Corinne Biderre

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.

Serodiagnostic Studies in an Immunocompetent Individual Infected with Encephalitozoon cuniculi

Little is known about the prevalence and clinical significance of infection with Encephalitozoon species in immunocompetent individuals. In the present study, by using indirect immunofluorescence technique (IFAT), Western blot, and recombinant antigens of the spore wall (SWP1) and polar tube (PTP1, PTP2, and PTP3 ), we analyzed the IgG antibody response of a laboratory worker who was infected with Encephalitozoon cuniculi. Serum samples were analyzed 1, 20, 32, and 38 months after infection. After 1 month, by use of IFAT, only spore-wall antigens were recognized, an antibody reaction that changed toward both the spore wall and polar tube in the following months. By use of Western blot analysis, a characteristic pattern that recognized multiple bands was noticed. Reaction against SWP1 was present in all 4 serum samples. The IgG response against PTP1, PTP2, and PTP3 was not detectable 1 month after infection, but became evident in the follow-up serum samples. Serum samples showed cross-reactivity with the spore wall of Encephalitozoon hellem and Encephalitozoon intestinalis, but only little cross-reactivity with the polar tube of these parasites. This is the first study to our knowledge that provides full details about the antibody response against a specified Encephalitozoon species in an immunocompetent person. The results strongly encourage the development and use of reliable serodiagnostic methods, which will provide information about the prevalence and clinical significance of Encephalitozoon species infection in humans.

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.

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.

Comparison of two isolates of Encephalitozoon hellem and E. intestinalis (microspora) by pulsed field gel electrophoresis

Summary Contour-clamped homogeneous electric field gel electrophoresis was used to separate chromosome-sized DNA from two strains each of Encephalitozoon hellem and Encephalitozoon intestinalis isolated in vitro from AIDS patients. The two isolates of E. hellem differed significantly in molecular karyotype (EhD with 8 bands from 207 to 281 kb, and EhC with 12 bands from 175 to 315 kb), indicating a marked genetic polymorphism. The two E. intestinalis isolates displayed the same molecular karyotype with 10 bands comprised of between 182 and 279 kb. Of microsporidia so far investigated, species of Encephalitozoon have the smallest nuclear genomes (2.3–2.9 Mb).

Homology modeling and calculation of the cobalt cluster charges of the Encephazlitozoon cuniculi methionine aminopeptidase, a potential target for drug design

Fumagillin is a potent anti-angiogenic drug used in cancer treatments. It is also one of the few molecules active against the Enterocytozoon and Encephalitozoon parasites responsible for various clinical syndromes in HIV-infected or immunosuppressive treated patients. Its toxicity, however, makes desirable the design of more specific molecules. The fumagillin target, as anti-angiogenic agent, is the methionine aminopeptidase, an ubiquitous metallo-enzyme responsible for the removing of the N-terminal methionine in nascent proteins. By analogy, it has been proposed that this enzyme could also be the target in the parasites. As a first approach to verify this and to determine if it would be possible to design a more specific derivative, we have built a homology model of the E. cuniculi aminopeptidase. The charges of the two cobalt ions present in the active site and of the side-chains involved in their binding were computed using ab-initio methods. A preliminary comparison of the interactions of the fumagillin and of a related compound, the TNP-470, with both the human and the parasitic enzymes strongly support the hypothesis that the parasitic aminopeptidase is indeed the target of the fumagillin. It also suggests that the TNP-470 interact identically with both enzymes while there could be small differences in case of the fumagillin.