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.     

Separation of soluble amoebicidal and tumoricidal activity of activated macrophages.

Macrophage-conditioned medium (M phi CM) prepared from mouse peritoneal macrophages activated in vivo with bacillus Calmette-Guérin (BCG) or Propionibacterium acnes and triggered with lipopolysaccharide in vitro contained tumoricidal and amoebicidal activity. The murine fibroblast cell line L929 was used as the indicator of tumoricidal activity and Naegleria fowleri amoeba was used to detect amoebicidal activity in M phi CM. The protease inhibitor, soybean trypsin inhibitor, decreased tumoricidal activity but had little effect on amoebicidal activity in M phi CM. Anti-TNF alpha antiserum inhibited tumoricidal activity in M phi CM. The antiserum reduced amoebicidal activity in BCG-activated M phi CM but had no effect on amoebicidal activity in P. acnes-activated M phi CM. Recombinant TNF alpha, rIL-1 alpha, or rIL-1 beta independently did not affect cytolysis of amoebae. Also, rTNF alpha had no effect on the growth of amoebae. Preparative flat-bed electrofocusing of BCG-activated M phi CM yielded fractions that exhibited different amoebicidal and tumoricidal activity profiles. Three domains of activity were analyzed (acidic, neutral, and basic). Anti-TNF alpha antiserum eliminated tumoricidal activity, but not amoebicidal activity, in fractions from the acidic domain. A combination of anti-TNF alpha and anti-IL-1 alpha antisera failed to eliminate amoebicidal activity in fractions from the basic domain. These results indicate that different factors are responsible for macrophage amoebicidal and tumoricidal activity. The amoebicidal factors in M phi CM affected cytolysis of several species of amoebae.     

Activated macrophages demonstrate direct cytotoxicity, antibody-dependent cellular cytotoxicity, and enhanced binding of Naegleria fowleri amoebae

Macrophages activated in vivo by injection of Corynebacterium parvum or bacillus Calmette-Guérin caused direct cytolysis of the pathogenic free-living amoeba, Naegleria fowleri, in vitro. Amoebicidal activity was time and cell density-dependent but was not dependent on the presence of specific antibody. Antibody-dependent cellular cytotoxicity for amoebae was also expressed by activated macrophages. Resident and thioglycolate-elicited macrophages demonstrated low cytolytic activity under all conditions tested. From scanning electron microscopy it appears that the degree of target cell binding is directly related to the degree of cytolysis expressed by the macrophage populations. Cell-cell contact was required for cytolysis of amoebae by activated macrophages since cytolysis did not occur when contact was blocked by a porous filter. For each macrophage population, the levels of amoebicidal activity and tumoricidal activity were comparable.     

Separation of Soluble Amoebicidal and Tumoricidal Activity of Activated Macrophages

ABSTRACT. Macrophage-conditioned medium (MøCM) prepared from mouse peritoneal macrophages activated in vivo with bacillus Calmette-Guérin (BCG) or Propionibacterium acnes and triggered with lipopolysaccharide in vitro contained tumoricidal and amoebicidal activity. The murine fibroblast cell line L929 was used as the indicator of tumoricidal activity and Naegleria fowleri amoeba was used to detect amoebicidal activity in MøCM. The protease inhibitor, soybean trypsin inhibitor, decreased tumoricidal activity but had little effect on amoebicidal activity in MøCM. Anti-TNF _α antiserum inhibited tumoricidal activity in MøCM. The antiserum reduced amoebicidal activity in BCG-activated MøCM but had no effect on amoebicidal activity in P. acnes -activated MøCM. Recombinant TNF _α , rIL-1 _α , or rIL-1 _β independently did not affect cytolysis of amoebae. Also, rTNF _α had no effect on the growth of amoebae. Preparative flat-bed electrofocusing of BCG-activated MøCM yielded fractions that exhibited different amoebicidal and tumoricidal activity profiles. Three domains of activity were analyzed (acidic, neutral, and basic). Anti-TNF _α antiserum eliminated tumoricidal activity, but not amoebicidal activity, in fractions from the acidic domain. A combination of anti-TNF _α and anti-IL-1 _α antisera failed to eliminate amoebicidal activity in fractions from the basic domain. These results indicate that different factors are responsible for macrophage amoebicidal and tumoricidal activity. The amoebicidal factors in MøCM affected cytolysis of several species of amoebae.     

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. fowler 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.     

