Naegleria genus pangenome reveals new structural and functional insights into the versatility of these free-living amoebae.

Introduction: Free-living amoebae of the Naegleria genus belong to the major protist clade Heterolobosea and are ubiquitously distributed in soil and freshwater habitats. Of the 47 Naegleria species described, N. fowleri is the only one being pathogenic to humans, causing a rare but fulminant primary amoebic meningoencephalitis. Some Naegleria genome sequences are publicly available, but the genetic basis for Naegleria diversity and ability to thrive in diverse environments (including human brain) remains unclear. Methods: Herein, we constructed a high-quality Naegleria genus pangenome to obtain a comprehensive catalog of genes encoded by these amoebae. For this, we first sequenced, assembled, and annotated six new Naegleria genomes. Results and Discussion: Genome architecture analyses revealed that Naegleria may use genome plasticity features such as ploidy/aneuploidy to modulate their behavior in different environments. When comparing 14 near-to-complete genome sequences, our results estimated the theoretical Naegleria pangenome as a closed genome, with 13,943 genes, including 3,563 core and 10,380 accessory genes. The functional annotations revealed that a large fraction of Naegleria genes show significant sequence similarity with those already described in other kingdoms, namely Animalia and Plantae. Comparative analyses highlighted a remarkable genomic heterogeneity, even for closely related strains and demonstrate that Naegleria harbors extensive genome variability, reflected in different metabolic repertoires. If Naegleria core genome was enriched in conserved genes essential for metabolic, regulatory and survival processes, the accessory genome revealed the presence of genes involved in stress response, macromolecule modifications, cell signaling and immune response. Commonly reported N. fowleri virulence-associated genes were present in both core and accessory genomes, suggesting that N. fowleri's ability to infect human brain could be related to its unique species-specific genes (mostly of unknown function) and/or to differential gene expression. The construction of Naegleria first pangenome allowed us to move away from a single reference genome (that does not necessarily represent each species as a whole) and to identify essential and dispensable genes in Naegleria evolution, diversity and biology, paving the way for further genomic and post-genomic studies.

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.

Heterogeneity in the genus Allovahlkampfia and the description of the new genus Parafumarolamoeba (Vahlkampfiidae; Heterolobosea).

Heterolobosean amoebae are common and diverse members of soil protist communities. In this study, we isolated seven strains of amoebae from soil samples taken in Tibet (at high altitude), Sardinia and the Netherlands, all resembling to belong to a similar heterolobosean morphospecies. However, sequences of the small subunit (SSU) rDNA and internal transcribed spacers, including the 5.8S rDNA, revealed a high heterogeneity in the genus Allovahlkampfia to which six of the isolates belong. Some unnamed strains, of which the sequences had been published before, are also included within the genus Allovahlkampfia. One Allovahlkampfia isolated in the Netherlands harbors a twin-ribozyme, containing a His-Cys box, similar to the one found in strain BA of Allovahlkampfia. The other SSU rDNA sequence grouped in phylogenetic analyses with sequences obtained in environmental sequencing studies as sister to the genus Fumarolamoeba. This phylogenetic placement was supported by analyses of the 5.8S rDNA leading us to describe it as a new genus Parafumarolamoeba.

Soil is the origin for the presence of Naegleria fowleri in the thermal recreational waters.

Naegleria fowleri is found in most geothermal baths of Guadeloupe and has been responsible for the death of a 9-year-old boy who swam in one of these baths in 2008. We wanted to determine the origin for the presence of this amoeba in the water. Water samples were taken at the origin of the geothermal sources and at the arrival in the baths. After filtration, cultures were made and the number of Naegleria present was determined using the most probable number method. Soil samples collected in the proximity of the baths were also tested for the presence of thermophilic amoebae. The species identification was obtained by PCR. During three consecutive months, no Naegleria could be found at the origin of any geothermal source tested. In contrast, N. fowleri was isolated at least once in all baths at the arrival of the water, except one. Thermophilic amoebae could be found in each soil sample, especially near the baths located at a lower altitude, but N. fowleri was only isolated near two baths, which were also the baths most often contaminated with this species. So it appears that the contamination of the water with N. fowleri occurs after emerging from the geothermal source when the water runs over the soil. Therefore, it should be possible to reduce the concentration of N. fowleri in the geothermal baths of Guadeloupe to for example less than 1 N. fowleri/10 L by installing a pipeline between the geothermal sources and the baths and by preventing flooding water from entering the baths after rainfall. By taking these measures, we were able to eliminate N. fowleri from a pool located inside a reeducation clinic.

