Expression of two cell wall proteins during the intracellular development of Encephalitozoon cuniculi: an immunocytochemical and in situ hybridization study with ultrathin frozen sections.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that develops asynchronously inside parasitophorous vacuoles. Spore differentiation involves the construction of a cell wall commonly divided into an outer layer (exospore) and a thicker, chitin-rich inner layer (endospore). The developmental patterns of protein deposition and mRNA expression for 2 different spore wall proteins were studied using immunocytochemical and in situ hybridization procedures with ultrathin frozen sections. The onset of deposition of an exospore-destined protein (SWP1) correlated with the formation of lamellar protuberances during meront-to-sporont conversion. No evidence for a release of SWP1 towards the parasitophorous vacuole lumen was obtained. An endospore-destined protein (EnP1) was detected early on the plasma membrane of meronts prior to extensive accumulation within the chitin-rich layer of sporoblasts. swp1 mRNA was preferentially synthesized in early sporogony while enp1 mRNA was transcribed during merogony and a large part of sporogony. The level of both mRNAs was reduced in mature spores. Considering the availability of the E. cuniculi genome sequence, the application of nucleic and/or protein probes to cryosections should facilitate the screening of various genes for stage-specific expression during microsporidian development.

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.

Molecular characteristics and physiology of microsporidia.

A survey of the molecular features of microsporidia is presented which attempts to comment on unresolved questions concerning the physiology of these amitochondrial intracellular parasites. Various transports of host-derived molecules can be predicted and trehalose appears as a potential reserve of glucose for energy metabolism. Significant insights into membrane lipids, polyamine metabolism and sporogony-specific proteins have been gained. Some species, such as Encephalitozoon cuniculi, are heterogeneous entities and harbor a small genome. Although showing a variation in genome size of 8.5-fold, microsporidia share reduced rDNA genes. Finally, data on gene organization and a possible evolutionary relationship with fungi are considered.

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 microsporidian Encephalitozoon.

Encephalitozoon cuniculi is an attractive model system for amitochondriate intracellular eukaryotic parasites. It is characterized by a very small genome (below 3 Mbp) and a unique invasion apparatus. Furthermore, as an infectious agent, it is important in human and veterinary medicine. The compactness of its genome involves the reduction of rDNA sequences as well as of some protein-coding genes and intergenic regions. Its highly differentiated apparatus to penetrate the host cell, an extrusome-like polar tube, is composed of novel proteins and may permit various pathways of infestation. Completion of the systematic E. cuniculi sequencing project should provide an important reference system for the comparative genomics of amitochondriate and mitochondriate parasites. Further analysis of orphan genes should help to identify factors that are responsible for its intracellular parasitic way of life.

Inter-strain variability of insertion/deletion events in the Encephalitozoon cuniculi genome: a comparative KARD-PFGE analysis.

We applied a two-dimensional pulsed-field gel electrophoresis procedure to the genomes of two karyotype variants assigned to two different strains of the microsporidian Encephalitozoon cuniculi, termed D (strain III) and F (strain II). Data obtained for BssHII and MluI restriction fragment length polymorphisms in each chromosome are compiled and compared to the reference strain I variant A. Six Insertion/Deletion (InDels) are found in subterminal position, some of these being characteristic of either D or F. Like in strain 1, the terminal fragments extending between each telomere and rDNA locus are conserved in length for each chromosome. They are however smaller than in reference variant. This size reduction is estimated to be 2.5 kbp for the strain III isolate and 3.5 kbp for the strain II isolate. We hypothesize that for the three E. cuniculi strains, all chromosome extremities are prone to a constant process of sequence homogenization through mitotic recombination between conserved regions.

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.

Towards the minimal eukaryotic parasitic genome.

Microsporidia are well-known to infect immunocompromised patients and are also responsible for clinical syndromes in immunocompetent individuals. In recent years, evidence has been obtained in support of a very close relationship between Microsporidia and Fungi. In some species, the compaction of the genome and genes is remarkable. Thus, a systematic sequencing project has been initiated for the 2.9 Mbp genome of Encephalitozoon cuniculi, which will be useful for future comparative genomic studies.