Evidence that a target-derived soluble factor is necessary for the selective lysis of SV40-transformed fibroblasts by activated mouse macrophages

Our laboratory is investigating the basis for the selective recognition of transformed cells by activated mouse macrophages. As targets we are using a panel of SV40-transformed, C3H.OL fibroblast cell lines (SV-COL) that display widely different levels of sensitivity to lysis, from highly sensitive to completely resistant. Our results show that adding conditioned medium from the macrophage-sensitive target SV-COL-E8 (CM(E8] to a cytolysis assay with the macrophage-resistant target SV-COL-F5f causes the macrophages to kill the resistant targets in a contact dependent fashion. We have termed this activity "macrophage cell lysis factor" (MCLF). MCLF activity was not detected in conditioned media from cells not killed by activated macrophages (i.e., 3T3-like cell lines or embryo fibroblasts) but was present in conditioned media from six other SV-COL cell lines at levels that were directly proportional to the sensitivity of those targets (r = 0.98). These data suggest that MCLF plays a key role in determining the lytic sensitivity of SV40-transformed fibroblasts. Finally, to ask whether the production of MCLF is sufficient to explain the selective recognition of SV40-transformed fibroblasts by activated macrophages we have tested whether CM(E8) will cause macrophages to kill normal cells. Our results show that in the presence of CM(E8) macrophages will kill immortalized, 3T3-like fibroblasts but will not kill normal embryo fibroblasts. These results suggest that the production of MCLF, or a similar activity, is necessary but not sufficient for macrophage cytolysis to occur and that a change in target cell phenotype that occurs during the process of immortalization is also required.     

Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A(2)

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A(2) (PLA(2)) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA(2)s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA(2) activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA(2), cPLA(2). However, unlike the cPLA(2) enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA(2) activity is sensitive to inhibitors that block the activities of cPLA(2)-alpha and the 80 kDa calcium-independent PLA(2), iPLA(2), that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [(3)H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA(2) that may play a role in the basal phospholipid metabolism of these cells.     

Modulation of Biological Functions of Naegleria fowleri Amoebae by Growth Medium

Two strains of Naegleria fowleri amoebae were studied when the amoebae were maintained in the same growth medium or in two different media. A weakly pathogenic strain of N. fowleri , LEE, and a highly pathogenic strain, LEEmpCl, were compared for growth properties, the presence or absence of surface structures termed food cups, cytopathogenicity, cellular locomotion, susceptibility to complement-mediated lysis and immunological relatedness by western immunoblot analysis when grown in Nelson medium or in Cline medium. The two different strains of N. fowleri , LEE and LEEmpCl, were more similar in protein profiles and functional activity when both strains were grown in the same nutritional medium. Differences in growth, proteins synthesized, cytopathogenicity, susceptibility to complement lysis and rate of locomotion were noted when the same strain was grown in different media. Naegleria fowleri grown in Cline medium demonstrated an increased rate of growth, an increase in its rate of locomotion, an increased resistance to complement lysis, and destroyed target nerve cells by contact-dependent lysis. In contrast, the same strain of amoeba grown in Nelson medium showed slower growth, destroyed target cells by trogocytosis, and was less resistant to complement-mediated lysis.     

Assessing the risk of primary amoebic meningoencephalitis from swimming in the presence of environmental Naegleria fowleri.

Free-living Naegleria fowleri amoebae cause primary amoebic meningoencephalitis (PAM). Because of the apparent conflict between their ubiquity and the rarity of cases observed, we sought to develop a model characterizing the risk of PAM after swimming as a function of the concentration of N. fowleri. The probability of death from PAM as a function of the number of amoebae inhaled is modeled according to results obtained from animals infected with amoeba strains. The calculation of the probability of inhaling one or more amoebae while swimming is based on a double hypothesis: that the distribution of amoebae in the water follows a Poisson distribution and that the mean quantity of water inhaled while swimming is 10 ml. The risk of PAM for a given concentration of amoebae is then obtained by summing the following products: the probability of inhaling n amoebae x the probability of PAM associated with inhaling these n amoebae. We chose the lognormal model to assess the risk of PAM because it yielded the best analysis of the studentized residuals. Nonetheless, the levels of risk thereby obtained cannot be applied to humans without correction, because they are substantially greater than those indicated by available epidemiologic data. The curve was thus adjusted by a factor calculated with the least-squares method. This provides the PAM risk in humans as a function of the N. fowleri concentration in the river. For example, the risk is 8.5 x 10(-8) at a concentration of 10 N. fowleri amoebae per liter.     

