Responses of unicellular predators to cope with the phototoxicity of photosynthetic prey.

Feeding on unicellular photosynthetic organisms by unicellular eukaryotes is the base of the aquatic food chain and evolutionarily led to the establishment of photosynthetic endosymbionts/organelles. Photosynthesis generates reactive oxygen species and damages cells; thus, photosynthetic organisms possess several mechanisms to cope with the stress. Here, we demonstrate that photosynthetic prey also exposes unicellular amoebozoan and excavates predators to photosynthetic oxidative stress. Upon illumination, there is a commonality in transcriptomic changes among evolutionarily distant organisms feeding on photosynthetic prey. One of the genes commonly upregulated is a horizontally transferred homolog of algal and plant genes for chlorophyll degradation/detoxification. In addition, the predators reduce their phagocytic uptake while accelerating digestion of photosynthetic prey upon illumination, reducing the number of photosynthetic cells inside the predator cells, as this also occurs in facultative endosymbiotic associations upon certain stresses. Thus, some mechanisms in predators observed here probably have been necessary for evolution of endosymbiotic associations.

Lipids Are the Preferred Substrate of the Protist Naegleria gruberi, Relative of a Human Brain Pathogen.

Naegleria gruberi is a free-living non-pathogenic amoeboflagellate and relative of Naegleria fowleri, a deadly pathogen causing primary amoebic meningoencephalitis (PAM). A genomic analysis of N. gruberi exists, but physiological evidence for its core energy metabolism or in vivo growth substrates is lacking. Here, we show that N. gruberi trophozoites need oxygen for normal functioning and growth and that they shun both glucose and amino acids as growth substrates. Trophozoite growth depends mainly upon lipid oxidation via a mitochondrial branched respiratory chain, both ends of which require oxygen as final electron acceptor. Growing N. gruberi trophozoites thus have a strictly aerobic energy metabolism with a marked substrate preference for the oxidation of fatty acids. Analyses of N. fowleri genome data and comparison with those of N. gruberi indicate that N. fowleri has the same type of metabolism. Specialization to oxygen-dependent lipid breakdown represents an additional metabolic strategy in protists.

Amphotericin B induces apoptosis-like programmed cell death in Naegleria fowleri and Naegleria gruberi.

Naegleria fowleri and Naegleria gruberi belong to the free-living amoebae group. It is widely known that the non-pathogenic species N. gruberi is usually employed as a model to describe molecular pathways in this genus, mainly because its genome has been recently described. However, N. fowleri is an aetiological agent of primary amoebic meningoencephalitis, an acute and fatal disease. Currently, the most widely used drug for its treatment is amphotericin B (AmB). It was previously reported that AmB has an amoebicidal effect in both N. fowleri and N. gruberi trophozoites by inducing morphological changes that resemble programmed cell death (PCD). PCD is a mechanism that activates morphological, biochemical and genetic changes. However, PCD has not yet been characterized in the genus Naegleria. The aim of the present work was to evaluate the typical markers to describe PCD in both amoebae. These results showed that treated trophozoites displayed several parameters of apoptosis-like PCD in both species. We observed ultrastructural changes, an increase in reactive oxygen species, phosphatidylserine externalization and a decrease in intracellular potassium, while DNA degradation was evaluated using the TUNEL assay and agarose gels, and all of these parameters are related to PCD. Finally, we analysed the expression of apoptosis-related genes, such as sir2 and atg8, in N. gruberi. Taken together, our results showed that AmB induces the morphological, biochemical and genetic changes of apoptosis-like PCD in the genus Naegleria.

Corifungin, a new drug lead against Naegleria, identified from a high-throughput screen.

Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

The prevalence, isolation and morphotyping of potentially pathogenic free-living amoebae from tap water and environmental water sources in Sivas.

OBJECTIVE: To our knowledge, there is no study dealing with the prevalence of free-living amoebas (FLA) in water sources in Turkey, previous studies were mostly case presentations. The aim of the present study was to investigate the prevalence of FLA from tap water and natural water sources in different parts of the city. METHODS: In the study, 250 samples were collected from the city centre, districts and villages. Two litres of water was collected from each source and filtered through a vacuum filtration system. The filter papers were washed in "Page's Amoeba Saline (PAS)" solution and incubated overnight. Filter papers were removed from the tubes and centrifuged; the final pellet was inoculated on non-nutrient agar (NNA) plates. The growth rate of FLA was checked after three days of inoculation and the flagellation test was performed to determine the presence of Naegleria spp. Heat tolerance of isolated strains was checked at 37, 42 and 52°C for the presence of pathogenic Acanthamoeba species. The cyst and trophozoite morphology of amoebas were examined under a light microscope and the genera was identified according to morphotyping keys. RESULTS: FLA were found in 75 (30.0%) of examined water samples. Eleven (4.4%) were identified as Acanthamoeba spp., 25 (10.0%) as Naegleria spp. and 39 (15.6%) as Hartmannella spp. after microscopic examination. CONCLUSION: Our study revealed that FLA are common inhabitants of household water as they are in the environment, so their own potential risks as well as transferring bacteria as other pathogens is important for human health.