Occurence of subtelomeric rearrangements in the genome of the microsporidian parasite Encephalitozoon cuniculi, as revealed by a new fingerprinting procedure based on two-dimensional pulsed field gel electrophoresis.

In Microsporidia, mitochondria-lacking eukaryotic intracellular parasites, genomic comparisons were so far based on molecular karyotyping. The mammal-infecting species Encephalitozoon cuniculi is characterized by a very low haploid genome size (approximately 2.8 Mbp) and rather high karyotype variability. Recently, we developed a two-dimensional pulsed field gel electrophoresis (2-D PFGE) fingerprinting technique useful for constructing a restriction map fo the genome of a mouse E. cuniculi isolate (karyotype variant A). The so-called karyotype and restriction display 2-D PFGE (KARD-PFGE) protocol involved 1-D chromosome separation, digestion with a rare cutter, Klenow radiolabeling of genomic DNA and 2-D separation of restriction fragments followed by autoradiography. In order to assess its suitability for detecting polymorphic loci in E. cuniculi, we applied KARD-PFGE with either BssHII or Mlul digestion to genome analysis of two rabbit isolates representative of two different karyotype variants (A and C). The 2-D spot pattern of the rabbit isolate variant A is identical to the reference mouse isolate but differs greatly from the rabbit isolate variant C. Chromosomal restriction fragment length polymorphisms (RFLPs) provide strong evidence for homologous chromosomes and frequent DNA rearrangements within subtelomeric regions just upstream of the dispersed rDNA units closely associated with each chromosomal end.

Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes.

A restriction map of the 2.8-Mb genome of the unicellular eukaryote Encephalitozoon cuniculi (phylum Microspora), a mammal-infecting intracellular parasite, has been constructed using two restriction enzymes with 6 bp recognition sites (Bss HII and Mlu I). The fragments resulting from either single digestions of the whole molecular karyotype or double digestions of 11 individual chromosomes have been separated by two-dimensional pulsed field gel electrophoresis (2D-PFGE) procedures. The average distance between successive restriction sites is approximately 19 kb. The terminal regions of the chromosomes show a common pattern covering approximately 15 kb and including one 16S-23S rDNA unit. Results of hybridisation and molecular combing experiments indicate a palindromic-like orientation of the two subtelomeric rDNA copies on each chromosome. We have also located 67 DNA markers (clones from a partial E. cuniculi genomic library) by hybridisation to restriction fragments. Partial or complete sequencing has revealed homologies with known protein-coding genes for 32 of these clones. Evidence for two homologous chromosomes III, with a size difference (3 kb) related to a subtelomeric deletion/insertion event, argues for diploidy of E.cuniculi. The physical map should be useful for both the whole genome sequencing project and studies on genome plasticity of this widespread parasite.

In-gel DNA radiolabelling and two-dimensional pulsed field gel electrophoresis procedures suitable for fingerprinting and mapping small eukaryotic genomes.

A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and [alpha-(32)P]dCTP incorporation mediated by the Klenow fragment. Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new two-dimensional pulsed field gel electrophoresis (2D-PFGE) protocols of genome analysis: (i) the KARD (karyotype and restriction display)-PFGE which provides a complete view of the fragments resulting from a single restriction of the whole genome and (ii) the DDIC (double digestion of isolated chromosome)-PFGE which is the eukaryotic counterpart of complete/complete 2D-PFGE in bacterial genomics.

Polymorphism of the gene encoding a major polar tube protein PTP1 in two microsporidia of the genus Encephalitozoon.

