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.     

Microsporidian polar tube proteins: highly divergent but closely linked genes encode PTP1 and PTP2 in members of the evolutionarily distant Antonospora and Encephalitozoon groups

The spore polar tube is a unique organelle required for cell invasion by fungi-related microsporidian parasites. Two major polar tube proteins (PTP1 and PTP2) are encoded by two tandemly arranged genes in Encephalitozoon species. A look at Antonospora (Nosema) locustae contigs (http://jbpc.mbl.edu/Nosema/Contigs/) revealed significant conservation in the order and orientation of various genes, despite high sequence divergence features, when comparing with Encephalitozoon cuniculi complete genome. This syntenic relationship between distantly related Encephalitozoon and Antonospora genera has been successfully exploited to identify ptp1 and ptp2 genes in two insect-infecting species assigned to the Antonospora clade (A. locustae and Paranosema grylli). Targeting of respective proteins to the polar tube was demonstrated through immunolocalization experiments with antibodies raised against recombinant proteins. Both PTPs were extracted from spores with 100mM dithiothreitol. Evidence for PTP1 mannosylation was obtained in studied species, supporting a key role of PTP1 in interactions with host cell surface.     

Identification of proteins in Encephalitozoon intestinalis, a microsporidian pathogen of immunocompromised humans: an immunoblotting and immunocytochemical study

Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube--the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55-60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.     

The microsporidian polar tube: a highly specialised invasion organelle

All of the members of the Microsporidia possess a unique, highly specialised structure, the polar tube. This article reviews the available data on the organisation, structure and function of this invasion organelle. It was over 100 years ago that Thelohan accurately described the microsporidian polar tube and the triggering of its discharge. In the spore, the polar tube is connected at the anterior end, and then coils around the sporoplasm. Upon appropriate environmental stimulation the polar tube rapidly discharges out of the spore pierces a cell membrane and serves as a conduit for sporoplasm passage into the new host cell. The mechanism of germination of spores, however, remains to be definitively determined. In addition, further studies on the characterisation of the early events in the rupture of the anterior attachment complex, eversion of the polar tube as well as the mechanism of host cell attachment and penetration are needed in order to clarify the function and assembly of this structure. The application of immunological and molecular techniques has resulted in the identification of three polar tube proteins referred to as PTP1, PTP2 and PTP3. The interactions of these identified proteins in the formation and function of the polar tube remain to be determined. Data suggest that PTP1 is an O-mannosylated glycoprotein, a post-translational modification that may be important for its function. With the availability of the Encephalitozoon cuniculi genome it is now possible to apply proteomic techniques to the characterisation of the components of the microsporidian spore and invasion organelle.     

A proteomic-based approach for the characterization of some major structural proteins involved in host-parasite relationships from the silkworm parasite Nosema bombycis (Microsporidia)

Nosema bombycis is the causative agent of the silkworm Bombyx mori pebrine disease which inflicts severe worldwide economical losses in sericulture. Little is known about host-parasite interactions at the molecular level for this spore-forming obligate intracellular parasite which belongs to the fungi-related Microsporidia phylum. Major microsporidian structural proteins from the spore wall (SW) and the polar tube (PT) are known to be involved in host invasion. We developed a proteomic-based approach to identify few N. bombycis proteins belonging to these cell structures. Protein extraction protocols were optimized and four N. bombycis spore protein extracts were compared by SDS-PAGE and 2-DE to establish complementary proteomic profiles. Three proteins were shown to be located at the parasite SW. Moreover, 17 polyclonal antibodies were raised against major N. bombycis proteins from all extracts, and three spots were shown to correspond to polar tube proteins (PTPs) by immunofluorescent assay and transmission electron microscopy immunocytochemistry on cryosections. Specific patterns for each PTP were obtained by MALDI-TOF-MS and MS/MS. Peptide sequence tags were deduced by de novo sequencing using Peaks Online and DeNovoX, then evaluated by MASCOT and SEQUEST searches. Identification parameters were higher than false-positive hits, strengthening our strategy that could be enlarged to a nongenomic context.     

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.     

Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia)

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-terminal amino acid residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.     

