Role of host cell integrins in the microsporidium Encephalitozoon intestinalis adherence and infection in vitro.

Microsporidia are obligate intracellular, spore-forming, fungal-related pathogens that employ a unique organelle, the polar tube, to transfer infectious spore contents into host cells to initiate infection. Spore adherence to host cells may provide the proximity required for polar tube/host cell interaction during in vivo infection. In previous in vitro studies, host sulfated glycosaminoglycans (GAGs) or recombinant microsporidia endospore protein (EnP1) was implicated in the pathogen adherence and infection process; however, complete ablation of spore adherence and infection could not be achieved, suggesting that additional or alternative spore and host cell determinants of adherence and infection may exist. Analysis of the Encephalitozoon intestinalis genome revealed about 100 predicted proteins containing the canonical integrin-binding motif arginine-glycine-aspartic acid (RGD); and, many pathogens have been shown to engage integrin molecules on cell surfaces. We hypothesized that host cell integrins play a role in microsporidia adherence and infection. In this study, we demonstrated that addition of exogenous integrin ligands or recombinant alpha 3 beta 1 integrin or alpha 5 beta 1 integrin to assays of E. intestinalis adherence and infection significantly reduced spore adherence and infection of host cells, supporting our hypothesis and implicating these specific integrins as putative host cell receptors for E. intestinalis spores.

The role of microsporidian polar tube protein 4 (PTP4) in host cell infection.

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens.

Microsporidiosis genital en mujeres con sida: hallazgos post-mortem / Genital microsporidiosis in women with AIDS: a post-mortem study

Antecedentes. La microsporidiosis es habitualmente una enfermedad oportunista fatal para los pacientes con sida y puede producir una infección localizada o sistémica en función de la especie infectante. La infección del tracto genital femenino por microsporidios ha sido escasamente reportada en la literatura. Objetivos. Describir las especies de microsporidios en el tracto genital femenino. Métodos. Se analizaron muestras de tejidos provenientes del aparato reproductor (ovario, trompa uterina y útero) de ocho mujeres fallecidas con síndrome de desgaste asociado al sida y microsporidiosis diseminada, en el período de 1997 a 2005 en el Instituto de Medicina Tropical Pedro Kourí. Para la identificación de las especies de microsporidios se utilizaron anticuerpos específicos mediante la técnica de inmunohistoquímica indirecta. Resultados. Se describe la infección por microsporidios en el tracto genital femenino. De las ocho mujeres estudiadas con la forma diseminada de estos parásitos, seis presentaron microsporidios en el tracto genital. Se identificaron Encephalitozoon cuniculi y Encephalitozoon hellem en el epitelio de revestimiento de la luz de trompas de Falopio y en endometrio. Conclusiones. Algunas especies de microsporidios pueden diseminarse a diversos órganos, especialmente cuando hay una profunda inmunodeficiencia como ocurre con el sida terminal. Según la literatura revisada esta es la mayor casuística recopilada de microsporidiosis genital(AU)

Background. Microsporidiosis is a life threatening opportunistic infection of AIDS patients. The infection is usually restricted to specific anatomical areas, but could become systemic depending on the involved species. Genital microsporidiosis in female patients is rare. Objective. To report genital microsporidiosis in female AIDS patients. Methods. Tissues samples from the genital tract (ovary, fallopian tubes and uterus) of eight deceased women who died of wasting syndrome associated to AIDS and disseminated microsporidiosis at the Institute of Tropical Medicine Pedro Kourí were collected between 1997 and 2005. Using an indirect immunohistochemistry assay the microsporidia species involved in those cases were identified. Results. We report several cases of microsporidial infection of the female genital tract. Six out of eight women with the disseminated form of the disease showed the presence of microsporidia in the genital tract. Encephalitozoon cuniculi and Encephalitozoon hellem were identified in the internal lining epithelium of the fallopian tubes and endometrium. Conclusions. Microsporidia species could disseminate to other organs and become systemic in severe immunocompromised cases. To our knowledge this is the greatest number of female genital tract microsporidiosis cases so far reported in humans(AU)

Diversity of microsporidia (Fungi: Microsporidia) among captive great apes in European zoos and African sanctuaries: evidence for zoonotic transmission?