Effect of thermal additions on the density and distribution of thermophilic amoebae and pathogenic Naegleria fowleri in a newly created cooling lake

Pathogenic Naegleria fowleri is the causative agent of fatal human amoebic meningoencephalitis. The protozoan is ubiquitous in nature, and its presence is enhanced by thermal additions. In this investigation, water and sediments from a newly created cooling lake were quantitatively analyzed for the presence of thermophilic amoebae, thermophilic Naegleria spp., and the pathogen Naegleria fowleri. During periods of thermal additions, the concentrations of thermophilic amoebae and thermophilic Naegleria spp. increased as much as 5 orders of magnitude, and the concentration of the pathogen N. fowleri increased as much as 2 orders of magnitude. Concentrations of amoebae returned to prior thermal perturbation levels within 30 to 60 days after cessation of thermal additions. Increases in the thermophilic amoeba concentrations were noted in Savannah River oxbows downriver from the Savannah River plant discharge streams as compared with oxbows upriver from the discharges. Concentrations of thermophilic amoebae and thermophilic Naegleria spp. correlated significantly with temperature and conductivity. Air samples taken proximal to the lake during periods of thermal addition showed no evidence of thermophilic Naegleria spp. Isoenzyme patterns of the N. fowleri isolated from the cooling lake were identical to patterns of N. fowleri isolated from other sites in the United States and Belgium.     

Effect of thermal additions on the density and distribution of thermophilic amoebae and pathogenic Naegleria fowleri in a newly created cooling lake.

Pathogenic Naegleria fowleri is the causative agent of fatal human amoebic meningoencephalitis. The protozoan is ubiquitous in nature, and its presence is enhanced by thermal additions. In this investigation, water and sediments from a newly created cooling lake were quantitatively analyzed for the presence of thermophilic amoebae, thermophilic Naegleria spp., and the pathogen Naegleria fowleri. During periods of thermal additions, the concentrations of thermophilic amoebae and thermophilic Naegleria spp. increased as much as 5 orders of magnitude, and the concentration of the pathogen N. fowleri increased as much as 2 orders of magnitude. Concentrations of amoebae returned to prior thermal perturbation levels within 30 to 60 days after cessation of thermal additions. Increases in the thermophilic amoeba concentrations were noted in Savannah River oxbows downriver from the Savannah River plant discharge streams as compared with oxbows upriver from the discharges. Concentrations of thermophilic amoebae and thermophilic Naegleria spp. correlated significantly with temperature and conductivity. Air samples taken proximal to the lake during periods of thermal addition showed no evidence of thermophilic Naegleria spp. Isoenzyme patterns of the N. fowleri isolated from the cooling lake were identical to patterns of N. fowleri isolated from other sites in the United States and Belgium.     

Inverse correlation between natural antitumor antibodies and tumor susceptibility in individual xid-bearing mice

Natural antibodies (NAb), natural killer (NK) cells and activated macrophages have all been implicated in the rejection of threshold syngeneic tumor inocula. Previous analysis of tumor susceptibility in normal versus inbred and F1 mice bearing the B cell deficiency associated with the xid mutation of CBA/N mice demonstrated an inverse relationship between the tumorigenicity of the RI-28, a radiation-induced leukemia of the CBA/H strain, and the pooled anti-RI-28 serum NAb levels in mice with the same genetic origins. No relationship with tumor susceptibility was seen with NK cell or in vivo activated macrophage cytolysis. Flow-cytometric determination of antitumor serum NAb bled from individual male and female (CBA/N X CBA/J)F1 mice 1 week prior to the threshold tumor inoculation has revealed extensive heterogeneity within the NAb levels of each sex. A comparative analysis of tumor fate with NAb activity revealed that tumors appeared in only 26.3% of animals with a mean fluorescence channel binding above 60 channels in contrast with 77.3% of animals with lower NAb levels. These data extend to the level of individual hosts the support for an inverse relationship between host NAb activity and tumor susceptibility. In addition, subsequent analysis of serum antitumor NAb levels, splenic NK cytolysis and in vitro lymphokine-activated macrophage activity with all three mediators originating from the same individual F1 mice showed no consistent correlations between these natural resistance activities, arguing for the exclusion of deficiencies in NK cell or macrophage function as the basis for the differential tumor susceptibility in individual F1 mice.     

