Heat-killed Malassezia pachydermatis suspension modulates the activity of macrophages challenged with Encephalitozoon cuniculi.

Phagocytic responses are critical for effective host defense against opportunistic fungal pathogens, such as Encephalitozoon cuniculi, an obligate intracellular fungus that causes emerging encephalitozoonosis in humans and other animals. Malassezia has immunomodulatory effects and can modulate the production of pro- and anti-inflammatory cytokines via keratinocytes and human monocytes. In this study, we evaluated the modulatory effects of heat-killed Malassezia pachydermatis suspension on macrophages challenged with Encephalitozoon cuniculi. Macrophages were treated with heat-killed M. pachydermatis suspension before being infected with spores of E. cuniculi. The cultures were stained with calcofluor, and the spores, internalized or not, were counted to determine their phagocytic capacity and index (PC and PI, respectively). Microbicidal and phagocytic activities were evaluated by transmission electron microscopy (TEM). The untreated macrophages had higher PC and PI and number of phagocytosed spores than treated macrophages. However, TEM revealed that treated macrophages had higher microbicidal activity because there were few spores in different degrees of degeneration and amorphous materials in the phagocytic vacuoles. Macrophages treated with heat-killed M. pachydermatis suspension had lower PC and PI and incipient presence of E. cuniculi in phagosomes. Treated macrophages had a mixed pattern of cytokine release with Th1, Th2, and Th17 profiles, with emphasis on interleukin (IL)-10, IL-4, IL-17, IL-6, and interferon (IFN)-γ secretion, and particularly high production of anti-inflammatory cytokines. Our results suggest that treatment with heat-killed M. pachydermatis suspension increases the release of cytokines and decreases the phagocytic activity of macrophages challenged with E. cuniculi.

Encephalitozoon cuniculi infection in cats: European guidelines from the ABCD on prevention and management.

OVERVIEW: Encephalitozoon cuniculi is a common obligate intracellular microsporidian parasite of rabbits that is increasingly recognised as a pathogen of cats and other mammalian species. These guidelines aim to review the literature on feline E cuniculi infection and provide recommendations on prevention and management. INFECTION IN CATS: E cuniculi infection should be considered as a differential diagnosis in cases of feline uveitis and cataract formation. It is not significantly associated with either chronic kidney disease or meningoencephalitis. E cuniculi infection is more common in stray or feral cats than in pet cats. DIAGNOSIS AND TREATMENT: Serological tests for antibody detection in the blood are easy to perform and can be useful for diagnosis, but their specificity is low as antibodies have been found in apparently healthy cats. PCR appears to be more sensitive than histopathology for diagnosis, and is more sensitive when performed on cataractous lenses compared with aqueous humour, although ease of sampling is an obvious limitation. Treatment is with fenbendazole for 3 weeks and phacoemulsification to remove microsporidia from cataractous lenses. ZOONOTIC RISK: E cuniculi is a potential zoonotic agent, and there is a particular risk to immunocompromised humans posed by infected rabbits. Albeit infrequent, spore shedding has been identified in cats, so care should be taken around infected cats.

Characterization of humoral and cell-mediated immunity in rabbits orally infected with Encephalitozoon cuniculi.

Encephalitozoonosis is a common infectious disease widely spread among rabbits. Encephalitozoon cuniculi, is considered as a zoonotic and emerging pathogen capable of infecting both immunocompetent and immunocompromised hosts. The aim of the study was to describe in detail the spread of the E. cuniculi in a rabbit organism after experimental infection and the host humoral and cellular immune response including cytokine production. For that purpose, healthy immunocompetent rabbits were infected orally in order to simulate the natural route of infection and euthanised at 2, 4, 6 and 8-weeks post-infection. Dissemination of E. cuniculi in the body of the rabbit was more rapid than previously reported. As early as 2 weeks post-infection, E. cuniculi was detected using immunohistochemistry not only in the intestine, mesenteric lymph nodes, spleen, liver, kidneys, lungs and heart, but also in nervous tissues, especially in medulla oblongata, cerebellum, and leptomeninges. Based on flow cytometry, no conspicuous changes in lymphocyte subpopulations were detected in the examined lymphoid organs of infected rabbits. Cell-mediated immunity was characterized by ability of both CD4+ and CD8+ T cells to proliferate after stimulation with specific antigens. Th1 polarization of immune response with a predominance of IFN-γ expression was detected in spleen, mesenteric lymph nodes and Peyer's patches. The increased expression of IL-4 and IL-10 mRNA in mixed samples from the small intestine is indicative of balanced control of IFN-γ, which prevents tissue damage. On the other hand, it can enable E. cuniculi to survive and persist in the host organism in a balanced host-parasite relationship. The Th17 immunity lineage seems to play only a minor role in E. cuniculi infection in rabbits.

