EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall

Microsporidia are obligate intracellular parasites forming environmentally resistant spores that harbour a rigid cell wall. This wall comprises an outer layer or exospore and a chitin-rich inner layer or endospore. So far, only a chitin deacetylase-like protein has been shown to localize to the Encephalitozoon cuniculi endospore and either one or two proteins have been clearly assigned to the exospore in two Encephalitozoon species: SWP1 in E. cuniculi, SWP1 and SWP2 in Encephalitozoon intestinalis. Here, we report the identification of two new spore wall proteins in E. cuniculi, EnP1 and EnP2, the genes of which are both located on chromosome I (ECU01_0820 and ECU01_1270, respectively) and have no known homologue. Detected by immunoscreening of an E. cuniculi cDNA library, enp1 is characterized by small-sized 5' and 3' untranslated regions and is highly expressed throughout the whole intracellular cycle. The encoded basic 40 kDa antigen displays a high proportion of cysteine residues, arguing for a significant role of disulfide bridges in spore wall assembly. EnP2 is a 22 kDa serine-rich protein that is predicted to be O-glycosylated and glycosylated phosphatidyl inositol-anchored. Although having been identified by mass spectrometry of a dithiothreitol-soluble fraction, this protein contains only two cysteine residues. Mouse polyclonal antibodies were raised against EnP1 and EnP2 recombinant proteins produced in Escherichia coli Our immunolocalisation data indicate that EnP1 and EnP2 are targeted to the cell surface as early as the onset of sporogony and are finally associated with the chitin-rich layer of the wall in mature spores.     

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 homeobox genes of <Emphasis Type="Italic">Encephalitozoon cuniculi</Emphasis> (Microsporidia) reveal a putative mating-type locus

Homeobox genes have been found in animals, fungi, and plants. Recently, the complete genomic sequence of Encephalitozoon cuniculi has become available and it was shown that this Microsporida species is related to fungi. Given this close relatedness to fungi, I have searched the genome of E. cuniculifor homeobox genes. There are 12 homeobox genes as well as one STE12 orthologue. The large number of homeobox genes when compared to the annotations, which only list one, suggests that possibly other smaller genes may have been overlooked in the published analysis and E. cuniculi may have more than 1,997 genes. Sequence and phylogenetic analyses show that the E. cuniculi homeobox genes also fall into two distinct groups, with two TALE and ten typical homeobox genes being present. Like in the mating-type locus of yeast and other fungi, one TALE and one typical homeobox are found in close proximity of each other on the chromosome, suggesting that Microsporidia also contain a mating-type locus.     

Class II photolyase in a microsporidian intracellular parasite

Photoreactivation is the repair of DNA damage induced by ultraviolet light radiation using the energy contained in visible-light photons. The process is carried out by a single enzyme, photolyase, which is part of a large and ancient photolyase/cryptochrome gene family. We have characterised a photolyase gene from the microsporidian parasite, Antonospora locustae (formerly Nosema locustae) and show that it encodes a functional photoreactivating enzyme and is expressed in the infectious spore stage of the parasite's life cycle. Sequence and phylogenetic analyses show that it belongs to the class II subfamily of cyclobutane pyrimidine dimer repair enzymes. No photolyase is present in the complete genome sequence of the distantly related microsporidian, Encephalitozoon cuniculi, and this class of photolyase has never yet been described in fungi, the closest relatives of Microsporidia, raising questions about the evolutionary origin of this enzyme. This is the second environmental stress enzyme to be found in A.locustae but absent in E.cuniculi, and in the other case (catalase), the gene is derived by lateral transfer from a bacterium. It appears that A.locustae spores deal with environmental stress differently from E.cuniculi, these results lead to the prediction that they are more robust to environmental damage.     

Reactive nitrogen and oxygen species, and iron sequestration contribute to macrophage-mediated control of Encephalitozoon cuniculi (Phylum Microsporidia) infection in vitro and in vivo

Encephalitozoon cuniculi (Phylum Microsporidia) infects a wide range of mammals, and replicates within resting macrophages. Activated macrophages, conversely, inhibit replication and destroy intracellular organisms. These studies were performed to assess mechanisms of innate immune responses expressed by macrophages to control E. cuniculi infection. Addition of reactive oxygen and nitrogen species inhibitors to activated murine peritoneal macrophages statistically significantly, rescued E. cuniculi infection ex vivo. Mice deficient in reactive oxygen species, reactive nitrogen species, or both survived ip inoculation of E. cuniculi, but carried significantly higher peritoneal parasite burdens than wild-type mice at 1 and 2 weeks post inoculation. Infected peritoneal macrophages could still be identified 4 weeks post inoculation in mice deficient in reactive nitrogen species. L-tryptophan supplementation of activated murine peritoneal macrophage cultures ex vivo failed to rescue microsporidia infection. Addition of ferric citrate to supplement iron, however, did significantly rescue E. cuniculi infection in activated macrophages and further increased parasite replication in non-activated macrophages over non-treated resting control macrophages. These results demonstrate the contribution of reactive oxygen and nitrogen species, as well as iron sequestration, to innate immune responses expressed by macrophages to control E. cuniculi infection.     

