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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.

Microsporidia (Fungi) have been repeatedly identified as the cause of opportunistic infections predominantly in immunodeficient individuals such as AIDS patients. However, the global epidemiology of human microsporidiosis is poorly understood and the ability of microsporidia to survive and multiply in immunocompetent hosts remains unsolved. To determine the presence of latent microsporidia infections in apparently healthy humans in the Czech Republic, the authors tested sera, urine and stool originating from fifteen persons within a three month period examined on a weekly basis. Sera, stool and urine samples originating from fifteen HIV-negative people at risk with occupational exposure to animals, aged 22-56 years, living in the Czech Republic were tested by indirect immunofluorescence assay (IFA) for the presence of specific anti-microsporidial antibodies, standard Calcofluor M2R staining for the detection of microsporidian spores in all urine sediments and stool smears and molecular methods for the microsporidial species determination. Specific anti-microsporidial antibodies were detected in fourteen individuals, asymptomatic Encephalitozoon spp. infection was found in thirteen and E. bieneusi infection was detected in seven of those examined. While E. hellem 1A and E. cuniculi II were the major causative agents identified, seven different genotypes of E. bieneusi were recorded. These findings clearly show that exposure to microsporidia is common and chronic microsporidiosis is not linked to any clinical manifestation in healthy population. Moreover, our results indicate much higher incidence of microsporidial infections among an apparently healthy population than previously reported. These results open the question about the potential risk of reactivation of latent microsporidiosis in cases of immunosupression causing life-threatening disease.

Microsporidia are a group of intracellular pathogens causing self-limited and severe diseases in immunocompetent and immunocompromised individuals, respectively. A cellular type 1 adaptive response, mediated by IL-12, IFNγ, CD4+, and CD8+ T cells has been shown to be essential for host resistance, and dendritic cells (DC) play a key role at eliciting anti-microsporidial immunity. We investigated the in vitro response of DC and DC precursors/progenitors to infection with Encephalitozoon intestinalis (Ei), a common agent of human microsporidosis. Ei-exposed DC cultures up-regulated the surface expression of MHC class II and the costimulatory molecules CD86 and CD40, only when high loads of spores were used. A vigorous secretion of IL-6 but not of IL-1β or IL-12p70 was also observed in these cultures. Ei-exposed DC cultures consisted of immature infected and mature bystander DC, as assessed by MHC class II and costimulatory molecules expression, suggesting that intracellular Ei spores deliver inhibitory signals in DC. Moreover, Ei selectively inhibited the secretion of IL-12p70 in LPS-stimulated DC. Whereas Ei-exposed DC promoted allogeneic naïve T cell proliferation and IL-2 and IFNγ secretion in DC-CD4+ T cell co-cultures, separated co-cultures with bystander or infected DCs showed stimulation or inhibition of IFNγ secretion, respectively. When DC precursors/progenitors were exposed to Ei spores, a significant inhibition of DC differentiation was observed without shifting the development toward cells phenotypically or functionally compatible with myeloid-derived suppressor cells. Neutralization experiments demonstrated that this inhibitory effect is IL-6-dependent. Altogether this investigation reveals a novel potential mechanism of immune escape of microsporidian parasites through the modulation of DC differentiation and maturation.

The cause of Crohn's Disease (CD) remains unknown. Recently a decrease in the global lymphocyte population in the peripheral blood of CD patients has been reported. This decrease was more evident in γδ T lymphocytes, especially γδ CD8+T subsets. Furthermore, a decrease of IL-7 was also observed in these patients. We propose the hypothesis that microsporidia, an obligate intracellular opportunistic parasite recently related to fungi, in CD patients can take advantage of the lymphocytes and IL-7 deficits to proliferate and to contribute to the pathophysiology of this disease. In this case-control study, serum samples were collected from 36 CD patients and from 36 healthy individuals (controls), IgE and IgG anti-Encephalitozoon antibodies were determined by ELISA; and forty-four intestinal tissue samples were analyzed through real time Polymerase Chain Reaction (PCR), twenty CD patients, nine with others diseases and 15 healthy subjects. We observed that IgE anti-Encephalitozoon levels were significantly higher in patients with CD: 0.386(±0.256) vs control group, 0.201(±0.147), P<0.001. However, IgG anti-Encephalitozoon values were significantly lower in CD patients: 0.361(±0.256) vs control group, 0.876(±0.380), P<0.001. In the group of CD patients, 6/20 (30%) were positive by real time PCR for microsporidia and, all the patients of the control group were negative by real time PCR. These results suggest that CD patients are a group at risk for microsporidiasis and, moreover that microsporidia may be involved as a possible etiologic factor of CD.

Advanced age is associated with immune system deficits that result in an increased susceptibility to infectious diseases; however, specific mediators of age-dependent immune dysfunction have not been fully elucidated. Here we demonstrated that aged mice exhibit poor effector CD8+ T cell polyfunctionality, primarily due to CD8+ T cell-extrinsic deficits, and that reduced CD8+ T cell polyfunctionality correlates with increased susceptibility to pathogenic diseases. In aged animals challenged with the parasite Encephalitozoon cuniculi, effector CD8+ T cell survival and polyfunctionality were suppressed by highly elevated TGF-β1. Furthermore, TGF-β depletion reduced effector CD8+ T cell apoptosis in both young and aged mice and enhanced effector CD8+ T cell polyfunctionality in aged mice. Surprisingly, intrinsic blockade of TGF-β signaling in CD8+ T cells was sufficient to rescue polyfunctionality in aged animals. Together, these data demonstrate that low levels of TGF-β1 promote apoptosis of CD8+ effector T cells and high TGF-β1 levels associated with age result in both CD8+ T cell apoptosis and an altered transcriptional profile, which correlates with loss of polyfunctionality. Furthermore, elevated TGF-β levels are observed in the elderly human population and in aged Drosophila, suggesting that TGF-β represents an evolutionarily conserved negative regulator of the immune response in aging organisms.