Dual infection of urinary tract with Enterocytozoon bieneusi and Encephalitozoon cuniculi in HIV/AIDS patients

Microsporidia are emerging pathogens which cause an opportunistic infections in immunocompromised patients, especially those with AIDS. Intestinal microsporidiosis is the most recognized infection, whereas urinary tract infections caused by microsporidia are rarely paid attention to either due to their subclinical course or diagnostic difficulties. In this report dual microsporidial infection of urinary tract, caused by Enterocytozoon bieneusi and Encephalitozoon cuniculi was described in HIV/AIDS patients under cART therapy. Since microsporidiosis can cause severe complications or even death in immunosuppressed patients, our results suggest that microsporidial infection should be included in routine investigation of HIV-positive patients, even asymptomatic.

Encephalitozoon cuniculi and Vittaforma corneae (Phylum Microsporidia) inhibit staurosporine-induced apoptosis in human THP-1 macrophages in vitro.

Obligately intracellular microsporidia regulate their host cell life cycles, including apoptosis, but this has not been evaluated in phagocytic host cells such as macrophages that can facilitate infection but also can be activated to kill microsporidia. We examined two biologically dissimilar human-infecting microsporidia species, Encephalitozoon cuniculi and Vittaforma corneae, for their effects on staurosporine-induced apoptosis in the human macrophage-differentiated cell line, THP1. Apoptosis was measured after exposure of THP-1 cells to live and dead mature organisms via direct fluorometric measurement of Caspase 3, colorimetric and fluorometric TUNEL assays, and mRNA gene expression profiles using Apoptosis RT2 Profiler PCR Array. Both species of microsporidia modulated the intrinsic apoptosis pathway. In particular, live E. cuniculi spores inhibited staurosporine-induced apoptosis as well as suppressed pro-apoptosis genes and upregulated anti-apoptosis genes more broadly than V. corneae. Exposure to dead spores induced an opposite effect. Vittaforma corneae, however, also induced inflammasome activation via Caspases 1 and 4. Of the 84 apoptosis-related genes assayed, 42 (i.e. 23 pro-apoptosis, nine anti-apoptosis, and 10 regulatory) genes were more affected including those encoding members of the Bcl2 family, caspases and their regulators, and members of the tumour necrosis factor (TNF)/TNF receptor R superfamily.

Differences in the intensity of infection caused by Encephalitozoon cuniculi genotype II and III - Comparison using quantitative real-time PCR.

Microsporidia are a group of obligate intracellular eukaryotic parasites, which are able to infect a wide range of animals, including humans. Four genotypes of Encephalitozoon cuniculi have been found to date. The different courses of microsporidiosis described in humans, which are dependent on immunological status of the host and genotype of E. cuniculi, have been successfully imitated in murine models. In the present study, we quantified the microsporidial burden in individual organs of a murine experimental model, using qPCR and we compared the parasitic load of two genotypes of E. cuniculi, namely genotype II and III (EC II and EC III). While the extent of microsporidiosis caused by EC II gradually increased over 35 days post infection (DPI) in both immunocompetent and immunodeficient mice and caused death in the latter at 28 DPI, EC III had spread into all host organs by seven DPI and was not lethal for either mouse strain during the experimental time period. Moreover, EC III persisted in many organs until termination of the experiment. The number of microsporidial spores in individual organs was ten times higher in EC III-infected animals compared to those infected with EC II. EC II infection also progressively shifted towards organs outside the gastrointestinal tract (GIT) in both monitored mouse strains; whereas, EC III infection equally remained in both the GIT and organs outside the GIT. With the increasing use of molecular methods in diagnostics, it is important to better understand the pathophysiology of microsporidia, including its ability to escape from the immune system and persist in host organisms. Our results indicate that pathogenicity is not directly connected to spore burden, as infection caused by E. cuniculi genotype II is less extensive and spreads more slowly within the host organism than infection caused by E. cuniculi genotype III, but which caused the earlier death of immunodeficient mice.

