Ricin B lectin-like proteins of the microsporidian Encephalitozoon cuniculi and Anncaliia algerae are involved in host-cell invasion.

Microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. The infection process requires a step of prior adherence of Microsporidia to the surface of host cells. A few studies demonstrated the involvement of proteins containing a ricin-B lectin (RBL) domain in parasite infection. In this study Anncalia algerae and Encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing RBL-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. Three proteins named AaRBLL-1 and AaRBLL-2 from A. algerae and EcRBLL-1 from E. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural RBL domain despite a significant amino acid sequence divergence. The production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. Adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both E. cuniculi and A. algerae, strongly suggesting that these proteins are involved in the infection process.

3-Dimensional organization and dynamics of the microsporidian polar tube invasion machinery.

Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 µm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.

A cullin-RING ubiquitin ligase promotes thermotolerance as part of the intracellular pathogen response in Caenorhabditis elegans.

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode Caenorhabditis elegans called the intracellular pathogen response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other cullin-ring ubiquitin ligase components, which comprise a greatly expanded gene family in C. elegans Here we use coimmunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4, and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that coimmunoprecipitate with CUL-6 and find that FBXA-158 and FBXA-75 promote thermotolerance. In summary, we have defined the three core components and two F-box adaptors of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.

Renal Microsporidiosis in Pediatric Bone Marrow Transplant Recipients: A Case Series.

Microsporidiosis is a rare, but emerging opportunistic infection in solid organ transplant and stem cell transplant recipients. Renal involvement in microsporidiosis is very rarely seen in these recipients. We describe two cases of pediatric renal microsporidiosis, diagnosed on renal biopsies, following bone marrow transplantation presenting as severe acute kidney injury. The first patient died, whereas the second survived due to early diagnosis based on high index of suspicion and prompt treatment with Albendazole. We believe these are the first such reported cases of renal microsporidiosis in pediatric bone marrow transplant recipients.

Invertebrate host responses to microsporidia infections.

Microsporidia are a group of fungi-like intracellular and unicellular parasites, which infect nearly all animals. As "master parasites", over 1400 microsporidian species have been described to date. Microsporidia infections in economical invertebrates (e.g., silkworm, shrimp) cause huge financial losses, while other microsporidia infections in daphnia, nematode, locust, honeybee and mosquito play important roles in the regulation of their population size. Research investigating invertebrate host responses following microsporidia infections has yielded numerous interesting results, especially pertaining to the innate immune response to these pathogens. In this review, we comparatively summarize the invertebrate host responses to various microsporidia infections. We discuss numerous critical events in host responses including ubiquitin-mediated resistance, production of reactive oxygen species, melanization and innate immune pathways, and the increased basic metabolism and the accumulation of juvenile hormone in infected hosts. Recent studies progressing our understanding of microsporidia infection are also highlighted. Collectively, these advances shed more light on general rules of invertebrate host immune responses and pathogenesis mechanisms of microsporidia, and concurrently offer valuable clues for further research on the crosstalk between hosts and intracellular pathogens.

PRODUCTION OF SINGLE CHAIN FRAGMENT VARIABLE (scFv) ANTIBODY AGAINST PARANOSEMA LOCUSTAE ALPHA/BETA-HYDROLASE AND IMMUNOLOCALIZATION OF MICROSPORIDIAN ENZYME IN THE INFECTED HOST CELL.

Microsporidia is a widespread group of fungi-related intracellular parasites. Direct contact of the most microsporidia species with host cytoplasm suggests that these parasites may control physiological processes of infected cells by secretion of various proteins. In previous experiments, secretion of significant amounts of microsporidia Paranosema locustae alpha/beta-hydrolase into infected cells of Locusta migratoria fat bodies was demonstrated using polyclonal antibodies against the enzyme. However, heterologous expression of microsporidian hydrolase in yeast Pichia pastoris cells was not accompanied by its secretion. In this study, we have constructed library of recombinant single chain antibodies (scFv-fragments) against proteins of fat bodies of infected locusts and isolated mini-antibody specifically recognizing the studied enzyme using phage display technology. Immunoblotting and immunofluorescent microscopy with selected scFv-fragment confirmed secretion of two different in size forms of P. locustae alpha/beta-hydrolase into infected host cell. Prospects of scFv-fragment use to explore the role of microsporidian hydrolase in host-parasite relations and mechanism of its secretion are discussed in the paper.

