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PubMedDate not supplied

The interaction of Naegleria fowleri amoebae with murine macrophage cell lines.

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D1, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowleri bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.

Animals↗
PubMed1994

Notes on protozoa in agricultural soil with emphasis on heterotrophic flagellates and naked amoebae and their ecology.

Heterotrophic flagellates and naked amoebae are usually very numerous in agricultural soils; with numbers in the magnitude of 10,000 to 100,000 (active+encysted) cells per gram of soil. In 'hotspots' influenced by living roots or by dead organic material, the number may occasionally be as high as several millions per gram of soil. An exact enumeration of these organisms is virtually impossible. As they most often adhere closely to the soil particles, direct counting will underestimate numbers since the organisms will be masked. The method usually applied for enumeration of these organisms, the 'most probable number (MPN) method', is based on the ability of the organisms to grow on particular culture media. This method will in many cases underestimate the total protozoan number (active+encysted). It is uncertain how many of the heterotrophic flagellates and naked amoebae are actively moving and how many are encysted at a particular time; the 'HCl-method' which has usually been used to discriminate between active and encysted has proven to be highly unreliable. Despite the methodological difficulties many investigations of these organisms indicate that they play an important role in agricultural soils as bacterial consumers, and to a minor extent as consumers of fungi. Because of their small size and their flexible body they are able to graze bacteria in small pores in the soil in which larger organisms are precluded from coming. Key factors restricting the number and activity of heterotrophic flagellates and naked amoebae in soils seem to be water potential and soil structure and texture. In micro-cosm experiments, small heterotrophic flagellates and naked amoebae regulate the size and composition of the bacterial community. Bacterial activity seems to be stimulated by these organisms in most cases as well as the mineralization of carbon and nitrogen and possibly other mineral nutrients. In the rhizosphere of living plants the activity of protozoa has proven to stimulate uptake of nitrogen in pot experiments, and it has been hypothesized that organic matter liberated by plants in the root zone will stimulate bacterial and protozoan activity, leading to mineralization of organic soil nitrogen which is subsequently taken up by the plants.

Animals↗
PubMed1987

Evidence for two mechanisms of human erythrocyte endocytosis by Entamoeba histolytica-like amoebae (Laredo strain).

A study of the ultrastructural aspects of endocytosis of human erythrocytes by Entamoeba histolytica-like amoebae (Laredo) revealed two different mechanisms of endocytosis. First, there is classical phagocytosis which consists of the formation of a phagocytotic pseudopod. This process begins with the engulfment of the red blood cell followed by its entrapment in a food vacuole of the same size as the erythrocyte. It is then digested in the food vacuole. The second means of endocytosis is achieved through a preliminary lytic attack on the red blood cell. Following attachment of the prey to the attacking cell, dendritic extensions elongate from the surface of the amoeba at the site of attachment. Intense folding and liquefaction of the red blood cell membrane is then observed. The fluid membrane is then sucked into the amoeba through a pinocytotic-like channel. The end result is the formation of small vacuoles in the amoeba's cytoplasm, filled with the digested red blood cell.

Animals↗
PubMed1988

Growth-supporting activity for Legionella pneumophila in tap water cultures and implication of hartmannellid amoebae as growth factors.

Photosynthetic cyanobacteria, heterotrophic bacteria, free-living amoebae, and ciliated protozoa may support growth of Legionella pneumophila. Studies were done with two tap water cultures (WS1 and WS2) containing L. pneumophila and associated microbiota to characterize growth-supporting activity and assess the relative importance of the microbiota in supporting multiplication of L. pneumophila. The water cultures were incubated in the dark at 35 degrees C. The growth-supporting factor(s) was separated from each culture by filtration through 1-micron-pore-size membrane filters. The retentate was then suspended in sterile tap water. Multiplication of L. pneumophila occurred when both the retentate suspension and the filtrate from either culture were inoculated into sterile tap water. L. pneumophila did not multiply in tap water inoculated with only the filtrate, even though filtration did not reduce the concentration of L. pneumophila or heterotrophic bacteria in either culture. Growth-supporting activity of the retentate suspension from WS1 was inactivated at 60 degrees C but unaffected at 0, 25, and 45 degrees C after 30-min incubations. Filtration experiments indicated that the growth-supporting factor(s) in WS1 was 2 to 5 micron in diameter. Ciliated protozoa were not detected in either culture. Hartmannellid amoebae were conclusively demonstrated in WS2 but not in WS1. L. pneumophila multiplied in tap water inoculated with the amoebae (10(3)/ml) and the 1-micron filtrate of WS2. No multiplication occurred in tap water inoculated with the filtrate only. Growth-supporting activity for L. pneumophila may be present in plumbing systems; hartmannellid amoebae appear to be important determinants of multiplication of L. pneumophila in some tap water cultures.

