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.

Free-Living Amoebae as Hosts for and Vectors of Intracellular Microorganisms with Public Health Significance.

Free-living amoebae (FLA) are parasites within both humans and animals causing a wide range of symptoms and act as hosts of, and vehicles for phylogenetically diverse microorganisms, called endocytobionts. The interaction of the FLA with sympatric microorganisms leads to an exceptional diversity within FLA. Some of these bacteria, viruses, and even eukaryotes, can live and replicate intracellularly within the FLA. This relationship provides protection to the microorganisms from external interventions and a dispersal mechanism across various habitats. Among those intracellularly-replicating or -residing organisms there are obligate and facultative pathogenic microorganisms affecting the health of humans or animals and are therefore of interest to Public Health Authorities. Mimiviruses, Pandoraviruses, and Pithoviruses are examples for interesting viral endocytobionts within FLA. Future research is expected to reveal further endocytobionts within free-living amoebae and other protozoa through co-cultivation studies, genomic, transcriptomic, and proteomic analyses.

Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones.

Legionella species are ubiquitous, waterborne bacteria that thrive in numerous ecological niches. Yet, in contrast to many other environmental bacteria, Legionella spp. are also able to grow intracellularly in predatory protozoa. This feature mainly accounts for the pathogenicity of Legionella pneumophila, which causes the majority of clinical cases of a severe pneumonia termed Legionnaires' disease. The pathomechanism underlying L. pneumophila infection is based on macrophage resistance, which in turn is largely defined by the opportunistic pathogen's resistance towards amoebae. L. pneumophila replicates in macrophages or amoebae in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial intracellular multiplication/defective for organelle trafficking (Icm/Dot) type IV secretion system and involves a plethora of translocated effector proteins, which subvert pivotal processes in the host cell. Of the ca. 300 different experimentally validated Icm/Dot substrates, about 50 have been studied and attributed a cellular function to date. The versatility and ingenuity of these effectors' mode of actions is striking. In this review, we summarize insight into the cellular functions and biochemical activities of well-characterized L. pneumophila effector proteins and the host pathways they target. Recent studies not only substantially increased our knowledge about pathogen-host interactions, but also shed light on novel biological mechanisms.

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.

Relevance of free-living amoebae as hosts for phylogenetically diverse microorganisms.

In addition to their role as parasites, free-living amoebae (FLA) can act as hosts of and vehicles for phylogentically diverse microorganisms while some of them replicate intracellularly. These microorganisms are adapted to the intracellular conditions in the amoeba, find suitable conditions and protection from negative environmental influences and take advantage of the dispersal in the environment by their amoebic host. It is expedient to call these organisms "endocytobionts", at least during the initial steps of any studies. By doing so, it is not necessary to go into potential characteristics of these relationships such as parasitism, phoresy, zoochory, or mutualism at an early stage of study. Among those organisms resisting the lysis within their amoebic host, there are obligate and facultative pathogenic microorganisms affecting the health of humans or animals. FLA-endocytobiont relationships are not only important for the tenacity of the involved microorganisms. Especially if FLA are present in biofilms and there are close ties with many other microorganisms, the odds are for some of these microorganisms to develop human pathogenic properties. Here, the amoebic passage seems to be a prerequisite for the development of virulence factors and it may have an impact on evolutionary processes.

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.

Acanthamoeba spp. en piscinas cubiertas de la ciudad de Bahía Blanca, provincia de Buenos Aires, Argentina / Acanthamoeba spp. in indoor swimming pools in Bahía Blanca city, Buenos Aires province, Argentina

Acanthamoeba ha sido aislada de numerosos hábitats incluyendo piletas de natación. Estudiar su distribución es importante ya que algunas especies causan enfermedad en el hombre. El objetivo del presente trabajo fue la búsqueda, aislamiento y caracterización de protozoos del género Acanthamoeba en piscinas cubiertas de la ciudad de Bahía Blanca, Argentina, en las cuatro estaciones del año durante 2007-2008. Se estudiaron 7 piscinas y en cada una se tomaron cuatro muestras: fondo, superficie, raspado de pared y para análisis bacteriológico. Las muestras se analizaron por observación directa y por cultivo en agar no nutritivo a 37 °C y a 42 °C. La identificación genérica se realizó de acuerdo con las características morfológicas de quistes y trofozoítos y para identificar Naegleria se realizó la prueba de transformación ameboflagelar. En 5 de las 7 piscinas se aislaron amebas de vida libre al menos en una época del año. La prueba de transformación ameboflagelar resultó negativa, descartando al género Naegleria. Todos los aislamientos correspondieron al género Acanthamoeba Grupos II y III de Pussard y Pons. Si bien la eliminación de Acanthamoeba en las aguas de natatorios resulta muy difícil por tratarse de un protozoario ubicuo y sumamente resistente a los desinfectantes comúnmente utilizados, se recomienda una limpieza profunda de las piscinas que minimice los riesgos de infección.