Morphological characteristics of developmental stages of Acanthamoeba and Naegleria species before and after staining by various techniques.

Seven stains were studied to determine the best color and contrast for staining the developmental stages of free living pathogenic Acanthamoeba and Naegleria species. The acid-fast bacilli stain (AFB) produced a blue color without contrast; trichrome-eosin and modified Field's showed various color contrasts; Giemsa, iron-hematoxylin, modified AFB and Gram produced only one color which distinguished the nucleus, nucleolus, cytoplasm, food- and water-vacuoles. The motile organs (acanthopodia, pseudopodia, lobopodia and flagella) were also clearly differentiated but produced a similar color as the cytoplasm. These motile organelles were first induced by incubating at 37 degrees C for at least 15 minutes and then fixing with methanol in order to preserve the protruding morphology prior to staining. The trichrome-eosin and iron-hematoxylin stains showed good color contrast for detecting all three stages, the trophozoite, cyst and flagellate; Giemsa and Gram stained the trophozoite and flagellate stages; the modified Field's and modified AFB stains stained only the trophozoite stage. Depending on the purpose, all these stains (except the AFB stain) can be used to identify the developmental stages of Acanthamoeba and Naegleria for clinical, epidemiological or public health use.

Detection of free living amoebae, Acanthamoeba and Naegleria, in swimming pools, Malaysia.

This study reports the detection of Acanthamoeba and Naegleria species in 14 swimming pools around Petaling Jaya and Kuala Lumpur, Malaysia. Sampling was carried out at 4 sites (the platforms (P), wall (W), 1 meter from the wall (1) and middle (2)) of each swimming pool. These free living amoebae (FLA) were detected under light and inverted microscopes after being cultured on the surface of non-nutrient agar lawned with Escherichia coli. Acanthamoeba species were detected in higher number of culture plates from all sampling sites of all the swimming pools. While Naegleria, were detected in fewer culture plates at 3 sampling sites (absent at site P) of 8 swimming pools. This suggested that the thick double-walled cysts of Acanthamoeba were more resistant, thus remaining viable in the dry-hot areas of the platforms and in chlorinated water of the swimming pools whereas Naegleria cysts, that are fragile and susceptible to desiccation, preferred watery or moist areas for growth and proliferation. The prevalence of both FLA was highest at site W (76.2%), followed by site 1 (64.7%), lowest at site 2 (19.4%), and could be detected at all 3 sampling levels (top, middle and bottom) of these 3 sites. The surface of site W might act as a bio-film that accumulated all kinds of microbes providing sufficient requirement for the FLA to develop and undergo many rounds of life cycles as well as moving from top to bottom in order to graze food. Other factors such as human activities, the circulating system which was fixed at all swimming pools, blowing wind which might carry the cysts from surroundings and the swimming flagellate stage of Naegleria could also contribute to the distribution of the FLA at these sampling sites. Both FLA showed highest growth (80.4%) at room temperature (25-28 ºC) and lesser (70.0%) at 37 ºC which might be due to the overgrowth of other microbes (E. coli, fungi, algae, etc). While at 44 ºC, only Acanthamoeba species could survive thus showing that our swimming pools are free from potentially pathogenic Naegleria species. However, further study is needed in order to confirm the virulence levels of these amoebae isolates.

Ultrastructural study of the encystation and excystation processes in Naegleria sp.