Immunity to gastrointestinal microparasites of fish.

Fish intestinal parasites cause direct mortalities and also morbidity, poor growth, higher susceptibility to opportunistic pathogens and lower resistance to stress. This review is focused on microscopic parasites (Protozoa and Metazoa) that invade the gastrointestinal tract of fish. Intracellular parasites (mainly Microsporidia and Apicomplexa) evoke almost no host immune reaction while they are concealed in the cytoplasmic and nuclear compartments, and can even use fish cells (macrophages) as Trojan horses to spread in the host. Inflammatory reaction only appears when the parasite bursts infected cells. Immunity against extracellular parasites is depicted for the myxozoans Ceratonova shasta and Enteromyxum spp. The cellular and humoral innate responses and the production of antibodies are crucial for resolving some of these myxozoonoses, but an excessive inflammatory reaction (concerted by cytokines) can become a fatal pathophysiological consequence. The local immune response plays a key role, with numerous genes more strongly regulated in the intestine than at lymphohaematopoietic organs.

Host-Parasite Interactions and Purifying Selection in a Microsporidian Parasite of Honey Bees.

To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection.

Effector CD8 T cell immunity in microsporidial infection: a lone defense mechanism.

Microsporidia is a group of pathogens, which can pose severe risks to the immunocompromised population such as HIV-infected individuals. The expertise to diagnose these pathogens is limited and therefore their prevalence is believed to be much higher than what is currently known. In a mouse model of infections, it has been reported that CD8 T cells are the primary effector cells responsible for protecting the infected host. As the infection is acquired via per-oral route, CD8 T cells in the gut compartment apparently act as a first line of defense against the pathogens. Thus, generation of a robust CD8 T cell response that exhibits polyfunctional ability is critical for host survival. In this review, we describe the effector CD8 T cells generated during microsporidial infection and underline the factors that may be essential for the elicitation of protective immunity against this understudied but significant pathogen. Overall, this review will highlight the necessity for a better understanding of the development of the CD8 T cell response in gut associated lymphoid tissue (GALT) and provide some insights into therapies that may be used to restore defective CD8 T cell functionality in an immunocompromised situation.

Microsporidia-host interactions.

Microsporidia comprise one of the largest groups of obligate intracellular pathogens and can infect virtually all animals, but host response to these fungal-related microbes has been poorly understood. Several new studies of the host transcriptional response to microsporidia infection have found infection-induced regulation of genes involved in innate immunity, ubiquitylation, metabolism, and hormonal signaling. In addition, microsporidia have recently been shown to exploit host recycling endocytosis for exit from intestinal cells, and to interact with host degradation pathways. Microsporidia infection has also been shown to profoundly affect behavior in insect hosts. Altogether, these and other recent findings are providing much-needed insight into the underlying mechanisms of microsporidia interaction with host animals.

A wild C. elegans strain has enhanced epithelial immunity to a natural microsporidian parasite.

Microbial pathogens impose selective pressures on their hosts, and combatting these pathogens is fundamental to the propagation of a species. Innate immunity is an ancient system that provides the foundation for pathogen resistance, with epithelial cells in humans increasingly appreciated to play key roles in innate defense. Here, we show that the nematode C. elegans displays genetic variation in epithelial immunity against intestinal infection by its natural pathogen, Nematocida parisii. This pathogen belongs to the microsporidia phylum, which comprises a large phylum of over 1400 species of fungal-related parasites that can infect all animals, including humans, but are poorly understood. Strikingly, we find that a wild C. elegans strain from Hawaii is able to clear intracellular infection by N. parisii, with this ability restricted to young larval animals. Notably, infection of older larvae does not impair progeny production, while infection of younger larvae does. The early-life immunity of Hawaiian larvae enables them to produce more progeny later in life, providing a selective advantage in a laboratory setting--in the presence of parasite it is able to out-compete a susceptible strain in just a few generations. We show that enhanced immunity is dominant to susceptibility, and we use quantitative trait locus mapping to identify four genomic loci associated with resistance. Furthermore, we generate near-isogenic strains to directly demonstrate that two of these loci influence resistance. Thus, our findings show that early-life immunity of C. elegans against microsporidia is a complex trait that enables the host to produce more progeny later in life, likely improving its evolutionary success.

