Molecular evidence for a new lineage within the Acanthamoeba T4 genotype.

Acanthamoeba is a widespread free-living amoeba capable of causing serious infections in humans and other animals, such as amoebic keratitis, disseminated infections, and fatal encephalitis. Strain identification is usually based on 18S rDNA sequencing, which allows the distinction of over twenty genotypes. Most sequences from environmental and clinical samples belong to the T4 genotype, which can be divided into seven subtypes, T4A to T4G, and by a nearly similar grouping of mitochondrial sequences into T4a to T4j subtypes. The co-clustering of nuclear and mitochondrial groups can be very useful for a better identification of lineages within the very rich T4 genotype. In this study, we provided molecular phylogenetic evidence for the delineation of a new nuclear subtype, hereafter labelled T4H, and its co-clustering with the mitochondrial T4j subtype. At least three cases of amoebic keratitis are due to strains belonging to this new group, present mainly in fresh water and detected in various countries (France, Iran, India and China).

On predatory fungi feeding on free-living amoebae harbouring yeast-like endoparasites.

Amoebae of the genus Vannella isolated from an ornamental fish aquarium were found to be infected with fungi. Upon plate culture, amoeba-trapping hyphal filaments were developed, and the amoeba trophozoites were found to harbour yeast-like parasites in their cytoplasm. Transfection of hyphae to a laboratory strain of Vannella resulted in the formation of conidia indicating the possible presence of zygomycetes of the genus Acaulopage, while efforts to culture the endoparasite remained unsuccessful. Biomolecular analysis based on rDNA revealed the presence of two distinct types of fungi, confirming the filamentous form as Acaulopage sp. (Zoopagomycota, Zoopagales) and identifying the yeast-like endoparasite as Cladosporium sp. (Ascomycota, Cladosporiales). To our knowledge, this is the first report of amoebae infected with Cladosporium.

On the diversity and clinical importance of Acanthamoeba spp. from Group 1.

Group 1 acanthamoebae are morphologically and phylogenetically distinct from all other Acanthamoeba species. They include five species, each labelled by its genotype: A. astronyxis (T7), A. tubiashi (T8), A. comandoni (T9), unnamed Acanthamoeba sp. (T17), and A. byersi (T18). Thought only environmental, they have recently attracted attention due to their recovery in cases of human keratitis and encephalitis, the main diseases caused by Acanthamoeba, where the usual causative agents are mainly species of Groups 2 and 3. Analysis of the available data confirms the pathogenic importance of these species, although it is probably minor compared to that of the species in Groups 2 and 3. In addition, it should be noted that there are difficulties in identifying genotypes by widely used molecular methods, and some misidentifications are revealed.

Putative group I introns in the eukaryote nuclear internal transcribed spacers.

Group I introns are mobile genetic elements that interrupt genes encoding proteins and RNAs. In the rRNA operon, introns can insert in the small subunit (SSU) and large subunit (LSU) of a wide variety of protists and various prokaryotes, but they were never found in the ITS region. In this study, unusually long ITS regions of fungi and closely related unicellular organisms (Polychytrium aggregatum, Mitosporidium daphniae, Amoeboaphelidium occidentale and Nuclearia simplex) were analysed. While the insertion of repeats is responsible for long ITS in other eukaryotes, the increased size of the sequences analysed herein seems rather due to the presence of introns in ITS-1 or ITS-2. The identified insertions can be folded in secondary structures according to group I intron models, and they cluster within introns in conserved core-based phylogeny. In addition, for Mitosporidium, Amoeboaphelidium and Nuclearia, more conventional ITS-2 structures can be deduced once spacer introns are removed. Sequences of five shark species were also analysed for their structure and included in phylogeny because of unpublished work reporting introns in their ITS, obtaining congruent results. Overall, the data presented herein indicate that spacer regions may contain introns.

Update on Acanthamoeba phylogeny.

The evolutionary history of Acanthamoeba has been substantially resolved by the 18S rDNA phylogeny which made it possible to delimit the main lines associated with some classical species. Some of them have proven to be polyphyletic, but the inappropriate use of treating under the same names unrelated strains persists. In this study, phylogenies based on the complete genes of nuclear and mitochondrial rDNA were compared, in order to verify the congruence of the different lines. Various groups can thus be identified, some of which associated with the type strains of given species. Recognizing them only by their species names would significantly reduce the current confusion, in addition to logically following basic taxonomic rules. In this manner, the well-known polyphyletic taxa A. castellanii and A. polyphaga, are restricted to the two lines specified by their type strains, while other widely used strains like Neff and Linc-AP1 that are often confused with the previous ones, can be assigned to their own lines. New species are potentially present in other groups and additional efforts are needed to delimit them.