An important aspect of the biology of Naegleria sp. is the differentiation processes that occur during encystation and excystation. We studied these using both fluorescence and transmission electron microscopy techniques. In the initial stages of encystation, the cisternae of the endoplasmic reticulum became densely filled with a fibrillar material. Vesicles with a similar content that appeared to be derived from the cisternae were also observed in close contact with the plasma membrane. As encystation progressed, the fibrillar material became localized on the surface of the amoeba. An irregular compaction was observed in some areas of the cyst wall, which contained thin extensions of the cyst wall fibrillar material. Completely formed cysts had two to three ostioles, each sealed by an operculum. The operculum contained two areas in which a differential compaction of the fibrillar structure was observed. When excystation was induced, small dense granules (DGs), which were in close contact with fibrillar material were observed in the cyst cytoplasm and in the peritrophic space. During excystation, the more compact component of the operculum moves to enable the pseudopod of the emerging trophozoite to penetrate the ostiole. Vacuoles containing a fibrillar material, probably derived from the cyst wall, were observed in the cytoplasm of the pseudopodia. Our results provide a platform for further studies using biochemical markers to investigate the origin of the cyst wall as well as the role of DGs during excystation in Naegleria.

Differential Salmonella survival against communities of intestinal amoebae.

Predation from intestinal amoebae may provide selective pressure for the maintenance of high genetic diversity at the Salmonella enterica rfb locus, whereby serovars better escape predators in particular environments depending on the O-antigens they express. Here, the hypothesis that amoebae from a particular intestinal environment collectively prefer one serovar over another is tested. Collections of Acanthamoeba, Tetramitus, Naegleria and Hartmannella were isolated from the intestinal tracts of several vertebrate hosts, including bullfrog tadpoles, goldfish, turtles and bearded dragons, and their feeding preferences were determined. Congeneric amoebae from the same environment had significantly similar feeding preferences. Strikingly, even unrelated amoebae - such as Naegleria and Tetramitus from goldfish - also had significantly similar feeding preferences. Yet amoebae isolated from different environments showed no similarity in prey choice. Thus, feeding preferences of amoebae appear to reflect their environment, not their taxonomic relationships. A mechanism mediating this phenotypic convergence is discussed.

De novo formation of basal bodies in Naegleria gruberi: regulation by phosphorylation.

The de novo formation of basal bodies in Naegleria gruberi was preceded by the transient formation of a microtubule (MT)-nucleating complex containing gamma-tubulin, pericentrin, and myosin II complex (GPM complex). The MT-nucleating activity of GPM complexes was maximal just before the formation of visible basal bodies and then rapidly decreased. The regulation of MT-nucleating activity of GPM complexes was accomplished by a transient phosphorylation of the complex. Inhibition of dephosphorylation after the formation of basal bodies resulted in the formation of multiple flagella. 2D-gel electrophoresis and Western blotting showed a parallel relationship between the MT-nucleating activity of GPM complexes and the presence of hyperphosphorylated gamma-tubulin in the complexes. These data suggest that the nucleation of MTs by GPM complexes precedes the de novo formation of basal bodies and that the regulation of MT-nucleating activity of GPM complexes is essential to the regulation of basal body number.

The isolation of Naegleria italica from Peru indicates that this potentially pathogenic species occurs worldwide.

The amoeboflagellate genus Naegleria includes a few species that are virulent in experimental animals. One of these species, Naegleria italica, has been isolated from the environment only in Italy and Australia. I report here the isolation of a strain of N. italica from a water sample collected in Peru. This broadens the occurrence of this species to encompass three different continents. This new N. italica isolate from Peru has the same ITS1, 5.8S rDNA and ITS2 sequence as that of the type strain from Italy and the isolate from Australia. From the same water body in Peru a Naegleria strain was isolated that differs from N. italica by only one additional base pair in the ITS2 sequence. The maximum growth temperature tolerated by this particular isolate is 40 degrees C, which is different from that of N. italica, which is 42 degrees C.

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

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

Transient concentration of a gamma-tubulin-related protein with a pericentrin-related protein in the formation of basal bodies and flagella during the differentiation of Naegleria gruberi.

The distribution of two proteins in Naegleria gruberi, N-gammaTRP (Naegleria gamma-tubulin-related protein) and N-PRP (Naegleria pericentrin-related protein), was examined during the de novo formation of basal bodies and flagella that occurs during the differentiation of N. gruberi. After the initiation of differentiation, N-gammaTRP and N-PRP began to concentrate at the same site within cells. The percentage of cells with a concentrated region of N-gammaTRP and N-PRP was maximal (68%) at 40 min when the synthesis of tubulin had just started but no assembled microtubules were visible. When concentrated tubulin became visible (60 min), the region of concentrated N-gammaTRP and N-PRP was co-localized with the tubulin spot and then flagella began to elongate from the region of concentrated tubulin. When cells had elongated flagella, the concentrated N-gammaTRP and N-PRP were translocated to the opposite end of the flagellated cells and disappeared. The transient concentration of N-gammaTRP coincided with the transient formation of an F-actin spot at which N-gammaTRP and alpha-tubulin mRNA were co-localized. The concentration of N-gammaTRP and formation of the F-actin spot occurred without the formation of microtubules but were inhibited by cytochalasin D. These observations suggest that the regional concentration of N-gammaTRP and N-PRP is mediated by actin filaments and might provide a site of microtubule nucleation for the assembly of newly synthesized tubulins into basal bodies and flagella.