Animals↗
PubMed2005

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.

Acanthamoeba↗
PubMed1991

Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells.

A cloned and axenically cultured strain of Hartmannella vermiformis was used as a model to study intracellular multiplication of Legionella pneumophila in amoebae. The growth of L. pneumophilia in both H. vermiformis and a human monocyte-like cell line (U937) was investigated with cytoskeletal and metabolic inhibitors. L. pneumophila replicated only intracellularly in these cellular models, and electron microscopy showed ultrastructural similarities in the initial phase of multiplication. Treatment of amoebae with an inhibitor of microfilament-dependent phagocytosis (cytochalasin D, 0.5 or 1.0 micrograms/ml) did not inhibit intracellular growth of L. pneumophila; however, intracellular multiplication was inhibited by treatment of U937 monocytes with the same concentrations of cytochalasin D. Methylamine (10 to 100 mM), an inhibitor of adsorptive pinocytosis, inhibited the replication of L. pneumophila in amoebae in a dose-dependent manner. All doses of methylamine tested (10 to 50 mM) inhibited growth of L. pneumophila in U937 monocytes. Cytochalasin D and methylamine had no effect on the multiplication of L. pneumophila in culture medium or on the viability of amoebae or U937 monocytes. Intracellular replication of L. pneumophila in H. vermiformis may be accomplished by a cytochalasin D-independent mechanism, such as adsorptive pinocytosis. In contrast, both cytochalasin D- and methylamine-sensitive mechanisms may be essential for the intracellular multiplication of L. pneumophila in U937 monocytes.

Analysis of Variance↗
PubMed2004

Icm/dot-independent entry of Legionella pneumophila into amoeba and macrophage hosts.

Legionella pneumophila, the causative agent of Legionnaires' disease, expresses a type IVB secretion apparatus that translocates bacterial proteins into amoeba and macrophage hosts. When stationary-phase cultures are used to infect hosts, the type IVB apparatus encoded by the icm/dot genes is required for entry, delay of phagosome-lysosome fusion, and intracellular multiplication within host cells. Null mutants with mutations in icm/dot genes are defective in these phenotypes. Here a new model is described in which hosts are infected with stationary-phase cultures that have been incubated overnight in pH 6.5 buffer. This model is called Ers treatment because it enhances the resistance to acid, hydrogen peroxide, and antibiotic stress beyond that of stationary-phase cultures. Following Ers treatment entry into amoeba and macrophage hosts does not require dotA, which is essential for Legionella virulence phenotypes when hosts are infected with stationary-phase cultures, dotB, icmF, icmV, or icmX. Defective host entry is also suppressed for null mutants with mutations in the KatA and KatB catalase-peroxidase enzymes, which are required for proper intracellular growth in amoeba and macrophage hosts. Ers treatment-induced suppression of defective entry is not associated with increased bacterial adhesion to host cells or with morphological changes in the bacterial envelope but is dependent on protein expression during Ers treatment. By using proteomic analysis, Ers treatment was shown to induce a protein predicted to contain eight tetratricopeptide repeats, a motif previously implicated in enhanced entry of L. pneumophila. Characterization of Ers treatment-dependent changes in expression is proposed as an avenue for identifying icm/dot-independent factors that function in the entry of Legionella into amoeba and macrophage hosts.

Acanthamoeba↗
PubMed1990

Pathogenic free-living amoebae.