Ubiquitin-mediated response to microsporidia and virus infection in C. elegans.

Microsporidia comprise a phylum of over 1400 species of obligate intracellular pathogens that can infect almost all animals, but little is known about the host response to these parasites. Here we use the whole-animal host C. elegans to show an in vivo role for ubiquitin-mediated response to the microsporidian species Nematocida parisii, as well to the Orsay virus, another natural intracellular pathogen of C. elegans. We analyze gene expression of C. elegans in response to N. parisii, and find that it is similar to response to viral infection. Notably, we find an upregulation of SCF ubiquitin ligase components, such as the cullin ortholog cul-6, which we show is important for ubiquitin targeting of N. parisii cells in the intestine. We show that ubiquitylation components, the proteasome, and the autophagy pathway are all important for defense against N. parisii infection. We also find that SCF ligase components like cul-6 promote defense against viral infection, where they have a more robust role than against N. parisii infection. This difference may be due to suppression of the host ubiquitylation system by N. parisii: when N. parisii is crippled by anti-microsporidia drugs, the host can more effectively target pathogen cells for ubiquitylation. Intriguingly, inhibition of the ubiquitin-proteasome system (UPS) increases expression of infection-upregulated SCF ligase components, indicating that a trigger for transcriptional response to intracellular infection by N. parisii and virus may be perturbation of the UPS. Altogether, our results demonstrate an in vivo role for ubiquitin-mediated defense against microsporidian and viral infections in C. elegans.

Microsporidia and its relation to Crohn's disease. A retrospective study.

BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSIONS: 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.

Paranucleospora theridion (Microsporidia) infection dynamics in farmed Atlantic salmon Salmo salar put to sea in spring and autumn.

The microsporidian Paranucleospora theridion (syn. Desmozoon lepeophtheirii) is a parasite of Atlantic salmon Salmo salar and also a hyperparasite of the salmon louse Lepeophtheirus salmonis. The parasite develops 2 types of spores in salmon, cytoplasmic spores in phagocytes and intranuclear spores in epidermal cells. The former type of development is assumed to be propagative (autoinfection), while the epidermal spores transfer the parasite to lice. Development in lice is extensive, with the formation of xenoma-like hypertrophic cells filled with microsporidian spores. We show that salmon are infected in the absence of lice, likely through waterborne spores that initiate infections in the gills. During summer and autumn the parasite propagates in the kidney, as evidenced by peaking normalised expression of P. theridion rRNA. Lice become infected during autumn, and develop extensive infections during winter. Lice mortality in winter and spring is likely responsible for a reservoir of spores in the water. Salmon transferred to sea in November (low temperature) did not show involvement of the kidney in parasite propagation and lice on such fish did not become infected. Apparently, low temperatures inhibit normal P. theridion development in salmon.

Prevalence of opportunistic intestinal parasitic infections among HIV-infected patients with low CD4 cells counts in France in the combination antiretroviral therapy era.