B-1 cells upregulate CD8 T lymphocytes and increase proinflammatory cytokines serum levels in oral encephalitozoonosis.

Microsporidia are intracellular pathogens that cause severe disease in immunocompromised humans and animals. We recently demonstrated that XID mice are more susceptible to Encephalitozoon cuniculi infection by intraperitoneal route, evidencing the role of B-1 cells in resistance against infection. The present study investigated the resistance and susceptibility against E. cuniculi oral infection, including the role of B-1 cells. BALB/c and BALB/c XID (B-1 cells deficient) mice were orally infected with E. cuniculi spores. No clinical symptoms were observed in infected animals; histopathology showed lymphoplasmocytic enteritis with degeneration of the apexes of the villi in all infected groups. Higher parasite burden was observed in infected BALB/c XID mice. In the spleen and peritoneum, all infected mice showed a decrease of lymphocytes, including CD8+ T cells, mostly in infected BALB/c XID mice. Adoptive transfer of B-1 cells (XID + B-1) was associated with a lower parasite burden. Pro-inflammatory cytokines (IFN-γ, TNF-α and IL-6) increased mostly in infected XID + B1 mice. Together, the present results showed that BALB/c XID mice infected by the oral route were more susceptible to encephalitozoonosis than BALB/c mice, demonstrating the B-1 cells importance in the control of the immune response against oral E. cuniculi infection.

The course of infection caused by Encephalitozoon cuniculi genotype III in immunocompetent and immunodeficient mice.

Encephalitozoon cuniculi is probably the most common microsporidia which infects a wide range of vertebrates, including human. So far, four genotypes of this parasite have been identified based on the rRNA internal transcribed spacer variations. The course of infection caused by E. cuniculi III had very massive onset in immunocompetent host characterized by the presence of this parasite in all organs and tissues within one week after peroral infection. Encephalitozoonosis caused by E. cuniculi III had very progressive spreading into all organs within first week post inoculation in immunocompromised SCID mice and led to the death of the host. The experimental treatment with albendazole of immunocompetent BALB/c mice infected with E. cuniculi III have shown very weak effect. Our findings clearly showed that the different course of infection and response to treatment depends not only on the immunological status of the host, but also on the genotype of microsporidia. It could be very important especially for individuals under chemotherapy and transplant recipients of organs originating from infected donors.

Dynamics of parasitophorous vacuoles formed by the microsporidian pathogen Encephalitozoon cuniculi.

It has been a long-standing debate if sexual development occurs in the microsporidian lineages. Previous studies, including morphological observations, ploidy analysis, and the presence of a sex-related locus, provided evidence of possible extant of sexual development. This study presents another line of evidence by monitoring the parasitophorous vacuoles (PVs) formed by Encephalitozoon cuniculi. Time lapse observations of infection cycles of E. cuniculi revealed that multiple PVs can be formed in a single host cell and the PVs in the single cell can merge (fusion) or split (fission). The dynamics of PVs may provide a route for interactions between genetically distinct microsporidian isolates during host infections.

Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1.

Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex.

Seroprevalence of Encephalitozoon cuniculi in wild rodents, foxes and domestic cats in three sites in the United Kingdom.

