Exploiting the architecture and the features of the microsporidian genomes to investigate diversity and impact of these parasites on ecosystems.

Fungal species play extremely important roles in ecosystems. Clustered at the base of the fungal kingdom are Microsporidia, a group of obligate intracellular eukaryotes infecting multiple animal lineages. Because of their large host spectrum and their implications in host population regulation, they influence food webs, and accordingly, ecosystem structure and function. Unfortunately, their ecological role is not well understood. Present also as highly resistant spores in the environment, their characterisation requires special attention. Different techniques based on direct isolation and/or molecular approaches can be considered to elucidate their role in the ecosystems, but integrating environmental and genomic data (for example, genome architecture, core genome, transcriptional and translational signals) is crucial to better understand the diversity and adaptive capacities of Microsporidia. Here, we review the current status of Microsporidia in trophic networks; the various genomics tools that could be used to ensure identification and evaluate diversity and abundance of these organisms; and how these tools could be used to explore the microsporidian life cycle in different environments. Our understanding of the evolution of these widespread parasites is currently impaired by limited sampling, and we have no doubt witnessed but a small subset of their diversity.

Functional characteristics of a reverse transcriptase encoded by an endogenous retrovirus from Drosophila melanogaster.

ZAM is an LTR-retrotransposon from Drosophila melanogaster that belongs to the genus errantivirus, viruses similar in structure and replication cycle to vertebrate retroviruses. A key component to its lifecycle is its reverse transcriptase which copies single-stranded genomic RNA into DNA. Here, we provide a detailed characterization of the enzymatic activities of the reverse transcriptase encoded by ZAM. When expressed in vitro, the reverse transcriptase domain associated with the RNase H domain encoded by the ZAM pol gene forms homodimers and displays an efficient RNA-dependent DNA-polymerase activity. It requires either Mg2+ or Mn2+ divalent cations, and works in basic pH, with a peak at around pH9. The so-called [RT-RH] polypeptide displays an optimal activity at 22 degrees C, a property that makes it well-adapted to the temperature of its host. This study contributes to our understanding of the general structures and functions of retroviral reverse transcriptases, a necessary process in the search for novel inhibitors.

Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi.

Microsporidia are obligate intracellular parasites infesting many animal groups. Lacking mitochondria and peroxysomes, these unicellular eukaryotes were first considered a deeply branching protist lineage that diverged before the endosymbiotic event that led to mitochondria. The discovery of a gene for a mitochondrial-type chaperone combined with molecular phylogenetic data later implied that microsporidia are atypical fungi that lost mitochondria during evolution. Here we report the DNA sequences of the 11 chromosomes of the approximately 2.9-megabase (Mb) genome of Encephalitozoon cuniculi (1,997 potential protein-coding genes). Genome compaction is reflected by reduced intergenic spacers and by the shortness of most putative proteins relative to their eukaryote orthologues. The strong host dependence is illustrated by the lack of genes for some biosynthetic pathways and for the tricarboxylic acid cycle. Phylogenetic analysis lends substantial credit to the fungal affiliation of microsporidia. Because the E. cuniculi genome contains genes related to some mitochondrial functions (for example, Fe-S cluster assembly), we hypothesize that microsporidia have retained a mitochondrion-derived organelle.

Microsporidian invasion apparatus: identification of a novel polar tube protein and evidence for clustering of ptp1 and ptp2 genes in three Encephalitozoon species.

Microsporidia are unicellular eukaryotes occurring as obligate intracellular parasites which produce resistant spores. A unique motile process is represented by the sudden extrusion of the sporal polar tube for initiating entry of the parasite into a new host cell. The complete sequence of an acidic proline-rich polar tube protein (renamed PTP1) has been previously reported for Encephalitozoon cuniculi and E. hellem. Our immunological investigations provided evidence for an additional PTP in E. cuniculi, termed PTP2. The corresponding gene was sequenced and then expressed in Escherichia coli. As expected, mouse antibodies raised against the recombinant protein reacted specifically with the polar tube. The single copy ptp1 and ptp2 genes of E. cuniculi were tandemly arranged on chromosome VI. Polyadenylation of the mRNAs was demonstrated. Identification and sequencing of homologous genes in the two other human-infecting Encephalitozoon species (ptp2 in E. hellem and ptp1 and ptp2 in E. intestinalis) were facilitated by conserved gene clustering. PTP2 appears as a novel structural protein (30 kDa) with a basic lysine-rich core and an acidic tail. Unlike PTP1, this protein is devoid of large tandem repeats. The interspecies conservation of cysteine residues supports a major role of disulfide bridges in polar tube assembly. The two PTPs should serve as both molecular markers of spore differentiation and diagnostic tools.

The identification of rRNA maturation sites in the microsporidian Encephalitozoon cuniculi argues against the full excision of presumed ITS1 sequence.