Solving an old enigma: Morellospora saccamoebae gen. nov., sp. nov. (Rozellomycota), a Sphaerita-like parasite of free-living amoebae.

The Rozellomycota form a lineage basal or sister to the Fungi, ancestor of Microsporidia. Their biodiversity is very rich but remains poorly characterized. The few known species are all parasites, whether of water molds and algae (Rozella), crustaceans (Mitosporidium), or as endonuclear parasites of amoebae (Nucleophaga, Paramicrosporidium). Since the nineteenth century, intracytoplasmic parasites of various protozoa have been described as species of the same genus Sphaerita. However, it was later thought possible to separate these parasites into at least two distinct groups, those forming flagellated zoospores, prevalent in Euglena and other flagellates, and those forming immobile spores, found mainly in free-living and endozoic amoebae. Herein, we report the recovery of a strain of the free-living amoeba species Saccamoeba lacustris, naturally infected by an intracytoplasmic parasite, which under light microscope has a morphology consistent with that of Sphaerita. Biomolecular analyses were thus performed. Our results show that the intracytoplasmic parasite of Saccamoeba belongs to the same subgroup of Mitosporidium and that it forms a new genus within Rozellomycota, Morellospora, that corresponds to the former spore-forming Sphaerita-like parasites of amoebae.

Unusual 18S rDNA of Acanthamoeba containing intron turned out to be a T5/T4 chimera.

The free-living amoebae of the genus Acanthamoeba are widely investigated for their diversity and evolution. Studies usually employ biomolecular methods targeting the 18S rRNA gene, assigning strains according to a well-established genotyping system. Strains from at least four genotypes contain introns in their rDNA. By retracing the evolutionary history of these introns within the amoebae, we found that the 18S rDNA of TUMSJ-341 strain (ATCC PRA-11), assigned to the genotype T5 (A. lenticulata), proved very unusual in our analyses, not corresponding to the characteristics of the group. The sequence contains a group I intron recovered only in A. lenticulata. At BLAST, however, the intron-less 18S rDNA of TUMSJ-341 does not match with T5 strains but shows some affinity with strains from genotype T4, suggesting a new genotype. Our accurate analysis of this sequence finally revealed a mixture of variable regions, showing that such discordant results are due to the insertion into the gene of a strain T5 of a DNA fragment containing hypervariable regions specific for a T4 strain. Data presented herein indicate that this sequence is probably a chimera.

Filling gaps in the microsporidian tree: rDNA phylogeny of Chytridiopsis typographi (Microsporidia: Chytridiopsida).

Microsporidia are intracellular eukaryotic parasites of animals, characterized by unusual morphological and genetic features. They can be divided in three main groups, the classical microsporidians presenting all the features of the phylum and two putative primitive groups, the chytridiopsids and metchnikovellids. Microsporidia originated from microsporidia-like organisms belonging to a lineage of chytrid-like endoparasites basal or sister to the Fungi. Genetic and genomic data are available for all members, except chytridiopsids. Herein, we filled this gap by obtaining the rDNA sequence (SSU-ITS-partial LSU) of Chytridiopsis typographi (Chytridiopsida), a parasite of bark beetles. Our rDNA molecular phylogenies indicate that Chytridiopsis branches earlier than metchnikovellids, commonly thought ancestral, forming the more basal lineage of the Microsporidia. Furthermore, our structural analyses showed that only classical microsporidians present 16S-like SSU rRNA and 5.8S/LSU rRNA gene fusion, whereas the standard eukaryote rRNA gene structure, although slightly reduced, is still preserved in the primitive microsporidians, including 18S-like SSU rRNA with conserved core helices, and ITS2-like separating 5.8S from LSU. Overall, our results are consistent with the scenario of an evolution from microsporidia-like rozellids to microsporidians, however suggesting for metchnikovellids a derived position, probably related to marine transition and adaptation to hyperparasitism. The genetic and genomic data of additional members of Chytridiopsida and Rozellomycota will be of great value, not only to resolve phylogenetic relationships but also to improve our understanding of the evolution of these fascinating organisms.

