Increased phagocytosis and growth inhibition of Encephalitozoon cuniculi by LPS-activated J774A.1 murine macrophages.

Encephalitozoon cuniculi is an obligate macrophage parasite of vertebrates that commonly infects rodents, monkeys, dogs, birds, and humans. In the present study, we aimed to assess the phagocytosis and intracellular survival of E. cuniculi spores using untreated and lipopolysaccharide (LPS)-activated J774A.1 murine macrophages and assess the macrophage viability. The experimental groups comprised untreated spores, spores killed by heat treatment at 90 °C, and spores killed by treatment with 10% formalin. LPS-activated macrophages significantly increased the phagocytosis of spores and reduced their intracellular growth after 24 and 48 h (P < 0.01); however, after 72 h, we observed an increase in spore replication but no detectable microbicidal activity. These results indicate that LPS activation enhanced E. cuniculi phagocytosis between 24 and 48 h of treatment, but the effect was lost after 72 h, enabling parasitic growth. This study contributes to the understanding of the phagocytosis and survival of E. cuniculi in murine macrophages.

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.

Limited effect of adaptive immune response to control encephalitozoonosis.

This study revises our understanding of the effectiveness of cell-mediated adaptive immunity and treatment against microsporidia using molecular detection and quantification of microsporidia in immunocompetent C57Bl/6 and immunodeficient CD4-/- and CD8-/- mice for the first time. We demonstrate an intense dissemination of microsporidia into most organs within the first weeks post-infection in all strains of mice, followed by a chronic infection characterized by microsporidia persistence in CD4-/- and C57Bl/6 mice and a lethal outcome for CD8-/- mice. Albendazole application reduces microsporidia burden in C57Bl/6 and CD4-/- mice, whereas CD8-/- mice experience only a temporary effect of the treatment. Surprisingly, treated CD8-/- mice survived the entire experimental duration despite enormous microsporidia burden. On the basis of our results, we conclude that microsporidia survive despite the presence of immune mechanisms and treatments that are currently considered to be effective and therefore that CD8 T lymphocytes represent a major, but not sole effector mechanism controlling microsporidiosis. Furthermore, the survival of mice does not correspond to spore burden, which provides new insight into latent microsporidiosis from an epidemiological point of view.

Identification of the microsporidian Encephalitozoon cuniculi as a new target of the IFNγ-inducible IRG resistance system.

The IRG system of IFNγ-inducible GTPases constitutes a powerful resistance mechanism in mice against Toxoplasma gondii and two Chlamydia strains but not against many other bacteria and protozoa. Why only T. gondii and Chlamydia? We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle. We examined another unicellular parasitic organism of mammals, member of an early-diverging group of Fungi, that bypasses the phagocytic mechanism when it enters the host cell: the microsporidian Encephalitozoon cuniculi. Consistent with the known susceptibility of IFNγ-deficient mice to E. cuniculi infection, we found that IFNγ treatment suppresses meront development and spore formation in mouse fibroblasts in vitro, and that this effect is mediated by IRG proteins. The process resembles that previously described in T. gondii and Chlamydia resistance. Effector (GKS subfamily) IRG proteins accumulate at the parasitophorous vacuole of E. cuniculi and the meronts are eliminated. The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function. In addition IFNγ-induced cells infected with E. cuniculi die by necrosis as previously shown for IFNγ-induced cells resisting T. gondii infection. Thus the IRG resistance system provides cell-autonomous immunity to specific parasites from three kingdoms of life: protozoa, bacteria and fungi. The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.

Encephalitozoon cuniculi takes advantage of efferocytosis to evade the immune response.