Encephalitozoon cuniculi is an obligate intracellular microsporidian that is the causal agent of encephalitozoonosis, an important and emerging disease in both humans and animals. Little is known about its occurrence in wildlife. In this study, serum samples from 793 wild rodents [178 bank voles (BV), 312 field voles (FV) and 303 wood mice (WM)], 96 foxes and 27 domestic cats from three study areas in the UK were tested for the presence of antibodies to E. cuniculi using a direct agglutination test (DAT). Seroprevalence in the wild rodents ranged from 1.00% to 10.67% depending on species (overall 5.31%) and was significantly higher in foxes [49.50% (50/96)]. None of the 27 cats sampled were found to be seropositive. This is the first report of seroprevalence to E. cuniculi in BV, FV, WM, foxes and cats in the UK and provides some evidence that foxes could act as sentinels for the presence of E. cuniculi in rodents. The study demonstrates that wildlife species could be significant reservoirs of infection for both domestic animals and humans.

Gain and loss of multiple functionally related, horizontally transferred genes in the reduced genomes of two microsporidian parasites.

Microsporidia of the genus Encephalitozoon are widespread pathogens of animals that harbor the smallest known nuclear genomes. Complete sequences from Encephalitozoon intestinalis (2.3 Mbp) and Encephalitozoon cuniculi (2.9 Mbp) revealed massive gene losses and reduction of intergenic regions as factors leading to their drastically reduced genome size. However, microsporidian genomes also have gained genes through horizontal gene transfers (HGT), a process that could allow the parasites to exploit their hosts more fully. Here, we describe the complete sequences of two intermediate-sized genomes (2.5 Mbp), from Encephalitozoon hellem and Encephalitozoon romaleae. Overall, the E. hellem and E. romaleae genomes are strikingly similar to those of Encephalitozoon cuniculi and Encephalitozoon intestinalis in both form and content. However, in addition to the expected expansions and contractions of known gene families in subtelomeric regions, both species also were found to harbor a number of protein-coding genes that are not found in any other microsporidian. All these genes are functionally related to the metabolism of folate and purines but appear to have originated by several independent HGT events from different eukaryotic and prokaryotic donors. Surprisingly, the genes are all intact in E. hellem, but in E. romaleae those involved in de novo synthesis of folate are all pseudogenes. Overall, these data suggest that a recent common ancestor of E. hellem and E. romaleae assembled a complete metabolic pathway from multiple independent HGT events and that one descendent already is dispensing with much of this new functionality, highlighting the transient nature of transferred genes.

Encephalitozoonosis in pharmacologically immunosuppressed mice.

Encephalitozoon cuniculi is a parasite that has been identified as a cause of opportunistic infections in immunocompromised individuals. This study was performed to evaluate E. cuniculi infection in pharmacologically immunosuppressed mice. Mice were immunosuppressed with cyclophosphamide (100mg/kg twice a week, IP) or cyclosporin (10mg/kg daily, IP) and inoculated with 10(7)E. cuniculi spores IP. The E. cuniculi spores were cultivated in MDCK cells. E. cuniculi identification was performed by light microscopy studies using Gram-Chromotrope, Hematoxylin-Eosin and Toluidine blue-fuchsin staining techniques, as well as by PCR at 15, 30 and 45 days post-inoculation (DPI). Cyclophosphamide-immunosuppressed mice have greatly reduced amounts of CD8(+), CD4(+) and CD3(+) T cells and CD19(+) B cells. The cells from these mice were analyzed by FACS and showed acute disseminated and fatal encephalitozoonosis. Mice treated with ciclosporin, which is both antiparasitic and immunosuppressive, have a milder, chronic, non-lethal infection and showed a significant reduction only in CD3(+) and CD4(+) T cell numbers. Our results support the role of CD8(+) T cells in controlling infection by E. cuniculi and show that preventive measures are essential for preventing this zoonosis in individuals undergoing chemotherapy for cancer or other immunosuppressive therapies.

Interactions of Encephalitozoon cuniculi polar tube proteins.

