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Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Kinderspitalgasse 15, 1095 Vienna, Austria
Rolf Michel
Johannes Lugauer
Central Institute of the Federal Armed Forces Medical Services, Department of Parasitology, Andernacher Straße 100, 56070 Koblenz, Germany
Claudia Wylezich
Department of Ecology and Limnology, Institute of Zoology, University of Cologne, Weyertal 119, 50923 Cologne, Germany
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ABSTRACT |
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Free-living amoebae can serve a great variety of organisms, predominantly bacteria but to a certain extent also fungi, as a suitable host supplying them with nutrients and protecting them from adverse environmental conditions. In the current study 18S rDNA sequencing was performed to identify a fungal parasite in a Thecamoeba quadrilineata isolate. This parasite morphologically resembled Cochlonema euryblastum, a member of the order Zoopagales, which comprises parasitic species on fungi and invertebrates. Sequence analysis corroborated the morphological identification and the fungal parasite clearly can be assigned to the Zoopagales. Phylogenetic analysis revealed C. euryblastum clustering with two representatives of the mycoparasitic family Piptocephalidaceae. This zooparasitic-mycoparasitic clade represents a sister group of a clade including another member of the Piptocephalidaceae and two other zooparasitic families. Thus, the addition of C. euryblastum to the zoopagalean tree further confirms the finding that molecular data do not support the traditional classification of the Zoopageles.
Key words: 18S rDNA, endocytobiont, free-living amoeba, parasite, Zoopagales, Zygomycota
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Based on their locomotive morphology they are divided into smooth, rough and wrinkled groups. Species of Thecamoeba are distributed widely in freshwater, can occur in saltwater and are among the largest protozoa in soil and mosses. Apparently no cysts are formed, despite their occurrence in habitats subject to drying (Page 1977, 1991).
FLA are known to serve a great variety of pathogenic and nonpathogenic bacteria as vehicles or hosts. In the past years particularly Acanthamoeba spp. harboring bacteria, such as Legionella pneumophila, Mycobacterium spp. or Chlamydia-like bacteria and protecting them against unfavourable environmental conditions, have been in the center of interest (Greub and Raoult 2004). However, other FLA are suitable hosts, such as Hartmannella vermiformis, Balamuthia mandrillaris and Naegleria spp. (Donlan et al 2005; Hoffmann and Michel 2001; Horn et al 2000; Michel et 1999, 2005; Shadrach et al 2005; Walochnik et al 2005).
Interactions of FLA with fungi have been described less frequently, nevertheless fungi are known to parasitize in FLA, usually killing their host. In 1879 Leidy described an amoeba with appendices resembling strings of sausages, which in fact belonged to the genus Mayorella, parasitized by Amoebophilus simplex (Brief 2005). Charles Drechsler described various Phytomyceta and Zoopagaceae parasitizing and killing terricolous amoebae (Drechsler 1938, 1942). Furthermore he identified a higher fungus belonging to the Basidiomycota as a parasite of Amoeba terricola (Drechsler 1969). More recently, a natural infection of Vannella spp. with a microsporidian species, growing and multiplying in its host, has been reported (Hoffman et al 1998). In an interaction of Acanthamoeba spp. with Cryptococcus neoformans the amoeba was shown to represent not only a nourishing host but to prime the fungus for an intracellular pathogenic strategy in macrophages (Steenbergen et al 2001).
In 1998 Michel reported on a yeast-like endocytobiont in Thecamoeba similis and in 1999 he described a fungus-like endocytobiont in a Thecamoeba quadrilineata isolate, which originally had been isolated from an eaves gutter. This endocytobiont appeared as a coil-shaped structure immobilizing and killing the amoebae. This endocytobiont morphologically resembled Cochlonema euryblastum, a parasitic fungus destructive to soil amoebae described by Drechsler in 1942 (Michel and Wylezich 2005).
The aim of the current study was to proof the morphological identification of this fungal parasite in T. quadrilineata by employing 18S rDNA sequencing and to reveal the position of this fungus within the order Zoopagales. To date no sequence data of the whole family Cochlonemataceae are available. Consequently 18S rDNA sequences of several other zoopagales were used for the construction of a phylogenetic tree.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Culture of amoebae/fungi.— – Amoebae were cultivated on nonnutrient agar plates, containing 0.1% sea salt and covered with a lawn of Escherichia coli. New subcultures of amoebae were prepared on a weekly basis. Because the fungal infection was lethal to the amoebae after a certain period of time, a parallel culture without fungus was installed to maintain the amoebal strain. This was achieved by adding an antimycotics suspension to the plate cultures until the amoebae were free of fungi. In addition, to maintain the fungus noninfected amoebae cultures were reinfected with the fungus when necessary. Re-infection was achieved by excising a piece of agar from a plate covered with conidia and carefully sliding the agar piece over amoebic cultures. Approximately 4 d after infection the majority of amoebae were dead and a large number of coil-shaped fungi surrounded by single conidia were observable. Amoebae were considered dead when the cell membrane was disrupted. About 1 wk after infection amoebae/fungi were harvested for DNA isolation.