Microsporidia are recognized as opportunistic pathogens in individuals with immunodeficiencies, especially related to T cells. Although the activity of CD8+ T lymphocytes is essential to eliminate these pathogens, earlier studies have shown significant participation of macrophages at the beginning of the infection. Macrophages and other innate immunity cells play a critical role in activating the acquired immunity. After programmed cell death, the cell fragments or apoptotic bodies are cleared by phagocytic cells, a phenomenon known as efferocytosis. This process has been recognized as a way of evading immunity by intracellular pathogens. The present study evaluated the impact of efferocytosis of apoptotic cells either infected or not on macrophages and subsequently challenged with Encephalitozoon cuniculi microsporidia. Macrophages were obtained from the bone marrow monocytes from C57BL mice, pre-incubated with apoptotic Jurkat cells (ACs), and were further challenged with E. cuniculi spores. The same procedures were performed using the previously infected Jurkat cells (IACs) and challenged with E. cuniculi spores before macrophage pre-incubation. The average number of spores internalized by macrophages in phagocytosis was counted. Macrophage expression of CD40, CD206, CD80, CD86, and MHCII, as well as the cytokines released in the culture supernatants, was measured by flow cytometry. The ultrastructural study was performed to analyze the multiplication types of pathogens. Macrophages pre-incubated with ACs and challenged with E. cuniculi showed a higher percentage of phagocytosis and an average number of internalized spores. Moreover, the presence of stages of multiplication of the pathogen inside the macrophages, particularly after efferocytosis of infected apoptotic bodies, was observed. In addition, pre-incubation with ACs or IACs and/or challenge with the pathogen decreased the viability of macrophages, reflected as high percentages of apoptosis. The marked expression of CD206 and the release of large amounts of IL-10 and IL-6 indicated the polarization of macrophages to an M2 profile, compatible with efferocytosis and favorable for pathogen development. We concluded that the pathogen favored efferocytosis and polarized the macrophages to an M2 profile, allowing the survival and multiplication of E. cuniculi inside the macrophages and explaining the possibility of macrophages acting as Trojan horses in microsporidiosis.

Germination of phagocytosed E. cuniculi spores does not significantly contribute to parasitophorous vacuole formation in J774 cells.

The obligate intracellular microsporidia have developed a unique invasion mechanism to infect their host cells. Spores explosively evert a tube-like structure and extrude the infectious spore content through this organelle into the host cell. Spores from species of the genus Encephalitozoon were also shown to be efficiently internalized by phagocytosis, which led to the hypothesis that spore germination from inside a phagosome might contribute to the infection process. Here, we challenge this hypothesis by quantifying Encephalitozoon cuniculi infection rates of J774 cells that were incubated with the phagocytosis inhibitor cytochalasin D. We demonstrate that the invasion rate in cytochalasin D-treated cells is identical to untreated controls, although phagocytic uptake of E. cuniculi spores was less than 10% of control samples. This study suggests that germination of phagocytosed spores is not a significant infection mode for E. cuniculi.

The microsporidian polar tube: evidence for a third polar tube protein (PTP3) in Encephalitozoon cuniculi.

The invasion strategy used by microsporidia is primarily related to spore germination. Small differentiated spores of these fungi-related parasites inject their contents into target cells through the lumen of a rapidly extruded polar tube, as a prerequisite to obligate intracellular development. Previous studies in Encephalitozoon species that infect mammals have identified two major antigenic polar tube proteins (PTP1 and PTP2) which are predicted to contribute to the high tensile strength of the polar tube via an assembly process dependent on disulfide linkages. By immunoscreening of a cDNA library, we found that a novel PTP is encoded by a single transcription unit (3990 bp) located on the chromosome XI of E. cuniculi. PTP3 is predicted to be synthesized as a 1256-amino acid precursor with a cleavable signal peptide. The mature protein lacks cysteine residue and its large acidic core is flanked by highly basic N- and C-terminal regions. Immunolocalization data indicated that PTP3 is involved in the sporoblast-to-spore polar tube biogenesis. A transcriptional up-regulation during sporogony is supported by a strong increase in the relative amount of Ecptp mRNAs within host cells sampled at late post-infection times. To begin to explore polar tube-associated protein interactions, spore proteins were extracted in the presence of SDS and dithiothreitol then incubated with a chemical cross-linker (DSP or sulfo-EGS). A large multimeric complex was formed and shown to contain PTP1, PTP2 and PTP3 with a few other proteins. PTP3 is hypothesized to play a role in the control of the polar tube extrusion as part of a specific response to ionic stimuli.

Disseminated lethal Encephalitozoon cuniculi (genotype III) infections in cotton-top tamarins (Oedipomidas oedipus)--a case report.

