Comparative genomic analysis of the 'pseudofungus' Hyphochytrium catenoides.

Eukaryotic microbes have three primary mechanisms for obtaining nutrients and energy: phagotrophy, photosynthesis and osmotrophy. Traits associated with the latter two functions arose independently multiple times in the eukaryotes. The Fungi successfully coupled osmotrophy with filamentous growth, and similar traits are also manifested in the Pseudofungi (oomycetes and hyphochytriomycetes). Both the Fungi and the Pseudofungi encompass a diversity of plant and animal parasites. Genome-sequencing efforts have focused on host-associated microbes (mutualistic symbionts or parasites), providing limited comparisons with free-living relatives. Here we report the first draft genome sequence of a hyphochytriomycete 'pseudofungus'; Hyphochytrium catenoides Using phylogenomic approaches, we identify genes of recent viral ancestry, with related viral derived genes also present on the genomes of oomycetes, suggesting a complex history of viral coevolution and integration across the Pseudofungi. H. catenoides has a complex life cycle involving diverse filamentous structures and a flagellated zoospore with a single anterior tinselate flagellum. We use genome comparisons, drug sensitivity analysis and high-throughput culture arrays to investigate the ancestry of oomycete/pseudofungal characteristics, demonstrating that many of the genetic features associated with parasitic traits evolved specifically within the oomycete radiation. Comparative genomics also identified differences in the repertoire of genes associated with filamentous growth between the Fungi and the Pseudofungi, including differences in vesicle trafficking systems, cell-wall synthesis pathways and motor protein repertoire, demonstrating that unique cellular systems underpinned the convergent evolution of filamentous osmotrophic growth in these two eukaryotic groups.

Elusive treatment for human rhinosporidiosis.

OBJECTIVES: The aim of this study was to clarify the contentious taxonomic classification of Rhinosporidium seeberi, the cause of human rhinosporidiosis, which may have treatment implications. METHODS: PCR was used to amplify the internal transcribed spacer (ITS)-2 region from the genomic DNA of the aetiological agent obtained from a sample of human rhinosporidiosis lesions. The amplicon was sequenced and the organism identified using the Basic Local Alignment Search Tools (BLAST). RESULTS: Phylogenetic analysis revealed that the aetiological agent clustered along with the R. seeberi isolated from humans and also with Amphibiocystidium ranae from frogs. This organism is a member of the order Dermocystida in the class Mesomycetozoea. A patient with disseminated rhinosporidiosis did not respond to conventional therapy with dapsone and surgical excision, and treatment with amphotericin B also proved futile. CONCLUSION: An effective treatment for R. seeberi-a eukaryote belonging to the class Mesomycetozoea-is still elusive.

Equine laryngeal rhinosporidiosis in western Canada.

A 12-year-old female Argentinean Warmblood mare was evaluated because of respiratory noise. The horse resided in Calgary, Alberta, Canada, but had been imported from Argentina 28 months prior to presentation. Endoscopy of the upper respiratory tract revealed a single polypoid mass on the left arytenoid. The mass was surgically excised and was diagnosed histologically as rhinosporidiosis. Polymerase chain reaction and DNA sequencing were used to confirm the etiological agent. Four weeks postoperatively, endoscopy was repeated, revealing recurrence of the original lesion with multiple additional polypoid masses on the larynx and in the oropharynx. Resolution of the disease had not been attained at the time of publication. The current report outlines a case of rhinosporidiosis in an unusual anatomical and geographic location. The infection most likely originated in Argentina, with a prolonged subclinical phase. Due to increased travel of human beings and animals, there is potential for the introduction of exotic diseases into nonendemic areas.

The taxonomy and phylogenetics of the human and animal pathogen Rhinosporidium seeberi: a critical review.