Protein glycosylation in the spores of the microsporidia Paranosema (Antonospora) grylli

Long adaptation of microsporidia, a large group of fungi-related protozoa, to intracellular lifestyle has resulted in drastic minimization of a parasite cell. Thus, diversity of carbohydrates in microsporidia glycoproteins and proteoglycans is expected to be restricted by O-linked manno-oligosaccharides because three genes involved in O-mannosylation of proteins and no components of N-linked glycosylation machinery were found in genome of human pathogen Encephalitozoon cuniculi. In this study we investigated glycosylation of spore proteins of microsporidia Paranosema (Antonospora) grylli infecting crickets Gryllus bimaculatus. Using periodic acid-Shiff reagent staining we have demonstrated that some P. grylli spore proteins are highly-glycosylated. The major polar tube protein (PTP1) of 56 kDa was shown as the most intensively decorated band. The experiments with N-glycosidase F and WGA lectin did not reveal any N-glycosylated proteins in P. grylli spores. At the same time, incubation of major spore wall protein of 40 kDa (p40) with mannose specific lectin GNA resulted in specific binding that was reduced by pretreatment of the protein with mannosidases. Interestingly, in spite of PTP1 glycosylation, polar tube proteins extracted from P. grylli spores were not precipitated by GNA-agarose. Since P. grylli and E. cuniculi are distantly related, our data suggest that dramatic reduction of protein glycosylation machinery is a common feature of microsporidia.     

SWP25, A Novel Protein Associated with the Nosema bombycis Endospore1

ABSTRACT. Microsporidia are eukaryotic, obligate intracellular, spore-forming parasites. The resistant spores, which harbor a rigid cell wall, are critical for their host-to-host transmission and persistence in the environment. The spore wall comprises two major layers: the exospore and the endospore. In Nosema bombycis, two spore wall proteins have been characterized—an endosporal protein, SWP30, and an exosporal protein, SWP32. Here, we report the identification of the third spore wall protein of N. bombycis, SWP25, the gene of which has no known homologue. SWP25 is predicted to posses a signal peptide and a heparin-binding motif. Immunoelectron microscopy analysis showed that this protein is localized to the endospore. This characterization of a new spore wall protein of N. bombycis may facilitate our investigation of the relationship between N. bombycis and its host, Bombyx mori.     

Identification of a Nosema bombycis (Microsporidia) spore wall protein corresponding to spore phagocytosis

Life-cycle stages of the microsporidia Nosema bombycis, the pathogen causing silkworm pebrine, were separated and purified by an improved method of Percoll-gradient centrifugation. Soluble protein fractions of late sporoblasts (spore precursor cells) and mature spores were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were recovered from gels and analysed by mass spectrometry. The most abundant differential protein spot was identified by database search to be a hypothetical spore wall protein. Using immunoelectron microscopy, we demonstrated that HSWP5 is localized to the exospore of mature spores and renamed it as spore wall protein 5 (NbSWP5). Further spore phagocytosis assays indicated that NbSWP5 can protect spores from phagocytic uptake by cultured insect cells. This spore wall protein may function both for structural integrity and in modulating host cell invasion.     

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.     

The microsporidian spore invasion tube. The ultrastructure, isolation, and characterization of the protein comprising the tube

The extrusion apparatus of the microsporidian parasitic protozoan Nosema michaelis discharges an invasion (or polar) tube with a velocity suitalbe for piercing cells and injecting infective sporoplasm. The tube is composed of a polar tube protein (PTP) which consists of a single, low molecular weight polypeptide slightly smaller than chymotrypsinogen-A. Assembled PTP tubes resist dissociation in sodium dodecyl sulfate and brief exposures in media at extreme ends of the pH range; however, the tubes are reduced by mercaptoethanol and dithiothreitol. When acidified, mercaptoethanol-reduced PTP self-assembles into plastic, two-dimensional monolayers. Dithiothreitol-reduced PTP will not reassemble when acidified. Evidence is presented which indicates that PTP is assembled as a tube within the spore; that the ejected tube has plasticity during sporoplasm passage; and, finally, that the subunits within the tube polymer are bound together, in part, by interprotein disulfide linkages.     

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.     

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.     

Comparative Analysis of the Proteins with Tandem Repeats from 8 Microsporidia and Characterization of a Novel Endospore Wall Protein Colocalizing with Polar Tube from Nosema bombycis

As a common feature of eukaryotic proteins, tandem amino acid repeat has been studied extensively in both animal and plant proteins. Here, a comparative analysis focusing on the proteins having tandem repeats was conducted in eight microsporidia, including four mammal‐infecting microsporidia (Encephalitozoon cuniculi, Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon bieneusi) and four insect‐infecting microsporidia (Nosema apis, Nosema ceranae, Vavraia culicis and Nosema bombycis). We found that the proteins with tandem repeats were abundant in these species. The quantity of these proteins in insect‐infecting microsporidia was larger than that of mammal‐infecting microsporidia. Additionally, the hydrophilic residues were overrepresented in the tandem repeats of these eight microsporidian proteins and the amino acids residues in these tandem repeat sequences tend to be encoded by GC‐rich codons. The tandem repeat position within proteins of insect‐infecting microsporidia was randomly distributed, whereas the tandem repeats within proteins of mammal‐infecting microsporidia rarely tend to be present in the N terminal regions, when compared with those present in the C terminal and middle regions. Finally, a hypothetical protein EOB14572 possessing four tandem repeats was successfully characterized as a novel endospore wall protein, which colocalized with polar tube of N. bombycis. Our study provided useful insight for the study of the proteins with tandem repeats in N. bombycis, but also further enriched the spore wall components of this obligate unicellular eukaryotic parasite.     