For the first time, Encephalitozoon (E.) cuniculi genotype III ('dog strain') was verified in two cotton-top tamarins (Oedipomidas oedipus) by light microscopy, immunohistochemistry, electron microscopy, PCR and sequencing. The animals had a disseminated lethal infection with this protist. In earlier reports, genotype III had been found only in domestic dogs, man, emperor tamarins (Saguinus imperator) and golden lion tamarins (Leontopithecus rosalia). This investigation establishes now that the 'dog strain' can occur in cotton-top tamarins too. This is further evidence for the zoonotic potential of E. cuniculi. Furthermore, free E. cuniculi spores were identified also in blood vessels of several tissues. These findings indicate that during a disseminated infection E. cuniculi spores can occur in peripheral blood, too. We propose that blood should also be included in the investigations for the detection of microsporidia, so that a possible disseminated course of an infection can be detected.

Humoral immune response in adult blue foxes (Alopex lagopus) after oral infection with Encephalitozoon cuniculi spores.

Encephalitozoon cuniculi causes severe diseases in blue fox puppies. When pregnant vixens are infected, parasites are transmitted over the placenta to the unborn that subsequently develop encephalitozoonosis. Adult foxes themselves do not have signs of disease, but show antibody titres to E. cuniculi. The purpose of the present study was to gain information on the immune response in adult foxes after experimental infection. Sixteen foxes were infected orally with E. cuniculi spores, eight of them twice and 28 days apart. The two groups of animals showed elevated serological values in both the carbon immunoassay and in the ELISA. Elevated serological levels were recorded up to 1 year after the infection took place. The control group (n=8) remained serologically negative throughout the trial. The results of the study showed that blue foxes could be seropositive for at least a year after oral infection with E. cuniculi.

An improved procedure for Percoll gradient separation of sporogonial stages in Encephalitozoon cuniculi (Microsporidia).

Intracellular development of microsporidian parasites comprises a proliferative phase (merogony) followed by a differentiation phase (sporogony) leading to the release of resistant spores. Sporogony implies, successively, meront-to-sporont transformation, sporont division into sporoblasts, and sporogenesis. We report a procedure improving the separation of sporogonial stages of Encephalitozoon cuniculi, a species that develops inside parasitophorous vacuoles of mammalian cells. Supernatants of E. cuniculi-infected Madin-Darby canine kidney cell cultures provided a large number of parasites mixed with host-cell debris. This material was gently homogenized in phosphate-buffered saline containing 0.05% saponin and 0.05% Triton X-100 then filtered through glass wool columns. Centrifugation of the filtrate on 70% Percoll-0.23 M sucrose gradient gave a reproducible pattern of bands at different densities. Transmission electron microscopy showed that three of the four collected fractions were free of visible contaminants. Corresponding prominent cell stages were early sporoblasts (fraction B), late sporoblasts plus immature spores (fraction C), and mature spores (fraction D). Further centrifugation of the lightest fraction (A) on 30% Percoll-0.23 M sucrose gradient generated a sporont-rich fraction (A2). First analysis of proteins from fractions A2 and D by two-dimensional gel electrophoresis suggested a potential use of the described method for proteomic profiling.

Disseminated encephalitozoonosis in captive, juvenile, cotton-top (Saguinus oedipus) and neonatal emperor (Saguinus imperator) tamarins in North America.