Electron microscopy.— – Amoebal host cells were harvested 3 d postinfection. The cell suspension was centrifuged at 600x g for 10 min. The resulting pellet was fixed in 3% glutaraldehyde (1 h), transferred to 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide and embedded in Spurr resin. Sections were stained with uranyl acetate and Reynold’s lead citrate and examined with a Leo EM 910 electron microscope.
Isolation of DNA.— –
Uninfected and infected amoebae were harvested directly from the plate cultures with sterile cotton-tipped applicators and resuspended in 200 µL sterile distilled water. Cells were disrupted by multiple freeze-thawing in liquid nitrogen before whole-cell DNA was isolated by a modified UNSET procedure (Hugo et al 1992). In brief, 500 µL of UNSET lysis buffer was added to the amoebae and fungi suspended in 200 µL distilled water. Then the suspension was overlaid with 700 µL of phenol-chloroform-isoamyl-alcohol (PCI) and shaken gently overnight. The suspension was centrifuged at 3000x g for 10 min, and the upper, aqueous phase was transferred to a new tube. PCI extraction was repeated two times for 10 min each time. Nucleic acids were precipitated by ethanol (overnight at –20 C), pelleted at 12 000x g for 30 min at 4 C, washed in 70% ethanol, air dried, and resuspended in 30 µL of sterile distilled water.
DNA Amplification.— –
For DNA amplification of the 18S rRNA gene, primers SSU1 (5'-CGACTGGTTGATCCTGCCAGTAG-3') and SSU2 (5'-GTGAACCTGCAGAAGGATCAGGA-3'), complementary to the 5'- and the 3'-end of the gene, respectively (Gast et al 1996), and six internal primers (Walochnik et al 2004), P1fw (5'-CAAGTCTG GTGCCAGCAGC-3'), P1rev (5'-GCTGCTGGCACCAGACTTG-3'), P2fw (5'-GATCAGATACCGTCGTAGTC-3'), P2rev (5'-GACTACGACGGTATCTGATC-3'), P3fw (5'-CAGGTCTGTGATGCCCTTAG-3'), P3rev (5'-CTAAGGGCATCACAGACCTG-3') were used, to obtain four gene fragments of 350–530 bp for sequencing. Primer locations were 531 (P1fw, P1rev), 995 (P2fw, P2rev) and 1343 bps (P3fw, P3rev) downstream of the 3' end of primer SSU1. We used 1 µL whole-cell DNA and a standard amplification program (30 cycles of 94 C for 1 min, 56 C for 2 min and 72 C for 3 min). Amplification of the 18S rRNA gene fragments was viewed by ethidium bromide staining in a 2% agarose gel electrophoresis. Amplification with two internal primer pairs (P1fw/P2rev, P2fw/P2rev) produced two PCR products, one Thecamoeba band and one Cochlonema band (FIG. 3). According to sequence data Thecamoeba fragments are supposed to be about 80 bp longer than zoopagalean fragments. Bands were separated on a 2% agarose gel and smaller bands were excised. Amplification of flanking fragments produced only one band, which were more likely to represent fungal bands considering the approximate size. A control PCR of uninfected Thecamoeba quadrilineata cultures was performed to rule out other fungal contaminations. Here amplification of the flanking fragments did not lead to a PCR product and amplification of the P1fw/P2rev and P2fw/P2rev fragments produced only one band identical in size to the bigger band produced for infected amoebae. In addition this was confirmed by sequencing and subsequent NCBI-BLAST search, showing highest identities with published fungal sequences.