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.     

Fine structure of a new human microsporidian, Encephalitozoon hellem, in culture

Encephalitozoon hellem is a new human microsporidian isolated from corneal biopsies and conjunctival scrapings of three AIDS patients and cultured in Madin Darby canine kidney (MDCK) cells. Encephalitozoon hellem and Encephalitozoon cuniculi display different protein profiles with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and unique antibody binding patterns with murine antisera against Western blots of each organism. Developmental stages of E. hellem in culture are similar to E. cuniculi. Meronts are 1.3-2.7 microns in diameter, develop within a parasitophorous vacuole adjacent to the vacuolar membrane, divide by binary fission, and contain one or two discrete nuclei. Sporonts measure 2 x 3 microns, separate from the vacuolar membrane, and have a thickened outer membrane. Sporoblasts display a tri-layered wall and possess the earliest recognized polar filaments. Mature spores measure 1 x 1.5 microns and are more electron-dense than other stages. Each spore contains a single nucleus, a polar tubule with four to nine coils, thin electron-dense exospore and thick, electron-lucent endospore. Although E. hellem and E. cuniculi differ biochemically and immunologically, their fine structure and development are indistinguishable.     

Fine Structure of a New Human Microsporidian, Encephalitozoon hellem, in Culture

Encephalitozoon hellem is a new human microsporidian isolated from corneal biopsies and conjunctival scrapings of three AIDS patients and cultured in Madin Darby canine kidney (MDCK) cells. Encephalitozoon hellem and Encephalitozoon cuniculi display different protein profiles with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and unique antibody binding patterns with murine antisera against Western blots of each organism. Developmental stages of E. hellem in culture are similar to E. cuniculi. Meronts are 1.3–2.7 μm in diameter, develop within a parasitophorous vacuole adjacent to the vacuolar membrane, divide by binary fission, and contain one or two discrete nuclei. Sporonts measure 2 × 3 μm, separate from the vacuolar membrane, and have a thickened outer membrane. Sporoblasts display a tri-layered wall and possess the earliest recognized polar filaments. Mature spores measure 1 × 1.5 μm and are more electron-dense than other stages. Each spore contains a single nucleus, a polar tubule with four to nine coils, thin electron-dense exospore and thick, electron-lucent endospore. Although E. hellem and E. cuniculi differ biochemically and immunologically, their fine structure and development are indistinguishable.     

Ultrastructural details of the xenoma of Loma myrophis (phylum Microsporidia) and extrusion of the polar tube during autoinfection

Xenomas of the recently described new microsporidian species Loma myrophis parasitizing the gut tissue of the Amazonian fish Myrophis platyrhynchus (family Ophichthidae) were described by light- and transmission-electron microscopy. The xenoma consisted of a thin fibrillar wall that surrounded a hypertrophic host cell cytoplasm containing numerous microsporidian developmental stages and spores. Several spores showed different stages of natural extrusion of the polar tube. Numerous longitudinal and transverse sections of the extruded polar tubes were observed in developing life-cycle stages (spores excepted), the nucleus of hypertrophic host cell, the xenoma wall and surrounding fibroblasts. The extruded polar tubes were projected in all directions with no preferential orientation. These aspects suggested that autoinfection occurred within this xenoma.     

Identification of a novel spore wall protein (SWP26) from microsporidia Nosema bombycis

Microsporidia are obligate intracellular parasites related to fungi with resistant spores against various environmental stresses. The rigid spore walls of these organisms are composed of two major layers, which are the exospore and the endospore. Two spore wall proteins (the endosporal protein-SWP30 and the exosporal protein-SWP32) have been previously identified in Nosema bombycis. In this study, using the MALDI-TOF-MS technique, we have characterised a new 25.7-kDa spore wall protein (SWP26) recognised by monoclonal antibody 2G10. SWP26 is predicted to have a signal peptide, four potential N-glycosylation sites, and a C-terminal heparin-binding motif (HBM) which is known to interact with extracellular glycosaminoglycans. By using a host cell binding assay, recombinant SWP26 protein (rSWP26) can inhibit spore adherence by 10%, resulting in decreased host cell infection. In contrast, the mutant rSWP26 (rΔSWP26, without HBM) was not effective in inhibiting spore adherence. Immuno-electron microscopy revealed that this protein was expressed largely in endospore and plasma membrane during endospore development, but sparsely distributed in the exospore of mature spores. The present results suggest that SWP26 is a microsporidia cell wall protein that is involved in endospore formation, host cell adherence and infection in vitro. Moreover, SWP26 could be used as a good prospective target for diagnostic research and drug design in controlling the silkworm, Bombyx mori, pebrine disease in sericulture.