Disseminated encephalitozoonosis was diagnosed in 2 sibling, juvenile, cotton-top tamarins (Saguinus oedipus) and 3 sibling, neonatal, emperor tamarins (S. imperator) by use of histologic examination, histochemical analysis, electron microscopy, and polymerase chain reaction (PCR) analysis with nucleotide sequencing. All tamarins were captive born at zoos in North America and died with no premonitory signs of disease. The main pathologic findings were myocarditis (4/5), hepatitis (3/5), interstitial pneumonia (3/5), skeletal myositis (3/5), meningoencephalitis (2/5), adrenalitis (2/5), tubulointerstitial nephritis (1/5), myelitis (1/5), sympathetic ganglioneuritis (1/5), and retinitis (1/5). Central nervous system lesions were the most prominent findings in cotton-top tamarins. The inflammation was predominantly lymphocytic and suppurative in cotton-top tamarins, whereas emperor tamarins had granulomatous or lymphoplasmacytic lesions. Intralesional periodic acid-Schiff-, gram-, or acid-fast (or all 3)-positive, oval-to-elliptical shaped organisms were found in 1 cotton-top and the 3 emperor tamarins. By electron microscopy, these organisms were consistent with microsporidia of the genus Encephalitozoon. E. cuniculi genotype III was detected by PCR analysis and sequencing in paraffin-embedded brain, lung, and bone marrow specimens from the cotton-top tamarins. Although PCR results were negative for one of the emperor tamarins, their dam was seropositive for E. cuniculi by ELISA and Western blot immunodetection. These findings and recent reports of encephalitozoonosis in tamarins in Europe suggest that E. cuniculi infection may be an emerging disease in callitrichids, causing high neonatal and juvenile mortality in some colonies. The death of 2 less than 1-day-old emperor tamarins from a seropositive dam supports the likelihood of vertical transmission in some of the cases reported here.

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.

Proteomic analysis of the eukaryotic parasite Encephalitozoon cuniculi (microsporidia): a reference map for proteins expressed in late sporogonial stages.

The microsporidian Encephalitozoon cuniculi is a unicellular obligate intracellular parasite considered as an emerging opportunistic human pathogen. The differentiation phase of its life cycle leads to the formation of stress-resistant spores. The E. cuniculi genome (2.9 Mbp) having been sequenced, we undertook a descriptive proteomic study of a spore-rich cell population isolated from culture supernatants. A combination of 2-DE and 2-DE-free techniques was applied to whole-cell protein extracts. Protein identification was performed using an automated MALDI-TOF-MS platform and a nanoLC-MS/MS instrument. A reference 2-DE map of about 350 major spots with multiple isoforms was obtained, and for the first time in microsporidia, a large set of unique proteins (177) including proteins with unknown function in a proportion of 25.6% was identified. The data are mainly discussed with reference to secretion and spore structural features, energy and carbohydrate metabolism, cell cycle control and parasite survival in the environment.

Encephalitozoon cuniculi: quantitation of parasites and evaluation of viability.

Two methods (manual and automated) for quantitation of viable versus dead Encephalitozoon cuniculi are reported. The manual method uses ethidium bromide and acridine orange to stain dead and viable organisms, respectively. The stained organisms are visually differentiated with the aid of a fluorescence microscope. The automated method uses propidium iodide to stain dead parasites, which are differentiated from viable unstained parasites with the aid of a flow cytometer. An automated cell counter (Coulter Counter) was used to count rapidly large numbers of samples and to improve the sensitivity of counting low concentrations of parasites. These methods will enhance investigators' abilities to conduct quantitative experiments on host defense mechanisms against E. cuniculi.

In vitro model to assess effect of antimicrobial agents on Encephalitozoon cuniculi.

We have developed a new micromethod to study the effect of drugs on microsporidia, using MRC5 fibroblasts infected by 10(5) spores of Encephalitozoon cuniculi. After 3 days of incubation with various concentrations of drugs, parasitic foci were counted in stained cultures. The inhibition of microsporidial growth exceeding 90% with albendazole (0.005 microgram/ml), fumagillin (0.001 microgram/ml), 5-fluorouracil (3 micrograms/ml), and sparfloxacin (30 micrograms/ml) was observed. Chloroquine, pefloxacin, azithromycin, and rifabutin were partially effective, at high concentrations. Arprinocid, metronidazole, minocycline, doxycycline, itraconazole, and difluoromethylornithine were not evaluable, since concentrations that inhibited microsporidia were also toxic for fibroblasts. Pyrimethamine, piritrexim, sulfonamides, paromomycin, roxithromycin, atovaquone, and flucytosine were ineffective. Our results confirm that albendazole and fumagillin have marked activity against E. cuniculi and show the antimicrosporidial activity of 5-fluorouracil and sparfloxacin. These data may form the basis for treatment of Encephalitozoon hellem and Septata intestinalis infections and represent an attempt to identify drugs effective against Enterocytozoon bieneusi.

Fractionation of sporogonial stages of the microsporidian Encephalitozoon cuniculi by Percoll gradients.

