organisms to be cultured in the laboratory seriously underestimate the true count. A direct counting method using acridine orange staining and epifluorescence microscopy was attempted to assess the possible magnitude of the error associated with indirect counting. While this direct method had limitations, notably the difficulty of unambiguously differentiating between small amoebae and nanoflagellates, the results suggested that the indirect method gave estimates that were close to the true count (within a factor of two). Mangrove waters are rich in heterotrophic protozoa (approximately 3 x 10 liter(-1)) and while the heterotrophic flagellates are by far the dominant group, naked amoebae outnumber ciliates some 20-fold. The ecological consequences of high numbers of amoebae, particularly the common small forms less than 10 microm in length, need to be examined for these important coastal sites.

Amoeba↗

PubMedDate not supplied

Genetic and physiological interactions in the amoeba-bacteria symbiosis.

Amoebae of the xD strain of Amoeba proteus that arose from the D strain by spontaneous infection of Legionella-like X-bacteria are now dependent on their symbionts for survival. Each xD amoeba contains about 42,000 symbionts within symbiosomes, and established xD amoebae die if their symbionts are removed. Thus, harmful infective bacteria changed into necessary cell components. As a result of harboring X-bacteria. xD amoebae exhibit various physiological and genetic characteristics that are different from those of symbiont-free D amoebae. One of the recent findings is that bacterial symbionts control the expression of a host's house-keeping gene. Thus, the expression of the normal amoeba sams gene (sams1) encoding one form of S-adenosylmethionine synthetase is switched to that of sams2 by endosymbiotic X-bacteria. Possible mechanisms for the switching of sams genes brought about by endosymbionts and its significance are discussed.

Amoeba↗

PubMed1975

Effect of near ultraviolet and visible light on amoeba.

The near ultraviolet and visible light (VL) impinging at an intensity of 2-5 x 10(2) J s-1 m-2 for 2-5 h kills the mitotic and the early S-phase (0- to 15-min-old) amoebae. At the mid- and late S-period only a fraction of cells are killed by VL and G2 phase cells are quite resistant. Amoebae of all cell cycle stages show a delay in the first mitotic division. DNA synthesis, as measured by [3H]thymidine incorporation, is depressed in the VL-exposed early-S amoebae. A concurrent but temporary inhibition in [3H]leucine incorporation also occurs in these cells. However, no significant change in [3H]uridine incorporation has been found. To localize the site of lethal damage, nuclear transplantation studies were undertaken between the control amoebae and the amoebae treated with VL. The nucleus of a VL-exposed early S-phase cell recovers when transplanted immediately after VL exposure into an enucleate G2 cytoplasm but dies if grafted into an enucleat S-phase cytoplasm. The therapeutic effect of the G2 cytoplasm, although at a lower level, is also evident even when the treated early S-phase nucleus is implanted 20 h later, but not after 48 h, into the G2 cytoplasm. The amoeba cytoplasm shows resistance to VL-irradiation, can accept a control nucleus from any cell cycle stage, and function normally. The G2 nucleus also remains apparently unaffected to VL exposure and can survive when it is transfered to the control cytoplasm of any cell-cycle phase. All these findings are discussed in the light of the possible existence of a repair system against VL-induced damage in the G2-phase amoeba.

Amoeba↗

PubMed2003

Amoeba-resisting bacteria and ventilator-associated pneumonia.

To evaluate the role of amoeba-associated bacteria as agents of ventilator-associated pneumonia (VAP), we tested the water from an intensive care unit (ICU) every week for 6 months for such bacteria isolates; serum samples and bronchoalveolar lavage samples (BAL) were also obtained from 30 ICU patients. BAL samples were examined for amoeba-associated bacteria DNA by suicide-polymerase chain reaction, and serum samples were tested against ICU amoeba-associated bacteria. A total of 310 amoeba-associated bacteria from 10 species were isolated. Twelve of 30 serum samples seroconverted to one amoeba-associated bacterium isolated in the ICU, mainly Legionella anisa and Bosea massiliensis, the most common isolates from water (p=0.021). Amoeba-associated bacteria DNA was detected in BAL samples from two patients whose samples later seroconverted. Seroconversion was significantly associated with VAP and systemic inflammatory response syndrome, especially in patients for whom no etiologic agent was found by usual microbiologic investigations. Amoeba-associated bacteria might be a cause of VAP in ICUs, especially when microbiologic investigations are negative.

Adult↗

PubMed2005

[Pathogenic properties of free-living amoebae isolated from natural and man-made bathing sites in the province of Western Pomerania].

