Interactions between Encephalitozoon cuniculi and macrophages. Parasitophorous vacuole growth and the absence of lysosomal fusion.

Encephalitozoon cuniculi grow within ever-increasing parasitophorous vacuoles (PV) in peritoneal macrophages. The PV boundary membrane conforms to a rich arrangement of blebs; similar, but free vesicles were observed within the PV space. An iron dextran-concanavalin A marker was used to express visually clustered distributions of Con A receptors on the PV boundary blebs and free vesicles; no marker was observed on other membrane surfaces within the PV. These results, combined with the observation that the PV grows while the host cytoplasm decreases in mass, implicate the PV boundary blebs of interiorizing into vesicles by a pinocytic mechanism. Phagocytic vacuoles, secondary lysosomes and pinocytic vesicles were labeled by incubating infected macrophages in minimum essential medium with ferritin. Ferritin readily accumulated in secondary lysosomes and phagocytic vacuoles; however, ferritin was excluded from parasitophorous vacuoles containing E. cuniculi. Acid phosphatase cytochemical reaction product was observed in lysosomes and phagocytic vacuoles; however, parasitophorous vacuoles with vegetative E. cuniculi were always negative.     

Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV). In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37 masculineC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.     

Opening of size-selective pores in endosomes during human rhinovirus serotype 2 in vivo uncoating monitored by single-organelle flow analysis

The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.     

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.     

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.     

Serological survey for antibodies to Encephalitozoon cuniculi in ownerless dogs from urban areas of Brazil and Colombia

There are 3 strains of Encephalitozoon cuniculi that occur in mammals. Strain III is associated with clinical disease in dogs, although some can be asymptomatic carriers and excrete spores in their urine. Several cases of human E. cuniculi infection caused by strain III have been observed in immunocompromised patients, indicating that E. cuniculi should be considered a zoonotic agent. Encephalitozoon cuniculi can cause fatal disease in maternally-infected or young dogs. Clinical signs in these animals included blindness, encephalitis, retarded growth rate, and nephritis. Encephalitozoon cuniculi has also been associated with primary renal failure in adult dogs. The present study used the direct agglutination test (DAT, cut-off 1:50) and the indirect fluorescent antibody test (IFAT, cut-off 1:10) to examine the prevalence of antibodies to E. cuniculi in dogs from Brazil and Colombia. Using the DAG, 31 (27.4%) of 113 dogs from Brazil and 47 (18.5%) of 254 dogs from Colombia were seropositive. Nine (14.3%) of 63 dogs from Brazil and 18 (35.3%) of the 51 dogs from Colombia were seropositive by indirect immunofluorescent antibody test. These results indicate that dogs from Brazil and Colombia are exposed to E. cuniculi.     

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.     

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L

Coxiella burnetii is a gram‐negative intracellular bacterium that forms a large, lysosome‐like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol‐binding protein‐related protein 1 long (ORP1L) is a mammalian lipid‐binding protein that plays a dual role in cholesterol‐dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N‐terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co‐localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L‐depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.     

Lipids of three microsporidian species and multivariate analysis of the host-parasite relationship.

Sporal lipids of 3 microsporidia, Encephalitozoon cuniculi from mammals and Glugea atherinae and Spraguea lophii from fishes, were investigated. High phospholipid levels were found (54.8-64.5% of total lipids), which is in agreement with the presence of highly developed internal membranes in microsporidian spores. Sphingomyelin was not detected in G. atherinae. Triglycerides (less than 10% of total lipids), cholesterol, and free fatty acids were identified in all species. Analysis of fatty acids from the phospholipid fraction revealed the predominance of docosahexaenoic acid (30-40% of total phospholipid fatty acids) in G. atherinae and S. lophii and oleic acid (25.8% of total phospholipid fatty acids) in E. cuniculi. The 3 microsporidia possessed a significant amount of branched-chain fatty acids (iso and anteiso forms) not found in the hosts, supporting the existence of some parasite-specific metabolic steps for these fatty acids. On the basis of phospholipid fatty acid profiles, host-parasite relationships were investigated through correspondence factorial analysis. It shows 3 distinct clusters with the first corresponding to fishes, the second to fish parasites, and the third to E. cuniculi and its host cell. These data suggest that the mammal microsporidia developing within parasitophorous vacuoles are more dependent on host cells than the fish microsporidia that induce cystlike structures.     

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.     

Fine Structure of Encephalitozoon cuniculi from Rabbits,Mice and Hamsters*

SYNOPSIS. Fine structure and development of Encephalitozoon cuniculi from rabbits were studied in rabbit choroid plexus (CP) cell cultures and were compared to hamster and mouse microsporida. Sporoplasms had a single limiting membrane and contained a large nucleus. Proliferative forms (schizonts) had double outer membranes, the outermost being associated with the formation of the limiting membrane of vacuoles formed within the host cell cytoplasm. These organisms were often binucleate and divided to form sporonts. Sporonts divided once to form 2 sporoblasts which developed into electron-dense spores. Spores had a thick, 3-layered wall and contained a polar filament. The developmental cycle of E. cuniculi in rabbit CP cultures progressed rapidly. Sporoplasms were observed in host cells at 3 hr postinoculation (PI). By 24 hr PI proliferative forms were associated with host cell cytoplasmic vacuoles which contained developing organisms. Mature spores were present in vacuoles by 2 days PI, indicating that the life cycle in the CP system is ∼ 48 hr. The fine structure and the sequential developmental cycle of the mouse and hamster isolates were observed to be identical to those of the rabbit isolate and different from those of the genus Nosema. It is proposed, therefore, that the 3 organisms represent the same species, Encephalitozoon cuniculi.     