Studies on pathogenic free-living amoebae performed in Korea were briefly reviewed. One strain of Naegleria fowleri was isolated from the sewage, and 3 strains of Acanthamoeba culbertsoni from a reservoir and the gill of fish. They were identified by morphological characteristics. Three strains among the 4 were experimentally proved pathogenic to cause primary amoebic meningoencephalitis in mice. The virulence of N. fowleri depended upon various factors such as strain, weight and sex of mice, and number of the inoculated amoebae. A. culbertsoni was found to retain cytolytic activities which were related to the pathogenicity. Also these amoebae were demonstrated to harbour the activities of acid phosphatase, peroxidase and ATPase. There is one case record of systemic Acanthamoeba infection in Korea, proved at autopsy. Cellular or humoral responses to these amoebae have been studied in vitro or in vivo. Immunization could reduce the mortality of experimentally infected mice.

Acanthamoeba↗
PubMed2005

Amphizoic amoebae: pathogenic free-living protozoa; review of the literature and review of cases in Thailand.

A large variety of species of free-living amoebae (FLA) caused an indefinite form of these protozoa. Non-fixed form, as indicated by amoeboid movement and possed the bacteria to survive in nature. Two species of pathogenic FLA: Naegleria fowleri and Acanthamoeba spp. were identified as the causative agents of Primary Amoebic Meningoencephalitis (PAM) and Granulomatous Amoebic Encephalitis (GAE) respectively. They were suggested to amphizoic protozoa, capable of living as parasites or as free-living and they were also considered to be distributed worldwide. These amoebae were detected in lakes, rivers and ponds. The first case of meningoencephalitis was observed in 1961 by Fowler Many cases were reported later on and the pathogenicity was tested by nasal inoculation of mice. In fact, quite a number of FLA were isolated but only a few species were pathogenic to humans. The three typical features which allow recognition of Naegleria spp. flagellate stage, round cyst and promitotic trophozoite. This promitosis distinguishes the Naegleria genus from Acanthamoeba spp. The disease caused by PAM usually occurs with acute onset, whereas chronic for GAE. The GAE cases mentioned are mostly in debilitated patients, chronic alcoholics or patients under treatment with immunosuppressive methods. About 6 cases of PAM were reported in Thailand during 1982-1997. Four cases of GAE were reported in 1994 and two isolated cases ofAcanthamoebafrom keratitis patients were reported in 2000. Finally one case of PAM and one case of GAE were reported in 2001. The surveys of FLA were set up to study the distribution of these pathogenic amoebae and determine the prevalence of amoebae in aquatic habitats of human environments. About 40% were identified as Acanthamoeba spp., 30% were Naegleria spp., 20% were Hartmanella and 10% were Vahlkampfia. Only 10% of Naegleria spp. belonged to Naegleria fowleri.

Acanthamoeba↗
PubMed2004

Detection of Hartmannella sp, a free-living amoeba from Sungai Setiu, Terengganu.

Hartmannella sp is one of the free-living amoebae that have the ability to infect animal tissues because it has been found in human's nasal mucosa, dog's bronchial and turkey's intestine. Treatment for diseases inflicted by free-living amoebae is difficult because most of them infect and damage the host's tissues, so preventive measures are better to take rather than to cure the diseases. In this study, water taken from several stations namely Kampung Padang, Kampung Besut, Ibu Bekalan Setiu, Kampung Tasik, Kampung Guntung, Kampung Nyatoh, Kampung Penarik and Kampung Mangkok) along Sungai Setiu, Terengganu was examined for the presence of Hartmannella sp. The results of this study indicated that only Ibu Bekalan Setiu station was found positive to have the amoeba. Detail results on the water quality and nutrient contents measured in relation to the distribution of the amoeba at Ibu Bekalan Setiu station are presented and discussed.

Journal Article↗
PubMed1968

Preparation and evaluation of standardized amoeba antigen from axenic cultures of Entamoeba histolytica.