INTRODUCTION: Pathogenic amoebae belonging to the genus of Acanthamoeba and Naegleria, as well as species such as Balamuthia mandrillaris and Sappinia diploidea cause harmful, life-threatening infections in humans and animals. These amoebae commonly occur in the natural environment. Free-living, pathogenic amoebae have hitherto been detected in water reservoirs near Poznań, Gdańsk, and Lublin. MATERIAL AND METHODS: The present work focused on the occurrence of thermophilic amoebae potentially pathogenic to humans. Natural and man-made bathing sites in the province of Western Pomerania were studied. A total of 299 water samples were collected between 2001 and 2003 from 86 sites (indoor swimming pools, seasonal pools, physiotherapy pool, fountains, lakes, ponds, rivers, lagoons, and coastal waters of the Baltic Sea). Amoebae were isolated at 37 degrees C from 223 samples, of which 34 (15.2%) demonstrated thermophilic properties. Tests using thermophilic isolates were carried out on mice through intranasal inoculation. Pathogenic properties and intensity of infection were determined on the basis of re-isolation of amoebae from agar substrates inoculated with fragments of internal organs. Organs of infected mice were examined histologically. RESULTS: Six isolates exhibited infective properties in mice. Five of them were obtained from two man-made water reservoirs belonging to the recreational complex of the city of Szczecin (AM 17, AM 148, AD 16, AD 148, AD 166). One isolate was from a natural reservoir (JG 172). Amoebae were isolated from the brain, lungs, liver, kidneys, spleen, and heart of the infected animals.

Amoeba↗

PubMed2001

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.

Journal Article↗

PubMed1983

Thermotaxis of Dictyostelium discoideum amoebae and its possible role in pseudoplasmodial thermotaxis.

Thermotaxis by individual amoebae of Dictyostelium discoideum on a temperature gradient is described. These amoebae show positive thermotaxis at temperatures between 14 degrees C and 28 degrees C shortly (3 hr) after food depletion. Increasing time on the gradient reduces the positive thermotactic response at the lower temperature gradients (midpoint temperatures of 14, 16, and 18 degrees C), and amoebae show an apparent negative thermotactic response after 12 hr on the gradient. The thermotaxis response curve for "wild-type" amoebae after 16 hr on the gradient is similar to that shown for the pseudoplasmodia. Growth of the amoebae at a different temperature causes a shift in the thermotaxis response curve for the amoebae. This adaptation is similar to that shown for the pseudoplasmodia. Two mutants in thermotaxis, HO428 and HO813, show changes in amoebal thermotaxis similar to the observed changes in pseudoplasmodial thermotaxis. On the basis of the similarities between these responses, thermotaxis by the amoebae is proposed to be the basis for thermotaxis by the multicellular pseudoplasmodium.

Journal Article↗

PubMedDate not supplied

A method for assessing the migratory response of Naegleria fowleri utilizing [3H]uridine-labeled amoebae.

A new procedure is described to assay the migratory response of Naegleria fowleri (ATCC 30894) amoebae to potential chemoattractants. The method utilizes a blind-well Boyden chemotaxis chamber, two micropore filters of different construction, and amoebae-labeled with [3H]uridine. The technique was standardized by determining the influence of incubation time, filter construction, filter pore size and geometry, amoebae to filter pore ratio, and chemoattractant concentration. Radiolabeled amoebae were placed in Boyden chambers that contained the combination of an upper polycarbonate filter with distinct pores with a diameter of 8 microns and a lower filter of nitrocellulose with a 150-micron depth to separate the wells. A ratio of two amoebae to one filter pore and a 2-h incubation period were chosen to obtain optimal migration conditions. Nerve cell extract was used as the chemoattractant. The migratory responses of both highly pathogenic and weakly pathogenic strains of N. fowleri to nerve cell extract were compared using either the radiolabel procedure or the conventional single filter, leading-front method. Using either method, a highly pathogenic cloned strain of N. fowleri amoebae moved in a directional manner (chemotactically) in vitro to B103 rat neuroblastoma cell extract. In contrast, a weakly pathogenic strain of amoebae responded in a nondirectional manner (chemokinetically) to nerve cell extract. While both the leading-front assay and the radiolabel assay give accurate results, the measurement of radiolabeled cells allows one to test a greater number of attractants in one assay and the procedure eliminates observer bias.

Animals↗

PubMed1993

[Natural killer cell activity in mice infected with free-living amoeba with reference to their pathogenicity].

The purpose of this observation was to investigate the natural killer cell activities in mice infected with pathogenic free-living amoeba, Naegleria fowleri and Acanthamoeba culbertsoni according to the infection doses, and infected with non-pathogenic free-living amoeba, Naegleria gruberi. The natural killer cell activity was examined by means of target binding capacity, active NK cell and maximum recycling capacity of the mice after inoculating free-living amoebae with low and high doses. The mice infected with 1,103, 1,105 A. culbertsoni trophozoites showed mortality rates of 6.9% and 65.5%, respectively. The mice infected with 1,104, 1,105 N. fowleri trophozoites showed mortality rates of 5.9% and 72.2%, respectively. The NK cell activities in all experimental groups increased significantly on day 1 after infection as compared with control group, and then remarkably declined thereafter, there was no difference of the cytotoxic activity of the NK cells in mice among inoculation doses of pathogenic free-living amoebae. The target binding capacities of NK cells and percentages of activated NK cells in mice infected with pathogenic free-living amoebae were significantly increased a day after infection, as compared with control group. There was no difference of the maximal recycling capacities of NK cells in all experimental groups as compared with control group. There was significant difference in the cytotoxic activity and single cell cytotoxicity of NK cells between the experimental groups infected with pathogenic free-living amoebae and that infected with non-pathogenic free-living amoebae.