Comparative analysis of sequences encoding ABC systems in the genome of the microsporidian Encephalitozoon cuniculi

Microsporidia are amitochondriate eukaryotic microbes with fungal affinities and a common status of obligate intracellular parasites. A set of 13 potential genes encoding ATP-binding cassette (ABC) systems was identified in the fully sequenced genome of Encephalitozoon cuniculi. Our analyses of multiple alignments, phylogenetic trees and conserved motifs support a distribution of E. cuniculi ABC systems within only four subfamilies. Six half transporters are homologous to the yeast ATM1 mitochondrial protein, a finding which is in agreement with the hypothesis of a cryptic mitochondrion-derived compartment playing a role in the synthesis and transport of Fe-S clusters. Five half transporters are similar to the human ABCG1 and ABCG2 proteins, involved in regulation of lipid trafficking and anthracyclin resistance respectively. Two proteins with duplicated ABC domains are clearly candidate to non-transport ABC systems: the first is homologous to mammalian RNase L inhibitor and the second to the yeast translation initiation regulator GCN20. An unusual feature of ABC systems in E. cuniculi is the lack of homologs of P-glycoprotein and other ABC transporters which are involved in multiple drug resistance in a large number of eukaryotic microorganisms.     

Distinct localization patterns of two putative mitochondrial proteins in the microsporidian Encephalitozoon cuniculi

Microsporidia were once considered amitochondriate, but have now been found to retain relict mitochondria called mitosomes. These organelles have been identified by immunolocalization in Trachipleistophora hominis, whereas most data on function have been inferred from the presence of mitochondrial protein-encoding sequences in the genome of Encephalitozoon cuniculi. Here we describe the localization of two such enzymes in E. cuniculi cells. Immunofluorescent localization of ferredoxin involved in mitochondrial iron-sulfur cluster assembly reveals a punctate distribution as expected for mitochondria. In contrast, localization of mitochondrial glycerol-3-phosphate dehydrogenase suggests a cytoplasmic distribution in E. cuniculi and possible relocalization of this typically mitochondrial protein.     

Microsporidian mitochondrial proteins: expression in Antonospora locustae spores and identification of genes coding for two further proteins

Microsporidia are obligate intracellular parasites, phylogenetically allied to the fungi. Once considered amitochondriate, now a number of mitochondrion-derived genes have been described from various species, and the relict organelle was recently identified in Trachipleistophora hominis. We have investigated the expression of potential mitochondrial targeted proteins in the spore stage to determine whether the organelle is likely to have a role in the spore or early infection stage. To investigate whether the Antonospora locustae genome codes for a different complement of mitochondrial proteins than Encephalitozoon cuniculi an EST library was searched for putative mitochondrial genes that have not been identified in the E. cuniculi genome project. The spore is the infectious stage of microsporidia, but is generally considered to be metabolically dormant. Fourteen genes for putatively mitochondrion-targeted proteins were shown to be present in purified spore mRNA by 3'-rapid amplification of cDNA ends and EST sequencing. Pyruvate dehydrogenase E1alpha and mitochondrial glycerol-3-phosphate dehydrogenase proteins were also shown to be present in A. locustae and E. cuniculi spores, respectively, suggesting a role for these proteins in the early stages of infection, or within the spore itself. EST sequencing also revealed two mitochondrial protein-encoding genes in A. locustae that are not found in the genome of E. cuniculi. One encodes a possible pyruvate transporter, the other a subunit of the mitochondrial inner membrane peptidase. In yeast mitochondria, this protein is part of a trimeric complex that processes proteins targeted to the inner membrane and the intermembrane space, and its substrate in A. locustae is presently unknown.     

DNA detection and genotypic identification of potentially human-pathogenic microsporidia from asymptomatic pet parrots in South Korea as a risk factor for zoonotic emergence

We detected and identified genotypes of human-pathogenic microsporidia in fecal samples from 51 asymptomatic captive-bred pet parrots in South Korea. Microsporidia were identified in 8 samples (15.7%); 7 parrots tested positive for Encephalitozoon hellem, and 1 parrot tested positive for both E. hellem and Encephalitozoon cuniculi. In genotypic identifications, E. hellem was present in genotypes 1A and 2B and E. cuniculi was present in genotype II. Pet parrots might be a source of human microsporidian infection.     