An apparent Acanthamoeba genotype is the product of a chimeric 18S rDNA artifact.

Free-living amoebae of the genus Acanthamoeba are potentially pathogenic protozoa widespread in the environment. The detection/diagnosis as well as environmental survey strategies is mainly based on the identification of the 18S rDNA sequences of the strains that allow the recovery of various distinct genotypes/subgenotypes. The accurate recording of such data is important to better know the environmental distribution of distinct genotypes and how they may be preferentially associated with disease. Recently, a putative new acanthamoebal genotype T99 was introduced, which comprises only environmental clones apparently with some anomalous features. Here, we analyze these sequences through partial treeing and BLAST analyses and find that they are actually chimeras. Our results show that the putative T99 genotype is very likely formed by chimeric sequences including a middle fragment from acanthamoebae of genotype T13, while the 5'- and 3'-end fragments came from a nematode and a cercozoan, respectively. Molecular phylogenies of Acanthamoeba including T99 are consequently erroneous as genotype T99 does not exist in nature. Careful identification of Acanthamoeba genotypes is therefore critical for both phylogenetic and diagnostic applications.

New insights from molecular phylogenetics of amoebophagous fungi (Zoopagomycota, Zoopagales).

Amoebophagous fungi are represented in all fungal groups: Basidiomycota, Ascomycota, Zygomycota, and Chytridiomycota. The amoebophagous fungi, within the zygomycota (Zoopagales, Zoopagomycota), mainly affect naked amoebae as ectoparasites or endoparasites. It is rather difficult to isolate members of the Zoopagales, because of their parasitic lifestyle, and to bring them into culture. Consequently, gene sequences of this group are undersampled, and its species composition and phylogeny are relatively unknown. In the present study, we were able to isolate amoebophagous fungi together with their amoeba hosts from various habitats (moss, pond, bark, and soil). Altogether, four fungal strains belonging to the genera Acaulopage and Stylopage plus one unidentified isolate were detected. Sequences of the 18S rDNA and the complete ITS region and partial 28S (LSU) rDNA were generated. Subsequent phylogenetic analyses showed that all new isolates diverge at one branch together with two environmental clonal sequences within the Zoopagomycota. Here, we provide the first molecular characterization of the genus Stylopage. Stylopage is closely related to the genus Acaulopage. In addition, taxonomy and phylogeny of amoebophagous fungi and their ecological importance are reviewed based on new sequence data, which includes environmental clonal sequences.

Molecular identification of bacterial endosymbionts of Sappinia strains.

The genus Sappinia comprises free-living amoebae occurring worldwide in a variety of habitats such as soils, plant matter and freshwater ponds, but also animal faeces, and includes at present three species, S. pedata, S. diploidea and S. platani. The genus is potentially pathogenic, as indicated by the identification of S. pedata in a case of human amoebic encephalitis. Electron microscopy studies on some strains already revealed intracellular bacteria in Sappinia. In the current study, we performed 16S ribosomal RNA gene (rDNA) analysis of these bacterial endosymbionts. We first inferred relationships among Sappinia strains on the basis of 18S rDNA, demonstrating that S. pedata emerged as sister to a larger clade including S. diploidea, S. platani and a few 'S. diploidea-like' strains. Thus, bacterial 16S rDNA was searched for in representative strains of each Sappinia species/subgroup. We found that Sappinia strains were associated to distinct species of Flavobacterium or Pedobacter (phylum Bacteroidetes). These appear to be distributed following the amoebal host subgroups, and are not directly related to other Bacteroidetes species known as interacting with free-living amoebae. While all the endosymbionts' close relatives are known to grow on agar, bacteriological media inoculated with amoebal extracts remained negative. Overall, results indicate that the recovered bacteria are likely specific obligate endosymbionts of Sappinia species. Further studies, including additional amoebal strains and deep morphological and molecular analyses, will be necessary to confirm this hypothesis.

Update on Acanthamoeba jacobsi genotype T15, including full-length 18S rDNA molecular phylogeny.