Acanthamoeba↗

PubMed1990

The amphiphilic action of vasopressin and analogues on the plasma membrane of Amoeba proteus.

Arginine (AVP) and lysine vasopressin induce a weak but statistically significant increase in the water permeability of Amoeba proteus plasmalemma. Vasotocin and deaminovasopressin, which share the hydroosmotic properties of AVP on classical vertebrate systems, are without effects on Amoeba while SKF 101926, a synthetic AVP antagonist, is even more effective than the parent compound. Theophyllin and dibutyryl-cAMP do not affect AVP action on Amoeba. Lithium, oxytocin, and carbachol are also without effect. Thus, it is unlikely that either V2 (cAMP) or V1 (phosphatidylinositol choline) receptors are involved. A clear correlation has been found between the amphiphilic character of tested peptides and their effect on Amoeba water permeability. Classical amphiphilic peptides, melittin, mastoparan, and fragment 1-8 of alpha-neoendorphin, also increased water permeability in Amoeba. It is known that vasopressin can interact with artificial lipid membranes, increasing their permeability to water. We propose that amphiphilic members of the AVP family interact directly with the lipid phase of the Amoeba membrane. Their incorporation within the lipid bilayer may cause local disruptions or may create micellar water channels as shown for other amphiphilic proteins. Our observations provide a model for the early evolution of peptide hormone systems, preceding the appearance of specific membrane receptors and associated second messenger amplifying mechanisms.

Amoeba↗

PubMed1993

A possible signal-coupling role for cyclic AMP during endocytosis in Amoeba proteus.

Cytoplasmic levels of cAMP in Amoeba proteus were measured utilizing radioimmunoassays under control conditions and when stimulated by inducers of either pinocytosis or phagocytosis. In control cells, cytoplasmic cAMP levels were approximately 0.39 pM/mg cells. When exposed to either chemotactic peptide or mannose which stimulate phagocytosis in the amoeba, there is a rapid doubling of the cAMP level within 45 sec of stimulation which then returns to the control level within 3-5 min. Theophylline prolongs the elevation of cytoplasmic cAMP in stimulated cells and is also capable of eliciting food vacuole formation in the amoeba. In addition isoproterenol also causes food vacuole formation in the amoeba as well as a large and prolonged increase in cytoplasmic cAMP levels. Inducers of pinocytosis (BSA and Na Cl) also elicit changes in cytoplasmic cAMP in the amoeba, but the response appears to differ from that elicited by inducers of phagocytosis in that the peak cAMP levels are broader and biphasic. It is concluded that cAMP plays a signal-coupling role during the early phases of both forms of endocytosis in Amoeba proteus.

Amoeba↗

PubMed1977

Binding of [125I]concanavalin A by interspecific amoeba hybrids.

Two types of interspecific amoeba hybrids, viz. PnIc (Amoeba proteus nucleus in A. indica cytoplasm) and InPc (A. indica nucleus in A. proteus cytoplasm), were tested for their [125I]Concanavalin A (Con A) binding activity at different periods of time. The cell surface binding of the labelled Con A was reduced to approx. 40 and 75% in the PnIc and InPc cells, respectively, 96 h after the cells were made hybrids. A significant increase in the binding of radioactive Con A was observed after the homologous nucleus was back transferred into InPc cells, but not in PnIc cells. No significant difference in the binding of the radioactive Con A was found between the progeny of the hybrid amoebae and the control amoebae. The Con A binding activity of the interspecific amoebae has been discussed in relation to the changes in the cytoplasmic membrane structures which presumably play a role in the formation of cell surface in the amoebae.

Amoeba↗

PubMed2004

Testate amoebae (Protista) communities in Hylocomium splendens (Hedw.) B.S.G. (Bryophyta): relationships with altitude, and moss elemental chemistry.

We studied the testate amoebae in the moss Hylocomium splendens along an altitudinal gradient from 1000 to 2200 m asl. in the south-eastern Alps of Italy in relation to micro- and macro-nutrient content of moss plants. Three mountainous areas were chosen, two of them characterised by calcareous bedrock, the third by siliceous bedrock. A total of 25 testate amoebae taxa were recorded, with a mean species richness of 9.3 per sampling plot. In a canonical correspondence analysis, 63.1% of the variation in the amoebae data was explained by moss tissue chemistry, namely by C, P, Ca, Mg, Al, Fe, and Na content and a binary site variable. We interpreted this result as an indirect effect of moss chemistry on testate amoebae through an influence on prey organisms. Although two species responded to altitude, there was no overall significant relationship between testate amoebae diversity or community structure and altitude, presumably because our sampling protocol aimed at minimizing the variability due to vegetation types and soil heterogeneity. This suggests that previous evidence of altitudinal or latitudinal effects on testate amoebae diversity may at least in part be due to a sampling bias, namely differences in soil type or moss species sampled.

Altitude↗