Evolution of HSP70 gene and its implications regarding relationships between archaebacteria,eubacteria, and eukaryotes

The 70-kDa heat-shock protein (HSP70) constitutes the most conserved protein present in all organisms that is known to date. Based on global alignment of HSP70 sequences from organisms representing all three domains, numerous sequence signatures that are specific for prokaryotic and eukaryotic homologs have been identified. HSP70s from the two archaebacterial species examined (viz., Halobacterium marismortui and Methanosarcina mazei) have been found to contain all eubacterial but no eukaryotic signature sequences. Based on several novel features of the HSP70 family of proteins (viz., presence of tandem repeats of a 9-amino-acid [a.a.] polypeptide sequence and structural similarity between the first and second quadrants of HSP70, homology of the N-terminal half of HSP70 to the bacterial MreB protein, presence of a conserved insert of 23–27 a.a. in all HSP70s except those from archaebacteria and gram-positive eubacteria) a model for the evolution of HSP70 gene from an early stage is proposed. The HSP70 homologs from archaebacteria and gram-positive bacteria lacking the insert in the N-terminal quadrants are indicated to be the ancestral form of the protein. Detailed phylogenetic analyses of HSP70 sequence data (viz., by bootstrap analyses, maximum parsimony, and maximum likelihood methods) provide evidence that archaebacteria are not monophyletic and show a close evolutionary linkage with the gram-positive eubacteria. These results do not support the traditional archaebacterial tree, where a close relationship between archaebacterial and eukaryotic homologs is observed. To explain the phylogenies based on HSP70 and other gene sequences, a model for the origin of eukaryotic cells involving fusion between archaebacteria and gram-negative eubacteria is proposed. Correspondence to: R. S. Gupta     

Polar tube proteins of microsporidia of the family encephalitozoonidae

Encephalitozoonidae are microsporidia associated with human infections including hepatitis, encephalitis, conjunctivitis, and disseminated disease. Microsporidia produce a small resistant spore containing a polar tube which serves as a unique vehicle of infection. Polar tube proteins (PTPs) from Encephalitozoon hellem. Encephalitozoon (Septata) intestinalis, and Encephalitozoon cuniculi were purified to homogeneity by HPLC. By SDS-PAGE, the Mr of E. hellem PTP was 55 kDa, while the Mr of E. intestinalis and E. cuniculi PTP was 45 kDa. Polyclonal rabbit antiserum to these purified PTPs localized to polar filaments by immunogold electron microscopy and immunofluorescence, and demonstrated cross-reactivity by both immunoblotting and immunogold electron microscopy. These PTPs have similar solubility properties, hydrophobicity, and proline content to a 43-kDa PTP we have previously purified from Glugea americanus, a fish microsporidium. As the polar tube is critical in the transmission of this organism, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.     

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.     

Characterization of aminopeptidase activity from three species of microsporidia: Encephalitozoon cuniculi,Encephalitozoon hellem,and Vittaforma corneae

Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.     

SWP5, a spore wall protein, interacts with polar tube proteins in the parasitic microsporidian Nosema bombycis

Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates. The microsporidian invasion process involves the extrusion of a unique polar tube into host cells. Both the spore wall and the polar tube play an important role in microsporidian pathogenesis. So far, five spore wall proteins (SWP1, SWP2, Enp1, Enp2, and EcCDA) from Encephalitozoon intestinalis and Encephalitozoon cuniculi and five spore wall proteins (SWP32, SWP30, SWP26, SWP25, and NbSWP5) from the silkworm pathogen Nosema bombycis have been identified. Here we report the identification and characterization of a spore wall protein (SWP5) with a molecular mass of 20.3 kDa in N. bombycis. This protein has low sequence similarity to other eukaryotic proteins. Immunolocalization analysis showed SWP5 localized to the exospore and the region of the polar tube in mature spores. Immunoprecipitation, mass spectrometry, and immunofluorescence analyses revealed that SWP5 interacts with the polar tube proteins PTP2 and PTP3. Anti-SWP5 serum pretreatment of mature spores significantly decreased their polar tube extrusion rate. Taken together, our results show that SWP5 is a spore wall protein localized to the spore wall and that it interacts with the polar tube, may play an important role in supporting the structural integrity of the spore wall, and potentially modulates the course of infection of N. bombycis.     

