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Microsporidia are spore-forming, obligate intracellular pathogens typically associated with opportunistic infections in immunocompromised individuals. Treatment options for microsporidia infections in humans are limited and additional research is necessary to create better therapeutic agents. For many pathogenic organisms, adhesion to the host cell surface is a prerequisite for tissue colonization and invasion. Our previous research has demonstrated a direct relationship between adherence of microsporidia spores to the surface of host cells and infectivity in vitro. In an effort to better understand adherence, we have turned our attention to determining what proteins may be involved in this process. Examination of the Encephalitozoon cuniculi genome database revealed a gene encoding a protein with sequence homology to members of the ADAM (a disintegrin and metalloprotease) family of type I transmembrane glycoproteins. The microsporidia ADAM (MADAM) protein is of interest because ADAMs are known to be involved in a variety of biological processes including cell adhesion, proteolysis, cell fusion, and signaling. The objectives for this study were to examine the localization of MADAM, analyze its potential involvement during adherence and/or host cell infection, and to identify potential binding partners or substrates. Through the use of immunoelectron transmission microscopy, we demonstrated that MADAM is localized to the surface exposed exospore, plasma membrane, and the polar sac-anchoring disk complex (a bell-shaped structure at the spore apex involved in the infection process). Location of MADAM within the exospore and polar sac-anchoring disk suggests that MADAM is in a position to facilitate spore adherence or host cell infection. Thus far, we have been unable to conclusively demonstrate that MADAM is involved in either event. Through the use of a yeast two-hybrid system, we were able to identify polar tube protein 3 (PTP3) as a potential binding partner or substrate for the MADAM protein. The interaction between MADAM and PTP3 was confirmed by in vitro co-immunoprecipitation. PTP3 is hypothesized to be involved in the process of polar tube extrusion by stabilizing the interaction between PTP1-PTP2 polymers. Further analysis of the interaction between MADAM and PTP3 may lead to a better understanding of the events that occur during polar tube extrusion.

Background: Microsporidia are obligate intracellular opportunistic fungi that cause significant pathology in immunocompromised hosts. However, 11 percent of immunocompetent individuals in the general population are microsporidia-seropositive, indicating that severe immune suppression may not be a prerequisite for infection. Encephalitozoon intestinalis is transmitted in contaminated water and initially infects gastro-intestinal enterocytes, leading to diarrheal disease. This organism can also disseminate to many other organs. A recent report suggests that microsporidia can establish persistent infections, which anti-fungal treatment does not eradicate. Like other intracellular pathogens, microsporidia infection stresses the host cell and infected individuals have elevated hydrogen peroxide and free radical levels. Findings. As oxidative stress can lead to DNA damage, we hypothesized that E. intestinalis-infection would increase host cell nuclear mutation rate. Embryo fibroblasts from Big Blue§ssup§TM§esup§ transgenic mice were E. intestinalis-infected and host nuclear mutation frequency was determined by selection of temperature-sensitive c-II gene mutant λ phage. The host mutation frequency in E. intestinalis-infected cultures was 2.5-fold higher than that observed in either mock-infected cells or cells infected with UV-inactivated E. intestinalis spores. Conclusions: These data provide the first evidence that microsporidia infection can directly increase host cellular mutation frequency. Additionally, some event in the microsporidia developmental cycle between host cell attachment and parasitophorous vacuole formation is required for the observed effect. As there is considerable evidence linking infection with other intracellular pathogens and cancer, future studies to dissect the mechanism by which E. intestinalis infection increases host mutation frequency are warranted.