Abstract: Two hundred and seventeen captive great apes (150 chimpanzees, Pan troglodytes; 14 bonobos, Pan paniscus; 53 western gorillas, Gorilla gorilla) and 20 personnel from thirteen European zoos and two African sanctuaries were sampled and examined in order to determine the occurrence ofEnterocytozoon bieneusi and species of Encephalitozoon in faecal specimens and to compare the epidemiological situation between zoos and sanctuaries. Microsporidia were detected at all sampling sites. Sequence analyses of ITS amplicons generated by using microsporidia-specific primers determined the presence ofmicrosporidia in 87 samples including 13 humans; since two cases of simultaneous occurrence of Encephalitozoon cuniculi and Enterocytozoon bieneusi were identified, 89 full-length ITS sequences were obtained, namely 78 Encephalitozoon cuniculi genotype I, five E. cuniculi genotype II, two E. hellem 1A and four Enterocytozoon bieneusi. No Encephalitozoon intestinalis-positive samples were identified. This is the first report of Encephalitozoon species and Enterocytozoon bieneusi genotypes in captive great apes kept under various conditions and the first record of natural infection with E. hellem in great apes. A comparison of zoos and sanctuaries showed a significantly higher prevalence of microsporidia in sanctuaries (P<0.001), raising a question about the factors affecting the occurrence of microsporidia in epidemiologically and sanitarily comparable types of facilities.

Effects of host temperature and gastric and duodenal environments on microsporidia spore germination and infectivity of intestinal epithelial cells.

Approximately 14 of the more than 1,000 species of microsporidia infect humans, only two of which, Enterocytozoon bieneusi and Encephalitozoon intestinalis, cause intestinal microsporidiosis. Clinical isolates of three microsporidia species, E. intestinalis, Encephalitozoon hellem, and the insect parasite, Anncaliia (Brachiola, Nosema) algerae were used in a spore germination assay, and enterocyte attachment and infection assays were performed to model the potential roles of gastric and duodenal environments and host temperature in determining why only one of these microsporidia species causes intestinal microsporidiosis. Enterocyte infection with A. algerae spores was 10% that of the Encephalitozoon species, a difference not attributable to differences in spore attachment to host cells. Prior spore treatment with pepsin in HCl, pancreatic enzymes, or ox bile did not inhibit germination or enterocyte infection by the three microsporidia species. While the Encephalitozoon species differentiated to mature spores within 3 days, the time taken for many enterocytes to turn over, A. algerae took 3-5 days to produce mature spores, near the upper limit for enterocyte turnover in vivo. Thus, host temperature may contribute to A. algerae not causing human intestinal microsporidiosis, but none of the factors tested account for the inability of E. hellem to cause such an infection.

Quantitative evaluation of the impact of bather density on levels of human-virulent microsporidian spores in recreational water.

Microsporidial gastroenteritis, a serious disease of immunocompromised people, can have a waterborne etiology. During summer months, samples of recreational bathing waters were tested weekly for human-virulent microsporidian spores and water quality parameters in association with high and low bather numbers during weekends and weekdays, respectively. Enterocytozoon bieneusi spores were detected in 59% of weekend (n = 27) and 30% of weekday (n = 33) samples, and Encephalitozoon intestinalis spores were concomitant in a single weekend sample; the overall prevalence was 43%. The numbers of bathers, water turbidity levels, prevalences of spore-positive samples, and concentrations of spores were significantly higher for weekend than for weekday samples; P values were <0.001, <0.04, <0.03, and <0.04, respectively. Water turbidity and the concentration of waterborne spores were significantly correlated with bather density, with P values of <0.001 and <0.01, respectively. As all water samples were collected on days deemed acceptable for bathing by fecal bacterial standards, this study reinforces the scientific doubt about the reliability of bacterial indicators in predicting human waterborne pathogens. The study provides evidence that bathing in public waters can result in exposure to potentially viable microsporidian spores and that body contact recreation in potable water can play a role in the epidemiology of microsporidiosis. The study indicates that resuspension of bottom sediments by bathers resulted in elevated turbidity values and implies that the microbial load from both sediments and bathers can act as nonpoint sources for the contamination of recreational waters with Enterocytozoon bieneusi spores. Both these mechanisms can be considered for implementation in predictive models for contamination with microsporidian spores.