What do we know by now about the genus Naegleria?

In this short overview of the genus Naegleria a brief historical sketch is given since the discovery of this amoeboflagellate in 1899 and the finding in 1970 that one species, Naegleria fowleri causes primary amoebic meningoencephalitis in man. Eight different types of this pathogen are known which have an uneven distribution over the world. Until now 47 different Naegleria spp. are described, of which two other species cause disease in experimental animals, and their geographical dispersal is indicated. The presence of group I introns in the SSU and in the LSU rDNA in the genus is discussed, as well as the possibility of sex or mating. It is also mentioned that the genome of N. fowleri should not be compared to that of Naegleria gruberi, to know why the former is pathogenic, but to the genome of its closest relative Naegleria lovaniensis.

Survey of Naegleria fowleri in geothermal recreational waters of Guadeloupe (French West Indies).

In 2008 a fatal case of primary amoebic meningoencephalitis, due to the amoeboflagellate Naegleria fowleri, occurred in Guadeloupe, French West Indies, after a child swam in a bath fed with geothermal water. In order to improve the knowledge on free-living amoebae in this tropical part of France, we investigated on a monthly basis, the presence of Naegleria spp. in the recreational baths, and stream waters which feed them. A total of 73 water samples, 48 sediments and 54 swabs samples were collected from 6 sampling points between June 2011 and July 2012. The water samples were filtered and the filters transferred to non-nutrient agar plates seeded with a heat-killed suspension of Escherichia coli while sediment and swab samples were placed directly on these plates. The plates were incubated at 44°C for the selective isolation of thermophilic Naegleria. To identify the Naegleria isolates the internal transcribed spacers, including the 5.8S rDNA, were amplified by polymerase chain reaction and the sequence of the PCR products was determined. Thermophilic amoebae were present at nearly all collection sites. The pathogenic N. fowleri was the most frequently encountered thermophilic species followed by N. lovaniensis. The concentration of N. fowleri was rather low in most water samples, ranging from 0 to 22 per liter. Sequencing revealed that all N. fowleri isolates belonged to a common Euro-American genotype, the same as detected in the human case in Guadeloupe. These investigations need to be continued in order to counsel the health authorities about prevention measures, because these recreational thermal baths are used daily by local people and tourists.

Characterization of Selenaion koniopes n. gen., n. sp., an amoeba that represents a new major lineage within heterolobosea, isolated from the Wieliczka salt mine.

A new heterolobosean amoeba, Selenaion koniopes n. gen., n. sp., was isolated from 73‰ saline water in the Wieliczka salt mine, Poland. The amoeba had eruptive pseudopodia, a prominent uroid, and a nucleus without central nucleolus. Cysts had multiple crater-like pore plugs. No flagellates were observed. Transmission electron microscopy revealed several typical heterolobosean features: flattened mitochondrial cristae, mitochondria associated with endoplasmic reticulum, and an absence of obvious Golgi dictyosomes. Two types of larger and smaller granules were sometimes abundant in the cytoplasm--these may be involved in cyst formation. Mature cysts had a fibrous endocyst that could be thick, plus an ectocyst that was covered with small granules. Pore plugs had a flattened dome shape, were bipartite, and penetrated only the endocyst. Phylogenies based on the 18S rRNA gene and the presence of 18S rRNA helix 17_1 strongly confirmed assignment to Heterolobosea. The organism was not closely related to any described genus, and instead formed the deepest branch within the Heterolobosea clade after Pharyngomonas, with support for this deep-branching position being moderate (i.e. maximum likelihood bootstrap support--67%; posterior probability--0.98). Cells grew at 15-150‰ salinity. Thus, S. koniopes is a halotolerant, probably moderately halophilic heterolobosean, with a potentially pivotal evolutionary position within this large eukaryote group.