Free-living amoebae of the genus Acanthamoeba are worldwide present in natural and artificial environments, and are also clinically important, as causative agents of diseases in humans and other animals. Acanthamoeba comprises several species, historically assigned to one of the three groups based on their cyst morphology, but presently recognized as at least 20 genotypes (T1-T20) on the basis of their nuclear 18S ribosomal RNA (rRNA) gene (18S rDNA) sequences. While strain identification may usually be achieved targeting short (<500 bp) 18S ribosomal DNA (rDNA) fragments, the use of full-length gene sequences (>2200 bp) is necessary for correct genotype description and reliable molecular phylogenetic inference. The genotype T15, corresponding to Acanthamoeba jacobsi, is the only genotype described on the basis of partial sequences (~1500 bp). While this feature does not prevent the correct identification of the strains, having only partial sequences renders the genotype T15 not completely defined and may furthermore affect its position in the Acanthamoeba molecular tree. Here, we complete this gap, by obtaining full-length 18S rDNA sequences from eight A. jacobsi strains, genotype T15. Morphologies and physiological features of isolated strains are reported. Molecular phylogeny based on full 18S rDNA confirms some previous suggestions for a genetic link between T15 and T13, T16, and T19, with T19 as sister-group to T15.

Molecular identification of Nucleophaga terricolae sp. nov. (Rozellomycota), and new insights on the origin of the Microsporidia.

Microsporidia are widespread endoparasites of animals, including humans. They are characterized by highly modified morphological and genetic features that cause difficulties in elucidating their enigmatic origin and evolution. Recent advances, however, indicate that the Microsporidia have emerged from the Rozellomycota, forming together either the most basal lineage of the Fungi or its closer relative. The Rozellomycota comprise a huge diversity of uncultured environmental clones, with a very few known species endoparasitic of algae and water moulds, like the chytrid-like Rozella, and of free-living amoebae, like Nucleophaga and the microsporidia-like Paramicrosporidium. A possible ancestral microsporidium, Mitosporidium, has recently been described from the water flea Daphnia, since the phylogenomic reconstruction showed that it branches to the root of the microsporidian tree, while the genome analysis revealed a fungal-like nuclear genome and the persistence of a mitochondrial genome. Here we report the 18S rDNA molecular phylogeny of an additional microsporidium-like endoparasite of amoebae, which has a developmental cycle almost identical to that of Nucleophaga amoebae. Our results show that the endoparasite is closely related to N. amoebae, forming a distinct species, for which we propose the name Nucleophaga terricolae. Furthermore, the Nucleophaga lineage is recovered as sister to the Microsporidia while Mitosporidium turns out to be member of a well-supported group of environmental clones. These results raise the question about the actual ancestry of the Microsporidia within the Rozellomycota. A precise and robust phylogeny will require further comparative genomic studies of these various strains, and should also consider the primitive microsporidia, for which genetic data are still lacking, because all these organisms are essentially morphologically similar.

Acanthamoeba misidentification and multiple labels: redefining genotypes T16, T19, and T20 and proposal for Acanthamoeba micheli sp. nov. (genotype T19).

Acanthamoeba species are ubiquitous amoebae able to cause important infections in humans and other vertebrates. The full/near-full sequences (>2000 bp) of the small subunit ribosomal RNA gene (SSU rDNA or 18S rDNA) are used to cluster Acanthamoeba as genotypes, labeled T1 to T20. Genotype T15 remains an exception, being described only partially on a 1500-bp fragment. Strains are thus usually identified based on their 18S identity matches with reference strains, often using shorter (<500 bp) diagnostic fragments of the gene. Nevertheless, short fragments (<1000 bp) have been used to propose genotypes. This has been criticized, and doubts arise therefore on possible confusion leading to classify distinct partial sequences with a same label(s). We demonstrate herein that several partial sequences misassigned either to T16 or to T4, actually belong to at least two separate and distinct genotypes. We obtained the full 18S rDNA of a strain previously typed as T16 on the basis of a small fragment and demonstrated that it actually belongs to the recently described T19. We propose the name Acanthamoeba micheli sp. nov., for this strain. Furthermore, partial molecular phylogenies were performed to show that several other misassigned T16 partial sequences belong to a new genotype. This latter includes also misassigned T4 partial sequences, only recently available as full sequences and labeled as T20. We thus reassign these partial sequences to the genotype T20. Longer sequences, ideally at least 90 % of the total gene length, should be obtained from strains to ensure reliable diagnostic and phylogenetic results.

Rediscovery of Nucleophaga amoebae, a novel member of the Rozellomycota.