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Alignment and Cluster analysis.— –
Multiple sequence alignment was performed by subsequent pairwise alignment with the Clustal X application (Thomson et al 1997). The alignment was imported into the GENEDOC sequence editor (Nicholas et al 1997) and manually refined to obtain a better consensus. For cluster analysis the 18S rDNA sequence of C. euryblastum was aligned with published zoopagalean sequences (Tanabe et al 2000). Primer sites, unique gaps, insertions and ambiguously aligned sites were excluded from the analysis. The dataset for phylogenetic analyses consisted of ~1480 aligned sites. Two representatives of the Kickxellales (AF007542
[GenBank]
, AF007543
[GenBank]
) were chosen as outgroup. Cluster analysis was performed by constructing a cladogram with the PHYLIP package (Felsenstein 1989). Analyses were performed with different evolutionary models including neighbor joining analysis of Kimura two-parameter distance estimates, maximum parsimony and maximum likelihood. The confidence of the branching order was assessed by the generation of 1000 bootstrap replicates for all three methods. Consensus trees were generated from the resulting trees with CONSENSE and prepared as a figure with TreeView (Page 1996). Sequence data was deposited in GenBank and is available at accession number No. DQ520640
[GenBank]
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Ultrathin sections of infected T. quadrilineata show the different developmental stages of C. euryblastum (FIG. 2A–D). Conidia were found outside the amoebae as well as within the cytoplasm of the host after ingestion. Young and intermediate stages (FIG. 2A–C) already contained nuclei and electron transparent vacuoles. Conidia germinated to produce coiled thalli as mature stages (FIG. 2D). When a thallus attained full size it began to produce conidiogenous hyphae, which initially were not segregated into conidia but which broke through the protoplasmic pellicle of the amoeba and then segregated after a considerable period of time in the environment. Of note, spores were not observed in any of the cultures.
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) with a GC-content of 49.35%. PCR products of Cochlonema euryblastum and Thecamoeba quadrilineata including a control PCR with uninfected amoebae is shown (FIG. 3). The sequence displayed highest similarity to Kuzuhaea moniliformis, a zoopagalean fungus. The calculated sequence similarity between K. moniliformis and C. euryblastum is 84.35%.
The phylogenetic analysis with three different tree-building programs resulted in homologous consensus trees with bootstrap supports of > 50% for all branches (FIG. 4). We have chosen Spiromyces aspiralis and Spiromyces minutus as outgroup because these two isolates have been shown to be a closely related group of the order Zoopagales based on prior 18S rDNA analyses. A previously detected mycoparasitic-zooparasitic clade of Syncephalis depressa, Thamnocephalis sphaerospora and Rhopalomyces elegans (Tanabe et al 2000) forms a sister group with a cluster of Piptocephalis corymbifera, Kuzuhaea moniliformis and C. euryblastum, also representing a mycoparasitic-zooparasitic clade in this study. These groupings are strongly supported by high bootstrap values. Zoophagus insidians exhibits the highest substitution rate within order Zoopagales reflected by its external position.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) with the only difference being that the conidia of C. euryblastum have a smooth surface compared to the conidia of C. odontosperma showing protuberances at the cell surface which appear as bumps on the conidia under the light microscope. Unfortunately no sequence data of C. odontosperma are available to date. Another interesting observation was that spores as described for C. euryblastum by Michel and Wylezich (2005) could not be observed in the present study. However this might be the result of parasite-host interactions. Such interactions between amoebae and their endocytobionts, bacteria as well as fungi, are well known. On one hand, amoebae can alter the shape and even the virulence of their endocytobionts and, on the other hand, endocytobionts can alter the morphological and physiological capabilities of their host amoebae (Barker et al 1993, Cirillo et al 1997, Steenbergen et al 2001, Walochnik et al 2005). The hypothesis of host-parasite interactions in the current case is corroborated by the conspicuous accumulation of mitochondria in the area between the two parasites (FIG. 2D), indicating high metabolism in this area.
The order Zoopagales (Zygomycota, Zygomycetes) comprises five families, with all members being obligatory parasites of other fungi or microscopic animals. Ectoparasitic and predacious species are characterized by the development of haustoria invading the host tissue, while endoparasitic species are characterized by an internal thallus without haustorium development. In addition to the family Cochlonemataceae, which represents parasites of different amoebae and nematodes, the Zoopagales include the Zoopagaceae, predators of nematodes, amoebae and rotifers, the Helicocephalidaceae, parasitic on nematode eggs, and the Piptocephalidaceae and Sigmoideomycetaceae, both mycoparasites. Cochlonemataceae either produce an internal thallus without development of a haustorium or an external thallus with haustoria formation only within the host (Benny et al 1992, Hawksworth et al 1995).