Knowledge of morphology is still required when identifying new amoeba isolates by molecular techniques.

We have isolated several free-living amoeba strains from the environment in Ghana, which have internal transcribed spacers, including the 5.8S rDNA, sequences similar to sequences attributed to Vahlkampfiidae (Heterolobosea) in databases. However, morphological examination shows that the isolates belong to the Hartmannellidae (Amoebozoa). We provide evidence that the sequences in the databases are wrongly classified as belonging to a genus or species of the Vahlkampfiidae, but rather belong to strains of the genus Hartmannella.

Origin and evolution of the worldwide distributed pathogenic amoeboflagellate Naegleria fowleri.

Naegleria fowleri, a worldwide distributed pathogen, is the causative agent of primary amoebic meningoencephalitis. Because it is such a fulminant disease, most patients do not survive the infection. This pathogen is a free-living amoeboflagellate present in warm water. To date, it is well established that there are several types of N. fowleri, which can be distinguished based on the length of the internal transcribed spacer 1 and a one bp transition in the 5.8S rDNA. Seven of the eight known types have been detected in Europe. Three types are present in the USA, of which one is unique to this country. Only one of the eight types occurs in Oceania (Australia and New Zealand) and Japan. In mainland Asia (India, China and Thailand) the two most common types are found, which are also present in Europe and the USA. There is strong indication that the pathogenic N. fowleri evolved from the nonpathogenic Naegleria lovaniensis on the American continent. There is no evidence of virulence differences between the types of N. fowleri. Two other Naegleria spp. are pathogenic for mice, but human infections due to these two other Naegleria spp. are not known.

Naegleria gruberi metabolism.

The completion of the genome project for Naegleria gruberi provides a unique insight into the metabolic capacities of an organism, for which there is an almost complete lack of experimental data. The metabolism of Naegleria seems to be extremely versatile, as can be expected for a free-living amoeboflagellate, but although considered to be fully aerobic, its genome also predicts important anaerobic traits. Other predictions are that carbohydrates are oxidised to carbon dioxide and water when oxygen is not limiting and that in the absence of oxygen the end-products will be succinate, acetate and minor quantities of ethanol and D-lactate. The hybrid mitochondrion/hydrogenosome has both cytochromes and an [Fe] hydrogenase, but seems to lack pyruvate-ferredoxin oxidoreductase. Genomic information also provides the possibility to identify drugs with a possible mode of action in the fatal primary amoebic meningoencephalitis caused by the closely related opportunistic pathogen Naegleria fowleri.

Oramoeba fumarolia gen. nov., sp. nov., a new marine heterolobosean amoeboflagellate growing at 54 °C.

An amoeba strain was isolated from marine sediment taken from the beach near a fumarole in Italy. The trophozoites of this new marine species transforms into flagellates with variable numbers of flagella, from 2 to 10. The strain forms round to oval cysts. This thermophilic amoeboflagellate grows at temperatures up to 54°C. Molecular phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) places the amoeboflagellate in the Heterolobosea. The closest relatives are Stachyamoeba sp. ATCC50324, a strain isolated from an ocean sample, and Vrihiamoeba italica, a recent isolate from a rice field. Like some other heterolobosean species, this new isolate has a group I intron in the SSU rDNA. Because of the unique place in the molecular phylogenetic tree, and because there is no species found in the literature with similar morphological and physiological characteristics, this isolate is considered to be a new genus and a new species, Oramoeba fumarolia gen. nov., sp. nov.

Isolation of a new heterolobosean amoeba from a rice field soil: Vrihiamoeba italica gen. nov., sp. nov.