Isolates of 2 microsporidian species from the genus Encephalitozoon (E. cuniculi and E. hellem) were compared by analysis of DNA amplified from a gene region encoding the repeat domain of a polar tube protein (PTP1). Sequence data obtained for 11 E. cuniculi isolates from 5 different mammalian hosts well support the existence of 3 previously designated strains. Strain type III was characterized by a lack of a 78 bp repeat, producing an amplicon of reduced size. Strain type II differed from strain type I by 3 nucleotide substitutions so that AvalI digestion of the corresponding PCR products provided distinct restriction patterns. Surprisingly, the comparison of 2 human isolates of E. hellem belonging to the same rDNA ITS genotype shows a high level of heterogeneity through numerous point mutations and variation in PTP1 repeat number. Further characterization of additional E. hellem isolates based on PTP1 sequence polymorphisms should be of interest for tracing sources of infection.

Molecular karyotype diversity in the microsporidian Encephalitozoon cuniculi.

The microsporidian Encephalitozoon cuniculi can infect numerous mammals, including man. Three strains of E. cuniculi have been identified so far, the major marker being the number of a tetranucleotide repeats in the rDNA internal transcribed spacer. We investigated diversity at the chromosomal level through the electrophoretic karyotypes obtained from 15 E. cuniculi isolates from 5 different host species. All preparations provided patterns with 9-12 bands within a narrow molecular size range. Six karyotype forms were distinguished, involving subdivision of strain I into 3 types (A, B, C) and strain II into 2 types (D, E). The types A, B and C were mainly associated with isolates from rabbits of different geographical origins. The types D, E and F were characterized by a reduced chromosome size range, 2 of these appearing specific to a carnivorous host species (D in dog and F in blue fox). Hybridization experiments showed that all E. cuniculi isolates possess 11 chromosomes, with a size polymorphism entailing occasional electrophoretic comigration of heterologous chromosomes and differential migration of homologous ones. DNA rearrangements should occur during mitosis and the hypothesis of diploidy for the basic state of E. cuniculi seems likely.

On proteins of the microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that can cause opportunistic infections in AIDS patients. Spore invasion of host cells involves extrusion of a polar tube. After immunocytochemical identification of several polar tube proteins (PTPs) in E. cuniculi, a major PTP was isolated from two-dimensional gels and two peptide fragments were sequenced. The complete nucleotide sequence of the corresponding gene was obtained using a combination of PCR amplification and cloning techniques. The gene exists as a single copy per haploid genome and encodes an acidic proline-rich protein, with a deduced molecular mass of 37 kDa, that contains four tandemly arranged 26-amino-acid repeats. An N-terminal region of 22 residues represents a cleaved signal peptide, probably involved in the targeting of the PTP. No similarity with known proteins has been found. The protein was expressed in Escherichia coli, purified and injected into mice. The antisera reacted specifically with the polar tube in indirect immunofluorescence assays and electron microscope immunocytochemistry. Further identification of conserved and variable PTP structural motifs should be useful for diagnostic purposes and new therapeutic strategies.

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.

Immunocytochemical identification of spore proteins in two microsporidia, with emphasis on extrusion apparatus.

Microsporidia can form small spores with a unique invasive apparatus featuring a long polar tube whose extrusion allows entry of infectious sporoplasm into a host cell. The reactivity of mouse polyclonal antibodies raised against sporal proteins from two microsporidian species belonging to different genera (Glugea atherinae and Encephalitozoon cuniculi) was studied by western blotting and indirect immunofluorescence. Whole protein antisera provided a few cross-reactions relatable to some proteins of the spore envelope or polar tube. Ultrastructural immunocytochemistry with murine antibodies against protein bands separated by sodium dodecylsulphate polyacrylamide gel electrophoresis allowed the assignment of several proteins to the polar tube (34, 75 and 170 kDa in Glugea, 35, 55 and 150 kDa in Encephalitozoon). Antigenic similarities were detected for the Glugea 34 kDa and Encephalitozoon 35 kDa polar tube proteins. Species-specific proteins were shown to be located in either the lamellar polaroplast of Glugea or the spore envelope of Encephalitozoon.