Modulation of Complement Resistance and Virulence of Naegleria fowleri Amoebae by Alterations in Growth Media

ABSTRACT. Highly-pathogenic, mouse-passaged Naegleria fowleri amoebae are complement resistant. The present study evaluates the effect of complement on N. fowleri and the virulence of the amoebae after animal passage and growth in two different axenic media. Pathogenic N. fowleri maintained in "enriched" Cline medium are virulent for mice and resistant to complement lysis. A rapid decline in resistance to complement and virulence for mice is observed when highly-pathogenic N. fowleri are grown in Nelson medium lacking hemin. N. fowleri maintained in Nelson medium can be rendered complement-resistant by shifting the amoebae to growth in Cline medium for 2 h prior to the addition of complement. Cycloheximide treatment of N. fowleri maintained in Nelson medium blocks the transition to a complement-resistant phenotype following a shift in growth medium. Proteins were radiolabeled with [³⁵S] during a shift from Nelson to Cline medium to identify specific polypeptides which may be associated with the functional activities related to virulence and resistance to complement.     

Cytopathogenicity of Naegleria fowleri for cultured rat neuroblastoma cells

The cytopathogenicity of Naegleria fowleri strain LEE (ATCC-30894) for cultured rat neuroblastoma cells (B-103) has been investigated. Both live N. fowleri amoebae and Naegleria lysates added to 51Cr-labeled B-103 cells caused release of radiolabel, which was dependent upon the ratio of amoebae to target cells or to the lysate concentration. Lysates of N. fowleri strains LEE, NF-66, NF-69, and HB-4 were equally injurious to B-103 target cells whereas lysates of strains 6088 and KUL were less cytotoxic. Highly pathogenic mouse-passaged strain LEE were less cytotoxic than axenically grown amoebae. Maximum cytotoxicity was observed in lysates from amoebae in late exponential or early stationary phase of growth. Cytopathogenicity of lysates was reduced after heating at 44 degrees C for 60 min or at 60 degrees C for 30 min. Cytotoxicity was stable during storage at 4 degrees C or at -20 degrees C for 26 h. Neither live amoebae nor lysates injured B-103 target cells at 4 degrees C. Live amoebae and lysates injured B-103 by a time, temperature, and concentration dependent process.     

Antagonistic Action of the Bacterium Bacillus Licheniformis M-4 Toward the Amoeba Naegleria Fowleri

ABSTRACT. Free-living amoebae belonging to the species Naegleria fowleri are known to be the etiological agents for a form of fulminant meningoencephalitis that is generally fatal (primary amoebic meningoencephalitis). In a broad bacterial screening from soil and water we have isolated three strains (M-4, D-13 and A-12) belonging to the species Bacillus licheniformis that have remarkable amoebicidal activity against Naegleria sp. and also against different Gram-positive and Gram-negative bacteria. Physical-chemical characteristics, partial purification and biological activities of a substance produced by the M-4 strain have been investigated. This substance (m-4) is stable at high temperature (up to 100°C) and extremes of pH (2.5-9.5) and also at -20°C for months. Its production is greatly influenced by oxygenation of the cultures and is probably related to the sporulation process of the bacterium. Scanning electron microscope observations reveal that amoebae are lysed after a few minutes contact with m-4.     

Peritonitis-induced antitumor activity of peritoneal macrophages from uremic patients

The macrophages belong to the effector cells of both nonspecific and specific immune response. These cells generally express little cytotoxicity unless activated. The present work was intended to determine if peritoneal macrophages collected from patients on Continuous Ambulatory Peritoneal Dialysis (CAPD) during episodes of peritonitis were active against human tumor cell lines without further in vitro stimulation. We also compared macrophage antitumor potential with effectiveness of drugs used in cancer therapy (taxol and suramin). Conditioned medium (CM) of macrophages collected during inflammation-free periods did not exhibit cytostatic and cytotoxic activity against both tumor (A549 and HTB44) and non-transformed (BEAS-2B and CRL2190) cells. Exposure of tumor cells to CM of macrophages harvested during peritonitis resulted in significant suppression of proliferation, impairment of viability and induction of apoptosis, in contrast to non-transformed cells, which remained unaffected. The efficacy of CM of inflammatory macrophages as an antitumor agent appeared to be comparable to cytostatic and cytotoxic potency of taxol and suramin or, in the case of HTB44 cells, even higher. The results obtained suggest that activated human macrophages might represent a useful tool for cancer immunotherapy.     