The successful mass cultivation of Entamoeba histolytica strains in pure culture has made it possible to produce pure amoeba antigens, which should improve the prospects for immunodiagnostic research and simplify the interpretation of the results obtained in tests for amoeba antibodies.The authors have mass-produced immunodiagnostic antigens for amoebiasis serology from 2 axenic strains of E. histolytica and standardized them as dry powders, which upon reconstitution contained approximately 1.8 mg N/ml, or roughly the equivalent of 10 x 10(6) amoebae per ml.The antigens were evaluated with 121 sera from subjects in Costa Rica, South Africa, Taiwan, Thailand and the USA, 49 of the sera being from cases of amoebic liver abscess, 41 from symptomatic intestinal amoebiasis, 19 from asymptomatic intestinal amoebiasis, and 12 from subjects presumed not to have amoebiasis.Positive results with the indirect haemagglutination test were obtained with 100% of amoebic liver abscess sera, 90.2% of those from symptomatic intestinal amoebiasis, 57.9% of those from asymptomatic intestinal amoebiasis, and 16.7% of the normal sera. Positive complement-fixation tests for the respective groups were obtained in 83.8%, 63.3%, 10.5% and 0%, and positive agar-gel diffusion in 79.6%, 53.7%, 0% and 0%.These results compare favourably with earlier reports of similar studies in which non-axenic cultures were used and agree well with those of investigators in different parts of the world to whom samples of the axenic antigens were sent.The authors conclude that the axenic antigens from either strain used, or from the 2 strains pooled, are of broad usefulness in the detection of amoebic antibodies, but point out that the limitations of the tests now used in amoeba serology are not yet clearly understood and that the pattern of antibody persistence after infection requires further study.

Antigens↗
PubMed1994

Entamoeba histolytica modulates the nitric oxide synthase gene and nitric oxide production by macrophages for cytotoxicity against amoebae and tumour cells.

Nitric oxide (NO) is the major cytotoxic molecule produced by activated macrophages for cytotoxicity against Entamoeba histolytica trophozoites. In the present study, we determined whether E. histolytica infection and soluble amoebic proteins affected macrophage cytotoxicity against amoebae and tumour cells by modulating the inducible NO synthase gene (iNOS) and NO (measured as nitrite, NO2-) and tumour necrosis factor-alpha (TNF-alpha) production. Amoebic liver abscess-derived macrophages [days 10, 20, 30 post-infection (p.i.)] stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) showed increased cytotoxicity against L929 cells (TNF-alpha-sensitive), but were refractory for killing amoebae and P815 cells (both NO-sensitive), concomitant with low NO2- production (< 4 microM/10(6) cells). In contrast, peritoneal and spleen macrophages at 10 and 20 days p.i. activated with IFN-gamma and LPS demonstrated increased killing of amoebae, and L929 and P815 cells concomitant with high NO2- production (> 12 microM/10(6) cells). Pretreatment of mouse bone marrow-derived macrophages with amoebic proteins suppressed IFN-gamma and LPS-induced amoebicidal (33%) and tumoricidal (44-49%) activities, with a corresponding decrease in TNF-alpha (56%) and NO (41%) production as well as TNF-alpha (41%) and iNOS (27%) mRNA by Northern blot analyses as compared to untreated activated controls. Inhibition of prostaglandin E2 (PGE2) biosynthesis in abscess and naive macrophages pretreated with amoebic proteins augmented IFN-gamma- and LPS-induced killing of L929 cells and TNF-alpha production, but failed to increase killing of P815 cells and amoebae as well as iNOS mRNA levels or NO production. These results suggest that E. histolytica selectively induces dysfunction of macrophage cytotoxicity by modulating iNOS mRNA expression and NO production independent from TNF-alpha and PGE2 allowing the parasites to survive within the host by impairing host immune responses.

Amino Acid Oxidoreductases↗
PubMed1996

Intracellular pressure is a motive force for cell motion in Amoeba proteus.

The cortical filament layer of free-living amoebae contains concentrated actomyosin, suggesting that it can contract and produce an internal hydrostatic pressure. We report here on direct and dynamic intracellular pressure (P(ic)) measurements in Amoeba proteus made using the servo-null technique. In resting apolar A. proteus, P(ic) increased while the cells remained immobile and at apparently constant volume. P(ic) then decreased approximately coincident with pseudopod formation. There was a positive correlation between P(ic) at the onset of movement and the rate of pseudopod formation. These results are the first direct evidence that hydrostatic pressure may be a motive force for cell motion. We postulate that contractile elements in the amoeba's cortical layer contract and increase P(ic) and that this P(ic) is utilized to overcome the viscous flow resistance of the intracellular contents during pseudopod formation.