Cultivation of pathogenic and opportunistic free-living amebas.

Free-living amebas are widely distributed in soil and water, particularly members of the genera Acanthamoeba and NAEGLERIA: Since the early 1960s, they have been recognized as opportunistic human pathogens, capable of causing infections of the central nervous system (CNS) in both immunocompetent and immunocompromised hosts. Naegleria is the causal agent of a fulminant CNS condition, primary amebic meningoencephalitis; Acanthamoeba is responsible for a more chronic and insidious infection of the CNS termed granulomatous amebic encephalitis, as well as amebic keratitis. Balamuthia sp. has been recognized in the past decade as another ameba implicated in CNS infections. Cultivation of these organisms in vitro provides the basis for a better understanding of the biology of these amebas, as well as an important means of isolating and identifying them from clinical samples. Naegleria and Acanthamoeba can be cultured axenically in cell-free media or on tissue culture cells as feeder layers and in cultures with bacteria as a food source. Balamuthia, which has yet to be isolated from the environment, will not grow on bacteria. Instead, it requires tissue culture cells as feeder layers or an enriched cell-free medium. The recent identification of another ameba, Sappinia diploidea, suggests that other free-living forms may also be involved as causal agents of human infections.

Distribution of pathogenic Naegleria spp in Thailand.

Research concerning the distribution, isolation, viability, ultrastructure, morphology and immunogenicity of Naegleria fowleri has been increasing in Thailand during 1988-2000. The distribution of the organism was carried out from 1985 to 1987 in Si Sa Ket and Ubon Rachathani Provinces, after the first fatal case was reported in Si Sa Ket. Since then in a 1998 survey of N. fowleri in stagnant water around industrial areas was carried out in Pathum Thani, Samut Prakan and Lopburi provinces. The results showed that 10% of pathogenic Naegleria belonged to species fowleri as characterized by morphology and the occurrence of pathogenesis in mice after nasal inoculation. In the same year, Nacapunchai et al (1999) determined the prevalence of amebae in aquatic habitat of human environments in five parts of Thailand during the summer. Fourteen percent of free living Naegleria spp were found in both soil and water resources. Recent studies of the ultrastructure, factors affecting the viability and SDS-PAGE electrophoretic patterns of 3 Thai strains of pathogenic Naegleria spp indicated their similarities in morphological characteristics of pathogenic reference control, Naegleria fowleri CDC VO 3081. Additional study using a genetic approach to species criteria using allozyme electrophoresis had been conducted.

The effects of diffusates from the spores of Aspergillus fumigatus and A. terreus on human neutrophils, Naegleria gruberi and Acanthamoeba castellanii.

Diffusates from dormant spores of Aspergillus fumigatus are inhibitory to certain functions of immune phagocytic cells and soil protozoa. An assay of human neutrophil phagocytosis and an in vitro method of measuring inhibition of the growth of free living amoebae are described. The anti-phagocytic and anti-amoebal effects of diffusates from clinical and environmental isolates of A. fumigatus and of A. terreus were measured using these methods. Spore diffusates from all isolates of A. fumigatus and A. terreus tested significantly inhibited neutrophil phagocytosis. Spore diffusates from A. fumigatus, but not A. terreus, significantly inhibited the growth of Naegleria gruberi. Spore diffusate from A. fumigatus did not inhibit the growth of Acanthamoeba castellanii. The relevance of these findings to the pathogenicity of A. fumigatus and A. terreus is discussed.

Stable intermediates and holdpoints in the rapid differentiation of Naegleria.