Keratinocyte-derived granulocyte/macrophage colony-stimulating factor induces DNA synthesis by peritoneal macrophages

Keratinocytes have been demonstrated to produce a number of cytokines, including growth factors such as the CSF IL-3. Circulating blood monocytes and some elicited macrophages retain a significant proliferative potential in response to colony-stimulating activity. Because a macrophage response is prominent in a variety of cutaneous immune reactions, we have studied the ability of conditioned media (CM) from a transformed murine keratinocyte cell line (PAM 212) and from normal murine keratinocytes to induce growth of peritoneal macrophages. CM from both normal and transformed keratinocyte cultures induces [3H]thymidine incorporation by thioglycollate-elicited, but not resident, peritoneal macrophages. IEF of PAM 212 CM reveals peaks of activity at pI 4.8 and less than or equal to 4.2. Analysis of CM by reversed-phase HPLC demonstrates active fractions that elute at 46 to 48% and 53 to 55% acetonitrile. The Mr of the 46 to 48% acetonitrile factor is 25 to 30 kDa by gel filtration HPLC. Polyclonal anti-granulocyte/macrophage (GM) CSF antibody blocks the induction of macrophage [3H]thymidine incorporation by factors with pI 4.8 and eluting at 46 to 48% acetonitrile but does not reduce the activity of crude CM or the factor eluting at 53 to 55% acetonitrile. Based on both physiochemical criteria and antibody neutralization, keratinocytes produce GM-CSF. Keratinocyte-derived factors, including GM-CSF, may play an important role in regulating cutaneous macrophage responses.     

Evaluation of the Indirect Fluorescent-Antibody Technique for Identification of Naegleria Species

The indirect fluorescent-antibody technique was used to assess a rapid method for identification of amoebae belonging to the genus Naegleria. Thirty-eight Naegleria and eight other limax amoeba strains were examined by using one N. gruberi and two N. fowleri antisera. All pathogenic Naegleriae, most of which originated from fatal cases of primary amoebic meningo-encephalitis, were identified as belonging to the fowleri species. Most of the N. gruberi strains showed irregular fluorescence. Other limax amoebae, such as Vahlkampfia, Acanthamoeba, Hartmannella, and Schizopyrenus sp. gave negative responses with the prepared antisera. The indirect fluorescent-antibody technique allows the identification of N. fowleri in a mixed culture of both N. fowleri and N. gruberi strains. Twenty-two Naegleria isolated from a suspected stream, other surface waters, and muddy soil could be excluded from the fowleri species with the indirect fluorescent-antibody technique. The results obtained demonstrate that this immunological technique is a valid method for the rapid identification of N. fowleri trophozoites.     

Membrane carbohydrate characterization of Acanthamoeba astronyxis, A. castellanii and Naegleria fowleri by fluorescein-conjugated lectins

A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.     

Biological factors affecting enflagellation of Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeboflagellate that can be evoked to transform from amoebae to flagellates by subculture to nonnutrient buffer. More than half of the amoebae of strains KUL, nN68, and Lovell became enflagellated 300 min after subculture to amoeba-saline, whereas no amoebae of strains NF66, NF69, and HB4 did. N. fowleri nN68 enflagellated best when grown at 32 or 37 degrees C and subcultured to amoeba-saline at 37 or 42 degrees C. Amoebae from the stationary phase of growth enflagellated more readily than did actively growing amoebae. Incubation in expended culture medium from stationary-phase cultures enhanced the capability of growing amoebae to enflagellate after subculture to amoebasaline. Enflagellation was more extensive when the population density in amoebasaline did not exceed 2 x 10(5) amoebae per ml. Cycloheximide at 1 microgram/ml and actinomycin D at 25 micrograms/ml inhibited growth of N. fowleri nN68. Cycloheximide at 0.5 microgram/ml and actinomycin D at 25 micrograms/ml completely prevented enflagellation when added at time zero. Cycloheximide at 0.5 microgram/ml, added 120 to 300 min after initiation of enflagellation, prevented further differentiation and caused existing flagellates to revert to amoeboid cells. Similarly, actinomycin D at 25 micrograms/ml, added 90 to 300 min after initiation of enflagellation, retarded differentiation and caused flagellates to revert. Radiolabeled precursors were incorporated into macromolecules during differentiation in nonnutrient buffer. Enflagellation of N. fowleri is a suitable model for studying regulation of a eucaryotic protist.