Immunocytochemical identification of the major exospore protein and three polar-tube proteins of the microsporidia Paranosema grylli

Microsporidia are intracellular eukaryotic parasites that can infect a wide range of animal hosts with several genera causing opportunistic infections in immunodeficient patients. Their spore wall and their unique extrusion apparatus, which has the form of a long polar tube, confer resistance of these parasites against the environment and during host-cell invasion. In contrast to parasites of vertebrates, the spore-wall and polar-tube proteins of many microsporidia species still remain to be characterized, even though a great number of microsporidia infect invertebrates. Here, we have identified one spore-wall protein and three polar-tube proteins of the microsporidia Paranosema grylli that infects the cricket Gryllus bimaculatus. Incubation of intact spores with an alkaline-saline solution resulted in the selective extraction of a major 40 kDa protein. A wash of the discharged (or destroyed) spores with SDS and the following solubilization of their polar tubes with 50-75% 2-mercaptoethanol extracted a major protein of ca. 56 kDa. When the polar tubes were solubilized in the presence of SDS, two additional proteins of 46 and 34 kDa were extracted. Antibodies specific for these extracted proteins were generated and isolated by incubation of immune sera with the protein bands that had been transferred to nitrocellulose. Western blotting demonstrated the cross-reactivity of the anti-p46 and anti-p34 antibodies. Immuno-electron microscopy with the anti-p40 antibody revealed specific decoration of the microsporidia exospore. The 56, 46 and 34 kDa proteins were characterized as polar-tube components due to the clear antibody labeling of the polar filament.     

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.     

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.     

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.     

Peculiarities of glycosylation of proteins in spores of microsporidia Paranosema (Antonospora) grylli

The long-term adaptation of microsporidia, a large group of fungi-related unicellular microorganisms, to intracellular parasitism has led to extreme minimization of the cell functional apparatus. For instance, diversity of carbohydrates in the composition of parasite glycoproteins and proteoglycans seems to be restricted to the presence of O-bound chains composed of mannose residues. This suggestion is based on the discovery in the genome of the human microsporidian Encephalitozoon cuniculi of three genes responsible for the O-mannosylation of proteins with a lack of enzymes participating in N-glycosylation. In the present work, peculiarities of protein glycosylation in spores of the microsporidian Paranosema grylli infecting the fat body of the Mediterranean field cricket Gryllus bimaculatus was studied. SDS-PAGE analysis of spore proteins with subsequent staining by periodate and Schiff reagent has shown that individual glycoproteins of P. grylli are highly glycosylated, while the maximal stain intensity was seen in the major polar-tube protein PTP1. Treatment of the extracted material with N-glycosidase F and hybridization with WGA lectin conjugated with horseradish peroxidase showed no presence of glycosylated proteins in the P. grylli spores. At the same time, the selectively extracted major protein of the exospore p40 was specifically recognized by lectin GNA conjugated with agarose balls. Pretreatment of p40 with α-and β-mannosidases decreased considerably the efficiency of binding. Since lectin GNA is specific towards mannose terminal residues, this indicates the O-mannosylation of the microsporidial exospore major protein. In spite of the intensive PTP1 glycosylation, extracted proteins of the P. grylli polar-tube had no specific binding with GNA-agarose, so the issue of peculiarities of their glycosylation remains an open question. Comparison of the obtained data with results of deciphering of the E. cuniuculi genome allows for the conclusion to be made that the minimization of the glycosylation apparatus of microsporidial proteins is the common peculiarity of this group of parasites.     