In Encephalitozoon cuniculi like in other microsporidia, the primary transcript for SSU and LSU rRNAs includes only one internal transcribed spacer (ITS1) which separates SSU rRNA from the 5.8S region associated with LSU rRNA. The extraction of total RNA from E. cuniculi-infected MRC5 cells using a hot phenol/chloroform procedure enabled us to perform primer extension and S1 nuclease protection experiments in the aim of identifying rRNA maturation sites. Our data support a simple processing (four cleavage sites) with elimination of only nine nucleotides between SSU and LSU rRNA regions. Most of the presumed ITS1 sequence characterized by strain-dependent polymorphism therefore remains linked to SSU rRNA 3' end. A new secondary structure for the sixth domain of E. cuniculi LSU rRNA is proposed following the identification of its 3' terminus.

Phylogenetic analysis of the small subunit ribosomal RNA of Marteilia refringens validates the existence of phylum Paramyxea (Desportes and Perkins, 1990).

Marteilia refringens is recognized as one of the most significant pathogens of bivalve molluscs. The nucleotide sequence of the small subunit ribosomal RNA gene of Marteilia refringens is used to elucidate the phylogenetic position of the phylum Paramyxea. Genomic DNA was extracted from sporangia of Marteilia, purified from infected blue mussels, Mytilus edulis, and flat oysters, Ostrea edulis. The sequences obtained from Marteilia species purified from both oysters and mussels were identical. The sequence identity was confirmed by in situ hybridization using a DNA probe targeted to a variable region of the ribosomal DNA. The small subunit ribosomal RNA gene sequence of M. refringens is very different from all known sequences of eukaryotic organisms, including those of myxosporeans and haplosporeans. Therefore, the phylum Paramyxea should continue to be recognized as an independent eukaryotic phylum.

Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core.

Microsporidia are eukaryotic parasites lacking mitochondria, the ribosomes of which present prokaryote-like features. In order to better understand the structural evolution of rRNA molecules in microsporidia, the 5S and rDNA genes were investigated in Encephalitozoon cuniculi . The genes are not in close proximity. Non-tandemly arranged rDNA units are on every one of the 11 chromosomes. Such a dispersion is also shown in two other Encephalitozoon species. Sequencing of the 5S rRNA coding region reveals a 120 nt long RNA which folds according to the eukaryotic consensus structural shape. In contrast, the LSU rRNA molecule is greatly reduced in length (2487 nt). This dramatic shortening is essentially due to truncation of divergent domains, most of them being removed. Most variable stems of the conserved core are also deleted, reducing the LSU rRNA to only those structural features preserved in all living cells. This suggests that the E.cuniculi LSU rRNA performs only the basic mechanisms of translation. LSU rRNA phylogenetic analysis with the BASEML program favours a relatively recent origin of the fast evolving microsporidian lineage. Therefore, the prokaryote-like ribosomal features, such as the absence of ITS2, may be derived rather than primitive characters.

Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of mitochondrial evolutionary origin.

An intronless gene encoding a protein of 592 amino acid residues with similarity to 70-kDa heat shock proteins (HSP70s) has been cloned and sequenced from the amitochondrial protist Encephalitozoon cuniculi (phylum Microsporidia). Southern blot analyses show the presence of a single gene copy located on chromosome XI. The encoded protein exhibits an N-terminal hydrophobic leader sequence and two motifs shared by proteobacterial and mitochondrially expressed HSP70 homologs. Phylogenetic analysis using maximum likelihood and evolutionary distances place the E. cuniculi sequence in the cluster of mitochondrially expressed HSP70s, with a higher evolutionary rate than those of homologous sequences. Similar results were obtained after cloning a fragment of the homologous gene in the closely related species E. hellem. The presence of a nuclear targeting signal-like sequence supports a role of the Encephalitozoon HSP70 as a molecular chaperone of nuclear proteins. No evidence for cytosolic or endoplasmic reticulum forms of HSP70 was obtained through PCR amplification. These data suggest that Encephalitozoon species have evolved from an ancestor bearing mitochondria, which is in disagreement with the postulated presymbiotic origin of Microsporidia. The specific role and intracellular localization of the mitochondrial HSP70-like protein remain to be elucidated.

Mapping of repetitive and non-repetitive DNA probes to chromosomes of the microsporidian Encephalitozoon cuniculi.

The molecular karyotype of a murine isolate of Encephalitozoon cuniculi, a microsporidian with a wide range of mammalian hosts, comprises eleven chromosomes ranging in size between 217 and 315 kb. To determine specific chromosomal markers, a partial genomic library was constructed and cloned DNA fragments were hybridized to chromosomal bands separated by pulsed-field gel electrophoresis. Most probes were assigned to single chromosomes, indicating prevalence of low-copy number nucleotide sequences within the very small genome of E. cuniculi (2.9 Mb). A few probes were shown to hybridize to all chromosomes. These repetitive DNA fragments corresponded to either rRNA genes or some non-coding regions whose sequences were characterized by short micro- and minisatellites. The chromosomal locations of beta-tubulin genes and six newly identified protein-encoding genes were determined. Genes encoding dihydrofolate reductase, thymidylate synthase, serine hydroxymethyl transferase, a cdc2 kinase-like protein and helicase ERCC6-like protein were each located on a single chromosome whereas genes for both beta-tubulin and aminopeptidase were on two different chromosomes. The mapping will serve as a reference for further analysis of intraspecific karyotype polymorphism in different isolates from different host species.