Acanthamoeba spp. has been isolated from many habitats, including swimming pools. Investigations on its distribution are relevant because many of its species cause human diseases. The aim of the present work was to investigate, isolate and characterize protozoan of the genus Acanthamoeba from indoor swimming pools in Bahía Blanca, Argentina, in the four seasons, during the 2007-2008 period. Seven pools were studied and samples were collected from the bottom, surface and wall by scraping them. Besides, samples for bacteriological analysis were taken from each pool. The samples were analyzed by direct observation and by culturing on non nutritive agar at 37 °C and at 42 °C. The generic identification was performed according to the morphologic characteristics of cysts and trofozoites, while the amoebo-flagellate transformation test was carried out to identify Naegleria. Free-living amoebas were isolated from five of the seven swimming pools examinated, in at least one season. Naegleria genus was not found as the amoebo-flagellate transformation tests were negative in all samples. All the isolations corresponded to the genus Acanthamoeba belonging to Pussard & Pons morphological Groups II and III. Although elimination of Acanthamoeba from the water is difficult because it is a ubiquitous protozoan. which is highly resistant to the commonly used disinfectants, a thorough cleaning of the pools to minimize the risks of infection is recommended.

Toxoplasma gondii: uptake and survival of oocysts in free-living amoebae.

Waterborne transmission of the oocyst stage of Toxoplasma gondii can cause outbreaks of clinical toxoplasmosis in humans and infection of marine mammals. In water-related environments and soil, free-living amoebae are considered potential carriers of various pathogens, but knowledge on interactions with parasitic protozoa remains elusive. In the present study, we assessed whether the free-living Acanthamoeba castellanii, due to its phagocytic activity, can interact with T. gondii oocysts. We report that amoebae can internalize T. gondii oocysts by active uptake. Intracellular oocysts in amoebae rarely underwent phagocytic lysis, retained viability and established infection in mice. Interaction of T. gondii with amoebae did not reduce the infectivity and pathogenicity of oocysts even after prolonged co-cultivation. Our results show that uptake of oocysts by A. castellanii does not restrain the transmission of T. gondii in a murine infection model.

Amibas con parasitismo facultativo en aguas termales de Trujillo, Venezuela / Amoebae with parasitic in termals water Trujillo, Venezuela

Experimentalmente se demuestra la presencia de amibas de vida libre potencialmente patógenas, en aguas termales de uso público en tres localidades de los didtritos Valera y Betijoque del Estado Trujillo, Venezuela

Acanthamoeba culbertsoni: sensibilidade aos diferentes métodos de desinfecçäo de lentes de contato hidrofílicas / Acanthamoeba culbertsoni: susceptibility to soft contact lens disinfection systems

Desde 1973, úlceras de córnea por Acanthamoeba spp têm sido descritas em número crescente e muitas vezes relacionados com o uso de lentes de contacto hidrofílicas (L.C.H.). Neste estudo foi testada a sensibilidade da Acanthamoeba culbertsoni aos diferentes métodos de desinfecçäo química (Hidrocare - Trietanol alquilamonio miramide e Flexcare - Clorixidine) e térmica (fervura simples por dez minutos com flutuador e aparelhos de asseptizaçäo - Contact Lens e Hydroset). Foram utilizadas 36 L.C.H. novas Soflens (Baush & Lomb) e 36 L.C.H. usadas Hydrosol (Solótica) que foram contaminadas com cistos e trofozoítos de Acanthamoeba culbertsoni por 12 horas. Após este período as lentes foram cortadas ao meio, ficando uma das metades semeada para controle e a outra submetida aos diferentes métodos de desinfecçäo, salientando-se que as lentes foram lavadas previamente com xampú apropriado por 20 segundos e enxaguadas com 5 ml de soluçäo fisiológica näo preservadas. As placas semeadas com as lentes eram observadas diariamente sob microspia óptica para detectar a passagem da forma cística para a trofozoítica, o que traduziria a viabilidade do parasita. Todos os resultados foram negativos. Todos os métodos testados se mostraram, eficientes. Concluímos que os métodos de desinfecçäo testados, desde que corretamente utilizados, säo eficazes na eliminaçäo da Acanthamoeba culbertsoni