Human-virulent microsporidian spores in solid waste landfill leachate and sewage sludge, and effects of sanitization treatments on their inactivation.

Solid waste landfill leachate and sewage sludge samples were quantitatively tested for viable Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi spores by the multiplexed fluorescence in situ hybridization (FISH) assay. The landfill leachate samples tested positive for E. bieneusi and the sludge samples for E. bieneusi and E. intestinalis. The effects of four sanitization treatments on the inactivation of these pathogens were assessed. Depending on the variations utilized in the ultrasound disintegration, sonication reduced the load of human-virulent microsporidian spores to nondetectable levels in 19 out of 27 samples (70.4%). Quicklime stabilization was 100% effective, whereas microwave energy disintegration was 100% ineffective against the spores of E. bieneusi and E. intestinalis. Top-soil stabilization treatment gradually reduced the load of both pathogens, consistent with the serial dilution of sewage sludge with the soil substrate. This study demonstrated that sewage sludge and landfill leachate contained high numbers of viable, human-virulent microsporidian spores, and that sonication and quicklime stabilization were the most effective treatments for the sanitization of sewage sludge and solid waste landfill leachates. Multiplexed FISH assay is a reliable quantitative molecular fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi spores in environmental samples.

Diagnosis and manifestation of encephalitozoonosis in mice after experimental infection with different species and application of dexamethasone.

Twenty-eight BALB/c mice were infected with different strains of Encephalitozoon species (Encephalitozoon cuniculi II - mouse type, E. cuniculi III - dog type, Encephalitozoon hellem, Encephalitozoon intestinalis). Five of them were infected with E. cuniculi II (mouse type) and simultaneously immunosuppressed with dexamethasone. Clinical signs of encephalitozoonosis were not remarkable. Ascites was found in two mice of dexamethasone-treated group 14 days post-infection (p.i.). The histopathological changes were found mainly in spleen and liver in the form of lymphoepithelioid granuloma. Spores were found in faeces since day 14 p.i. and visualized by Calcoflour White M2R. After cultivation on cellular cultures (VERO E6 - monkey kidney cells, RK-13 - rabbit kidney fibroblasts), the species differentiation was performed by PCR using panmicrosporidial primers (PMP1, PMP2) and specific primers (ECUN-F, ECUN-R, V1, SI-500). The differences were recorded in the immune response of immunocompetent and immunosuppressed mice. At day 60 p.i., the titres of specific antibodies measured by indirect immunofluorescence antibody test were lower (1:4096) in dexamethasone-treated mice when compared with non-immunosuppressed animals (1:8196). The significant increases of antibody titres were recorded in particular infected groups within the experiment (P < 0.01 between day 14 p.i. and day 30 p.i., P < 0.001 between day 14 p.i. and day 60 p.i.). Experimental encephalitozoonosis in non-immunosuppressed and immunosuppressed mice provides a useful model for the study of immune response and lesions associated with these protozoans.

Augmentation of microsporidia adherence and host cell infection by divalent cations.