Rhinosporidum seeberi is the etiologic agent of rhinosporidiosis, a disease of mucous membranes and infrequent of the skin and other tissues of humans and animals. Because it resists culture, for more than 100 years true taxonomic identity of R. seeberi has been controversial. Three hypotheses in a long list of related views have been recently introduced: 1) a prokaryote cyanobacterium in the genus Microcystis is the etiologic agent of rhinosporidiosis, 2) R. seeberi is a eukaryote pathogen in the Mesomycetozoa and 3) R. seeberi is a fungus. The reviewed literature on the electron microscopic, the histopathological and more recently the data from several molecular studies strongly support the view that R. seeberi is a eukaryote pathogen, but not a fungus. The suggested morphological resemblance of R. seeberi with the genera Microcystis (bacteria), Synchytrium and Colletotrichum (fungi) by different teams is merely hypothetical and lacked the scientific rigor needed to validate the proposed systems. A fundamental aspect against the prokaryote theory is the presence of nuclei reported by numerous authors and updated in this review. Moreover, Microcystis's and Synchytrium's ultra-structural and key cell cycle traits cannot be found in R. seeberi parasitic phase. The PCR amplification of a cyanobacteria 16S rDNA sequence from cases of rhinosporidiosis, while intriguing, will be viewed here as an anomaly due to contamination with environmental Microcystis or perhaps as an endosymbiotic acquisition of plastids from cyanobacteria ancestors. Thus, even if R. seeberi possesses prokaryote DNA, this does not prove that R. seeberi is a cyanobacterium. The placement of R. seeberi within the fungi is scientifically untenable. The isolation and the DNA analysis performed in a fungal strain, and the lack of appropriate controls are the main problems of this claim. Further studies are needed to validate R. seeberi's acquisition of prokaryote plastids and other issues that still need careful scrutiny.

The regional sero-epidemiology of rhinosporidiosis in Sri Lankan humans and animals.

No data is available in the world literature on serum anti-rhinosporidial antibody levels in animals, and as far as we aware this is the first report. Although rhinosporidiosis in farm and domestic animals has been widely reported from other countries, rhinosporidiosis in animals has not been reported in Sri Lanka, though this country has the highest world-wide prevalence of human rhinosporidiosis on a unit-population basis. Serum IgG titres in 6 species of Sri Lankan animals (buffalo, cat, cattle, dog, goat, horse; total 291) were assayed by the Immuno blot (dot-ELISA) method on nitrocellulose paper and were compared with serum IgG titres in normal Sri Lankan human subjects (total 211) in different geographical areas, and in human Sri Lankan patients with rhinosporidiosis as reference values (total 36). Sensitization to rhinosporidial antigen(s) was detected in all 6 species of animals and the highest titres (1/3200) were found in cats, and free-grazing horses. Cattle showed higher levels of antibody than buffaloes. The titres in these animals are compared with world reports on overt rhinosporidiosis in these species, and with titres in normal Sri Lankan humans. Human, but not animal titres showed variations compatible with the regional prevalence of rhinosporidiosis. The variations in titres in animals especially horses, were probably more related to their mode of feeding, while in humans the titres in normal persons were probably related to the rhinosporidial-endemicity of their respective regions. No conclusions from sero-positivity in animals could be made regarding the absence of reports on rhinosporidiosis as an overt disease in these Sri Lankan animal species but the possibility of a genetically-determined insusceptibility to rhinosporidiosis in Sri Lanka, is considered. Rhinosporidium seeberi-specific PCR positive reactions were obtained with nasal scrapings from cattle that microscopically showed PAS+ bodies that were compatible with rhinosporidial sporangia. Sequence-analysis of the reactions products from five positive R. seeberi-specific PCR samples (four in this study and 1 in a previous study) gave results confirmatory of R. seeberi.

Detection of 16S rRNA gene in round bodies isolated from polyps of rhinosporidiosis.

Polyps of rhinosporidiosis contain microscopic round bodies, believed to be causative agents of the disease. The source and identity of these round bodies has been debated ever since the first case was described in the year 1900. Most patients have a history of exposure to pond water. Collection and microscopic analysis of pond water samples where patients had been dipping led to the idea that a cyanobacterium could possibly develop into round bodies after entering the patient's nose. Molecular genomic studies based on PCR, cloning, and sequencing were, therefore, carried out using purified DNA, extracted from individually separated round bodies from polyp samples. Parallel investigations were also conducted on DNA extracted from cyanobacterium Microcystis isolated from pond water samples. The results of these experiments presented here provide evidence of the presence of 1458 bp 16S rRNA gene in round bodies. This is the first molecular study indicating the prokaryotic identity of round bodies in rhinosporidiosis.

Molecular evidence for multiple host-specific strains in the genus Rhinosporidium.