[Zoophagous fungi]. / Zoophage Pilze.

Fungi which kill microscopic animals of different systematic origin by capturing, invading, and digesting them are described. The focal point is nematode destroying fungi, as being the most abundant, most often isolated and most intensively studied. Their ecology and their probable role in the control of nematodes are discussed.

Amebiasis fulminante / Invasive amebiasis

La amebiasis constituye una parasitosis de alta prevalencia en nuestro país; una de sus presentaciones clínicas es la colitis amebiana fulminante, con una mortalidad de 100% en casos no tratado quirúrgicamente y de más de 50% en los casos con tratamiento quirúrgico. Se analiza la casuística de colitis aguda necrosante amebiana en el Hospital Eugenio Espejo, en un período de cinco años (1980-1985) encontrándose una mortalidad del 100% en nueve casos operados y tres sin cirugía. Sólo en dos casos el coproparasitario fue positivo para ameba histolítica; el síntomas más común fue síndrome disenterico. Al examen patológico se observó vulcus confluentes especialmente en ciego y colon. En todos los casos las lesiones abarcaron toda la extensión del colon y se encontró mayor compromiso en los casos tratados quirúrgicamente (AU)nte amebiana en el Hospital Eugenio Espejo, en un período de cinco años (1980-1985) encontrándose una mortalidad del 100% en nueve casos operados y tres sin cirugía. Sólo en dos casos el coproparasitario fue positivo para ameba histolítica; el síntomas más común fue síndrome disenterico. Al examen patológico se observó vulcus confluentes especialmente en ciego y colon. En todos los casos las lesiones abarcaron toda la extensión del colon y se encontró mayor compromiso en los casos tratados quirúrgicamente

[Acanthamoebae, Naegleria and other free-living Amoebae in cooling and rinsing water of dental treatment units]. / Acanthamoeben, Naeglerien und andere freilebende Amöben in Kühl- und Spülwasser von Zahnbehandlungseinheiten.

215 water samples were taken from 49 dental treatment units and investigated for the existence of free-living amoebae. In all water-carrying systems of the dental treatment units it was possible to verify the incidence of one or more amoeba species. In 8.2 per cent of the units Naegleria species was found and in 12.2 per cent Acanthamoeba species was present. Seven Naegleria and six Acanthamoeba strains (2 A. castellanii and 4 A. polyphaga) were isolated. From samples originating from 12 dental treatment units (DTU) another 42 amoeba strains were isolated which consisted of 14 different species within 9 classes. Among them Vannella mira (in 19 per cent of samples) and Hartmannella vermiformis (10.6 per cent) were found to be the most frequent species, followed by H. cantabrigensis (9.5 per cent), V. platypodia, Platyamoeba stenopodia and V. simplex (7.1 per cent each). In 10 per cent of samples monotrichous and bitrichous flagellates such as the Bodo species were found, whereas two samples contained ova, larvae and adult free-living nematodes. Among the isolated Naegleria strains no thermophilic strain was present. Consequently they belong to the N. gruberi complex. Among the Acanthamoebae five of the six strains were thermophilic. All strains were investigated for pathogenic properties by means of the mice inoculation test. Two strains proved pathogenic - it was possible to isolate them from the brain and lung of dead mice. Another two strains proved to have invasive properties because they were isolated from the brain of infected animals; however, they did not give rise to disease or death of the respective animals. Supplementary microbiological tests demonstrated the existence of bacteria and fungi in 84 per cent of dental treatment units. Pseudomonas spec. were detected in 75% of dental units, Serratia marcescens in 2% and fungi in nearly 3%. 58.3% of all water samples contained total germ counts of more than 100/ml.