The infection process of intracellular opportunistic microsporidia involves the forcible eversion of a coiled hollow polar filament that pierces the host cell membrane, allowing the passage of infectious sporoplasm into the host cell cytoplasm. Although the exact mechanism of spore activation leading to polar filament discharge is unknown, we have shown that spore adherence to host cells, which is mediated by sulfated glycosaminoglycans, may play a vital role. When adherence is inhibited, host cell infection decreases, indicating a direct link between adherence and infection. The goal of this study was to evaluate the effects of exogenous divalent cations on microsporidia spore adherence and infection. Data generated using an in vitro spore adherence assay show that spore adherence is augmented by manganese (Mn2+) and magnesium (Mg2+), but not by calcium (Ca2+). However, each of the three divalent cations contributed to increased host cell infection when included in the assay. Finally, we show that Mn2+ and Mg2+ may activate a constituent on the microsporidia spore, not on the host cell, leading to higher infection efficiency. This report further supports recent evidence that spore adherence to the host cell surface is an important aspect of the microsporidial infection process.

Encephalitozoon hellem infection as the cause of a unilateral chronic keratoconjunctivitis in an umbrella cockatoo (Cacatua alba).

An umbrella cockatoo (Cacatua alba) was presented with a keratoconjunctivitis of 9 months' duration that was refractory to antibacterial and antifungal treatments. Microsporidian-type spores were observed in calcofluor M2R-stained cytological preparations of debrided corneal epithelium. Hematoxylin and eosin-stained sections of a conjunctival biopsy also showed a small number of intracellular parasites. Microsporidial DNA was detected by polymerase chain reaction in DNA extracted from the remaining biopsy material. Sequence analysis of the small subunit ribosomal RNA gene identified the microsporidium as Encephalitozoon hellem. The cockatoo was treated with oral albendazole for 3 months with resolution of the signs, and then again for 4 months when the signs returned. Since then, the bird has remained healthy for 2 years. This is the second report of a microsporidial keratoconjunctivitis in a bird, and the first report of an E. hellem infection in a cockatoo. Ophthalmologists should consider E. hellem as a possible cause of a keratoconjunctivitis in parrots that is refractory to treatment with traditional antimicrobials.

Variability in infection efficiency in vitro of different strains of the microsporidian Encephalitozoon hellem.

The infection efficiency of different strains of Encephalitozoon hellem of human origin was tested in Vero E6 cell cultures, scoring the number of infection foci (NIF) after 9, 14, 20, and 24 days of inoculation. The results revealed a strong interaction of the strain type with time: different strains showed different proliferative dynamics. Number of infection foci was lower on the first sampling day for CDC: V257, EHVS-96, and PV6-96, with a subsequent increase at a higher rate for the first strain and lower for the latter. In contrast, PV7-96 showed the highest NIF at the first sampling, followed by a slight decrease. Since these strains were selected by their genotype for the polar tube protein (PTP)-1A, 1B, 1C, and 2C, respectively, it is tempting to suggest a major role of this protein in the differences detected, although the influence of other genes that hypothetically may also differ among the strains employed cannot be discarded. The different in vitro infection efficiencies raise the possibility that some strains of E. hellem will also produce more aggressive features in infected patients.

Role of sulfated glycans in adherence of the microsporidian Encephalitozoon intestinalis to host cells in vitro.

Microsporidia are obligate intracellular opportunistic protists that infect a wide variety of animals, including humans, via environmentally resistant spores. Infection requires that spores be in close proximity to host cells so that the hollow polar tube can pierce the cell membrane and inject the spore contents into the cell cytoplasm. Like other eukaryotic microbes, microsporidia may use specific mechanisms for adherence in order to achieve target cell proximity and increase the likelihood of successful infection. Our data show that Encephalitozoon intestinalis exploits sulfated glycans such as the cell surface glycosaminoglycans (GAGs) in selection of and attachment to host cells. When exogenous sulfated glycans are used as inhibitors in spore adherence assays, E. intestinalis spore adherence is reduced by as much as 88%. However, there is no inhibition when nonsulfated glycans are used, suggesting that E. intestinalis spores utilize sulfated host cell glycans in adherence. These studies were confirmed by exposure of host cells to xylopyranoside, which limits host cell surface GAGs, and sodium chlorate, which decreases surface sulfation. Spore adherence studies with CHO mutant cell lines that are deficient in either surface GAGs or surface heparan sulfate also confirmed the necessity of sulfated glycans. Furthermore, when spore adherence is inhibited, host cell infection is reduced, indicating a direct association between spore adherence and infectivity. These data show that E. intestinalis specifically adheres to target cells by way of sulfated host cell surface GAGs and that this mechanism serves to enhance infectivity.