Recent studies showed that the huge diversity branching at or near the phylogenetic root of the fungal kingdom, mostly constituted by uncultured environmental clones, is actually characterized by intracellular predators/parasites of various eukaryotes. These form three related lineages: the Aphelidea, endoparasites of algae; the Rozellomycota, with Rozella species mainly endoparasites of water moulds, and Paramicrosporidium species endonuclear parasites of amoebae; and the Microsporidia, mainly endoparasites of animals. Increasing evidence suggests the emergence of Microsporidia from within Rozellomycota; however, their fungal or protistan nature is still unclear. Here, we report the molecular phylogeny based on the small subunit ribosomal RNA (SSU rDNA) gene, of an additional endoparasite of amoebae, corresponding to the old enigmatic chytrid Nucleophaga amoebae described in the nineteenth century. Our results show that Nucleophaga, possessing a morphotype intermediate between Rozella and Paramicrosporidium, emerges as a unique lineage within the Rozellomycota. The recovery and characterization of new members of Rozellomycota are of high value for the understanding of the early evolutionary history of the Fungi and related lineages.

Microsporidia-like parasites of amoebae belong to the early fungal lineage Rozellomycota.

Molecular phylogenies based on the small subunit ribosomal RNA gene (SSU or 18S ribosomal DNA (rDNA)) revealed recently the existence of a relatively large and widespread group of eukaryotes, branching at the base of the fungal tree. This group, comprising almost exclusively environmental clones, includes the endoparasitic chytrid Rozella as the unique known representative. Rozella emerged as the first fungal lineage in molecular phylogenies and as the sister group of the Microsporidia. Here we report rDNA molecular phylogenetic analyses of two endonuclear parasites of free-living naked amoebae having microsporidia-like ultrastructural features but belonging to the rozellids. Similar to microsporidia, these endoparasites form unflagellated walled spores and grow inside the host cells as unwalled nonphagotrophic meronts. Our endonuclear parasites are microsporidia-like rozellids, for which we propose the name Paramicrosporidium, appearing to be the until now lacking morphological missing link between Fungi and Microsporidia. These features contrast with the recent description of the rozellids as an intermediate wall-less lineage of organisms between protists and true Fungi. We thus reconsider the rozellid clade as the most basal fungal lineage, naming it Rozellomycota.

Editorial for the Special Issue "Advances in Acanthamoeba".

Some free-living amoebae can behave as opportunistic parasites, causing rare but dangerous diseases in humans and animals, primarily amoebic keratitis, with loss of vision, and encephalitis, which is almost always fatal [...].

Validation and redescription of Acanthamoeba terricola Pussard, 1964 (Amoebozoa: Acanthamoebidae).

Acanthamoeba castellanii (Douglas, 1930) Page, 1967 is the type species of a widespread genus of free-living amoebae, potentially pathogenic for humans and animals. The Neff strain is one of the most widely used in biological research, serving as a model for both A. castellanii and the whole genus in general. The Neff strain, isolated in California, closely resembles another strain found in France and originally described as a separate species, Acanthamoeba terricola Pussard, 1964, but both were successively synonymized with A. castellanii. Molecular sequence analysis has largely replaced morphological diagnosis for species identification in Acanthamoeba, and rDNA phylogenies show that the Neff strain forms a distinct lineage from that of the type strain of A. castellanii. In this study, we compared the type strain of A. terricola with the Neff strain and A. castellanii, and analysed the available molecular data including new sequences obtained from A. terricola. Here we provide molecular evidence to validate the species A. terricola. The Neff strain is therefore transferred to A. terricola and should no longer be considered as belonging to A. castellanii.

Molecular characterization and ultrastructure of a new amoeba endoparasite belonging to the Stenotrophomonas maltophilia complex.

Naegleria and Acanthamoeba spp. were recovered from biofilm of a flushing cistern in a lavatory and both were found to be infected by rod-shaped bacteria enclosed within a vacuole. These intracellular bacteria behave like parasites, causing lysis of host amoebae. The bacteria proved unculturable on bacteriological media, and but could be maintained as endocytobionts within Acanthamoeba on agar plates. A marked differential host preference was observed in co-culture assays with various strains of amoebae. Molecular phylogenetic analyses performed on almost complete 16S rDNA sequences showed that the bacteria emerged as an atypical rapidly-evolving strain within the Stenotrophomonas maltophilia complex (Gamma-Proteobacteria, Xanthomonadales).