Microsporidia are eukaryotic, obligate intracellular organisms defined by small spores that contain a single invasion organelle, the polar tube, which coils around the interior of the spore. When these parasites infect host cells, the polar tube is discharged from the anterior pole of the spore, pierces the cell, and transfers sporoplasm into the cytoplasm of the host. Three polar tube proteins (PTP1, PTP2, and PTP3) have been identified in this structure. The interactions of these proteins in the assembly and function of the polar tube are not known. This study was undertaken to examine the protein interactions of the Encephalitozoon cuniculi polar tube proteins (EcPTPs). Immunofluorescence and immunoelectron microscopy confirmed the colocalization of EcPTP1, EcPTP2, and EcPTP3 to the polar tube. Experiments using cross-linkers indicated that EcPTP1, EcPTP2, and EcPTP3 form a complex in the polar tube, which was confirmed by immunoprecipitation using EcPTP1 antiserum. Yeast two-hybrid analysis revealed that full-length EcPTP1, EcPTP2, and EcPTP3 interact with each other in vivo. Both the N and C termini of EcPTP1 were involved in these interactions, but the central region of this protein, which contains a repetitive motif, was not. Further studies of polar tube proteins and their structural interactions may help elucidate the formation of the polar tube during the invasion process.

Evaluation of the usefulness of an ELISA and protein electrophoresis in the diagnosis of Encephalitozoon cuniculi infection in rabbits.

OBJECTIVE-To evaluate the usefulness of an antibody detection ELISA and protein electrophoresis (PE) for diagnosing Encephalitozoon cuniculi (ECUN) infection in pet rabbits. ANIMALS-203 pet rabbits. PROCEDURES-Serum and plasma samples from pet rabbits were submitted from veterinary clinics within the United States. Participating veterinarians completed a questionnaire that was used to classify rabbits as clinically normal (n=33), suspected of having an ECUN infection (103), or clinically abnormal but not suspected of having an ECUN infection (67). An ELISA for detection of serum or plasma IgG against ECUN was developed by use of commercially available reagents. Results of the ELISA and PE were used to detect ECUN infection. RESULTS-A high seroprevalence of antibody against ECUN was detected in all 3 groups of rabbits. In rabbits suspected of having an ECUN infection, the mean IgG titer was 1.7 times as high as the values in the other rabbit groups. Rabbits suspected of having an ECUN infection and those that were simply clinically abnormal had a higher concentration of gamma-globulins than clinically normal rabbits. This increase in globulins concentration was accompanied by a decrease in the albumin-to-globulin ratio. Results of the ELISA and PE were significantly different between clinically normal rabbits and those suspected of having an ECUN infection. CONCLUSIONS AND CLINICAL RELEVANCE-The combination of an ELISA and PE may aid in the diagnosis of ECUN infection in pet rabbits. IMPACT FOR HUMAN MEDICINE-Because ECUN is a potential zoonotic agent, diagnostic methods for pet rabbits need to be improved to protect human health.

Effects of interferon gamma and specific polyclonal antibody on the infection of murine peritoneal macrophages and murine macrophage cell line PMJ2-R with Encephalitozoon cuniculi.

Experimental activation of peritoneal macrophages by interferon gamma (IFN-gamma) resulted in the inhibition of Encephalitozoon cuniculi replication. However, E. cuniculi could replicate either in a non-activated cell line of murine macrophages PMJ2-R or in IFN-gamma-activated PMJ2-R cells. Moreover, activation with IFN-gamma led to faster replication of E. cuniculi in these cells. Opsonisation of E. cuniculi spores with anti-E. cuniculi polyclonal antibody did not affect E. cuniculi replication in both, non-activated and activated murine macrophages. In contrast, opsonisation of E. cuniculi spores caused the most effective replication of E. cuniculi in activated PMJ2-R cells. However, production of nitric oxide by these cells was significantly more intensive than that in non-activated, infected cells, where the parasite replicated to a much lesser extent. Our results support the hypothesis that E. cuniculi uses phagocytosis for the infection of host cells. They also indicate that the mechanism by which spores of E. cuniculi are killed by macrophages is not dependent on nitric oxide and they reveal that PMJ2-R cells cannot substitute peritoneal murine macrophages in immunological studies on E. cuniculi.