Background: Microsporidia are obligate intracellular opportunistic fungi that cause significant pathology in immunocompromised hosts. However, 11 percent of immunocompetent individuals in the general population are microsporidia-seropositive, indicating that severe immune suppression may not be a prerequisite for infection. Encephalitozoon intestinalis is transmitted in contaminated water and initially infects gastro-intestinal enterocytes, leading to diarrheal disease. This organism can also disseminate to many other organs. A recent report suggests that microsporidia can establish persistent infections, which anti-fungal treatment does not eradicate. Like other intracellular pathogens, microsporidia infection stresses the host cell and infected individuals have elevated hydrogen peroxide and free radical levels. Findings. As oxidative stress can lead to DNA damage, we hypothesized that E. intestinalis-infection would increase host cell nuclear mutation rate. Embryo fibroblasts from Big Blue§ssup§TM§esup§ transgenic mice were E. intestinalis-infected and host nuclear mutation frequency was determined by selection of temperature-sensitive c-II gene mutant λ phage. The host mutation frequency in E. intestinalis-infected cultures was 2.5-fold higher than that observed in either mock-infected cells or cells infected with UV-inactivated E. intestinalis spores. Conclusions: These data provide the first evidence that microsporidia infection can directly increase host cellular mutation frequency. Additionally, some event in the microsporidia developmental cycle between host cell attachment and parasitophorous vacuole formation is required for the observed effect. As there is considerable evidence linking infection with other intracellular pathogens and cancer, future studies to dissect the mechanism by which E. intestinalis infection increases host mutation frequency are warranted.

La microsporidie Encephalitozoon cuniculi, parasite intracellulaire obligatoire, pathogène de l'homme, est responsable d'infections opportunistes chez des sujets immunodéprimés. Sa spore est protégée par une épaisse paroi protéo-chitineuse pour laquelle peu de données sur les constituants protéiques sont disponibles. Dans ces travaux, nous avons décrit le protéome exprimé dans les stades tardifs de développement d'E. cuniculi. Grâce à des extractions protéiques séquentielles et une double stratégie d'analyse protéomique, après électrophorèse et "Shotgun", 177 protéines différentes ont pû être identifiées, permettant d'obtenir une vision globale de la physiologie de la spore. L'exploitation de ces données et le développement d'un crible bioinformatique a permis l'identification de 4 protéines de paroi. Le premier modèle dynamique de morphogenèse de la paroi microsporidienne a été proposé grâce au suivi de la localisation de ces protéines durant le cycle de développement

The influence of interferon gamma and specific antibodies on the infection with E. cuniculi in vivo has been studied. Reconstruction of SCID mice with CD4+ T-lymphocytes from BALB/c mice and from mice with defect gene for interferon gamma was used. Effects of the treatment with mouse recombinant interferon gamma and anti-E. cuniculi sera on survival of E. cuniculi infected SCID mice were monitored. The influence of the immunization with E. cuniculi antigen on the survival of E. cuniculi infected mice with defect gene for interferon gamma was examined.

The course of microsporidiosis caused by Encephalitozoon cuniculi in immunocompetent BALB/c mice and immunodeficient SCID mice was screened using molecular methods. The site of infection in organs was located using molecular and histology methods. The effectiveness of albendazole treatement and possibility of infection relapse after immunosuppresion (cyclosporine A, tacrolimus, mycofenolate mofetil) was also studied. Moreover, the course of excretion of microsporidial spores in feces was monitored during the whole time of experiment.

A total of 587 faecal samples were collected from 98 horses of different age from three farms with various breeding system (pasture, stable and combination) in the South Bohemia, Czech Republic during three consecutive years (from 2009 to 2011), and screened for the presence of endoparasites, especially these inhabiting gastrointestinal tract and lung, using standard parasitological methods including flotation, sedimentation and staining methods. Moreover presence of Encephalitozoon spp. was detected using genus specific nested PCR. Small strongyles were the most common nematodes among studied horses. The age of animals was evaluated as a risk factor in relation to Eimeria leuckarti, Strongyloides westeri and Parascaris equorum infection in foals. The resistance of small strongyles (Cyathostominae) to benzimidazol anthelmintics has been revealed only on one of the visited farm. Most of examined animals were mono-infected. The breeding of horses in the stable represent management system with lowest risk in relation to parasitic infection of animals. Molecular analysis revealed the presence of human pathogenic Encephalitozoon cuniculi genotype I in horses.