The taxonomic relationship of Rhinosporidium seeberi with other organisms remained controversial for over a century. Recently, molecular studies have shown R. seeberi to be a protistal microbe in the newly described class Mesomycetozoea at the animal-fungal boundary. Phylogenetic analyses of R. seeberi using 18S small-subunit (SSU) rRNA genes from several hosts suggested Rhinosporidium as a monotypic genus. To test this hypothesis, the internal transcribed spacer 1 (ITS1), 5.8S, and ITS2 from eight humans, two swans, and a dog with rhinosporidiosis were sequenced. The ITS regions were amplified by PCR using a primer designed from a unique region of R. seeberi's 18S SSU rRNA genes in combination with the ITS4 universal primer. In addition, the universal ITS4 and ITS5 primers were also used. R. seeberi's ITS sequences showed differences in the numbers of nucleotides among strains. For instance, the eight human ITS sequences were uniformly similar with only a few mismatches and approximately 1,060 bp long. In contrast, sequences from one of the swans and the dog were 1,356 bp and approximately 1,147 bp long, respectively. Clustal analysis of all of the ITS sequences showed multiple 50- to 60-bp gaps and several mismatches among them. Parsimony analysis placed the Rhinosporidium ITS sequences in three well-supported sister groups according to the hosts' identities. This analysis strongly suggests that the genus Rhinosporidium may possess multiple host-specific strains. No correlation was found between this finding and the phenotypic features of R. seeberi in the studied samples.

Lacazia loboi and Rhinosporidium seeberi: a genomic perspective.

In the past five years, with the use of molecular strategies the phylogenetic affinities of the two more resilient pathogens studied in medical mycology, Lacazia loboi and Rhinosporidium seeberi were finally deciphered. These studies found that L. loboi was the sister taxon to Paraccidioides brasiliensis, and R. seeberi was closely related to protistan spherical aquatic fish pathogens, located at the point were animals diverged from the fungi, in the class Mesomycetozoea. These initial studies indicated that a molecular strategy was the ideal approach to further understand these anomalous pathogens. However, the limited amount of information gathered so far from few DNA sequences, although crucial to place these organisms in the tree of life and to take a glance to their ecological preferences, did not provide answers to other important traits. In the following pages we discuss a genomic perspective for both pathogens and the benefit that such information could generate to understand more about these two uncultivated pathogens.

Lacazia loboi and Rhinosporidium seeberi: a genomic perspective

In the past five years, with the use of molecular strategies the phylogenetic affinities of the two more resilient pathogens studied in medical mycology, Lacazia loboi and Rhinosporidium seeberi were finally deciphered. These studies found that L. loboi was the sister taxon to Paraccidioides brasiliensis, and R. seeberi was closely related to protistan spherical aquatic fish pathogens, located at the point were animals diverged from the fungi, in the class Mesomycetozoea. These initial studies indicated that a molecular strategy was the ideal approach to further understand these anomalous pathogens. However, the limited amount of information gathered so far from few DNA sequences, although crucial to place these organisms in the tree of life and to take a glance to their ecological preferences, did not provide answers to other important traits. In the following pages we discuss a genomic perspective for both pathogens and the benefit that such information could generate to understand more about these two uncultivated pathogens.

Rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites.

Rhinosporidium seeberi, a microorganism that can infect the mucosal surfaces of humans and animals, has been classified as a fungus on the basis of morphologic and histochemical characteristics. Using consensus polymerase chain reaction (PCR), we amplified a portion of the R. seeberi 18S rRNA gene directly from infected tissue. Analysis of the aligned sequence and inference of phylogenetic relationships showed that R. seeberi is a protist from a novel clade of parasites that infect fish and amphibians. Fluorescence in situ hybridization and R. seeberi- specific PCR showed that this unique 18S rRNA sequence is also present in other tissues infected with R. seeberi. Our data support the R. seeberi phylogeny recently suggested by another group. R. seeberi is not a classic fungus, but rather the first known human pathogen from the DRIPs clade, a novel clade of aquatic protistan parasites (Ichthyosporea).

Phylogenetic analysis of Rhinosporidium seeberi's 18S small-subunit ribosomal DNA groups this pathogen among members of the protoctistan Mesomycetozoa clade.

For the past 100 years the phylogenetic affinities of Rhinosporidium seeberi have been controversial. Based on its morphological features, it has been classified as a protozoan or as a member of the kingdom Fungi. We have amplified and sequenced nearly a full-length 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence from R. seeberi. Using phylogenetic analysis, by parsimony and distance methods, of R. seeberi's 18S SSU rDNA and that of other eukaryotes, we found that this enigmatic pathogen of humans and animals clusters with a novel group of fish parasites referred to as the DRIP clade (Dermocystidium, rossete agent, Ichthyophonus, and Psorospermium), near the animal-fungal divergence. Our phylogenetic analyses also indicate that R. seeberi is the sister taxon of the two Dermocystidium species used in this study. This molecular affinity is remarkable since members of the genus Dermocystidium form spherical structures in infected hosts, produce endospores, have not been cultured, and possess mitochondria with flat cristae. With the addition of R. seeberi to this clade, the acronym DRIP is no longer appropriate. We propose to name this monophyletic clade Mesomycetozoa to reflect the group's phylogenetic association within the Eucarya.