Expression of two cell wall proteins during the intracellular development of Encephalitozoon cuniculi: an immunocytochemical and in situ hybridization study with ultrathin frozen sections.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that develops asynchronously inside parasitophorous vacuoles. Spore differentiation involves the construction of a cell wall commonly divided into an outer layer (exospore) and a thicker, chitin-rich inner layer (endospore). The developmental patterns of protein deposition and mRNA expression for 2 different spore wall proteins were studied using immunocytochemical and in situ hybridization procedures with ultrathin frozen sections. The onset of deposition of an exospore-destined protein (SWP1) correlated with the formation of lamellar protuberances during meront-to-sporont conversion. No evidence for a release of SWP1 towards the parasitophorous vacuole lumen was obtained. An endospore-destined protein (EnP1) was detected early on the plasma membrane of meronts prior to extensive accumulation within the chitin-rich layer of sporoblasts. swp1 mRNA was preferentially synthesized in early sporogony while enp1 mRNA was transcribed during merogony and a large part of sporogony. The level of both mRNAs was reduced in mature spores. Considering the availability of the E. cuniculi genome sequence, the application of nucleic and/or protein probes to cryosections should facilitate the screening of various genes for stage-specific expression during microsporidian development.

Proteolytic activity in Encephalitozoon cuniculi sporogonial stages: predominance of metallopeptidases including an aminopeptidase-P-like enzyme.

A fraction enriched in spore precursor cells (sporoblasts) of the microsporidian Encephalitozoon cuniculi, an intracellular parasite of mammals, was obtained by Percoll gradient centrifugation. Soluble extracts of these cells exhibited proteolytic activity towards azocasein, with an alkaline optimum pH range (9-10). Prevalence of some metallopeptidases was supported by the stimulating effect of Ca2+, Mg2+, Mn2+ and Zn2+ ions, and inhibition by two chelating agents (EDTA and 1,10-phenanthroline), a thiol reductant (dithiothreitol) and two aminopeptidase inhibitors (bestatin and apstatin). Zymographic analysis revealed four caseinolytic bands at about 76, 70, 55 and 50 kDa. Mass spectrometry of tryptic peptides from one-dimensional gel slices identified a cytosol (leucine) aminopeptidase homologue (M17 family) in 50-kDa band and an enzyme similar to aminopeptidase P (AP-P) of cytosolic type (M24B subfamily) in 70-kDa band. Multiple sequence alignments showed conservation of critical residues for catalysis and metal binding. A long insertion in a common position was found in AP-P sequences from E. cuniculi and Nosema locustae, an insect-infecting microsporidian. The expression of cytosolic AP-P in sporogonial stages of microsporidia may suggest a key role in the attack of proline-containing peptides as a prerequisite to long-duration biosynthesis of structural proteins destined to the sporal polar tube.

The putative chitin deacetylase of Encephalitozoon cuniculi: a surface protein implicated in microsporidian spore-wall formation.

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide deacetylase family with a suspected chitin deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.

Cell invasion and intracellular fate of Encephalitozoon cuniculi (Microsporidia).

Microsporidia are obligate intracellular parasites that utilize a unique mechanism to infect host cells, which is one of the most sophisticated infection mechanisms in biology. Microsporidian spores contain a long coiled polar tube that extrudes from the spores and penetrates the membranes of new host cells. We have initiated a study to investigate the invasive process and intracellular fate of the microsporidium Encephalitozoon cuniculi. Here we show that relatively few cells were infected through the traditional penetration of the polar tube from outside. Rather, phagocytosis of spores occurred at least 10 times more frequently than injection of sporoplasms. Some spores extruded their polar tube inside the cells following phagocytosis. Membranes of the vacuoles surrounding the internalized spores were positive for late endosomal and lysosomal markers. Spores that remained inside these compartments disappeared within 3 days. Thus, our studies demonstrate that in addition to the unique way in which microsporidia infect host cells, E. cuniculi spores can also gain access to host cells by phagocytosis. The presence of intracellular spores that have extruded their polar tube shows that some spores germinate after phagocytosis, thus escaping from the phagosomes that mature into lysosomes.