Amoeba↗
PubMed1997

Chemotaxis of Amoeba proteus in the developing pH gradient within a pocket-like chamber studied with the computer assisted method.

A new "U" shaped, pocket-like chamber was used to observe the chemotactic responses of individual cells. This method permits monitoring of both the development of the concentration gradient of a tested substance and cell locomotion. We investigated the chemotactic responses of Amoeba proteus and observed that the amoebae moved in positively and negatively developing [H+] gradients towards the solution of lower pH in a pH range 5.75-7.75. The chemotactic response of amoebae to [H+] gradients required the presence of extracellular calcium ions. It was blocked and random locomotion was restored by the replacement of calcium with magnesium in the cell medium. Time-lapse video recording and data processing were accomplished with computer-assisted methods. This made it possible to compare selected methods of data presentation and analysis for cells locomoting in isotropic and anisotropic conditions. The cell trajectories were determined and displayed in circular diagrams, lengths of cell tracks and final cell displacements were estimated and a few parameters characterizing cell locomotion were computed.

Amoeba↗
PubMed2000

Immediate and long-term galvanotactic responses of Amoeba proteus to dc electric fields.

The long-term and immediate galvanotactic responses of Amoeba proteus to the direct current electric fields (dcEFs) were studied with the methods of computer-aided image analysis. It was found that in contrast to earlier reports, amoebae continued locomotion towards cathode (the negative pole) for hours and the increase in the field strength in the range 300-600 mV/mm caused the straightening of cell trajectories accompanied by the decreased frequency of the lateral pseudopods formation and lesser change in the speed of cell movement. In the cell regions pointing to the anode, the formation of new pseudopodia was prevented and the higher cEFs strength the more extended were the regions in which formation of new pseudopods was inhibited. Replacement of calcium with magnesium in the extracellular medium reduced the galvanotactic cell responses. Research on the localisation and kinetics of the primary cell responses to the dcEF or to change in its direction revealed that the primary cell responses occurred at the anode oriented cell regions. The cell response to the field reversal appeared to be localised and to take place in less than 1 sec. First the retraction and withdrawal of the anode-directed pseudopodium was observed whereas the uroid (cell tail) moved for 10-40 sec in the original direction before it begun to react to the field reversal. The exposure of amoebae to the dcEFs sensitised them to the reversion in the field direction and induced an acceleration of cell responses. The results presented are difficult to reconcile with the attempt to explain the cell galvanotaxis as a consequence of the membrane protein lateral electrophoresis or electroosmosis. It is suggested that the lateral electrophoresis of ions and the modification of ionic conditions at the vicinity of ion channels may be involved in the induction of fast responses of cells to external dcEFs.

Amoeba↗
PubMed2005

Characterization of Amoeba proteus myosin VI immunoanalog.

Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.

Actins↗
PubMed1984

ATP and the autonomy of the contractile vacuole in Amoeba proteus.

Contractile vacuole function in amoebae treated with immobilizing (5 mM) and nonimmobilizing (0.125 mM) concentrations of ATP has been studied. In ATP-immobilized amoebae, most vacuolar parameters are accelerated, especially the rate of output which passes from 30 to 70 micron3/sec. This favors the concept of an autonomous vacuole, fully functional in the absence of any bulk contribution to it from remote points of the cell. A lower concentration of ATP (0.125 mM), which does not inhibit movement, causes a still greater acceleration of vacuolar function. Work is in progress to elucidate the site and mode of action of exogenous ATP on Amoeba.

Adenosine Triphosphate↗
PubMed1993

Is actin involved in the nuclear division in Amoeba proteus?

During semi-open mitosis of Amoeba proteus the nuclear envelope is not dispersed and nucleus divides by fission. The presence of actin layer close to nuclear envelope was demonstrated in interphase and telophase nuclei of that amoeba stained with rhodamine labelled phalloidin. In telophase, an accumulation of actin arises in the space between the future daughter nuclei. It appears to be comparable with the contractile ring of dividing cells. This suggests that actin associated with the nuclear envelope of Amoeba proteus may be involved in final separation of the daughter nuclei, forming a constriction ring at the middle of dividing nucleus.

Actins↗