EnP1, a microsporidian spore wall protein that enables spores to adhere to and infect host cells in vitro

Microsporidia are spore-forming fungal pathogens that require the intracellular environment of host cells for propagation. We have shown that spores of the genus Encephalitozoon adhere to host cell surface glycosaminoglycans (GAGs) in vitro and that this adherence serves to modulate the infection process. In this study, a spore wall protein (EnP1; Encephalitozoon cuniculi ECU01_0820) from E. cuniculi and Encephalitozoon intestinalis is found to interact with the host cell surface. Analysis of the amino acid sequence reveals multiple heparin-binding motifs, which are known to interact with extracellular matrices. Both recombinant EnP1 protein and purified EnP1 antibody inhibit spore adherence, resulting in decreased host cell infection. Furthermore, when the N-terminal heparin-binding motif is deleted by site-directed mutagenesis, inhibition of adherence is ablated. Our transmission immunoelectron microscopy reveals that EnP1 is embedded in the microsporidial endospore and exospore and is found in high abundance in the polar sac/anchoring disk region, an area from which the everting polar tube is released. Finally, by using a host cell binding assay, EnP1 is shown to bind host cell surfaces but not to those that lack surface GAGs. Collectively, these data show that given its expression in both the endospore and the exospore, EnP1 is a microsporidian cell wall protein that may function both in a structural capacity and in modulating in vitro host cell adherence and infection.     

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.     

Encephalitozoon cuniculi Isolated from the Urine of an AIDS Patient, which Differs from Canine and Murine Isolates

ABSTRACT. A species of Encephalitozoon has been isolated from the urine of a patient with the acquired immunodeficiency syndrome and maintained in vitro in Madin Darby Canine Kidney cells. When examined by random amplified polymoprhic DNA polymerase chain reaction the new isolate was found to differ from E. hellem and to have amplified products in common with murine and canine E. cuniculi . However, it more closely resembled the canine than the murine isolate. Sodium dodecylsulphate polyacrylamide gel electrophoresis differentiated between all three isolates of E. cuniculi , with a band at 42–45 kDa present in the murine isolate only, bands at 52 kDa present in the canine and human isolates but not the murine, and a single band at 60 kDa (murine) and 65 kDa (canine) replaced by two bands at 55 and 70 kDa in the human isolate. The 55 kDa and 70 kDa antigens were also revealed as characteristic bands of the human isolate by Western blotting. The study has thus revealed that the species Encephalitozoon cuniculi is not a homogeneous entity.     

Genetically unique microsporidian Encephalitozoon cuniculi strain type III isolated from squirrel monkeys

Microsporidian spores were isolated from two squirrel monkeys (Saimiri sciureus) that had been bred at an animal-breeding colony in Japan. The spores were identified as Encephalitozoon cuniculi on the basis of nucleotide sequence analysis of the small-subunit (SSU) rRNA gene. The internal transcribed spacer (ITS) gene sequence revealed that these isolates were classified into genotype III because it contained tetrarepeats of 5'-GTTT-3'. However, the sequences of the polar tube protein (PTP) gene of the monkey isolates were not identical to a reported sequence of genotype III but were quite similar to a reported sequence of genotype II. On the other hand, sequence analysis of the spore wall protein 1 (SWP-1) gene revealed that the monkey isolates did not belong to any of genotypes I, II and III. These results suggest that the present E. cuniculi isolates of squirrel monkey origin are a new subtype of E. cuniculi ITS genotype III that can cause a disseminated infection.     

Prevalence and Genotyping of Microsporidian Parasites in Dogs in Turkey: Zoonotic Concerns

Microsporidia are opportunistic pathogens that infect a wide range of invertebrates and vertebrates. To assess the potential role of dogs in the transmission of these zoonotic pathogens, a total of 282 fecal samples from dogs in the Central Anatolia Region of Turkey were analyzed by utilizing species specific polymerase chain reaction for the four most frequent human microsporidia. Two microsporidia species were recognized in 41 samples (14.5%). Encephalitozoon intestinalis was detected in 35 samples (12.4%) and it was the most common microsporidium. The second microsporidium, E. cuniculi, was identified in six (2.1%) of the samples. Sequence analysis of the intergenic spacer of the ribosomal ribonucleic acid (RNA) internal transcribed spacer (ITS) gene revealed the presence of three E. intestinalis haplotypes closely associated with each other. No polymorphic region was found among the ITS sequences of E. cuniculi isolates and they were characterized as genotype III. This study provides the first data on the zoonotic microsporidia species from dogs in Turkey.