The spore wall and polar tube proteins of the microsporidian Nosema grylli: the major spore wall protein is released before spore extrusion

Incubation of Nosema grylli spores in alkaline--saline solution (10 mM KOH, 170 mM KCl) leads to solubilization of the major spore wall protein of 40 kDa (p40). Both the compounds of this solution are crucial for p40 solubilization. After spore incubation in 170 mM KCl no proteins were released in the medium. In contrast, 10 mM KOH causes a release of many spore proteins but only a small amount of p40. A long storage of spores (over a year) in water or 0.02% sodium azide results in a sharp decrease of p40 content. Specific polyclonal antibodies were obtained by immunization of rabbits with isolated p40. The specificity of serum was confirmed by immunoblotting. IFA showed reliable reaction on the envelopes of sporonts and sporoblasts, whereas only part of spores reacted with antibodies. This distinction may be due to changing surface antigens during spore maturation. Solubilization of p40 under alkaline conditions could be associated with spore extrusion, since a subsequent transfer of spores to neutral solution leads to their discharge. Subsequent wash of discharged spores with 1-3% SDS, 9 M urea and treatment by 100% 2-ME result in solubilization of protein of 56 kDa (p56). The maximum concentration of 2-ME is important for isolation of pure p56. Evidence has been provided that p56 is a protein of N. grylli polar tubes. Treatment of discharged spores by 2-ME in the presence of SDS results in solubilization of four additional proteins with molecular weights about 46, 34, 21 and 15 kDa.     

Major O-glycans in the spores of two microsporidian parasites are represented by unbranched manno-oligosaccharides containing alpha-1,2 linkages

Protein glycosylation in microsporidia, a fungi-related group comprising exclusively obligate intracellular parasitic species, is still poorly documented. Here, we have studied glycoconjugate localization and glycan structures in spores of Encephalitozoon cuniculi and Antonospora locustae, two distantly related microsporidians invading mammalian and insect hosts, respectively. The polar sac-anchoring disc complex or polar cap, an apical element of the sporal invasion apparatus, was strongly periodic acid-thiocarbohydrazide-Ag proteinate-positive. Mannose-binding lectins reacted with the polar cap and recognized several bands (from 20 to 160 kDa) on blots of E. cuniculi protein extracts. Physicochemical analyses provided the first determination of major glycostructures in microsporidia. O-linked glycans were demonstrated to be linear manno-oligosaccharides containing up to eight alpha1, 2-linked mannose residues, thus resembling those reported in some fungi such as Candida albicans. No N-linked glycans were detected. The data are in accordance with gene-based prediction of a minimal O-mannosylation pathway. Further identification of individual mannoproteins should help in the understanding of spore germination mechanism and host-microsporidia interactions.     

Identification of a Microsporidian Polar Tube Protein Reactive Monoclonal Antibody

ABSTRACT. The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analyis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents.     

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 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.     

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.     

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.     

Trehalose catabolism in microsporidia Nosema grylli spores

Some differences in trehalose catabolism were found for terrestrial and aquatic microsporidian species (Undeen, Van der Meer, 1999). In microsporidia species from aquatic hosts, the spore extrusion causes the intrasporal trehalose hydrolysis by trehalase that is followed by the drastic rise of reducing sugars (glucose) concentration. On the contrary, in tested terrestrial microsporidian species, total and reducing sugars remain unchanged through the germination. In this study we demonstrate by means of the enzymatic and paper chromatography methods, that in spores of microsporidia Nosema grylli, infecting fat bodies of crickets Gryllus bimaculatus, neither an increase of glucose concentration nor a reduction in intrasporal trehalose content takes place during the spore discharge. In this respect N. grylli is close to other terrestrial species. However, we have revealed in N. grylli spores activity of alpha,alpha-trehalase (EC 3.2.1.28) with acid pH-optimum like it was found by other authors in spores of aquatic microsporidia N. algerae. This result differs from the neutral pH-optimum (7.0) of trehalse of other terrestrial microsporidia N. apis. Concentration of trehalose in N. grylli spores reduces during long-term storage. All attempts to detect an activity of trehalose phosphorylase (synthase) (K phi 2.4.1.64), other potential key enzyme for trehalose catabolism in N. grylli spores have failed. The absence of changes of the sugar content in terrestrial microsporidian spores during the extrusion indicates, that the main physiological role of trehalose hydrolysis by trehalase in these species is catabolism of energy reserves for providing the long-term survival in the environment.     