A heterolobosean amoeba strain 6_5F was isolated from an Italian rice field soil. Although 18S rRNA gene sequence analysis demonstrated that the new isolate was closely related to Stachyamoeba sp. ATCC 50324, further molecular analysis and morphological observation showed distinct differences amongst the two. The 5.8S rRNA gene was successfully amplified and sequenced for strain 6_5F but not for strain ATCC 50324. Trophozoites of strain ATCC 50324 transform into flagellate forms in the late stage of incubation before encystment, while strain 6_5F do not show flagellate forms under different conditions of the flagellation test. Light and electron microscopic observation showed the structural difference of cysts of strain 6_5F from strain ATCC 50324 and also from the type strain Stachyamoeba lipophora. The results show that the strain 6_5F is distinct from Stachyamoeba spp. and we propose a new genus and species for this isolate, Vrihiamoeba italica gen. nov., sp. nov.

Marinamoeba thermophila, a new marine heterolobosean amoeba growing at 50 degrees C.

Two amoeba strains were isolated from marine sediment taken at the same place with 18 months interval from a region of the sea floor heated by extended submarine hot springs and fumaroles. These thermophilic amoebae grow at temperatures up to 50 degrees C. Sequences of the internal transcribed spacer demonstrated that the two strains belong to the same species and are different from any genus for which sequences are known. Phylogeny using small subunit ribosomal RNA places the amoeba in the Heterolobosea. Their closest relatives are the hypersaline flagellate Pleurostomum flabellatum and the hypersaline amoeba Tulamoeba peronaphora. The freshwater amoeboflagellate genera Naegleria and Willaertia belong to the same phylogenetic clade in the Vahlkampfiidae. The new marine species does not transform into flagellates. It forms cysts, which are round to ellipsoidal with few pores. Because of their unique place in the molecular phylogenetic tree, and because there is no morphologically identical species found in the literature, these isolates are considered to be a new species and a new genus, Marinamoeba thermophila.

Tetramitus thermacidophilus n. sp., an amoeboflagellate from acidic hot springs.

Tetramitus thermacidophilus n. sp. is a novel thermophilic and acidophilic amoeboflagellate isolated from acidic hot springs in the Caldera Uzon (Kamchatka, Russia) and in Pisciarelli Solfatara (Naples, Italy). We describe it based on physiological, morphological, and sequence data. It was grown in monoxenic culture on the archaeon Acidianus brierleyi as food. Tetramitus thermacidophilus multiplies in a pH range from 1.2 to 5 and in a temperature range from 28 °C to 54 °C. The shortest doubling time was 4.5 h at pH 3 at 45 °C. Its spindle-shaped biflagellated stage was only rarely found in culture. The amoeboid stage shows the typical locomotive form of vahlkampfiid amoebae. Sequence comparisons of the internal transcribed spacer sequences and the small subunit rRNA genes confirm that T. thermacidophilus is a novel species within the genus Tetramitus and that both isolates belong to that species.

Occurrence of free-living amoebae in communities of low and high endemicity for Buruli ulcer in southern Benin.

Buruli ulcer or Mycobacterium ulcerans disease occurs mainly in areas in proximity to standing or slowly running freshwater, habitats in which free-living amoebae occur. For this reason, a possible link between the habitat of M. ulcerans and free-living amoebae was investigated. Free-living amoebae and mycobacteria were isolated from water and biofilm specimens taken from protected and unprotected sources of water in villages known to have either high or low endemicity for Buruli ulcer in Benin. Amoebae were isolated from 78.8% of samples. A greater proportion of water bodies in areas of high endemicity had amoebae than in areas of low endemicity (83.3% versus 66.7%). Protected sources of water were significantly more likely to contain amoebae in areas of high endemicity than in areas of low endemicity (88.0% versus 11.1%). Several pathogenic free-living amoebae and mycobacteria were isolated. However, no M. ulcerans was isolated and no specimen was positive for IS2404 PCR. Our results show that the study area has a water hygiene problem, which is greater in areas of high Buruli ulcer endemicity than in areas of low endemicity. Our observations indicate that additional studies are required to explore the possible link between free-living amoebae and mycobacteria.