Glycosylation of the major polar tube protein of Encephalitozoon hellem, a microsporidian parasite that infects humans.

The microsporidia are ubiquitous, obligate intracellular eukaryotic spore-forming parasites infecting a wide range of invertebrates and vertebrates, including humans. The defining structure of microsporidia is the polar tube, which forms a hollow tube through which the sporoplasm is transferred to the host cell. Research on the molecular and cellular biology of the polar tube has resulted in the identification of three polar tube proteins: PTP1, PTP2, and PTP3. The major polar tube protein, PTP1, accounts for at least 70% of the mass of the polar tube. In the present study, PTP1 was found to be posttranslationally modified. Concanavalin A (ConA) bound to PTP1 and to the polar tube of several different microsporidia species. Analysis of the glycosylation of Encephalitozoon hellem PTP1 suggested that it is modified by O-linked mannosylation, and ConA binds to these O-linked mannose residues. Mannose pretreatment of RK13 host cells decreased their infection by E. hellem, consistent with an interaction between the mannosylation of PTP1 and some unknown host cell mannose-binding molecule. A CHO cell line (Lec1) that is unable to synthesize complex-type N-linked oligosaccharides had an increased susceptibility to E. hellem infection compared to wild-type CHO cells. These data suggest that the O-mannosylation of PTP1 may have functional significance for the ability of microsporidia to invade their host cells.

The genes encoding cAMP-dependent protein kinase catalytic subunit homologues of the microsporidia Encephalitozoon intestinalis and E. cuniculi: molecular characterisation and phylogenetic analysis.

A gene encoding a protein kinase was identified by homology-based PCR amplification in Encephalitozoon intestinalis, a microsporidian parasite pathogenic to humans, and its orthologue has been identified by database mining in the genome of the related species E. cuniculi, whose sequence has been recently published. Phylogenetic analysis revealed that the proteins encoded by these genes are homologues of the cAMP-dependent protein kinase catalytic subunits (PKAc). Southern blot analysis indicated that the EiPKAc gene is present in two copies in the E. intestinalis genome, whereas the E. cuniculi orthologue (EcPKAc) is a single copy gene. RT-PCR data showed that the EiPKAc gene is expressed in at least one of the intracellular stages during infection of the mammalian host cell by E. intestinalis.

Infectivity of microsporidia spores stored in seawater at environmental temperatures.

To determine how long spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis remain viable in seawater at environmental temperatures, culture-derived spores were stored in 10, 20, and 30 ppt artificial seawater at 10 and 20 C. At intervals of 1, 2, 4, 8, and 12 wk, spores were tested for infectivity in monolayer cultures of Madin Darby bovine kidney cells. Spores of E. hellem appeared the most robust, some remaining infectious in 30 ppt seawater at 10 C for 12 wk and in 30 ppt seawater at 20 C for 2 wk. Those of E. intestinalis were slightly less robust, remaining infectious in 30 ppt seawater at 10 and 20 C for 1 and 2 wk, respectively. Spores of E. cuniculi remained infectious in 10 ppt seawater at 10 and 20 C for 2 wk but not at higher salinities. These findings indicate that the spores of the 3 species of Encephalitozoon vary in their ability to remain viable when exposed to a conservative range of salinities and temperatures found in nature but, based strictly on salinity and temperature, can potentially remain infectious long enough to become widely dispersed in estuarine and coastal waters.

Chlorine inactivation of spores of Encephalitozoon spp.

This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis and to investigate the effect of chlorine on two other species, E cuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.

Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation.

Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal-oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log(10) or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm(2).