Spores of two fish microsporidia (Pseudoloma neurophilia and Glugea anomala) are highly resistant to chlorine

Pseudoloma neurophilia (Microsporidia) is the most common pathogen found in zebrafish Danio rerio research facilities. The parasite is associated with marked emaciation. Zebrafish laboratories usually disinfect eggs to prevent transmission of pathogens, typically with chlorine at 25 to 50 ppm for 10 min. The ability of chlorine to kill spores of P. neurophilia and 2 other microsporidia, Glugea anomala and Encephalitozoon cuniculi, was evaluated using 2 viability stains. SYTOX Green was used to visualize dead spores, and live spores were identified by their ability to extrude polar tubes in Fungi-Fluor solution following UV exposure. Results with both stains were similar at various chlorine concentrations for P. neurophilia and G. anomala, but Fungi-Fluor was not useful for E. cuniculi, due to the much smaller spore size. Using the SYTOX stain, we found that 5 ppm chlorine for 10 min causes 100% death in spores of E. cuniculi, which was similar to findings in other studies. In contrast, the spores of P. neurophilia and G. anomala were much more resistant to chlorine, requiring >100 or 1500 ppm chlorine, respectively, to achieve >95% spore death. Repeating chlorine exposures with spores of P. neurophilia using solutions adjusted to pH 7 increased the efficacy of 100 ppm chlorine, achieving >99% spore inactivation. We corroborated our viability staining results with experimental exposures of zebrafish fry, achieving heavy infections in fry at 5 to 7 d post-exposure in fish fed spores treated at 50 ppm (pH 9). Some fish still became infected with spores exposed to 100 ppm chlorine (pH 9.5). This study demonstrates that spores of certain fish microsporidia are highly resistant to chlorine, and indicates that the egg disinfection protocols presently used by most zebrafish research facilities will not prevent transmission of P. neurophilia to progeny.     

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.     

Nbseptin2 Expression Pattern and Its Interaction with NbPTP1 during Microsporidia Nosema bombycis Polar Tube Extrusion

Nosema bombycis (Nb) is a deadly species of microsporidia capable of causing pébrine, leading to heavy losses in sericulture. Germination is an important biological event in the invasion process of microsporidia. Septins, a family of membrane‐associated proteins, play a critical role in tissue invasion and have been recognized as a virulence factor in numerous pathogens. Previous work in our laboratory has shown that Nosema bombycis septin2 (Nbseptin2) interacts with subtilisin‐like protease 2 (NbSLP2). Herein, we found that Nbseptin2 was mainly associated with the plasma membrane in spores. Following spore germination, Nbseptin2 was found to co‐localize with polar tube protein 1 (NbPTP1) at the polar cap and proximal zone of the polar tube. Co‐immunoprecipitation and yeast two‐hybrid analysis further confirmed that Nbseptin2 interacted with NbPTP1. The translocation and interaction of Nbseptin2 in the spores suggest that Nbseptin2 may play a significant role in microsporidia polar tube extrusion process. Our findings improve understanding of the mechanisms underlying microsporidia germination.     

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.     

家蚕微孢子虫极管蛋白1 (NbPTP1) 在果蝇S2细胞中的表达与糖基化修饰

极管蛋白(Polar tube protein)是极管的主要成分,能特异性定位于微孢子虫极管,在微孢子虫侵染宿主过程中发挥重要作用。文中分析了家蚕微孢子虫极管蛋白1中潜在的O-、N-糖基化修饰位点,克隆了家蚕微孢子虫极管蛋白1全基因序列,并将其插入带有V5和His标签的真核表达载体pMT/Bip/V5-His A中,成功构建了pMT/Bip/V5-His A-NbPTP1重组质粒,经转染果蝇S2细胞后,发现NbPTP1基因能在果蝇细胞中高效表达。此外,Lectin blotting和β-消除反应分析结果表明:果蝇S2细胞内表达的NbPTP1具有O-糖基化修饰特征。以上结果为研究NbPTP1的糖基化修饰特征与其功能之间的关系提供了基础,有助于揭示微孢子虫侵染机制,建立可行有效的微孢子虫病诊断和防治措施。