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.

Thermodesulfobium sp. strain 3baa, an acidophilic sulfate reducing bacterium forming biofilms triggered by mineral precipitation.

Sulfate reducing prokaryotes are promising candidates for the remediation of acidic metal-rich waste waters. However, only few acidophilic species have been described to date. Chemolithoautotrophic strain 3baa was isolated from sediments of an acidic mine pit lake. Based on its 16S-rRNA gene sequence it belongs to the genus Thermodesulfobium. It was identified as an acidophile growing in artificial pore water medium in the range of pH 2.6-6.6. Though the highest sulfate reduction rates were obtained at the lower end of this range, elongated cells and extended lag phases demonstrated acid stress. Sulfate reduction at low pH was accompanied by the formation of mineral precipitates strongly adhering to solid surfaces. A structural investigation by laser scanning microscopy, electron microscopy and X-ray microanalysis revealed the formation of Al hydroxides and Fe sulfides which were densely populated by cells. Al hydroxides precipitated first, enabling initial cell attachment. Colonization of solid surfaces coincided with increased sulfate reducing activity indicating more favourable growth conditions within biofilms compared with free-living cells. These findings point out the importance of cell-mineral interaction for biofilm formation and contribute to our understanding how sulfate reducing prokaryotes thrive in both natural and engineered systems at low pH.

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.

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.

Investigations of an extraordinary endocytobiont in Acanthamoeba sp.: development and replication.

In this article, the results of investigations concerning a parasitic endocytobiont within the host amoebae (Acanthamoeba sp.) are presented. The endocytobiont was recently isolated from the contact lens and the inflamed eye of a patient with keratitis. Light microscopy and electron microscopy were performed to provide morphological details: Light microscopy revealed the presence of ovoid microorganisms developing and proliferating within the cytoplasm of the amoebic trophozoites. Details of the unusual development of these endocytobionts within the amoebae could be studied and demonstrated by means of electron microscopy. Foldings and morphological reorganization of the microorganisms took place exclusively within the host cytoplasm. The intracellularly aggregating organisms led to the rupture of the Acanthamoeba trophozoites after proliferation. Numerous microorganisms were released, which were infectious and were subsequently ingested by hitherto uninfected acanthamoebic trophozoites. To evaluate the in vitro growth of the isolated endocytobionts (without their hosts), they were transferred to several different culture plates. There was no growth of these unique organisms on five different common cultural plates suitable for the growth of bacteria and fungi.

The varicella-zoster virus induces apoptosis in vitro in subpopulations of primary human peripheral blood mononuclear cells.

Varicella-zoster virus (VZV), a member of Herpesviridae, subfamily alpha-Herpesvirinae, is pathogenic exclusively in the human. Chickenpox is the result of primary infection of VZV. During the viremic stage, VZV infects peripheral blood mononuclear cells (PBMC) and spreads to the periphery. In skin cells it causes typical lesions. Apoptosis has been demonstrated in different cell types by other alpha-herpesviruses. VZV-infected T lymphocytes, B lymphocytes, and monocytes, respectively, were examined in this in vitro study by flow cytometry, immunofluorescence and electron microscopy. All infected cell types showed signs of apoptosis: a lower DNA content, DNA fragmentation, loss of membrane integrity, and an altered nuclear morphology. The results observed led to the suggestion that VZV can induce apoptosis during infection in vivo in the PBMC subpopulations.

Intracellular survival and saprophytic growth of isolates from the Burkholderia cepacia complex in free-living amoebae.

Members of the taxonomically diverse Burkholderia cepacia complex have become a major health risk for patients with cystic fibrosis (CF). Although patient-to-patient transmission of B. cepacia strains has been well-documented, very little is known about possible vehicles of transmission and reservoirs for these micro-organisms. In this work, it is shown that strains of the B. cepacia complex can survive within different isolates of the genus Acanthamoeba. Trophozoites containing bacteria developed profuse cytoplasmic vacuolization. Vacuolization was not detected in trophozoites infected with live Escherichia coli or heat-killed B. cepacia, or by incubation of trophozoites with filter-sterilized culture supernatants, indicating that metabolically active intracellular bacteria are required for the formation of vacuoles. Experiments with two different B. cepacia strains and two different Acanthamoeba isolates revealed that bacteria display a low level of intracellular replication approximately 72-96 h following infection. In contrast, extracellular bacteria multiplied efficiently on by-products released by amoebae. The findings suggest that amoebae may be a reservoir for B. cepacia and possibly a vehicle for transmission of this opportunistic pathogen among CF patients.