Microsporidia are obligate intracellular parasites that are increasingly recognized as a cause of opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi has been identified in humans with AIDS and infects a wide range of mammalian hosts. Little is known about the metabolic processes that regulate growth and replication of microsporidia. Examination of the individual stages of development will facilitate such studies and reveal possible targets for drug therapy. The purpose of this study was to fractionate and purify stages of the microsporidian life cycle. Encephalitozoon cuniculi were cultured in RK-13 cells. The tissue supernatants containing multiple parasite stages, empty microsporidial husks and host cell debris were collected, washed, and subjected to differential centrifugation in 80% stock isotonic Percoll. Transmission electron microscopy and SDS-polyacrylamide gel electrophoresis were used to compare the content and purity of each fraction. Mature spores formed a band at a density of approximately 1.138 g/ml. Sporoblasts were found at densities between 1.102 g/ml and 1.119 g/ml. A mixture of sporonts, sporoblasts, microsporidial husks, and cell debris remained at the top of the gradient and additional centrifugation in 30% and 50% Percoll resulted in separation of these stages. These results represent the first step toward fractionating stages of microsporidia infecting humans.

The infectivity of Encephalitozoon cuniculi in vivo and in vitro.

The infectivity of Encephalitozoon cuniculi grown in cell cultures was determined in cultured cells and in wild and domestic rabbits. The ratio of the total to tissue culture viable count was 1,300 (median of seven determinations). The mean ratio of intact spore count to total count, as determined by electron microscopy was 0.12. Although variation between infectivity experiments was large, the median animal infective dose contained 51 FFU (cell culture focus-forming units) for wild rabbits (Oryctolagus cuniculus) and 40 FFU for domestic rabbits. These two infectivities were not statistically different.

In vitro culture, ultrastructure, antigenic, and molecular characterization of Encephalitozoon cuniculi isolated from urine and sputum samples from a Spanish patient with AIDS.

In this report we describe the cultivation of two isolates of microsporidia, one from urine and the other from sputum samples from a Spanish AIDS patient. We identified them as Encephalitozoon cuniculi, type strain III (the dog genotype), based on ultrastructure, antigenic characteristics, PCR, and the sequence of the ribosomal DNA internal transcribed spacer region.

Rabbit intestinal xenograft model for human Encephalitozoon infections in mice.

BACKGROUND AND PURPOSE: The gastrointestinal tract is a common portal of entry for Encephalitozoon cuniculi, one of several microsporidial organisms emerging as opportunistic pathogens in immunocompromised humans. Although most human microsporidial pathogens can be propagated in vitro and in a variety of laboratory animals, an experimental animal system to specifically study intestinal uptake and systemic spread of these organisms does not exist. METHODS: Paired segments of near-term fetal rabbit small intestine were implanted subcutaneously into 25 athymic nude or 10 severe combined immune deficient mice. Five weeks after surgery, 65 xenografts were inoculated intraluminally with E. cuniculi (n = 14), E. intestinalis (n = 27), E. hellem (n = 20), or RK-13 cells (n = 2), or were left uninoculated (n = 2). RESULTS: Intestinal xenograft infection with E. cuniculi (n = 11), E. intestinalis (n = 17), and E. hellem (n = 18) was determined by light microscopy; control xenografts remained uninfected. Extraintestinal infection with E. cuniculi developed in host mouse brain, respiratory tract, spleen, salivary glands, and gastrointestinal tract (3 of 3 mice), and infection with E. intestinalis developed in the liver (8 of 15 mice). CONCLUSION: Intestinal xenografts provide a unique, sterile, and biologically relevant animal model system for studying host enterocyte/parasite interactions, mechanisms of microsporidial pathogenicity, antimicrosporidial chemotherapeutic agents, and immune effector mechanisms. This model provides evidence for persistent graft infection with three Encephalitozoon spp., and for intestinal spread of E. cuniculi and E. intestinalis from infected enterocytes in immunoincompetent mice.

Evidence of actin in the cytoskeleton of microsporidia.

Using transmission electron microscopy, immuno-electron microscopy, and biochemical techniques such as 2-D electrophoresis and immunoblotting, actin was found in all biological stages of the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi.