Guy Méténier

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 ∼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.

Functional and evolutionary analysis of a eukaryotic parasitic genome.

The DNA sequences of the 11 linear chromosomes of the approximately 2.9 Mbp genome of Encephalitozoon cuniculi, an obligate intracellular parasite of mammals, include approximately 2000 putative protein-coding genes. The compactness of this genome is associated with the length reduction of various genes. Essential functions are dependent on a minimal set of genes. Phylogenetic analysis supports the hypotheses that microsporidia are related to fungi and have retained a mitochondrion-derived organelle, the mitosome.

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

ABSTRACT Microsporidia are unicellular eukaryotes occuring 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 cuniculiand E. hellem. Our immunological investigations provided evidence for an additional PTP in E. cuniculi, termed PTP2. The corresponding gene was sequenced and then expressed inEscherichia coli. As expected, mouse antibodies raised against the recombinant protein reacted specifically with the polar tube. The singlecopy ptp1 and ptp2 genes ofE. cuniculi were tandemly arranged on chromosome VI. Polyadenylation of the mRNAs was demonstrated. Identification and sequencing of homologous genes in the two other human-infectingEncephalitozoon 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.

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.

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.

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.

A small spliceosomal-type intron occurs in a ribosomal protein gene of the microsporidian, Encephalitozoon cuniculi

Among unicellular eukaryotes, microsporidia are obligately intracellular amitochondrial parasites that are considered to be of very ancient origin as deduced from the prokaryotic features of their ribosomes [1–3], and rRNA [4] and EF1a /EF2 phylogenies [5]. Microsporidia are also characterized by small nuclear genomes ranging from 19.6 Mb in Glugea atherinae [6] to only 2.9 Mb in Encephalitozoon cuniculi [7], and thus partially spanning the prokaryotic range. Sequencing data on the E. cuniculi genome have supplied information on noncoding regions but have not allowed identification of introns [8]. However, the relatively small sample of genes sequenced in microsporidia may be insufficiently representative for detection of potential intervening sequences. During screening of a partial plasmid library from E. cuniculi genomic DNA, we isolated a clone (c141) coding for a homolog of a large subunit ribosomal protein (L27a or L29 in yeast) gene known to contain usually one or more introns. Blast alignments first revealed that a single reading frame accounts for the entire putative L27a coding region with a termination codon UAA. However, an AUG start codon could not be placed in the position expected on the basis of the conserved character of the L27a amino-terminal region. A thorough examination of this region identified an AT-rich (61%) intervening sequence, with a size of only 28 bp, that creates a frameshift and extends from just after an AUG codon (Fig. 1A). This intron harbors consensus spliceosomal boundaries (5%-GT...AG-3%) with a 5% region identical to the consensus of higher eukaryotes (GTAAGT). In the mould Neurospora crassa, one Abbre6iations: NLS, nuclear localization signal. * Corresponding author. Tel.: +33 473 407457; fax: +33 473 407455; e-mail: vivares@cicsun.univ-bpclermont.fr 1 Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB data bases under the accession number AF054829.

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.

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.