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.

The ecological role and overlap with urban environments make wild carnivores useful epidemiological sentinels for several pathogens. The present study aimed to investigate the seroprevalence of Toxoplasma gondii, Neospora caninum and Encephalitozoon cuniculi in red foxes (Vulpes vulpes) from Central and Southern Italy. Sera from 120 foxes were analyzed using IFAT with a 1:20 cut-off value. Overall, seropositivity was highest for T. gondii (68.5%), followed by E. cuniculi (15.0%) and N. caninum (3.3%). Multivariable logistic regression models with stepwise selection identified age class and location as significant predictor factors for T. gondii exposure, with adults and red foxes from Southern Italy showing higher levels of prevalence. No significant associations with epidemiological risk factors were detected for E. cuniculi or N. caninum. Co-infections were detected in 15% of red foxes with a statistically significant positive association between T. gondii and E. cuniculi. These findings highlight that red foxes, being scavengers, are particularly exposed to food-borne pathogens, especially to T. gondii, and prove once again that they are reliable epidemiological sentinels for parasites that circulate at the wild–domestic interface.

The microbiota contributes to the induction of both effector and regulatory responses in the gastrointestinal (GI) tract. However, the mechanisms controlling these distinct properties remain poorly understood. We previously showed that commensal DNA promotes intestinal immunity. Here, we find that the capacity of bacterial DNA to stimulate immune responses is species specific and correlated with the frequency of motifs known to exert immunosuppressive function. In particular, we show that the DNA of Lactobacillus species, including various probiotics, is enriched in suppressive motifs able to inhibit lamina propria dendritic cell activation. In addition, immunosuppressive oligonucleotides sustain Treg cell conversion during inflammation and limit pathogen-induced immunopathology and colitis. Altogether, our findings identify DNA-suppressive motifs as a molecular ligand expressed by commensals and support the idea that a balance between stimulatory and regulatory DNA motifs contributes to the induction of controlled immune responses in the GI tract and gut immune homeostasis. Further, our findings suggest that the endogenous regulatory capacity of DNA motifs enriched in some commensal bacteria could be exploited for therapeutic purposes.

Here, we have investigated the possible effect of B-1 cells on the activity of peritoneal macrophages in E. cuniculi infection. In the presence of B-1 cells, peritoneal macrophages had an M1 profile with showed increased phagocytic capacity and index, associated with the intense microbicidal activity and a higher percentage of apoptotic death. The absence of B-1 cells was associated with a predominance of the M2 macrophages, reduced phagocytic capacity and index and microbicidal activity, increased pro-inflammatory and anti-inflammatory cytokines production, and higher percentual of necrosis death. In addition, in the M2 macrophages, spore of phagocytic E. cuniculi with polar tubular extrusion was observed, which is an important mechanism of evasion of the immune response. The results showed the importance of B-1 cells in the modulation of macrophage function against E. cuniculi infection, increasing microbicidal activity, and reducing the fungal mechanisms involved in the evasion of the immune response.

Rabbits ( Oryctolagus cuniculus ) are frequently reared for meat production in China. The aim of this study was to assess the seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi , and risk factors of infection in domestic rabbits raised in Henan province, central China. 1,213 serum samples of domestic rabbits were collected and tested for anti- T. gondii and anti- E. cuniculi antibodies using a modified agglutination test (MAT) and an enzyme-linked immunosorbent assay (ELISA), respectively. The serum positive rates of T. gondii and E. cuniculi were 128/1,213 (10.55%) and 235/1,213 (19.37%), respectively. Co-infection of T. gondii and E. cuniculi was demonstrated in 84 specimens; 44 rabbits were seropositive for T. gondii alone, while 151 rabbits were seropositive for E. cuniculi alone. The main risk factors simultaneously associated with T. gondii and E. cuniculi infection were the age of the rabbit, the type of food, and the rabbit rearing system. Serum positive rates of T. gondii and E. cuniculi among domestic rabbits were high, indicating the possibility of public health issues. Les lapins ( Oryctolagus cuniculus ) sont fréquemment élevés pour la production de viande en Chine. Le but de l’étude était de déterminer la séroprévalence de Toxoplasma gondii et d’ Encephalitozoon cuniculi ainsi que les facteurs de risque d’infection chez les lapins domestiques élevés dans la province du Henan, en Chine centrale. 1 213 échantillons de sérum de lapins domestiques ont été collectés et testés pour les anticorps anti- T. gondii et anti- E. cuniculi en utilisant un test d’agglutination modifié (MAT) et un dosage immuno-enzymatique (ELISA), respectivement. Les taux sériques positifs de T. gondii et E. cuniculi étaient de 128/1 213 (10,55%) et de 235/1 213 (19,37%), respectivement. La co-infection par T. gondii et E. cuniculi a été démontrée chez 84 spécimens ; 44 lapins étaient séropositifs pour T. gondii seul, alors que 151 lapins étaient séropositifs pour E. cuniculi seul. Les principaux facteurs de risque associés simultanément à l’infection par T. gondii et E. cuniculi étaient l’âge du lapin, le type d’aliment et le système d’élevage du lapin. Les taux sériques positifs de T. gondii et d’E . cuniculi chez les lapins domestiques étaient élevés, ce qui indique la possibilité de problèmes de santé publique.