Indirect evidence of submicroscopic pores in giant unilamellar [correction of unilamelar] vesicles

Formation of pore-like structures in cell membranes could participate in exchange of matter between cell compartments and modify the lipid distribution between the leaflets of a bilayer. We present experiments on two model systems in which major lipid redistribution is attributed to few submicroscopic transient pores. The first kind of experiments consists in destabilizing the membrane of a giant unilamellar vesicle by inserting conic-shaped fluorescent lipids from the outer medium. The inserted lipids (10% of the vesicle lipids) should lead to membrane rupture if segregated on the outer leaflet. We show that a 5-nm diameter pore is sufficient to ease the stress on the membrane by redistributing the lipids. The second kind of experiments consists in forcing giant vesicles containing functionalized lipids to adhere. This adhesion leads to hemifusion (merging of the outer leaflets). In certain cases, the formation of pores in one of the vesicles is attested by contrast loss on this vesicle and redistribution of fluorescent labels between the leaflets. The kinetics of these phenomena is compatible with transient submicroscopic pores and long-lived membrane defects.     

Extracellular transport and integration of plant secretory proteins into pathogen-induced cell wall compartments

Many fungal parasites enter plant cells by penetrating the host cell wall and, thereafter, differentiate specialized intracellular feeding structures, called haustoria, by invagination of the plant's plasma membrane. Arabidopsis PEN gene products are known to act at the cell periphery and function in the execution of apoplastic immune responses to limit fungal entry. This response underneath fungal contact sites is tightly linked with the deposition of plant cell wall polymers, including PMR4/GSL5-dependent callose, in the paramural space, thereby producing localized wall thickenings called papillae. We show that powdery mildew fungi specifically induce the extracellular transport and entrapment of the fusion protein GFP–PEN1 syntaxin and its interacting partner monomeric yellow fluorescent protein (mYFP)–SNAP33 within the papillary matrix. Remarkably, PMR4/GSL5 callose, GFP–PEN1, mYFP–SNAP33, and the ABC transporter GFP–PEN3 are selectively incorporated into extracellular encasements surrounding haustoria of the powdery mildew Golovinomyces orontii , suggesting that the same secretory defense responses become activated during the formation of papillae and haustorial encasements. This is consistent with a time-course analysis of the encasement process, indicating that these extracellular structures are generated through the extension of papillae. We show that PMR4/GSL5 callose accumulation in papillae and haustorial encasements occurs independently of PEN1 syntaxin. We propose a model in which exosome biogenesis/release serves as a common transport mechanism by which the proteins PEN1 and PEN3, otherwise resident in the plasma membrane, together with membrane lipids, become stably incorporated into both pathogen-induced cell wall compartments.     

A new model for nuclear envelope breakdown

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.     

Microscopy and cytochemistry of the biogenesis of the parasitophorous vacuole

Some parasitic protozoa are able to penetrate into host cells where they multiply. The process of penetration involves steps such as attachment to the host cell surface, internalization of the protozoan through an endocytic process with the formation of a parasitophorous vacuole (PV), and the subsequent interaction of the protozoan with the membrane lining the PV. This review analyzes the biogenesis of the PV from a morphological and cytochemical perspective. Special emphasis is given to (a) the localization of plasma membrane-associated enzymes such as Na⁺-K⁺-ATPase, Ca²⁺-ATPase, 5-nucleotidase, and NAD(P)H-oxidase, (b) glycoconjugates, detected using labeled lectins, (c) anionic sites, detected using cationic particles, and (d) integral membrane proteins, using freeze-fracture replicas, and lipids during the formation of the PV containing Trypanosoma cruzi, Leishmania, Toxoplasma gondii, and Plasmodium.     

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.     

CD8+ CTLs are essential for protective immunity against Encephalitozoon cuniculi infection

Encephalitozoon cuniculi is a protozoan parasite that has been implicated recently as a cause of opportunistic infection in immunocompromised individuals. Protective immunity in the normal host is T cell-dependent. In the present study, the role of individual T cell subtypes in immunity against this parasite has been studied using gene knockout mice. Whereas CD4-/- animals resolved the infection, mice lacking CD8+ T cells or perforin gene succumbed to parasite challenge. The data obtained in these studies suggest that E. cuniculi infection induces a strong and early CD8+ T response that is important for host protection. The CD8+ T cell-mediated protection depends upon the CTL activity of this cell subset, as the host is rendered susceptible to infection in the absence of this function. This is the first report in which a strong dependence upon the cytolytic activity of host CD8+ T cells has been shown to be important in a parasite 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 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.