The rapidity of the optional 90-min differentiation of Naegleria gruberi from amoebae to flagellates suggests the possibility of a free-running cascade of events from initiating stimulus through gene expression to organelle assembly and cell morphogenesis. Instead our experiments reveal two points early in the differentiation at which the strength of the inducing stimulus is reevaluated by the cells. Two new physical start signals for differentiation, temperature downshift (DeltaT) and mechanical agitation, are shown to regulate differentiation synergistically with each other and with previously defined signals. A DeltaT of -10 degrees C induces complete differentiation directly in the growth environment, whereas smaller DeltaTs initiate differentiation and allow it to progress for a short time, after which the cells "hold" for up to 4 h, awaiting a stimulus to continue differentiation. Our work defines two "holdpoints," optional points in development where progress can stop, awaiting a suitable signal, while cells retain whatever intermediates represent progress. We propose that such holdpoints, which can be detected in this system because of the temporal reproducibility of the differentiation, are likely to be found in other differentiating cells.

Dependence of growth, metabolic expression, and pathogenicity of Naegleria fowleri on exogenous porphyrins.

The pathogenicity of the free-living amoeba Naegleria fowleri is modulated by the composition of the medium used for cultivation. The constituents that determine the level of pathogenicity of N. fowleri, however, have not been definitively established. The present study examined the effects of selected porphyrins on N. fowleri amoebae. The iron-containing porphyrins, hemin or hematin, or the iron-free porphyrin, protoporphyrin IX, were effective in supporting growth of N. fowleri in Cline medium lacking serum. Iron-binding proteins, including hemoglobin, could not satisfy the growth requirement of the amoebae for exogenous porphyrin. Expression of biological functions including azocaseinase activity, agglutination, mobility, complement susceptibility, and virulence were altered by the composition of the growth medium. Amoebae grown in Cline medium supplemented with either hemin or protoporphyrin IX displayed greater mobility and were more resistant to lysis by complement than those grown in Nelson medium. Similarly, amoebae grown in Cline medium supplemented with either hemin or protoporphyrin IX were more pathogenic for B6C3F1 mice than those grown in Nelson medium. The addition of protoporphyrin IX to Nelson medium resulted in a modest increase in mobility, resistance to complement lysis and virulence when compared to N. fowleri amoebae grown in Nelson medium without added porphyrin.

Viability of pathogenic Acanthamoeba and Naegleria and virulence of N. fowleri during long-term cryopreservation.

This is a followup report on the viability of pathogenic Acanthamoeba castellanii, Naegleria australiensis, and N. fowleri during 5 years of cryopreservation and the virulence of N. fowleri during 30 months of cryostorage, all at -70 degrees C. The greatest decrease in viability occurred during the first year of freezing and was 10-fold greater than the average yearly decrease during years 2-5. At 5 years of cryostorage viability was 33% for A. castellanii, 38% for N. fowleri and 51% for N. australiensis. Virulence of N. fowleri did not decrease during 30 months of freezing and what appeared to be an increase in virulence during cryopreservation may be the result of reduced viability of the less virulent amebae in a culture.

Independent mechanisms are utilized for the coordinate and transient accumulation of two differentiation-specific mRNAs during differentiation of Naegleria gruberi amoebae into flagellates.

During the differentiation of Naegleria gruberi amoebae into flagellates, four differentiation-specific (DS) mRNAs are transiently and coordinately accumulated. Three of the four DS mRNAs, Class II, III, and IV, encode alpha-tubulin, beta-tubulin, and flagellar calmodulin, respectively. The protein product of the Class I mRNA has not been identified. We examined the effects of inhibition of protein synthesis on transcription and accumulation of beta-tubulin mRNA and Class I mRNA to understand the mechanism of coordinate regulation. Inhibition of protein synthesis at the beginning of differentiation completely blocked transcription of the beta-tubulin gene. Addition of cycloheximide at 30 or 40 min after initiation of differentiation inactivated transcription of the beta-tubulin gene in less than 10 min as judged by nuclear run-on experiments. However, once differentiation had proceeded for more than 50 min, inhibition of protein synthesis did not inactivate transcription of beta-tubulin mRNA was more active in cycloheximide-treated cells than in control cells. Cycloheximide treatment at the initiation of the differentiation also blocked transcription of the Class I gene. However, addition of the drug after 30 min had no significant effect on the transcription of the Class I gene. Cycloheximide treatment also increased the half-lives of beta-tubulin and Class I mRNA drastically. These data suggest that: (1) the transient accumulation of the two DS mRNAs during differentiation are regulated by changing both the rate of transcription and the stability of the mRNAs; (2) protein synthesis is required for the transcriptional and post-transcriptional regulations; (3) the transcriptional regulation mechanisms of the beta-tubulin gene and that of the Class I gene are distinct; and (4) the transcription of the beta-tubulin gene is regulated by different mechanisms during differentiation.