Fatal disseminated Acanthamoeba lenticulata infection in a heart transplant patient.

We report a fatal case of disseminated acanthamebiasis caused by Acanthamoeba lenticulata (genotype T5) in a 39-year-old heart transplant recipient. The diagnosis was based on skin histopathologic results and confirmed by isolation of the ameba from involved skin and molecular analysis of a partial 18S rRNA gene sequence (DF3).

Two new Tetramitus species (Heterolobosea, Vahlkampfiidae) from cold aquatic environments.

Characterisation of the protists of cold environments provides important background for assessing the effects of climate change on microbial communities. Tetramitus angularis n. sp., from aquatic environments in Iceland and Switzerland, is the first vahlkampfiid recognised to have a characteristic Tetramitus flagellate stage combined with pre-formed excystment pores, which are not typical of this genus. T. angularis amoebae have a typical vahlkampfiid locomotive form and contain prominent lipid inclusions. Flagellates have a collar and cytostome, and can be mono- to multi-nucleate with corresponding change in cell shape from cylindrical to ellipsoidal and variable number of flagella. Cysts are round to semi-angular and have 2-5 pores closed by protruding, translucent plugs. A second organism, T. parangularis n. sp. from Alaska, has similar cysts but a flagellate stage has not been recognised; ITS sequence divergence is consistent with species criteria in the Vahlkampfiidae. Phylogenetic analysis of sequence data for the 5.8S rDNA region clusters the new spp. with T. rostratus, T. entericus and T. waccamawensis.

Molecular identification of free-living amoebae of the Vahlkampfiidae and Acanthamoebidae isolated in Arizona (USA).

Sediment samples from rivers, canals and lakes in Arizona (USA) were cultured for free-living amoebae at three different incubation temperatures (22, 37 and 40 degrees C). Isolates belonging to the Vahlkampfiidae were identified by sequencing the PCR-amplified ITS1, 5.8S and ITS2 rDNA. With this molecular method three Naegleria spp. were identified, N. gruberi sensu stricto, N. australiensis and N. tihangensis. Also a strain each of Willaertia magna and Vahlkampfia avara were identified. Three samples yielded two new Tetramitus spp. of which the closest relative is T. ovis. Many Acanthamoeba strains were also isolated. The genotype of these strains was identified using Acanthamoeba-specific primers (JDP1 and JDP2) amplifying a part of the SSUrDNA and sequencing with an internal primer (892c). Five of the Acanthamoeba isolates belong to genotype T5 (A. lenticulata), while five are genotype T4.

Isolation and molecular identification of free-living amoebae of the genus Naegleria from Arctic and sub-Antarctic regions.

Twenty-three freshwater samples with sediment taken from two regions in the Arctic, Spitzbergen and Greenland, and one region in sub-Antarctica, Ile de la Possession, were cultured for amoebae at 37 degrees C and room temperature (RT). Only two samples yielded amoebae at 37 degrees C and the two isolates were identified from their morphological features to belong to the genus Acanthamoeba. Vahlkampfiid amoebae were isolated from 11 samples at RT. Morphological analysis of the cysts identified all 11 isolates as belonging to the genus Naegleria, although only about half of them (45%) transformed into flagellates. Ribosomal DNA sequence analysis demonstrated that these isolates represent novel species and that N. antarctica, N. dobsoni and N. chilensis are their closest relatives. Not surprisingly, these three species also grow at lower temperatures (<37 degrees C) than the majority of described Naegleria spp. Two of the eight new species were found in both Arctic and sub-Antarctic regions, and other new species from the Arctic are closely related to new species from the sub-Antarctic. Therefore, it seems the Naegleria gene pool present in the polar regions is different from that found in temperate and tropical regions.