C. euryblastum was first described by Charles Drechsler (1942), who characterized it as a parasitic fungus destructive to soil amoebae. C. euryblastum conidia are ingested by the amoebic host by phagocytosis and germinate to produce a longish-ovoid thallus in the cytoplasm, which develops into a characteristic coil-shaped thallus. At that stage amoebae become rounded and immotile. The thallus subsequently produces hyphae that branch several times after growing through the host pellicle. The conidiogenous hyphae become fragmented by cross wall formation resulting in a chain of conidia. The fragmented hyphae are fragile and release single conidia, which can be observed near the dead amoebae (Drechsler 1942, Michel 1999, Saikawa and Sato 1991). Cochlonemataceae and Zoopagaceae originally were considered to be one family, until Duddington (1973) transferred the parasitic forms to the Cochlonemataceae and left the predacious taxa in the Zoopagaceae. In 1979 Benjamin added the Piptocephalidaceae and the Helicocephalidaceae to this order. The Sigmoideomycetaceae were transferred from the Mucorales to the Zoopagales in 1995 (Hawksworth et al 1995), which was confirmed in 2000, when Chien reported on the formation of haustoria within this family.
To date little sequence information is available from this order and there are no sequences of the family Cochlonemataceae available, mainly due to difficulties in culturing these fungi. Tanabe et al (2000) investigated the molecular phylogeny of parasitic zygomycota and sequenced among others six zoopagalean fungi. In their study a monophyletic cluster of Zoopagales, Kickxellaes and Harpellales was supported. Furthermore they identified a monophyletic mycoparasitic-zooparasitic clade within the Zoopagales, consisting of Syncephalis depressa, traditionally classified as Piptocephalidaceae, Rhopalomyces elegans (Helicocephalidaceae) and Thamnocephalis sphaerospora (Sigmoideeomycetaceae). This clade formed a sister group of a clade comprising two Piptocephalidaceae isolates. These findings interfered with traditional classification,
Phylogentic analyses in the current study are concordant with Tanabe’s observations with S. depressa clustering with R. elegans and T. sphaerospora. Again two members of the Piptocephalidaceae, namely Kuzuhaea moniliformis and Piptocephalis corymbifera, form a sister group but now including the zooparasitic C. euryblastum. Furthermore, the previously observed external position of Zoophagus insidians was confirmed with all tree-building methods.
It has been shown that the clade of S. depressa, T. sphaerospora and R. elegans is not the only zooparasitic-mycoparasitic clade within the Zoopagales but that K. moniliforms, P. corymbifera (Piptocephalidaceae) and C. euryblastum also represent a clade of zooparasitic and mycoparasitic fungi. Adding C. euryblastum to the phylogenetic tree corroborates Tanabe’s findings that the molecular phylogeny of the order Zoopagales based on the 18S rDNA does not correlate with the traditional classification. It was demonstrated that S. depressa not only clusters with members of other families but also that member of the Cochlonemataceae appears to be more closely related to members of the Piptocephalidaceae.
When constructing a second phylogenetic tree with more families of the Zygomycota, also this was concordant with Tanabe’s observations. The Zoopagales, Kickxellaes and Harpellales distinctly formed a monophyletic group supported by high bootstrap values (>70%) in all tree building methods (data not shown).
To address questions on the relationships within the order Zoopagales more sequence data clearly are required. Groupings interfering with traditional classification based on morphology (e.g. the zooparasitic and mycoparasitic clades reported in this study and in prior studies) are a major issue, as are highly divergent isolates within the order, such as Zoophagus insidians. Tanabe et al (2004) reported that RPB1 (RNA polymerase II largest subunit) analyses outperform 18S rRNA gene analyses, with RPB1 analyses basically corroborating 18S rRNA gene phylogeny and partly reflecting intra-ordinal relationships within the Zygomycota more accurately. In that study the monophyletic clade of the Zoopagales, Kickxellales and Harpellales was not supported and a closer relationship of Zoopagales with Entomophtohorales and Blastocladiales was revealed. However, in contrast to the 18S rRNA gene analyses, the Zoopagales represented a monophyletic clade in the RPB1 analysis. Nevertheless phylogenetic studies based on either of these loci leave specific questions unanswered and show several inconsistencies.
Altogether, our 18S rDNA analysis clearly corroborates the morphological identification of the fungus parasitizing an isolate of Thecamoeba quadrilineata as Cochlonema euryblastum. To our knowledge these are the first sequence data for the Cochlonemataceae and cluster analyses supported the grouping with other zoopagaleans with high bootstrap values.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: Julia.walochnic{at}meduniwien.ac.at
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LITERATURE CITED |
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