BACKGROUND: The use of combination antiretroviral therapy (cART) has dramatically reduced the prevalence of opportunistic infections, however data on the prevalence of intestinal parasitic infections in HIV-infected patients with low CD4 cell counts in the cART era are scarce. METHODS: We performed a prospective cohort study among HIV-infected patients with CD4 cell counts <100/mm(3) seen at a university hospital in Paris. Medical records were reviewed and stool samples were obtained for macroscopic examination and detection of parasites including cryptosporidia and microsporidia, whether or not the patient had diarrhea. Stool cultures were performed for patients with diarrhea. Factors associated with the detection of parasites were then identified. RESULTS: Stools samples from 143 consecutive patients were analyzed. Patients were mostly men (76%), and the median patient age was 41 years. The median CD4 cell count was 32/mm(3), and 59% were receiving cART. Diarrhea was present in 85 patients (59%), 19 of whom (22%) had intestinal parasites detected in stools. Three patients with diarrhea were diagnosed with Salmonella typhimurium, Campylobacter coli, and Clostridium difficile infections. Among the 58 patients without diarrhea, parasitic intestinal pathogens were still identified in six (10%). The overall prevalence of intestinal parasites was 17%, with cryptosporidia (n=8), microsporidia (n=6), and Giardia duodenalis (n=5) being the most frequent pathogens. Patients with intestinal parasites had diarrhea more often (76% vs. 56%, p=0.025) and were more often at US Centers for Disease Control and Prevention (CDC) clinical stage C (84% vs. 69%, p=0.024) than patients without parasites. CONCLUSIONS: The prevalence of intestinal parasitic infections remains significant in HIV-infected patients with low CD4 counts in the cART era. A systematic search for parasitic pathogens including microsporidia, cryptosporidia, and G. duodenalis should be performed even in the absence of diarrhea.

Infections of Blastocystis hominis and microsporidia in cancer patients: are they opportunistic?

Chemotherapy can cause immunosuppression, which may trigger latent intestinal parasitic infections in stools to emerge. This study investigated whether intestinal parasites can emerge as opportunistic infections in breast and colorectal cancer patients (n=46 and n=15, respectively) undergoing chemotherapy treatment. Breast cancer patients were receiving a 5-fluorouracil/epirubicin/cyclophosphamide (FEC) regimen (6 chemotherapy cycles), and colorectal cancer patients were receiving either an oxaliplatin/5-fluorouracil/folinic acid (FOLFOX) regimen (12 cycles) or a 5-fluorouracil/folinic acid (Mayo) regimen (6 cycles). Patients had Blastocystis hominis and microsporidia infections that were only present during the intermediate chemotherapy cycles. Thus, cancer patients undergoing chemotherapy should be screened repeatedly for intestinal parasites, namely B. hominis and microsporidia, as they may reduce the efficacy of chemotherapy treatments.

T cell response and persistence of the microsporidia.

The microsporidia are a diverse phylum of obligate intracellular parasites related to the fungi that cause significant and sometimes life-threatening disease in immune-compromised hosts, such as AIDS and organ transplant patients. More recently, their role in causing pathology in immune-competent populations has also been appreciated. Interestingly, in several instances, the microsporidia have been shown to persist in their hosts long term, causing at opposite ends of the spectrum either an intractable chronic diarrhea and wasting in patients with advanced-stage AIDS or asymptomatic shedding of spores in healthy populations. Much remains to be studied regarding the immune response to these pathogens, but it seems clear that CD8+ T cells are essential in clearing infection. However, in the infection models examined thus far, the role for CD4+ T cells is unclear at best. Here, we discuss the possible reasons and ramifications of what may be a weak primary CD4+ T cell response against Encephalitozoon cuniculi. Given the central role of the CD4+ T cell in other models of adaptive immunity, a better appreciation of its role in responding to microsporidia may provide insight into the survival strategies of these pathogens, which allow them to persist in hosts of varied immune status.

[Using of antibodies against Microsporia Hsp70 family proteins for analysis of secretome of intracellular parasites].

Microsporidia is a large group of fungi-related unicellular parasites with obligate intracellular lifestyle. Unlike other protozoan intracellular parasites (Kinetoplastida and Apicomplexa), most microsporidian species develop in direct contact with the host cell cytoplasm. This fact, acquisition of unique transporters to exploit host metabolic system (alongside the strong minimization of own machinery) and predicted repertoire of microsporidia secretome altogether suggest an active role of parasite proteins in the control of infected cell. Lack of information about secretome of microsporidia intracellular stages is largely due to the methodological difficulties of working with the obligate intracellular parasites. An important problem of such study is the contamination of preparations of host cell cytoplasm by inner (nonsecreted) parasite proteins. Even the homogenization of infected tissue in mild conditions and removal of parasite cells by low-speed centrifugation may result in their partial disruption. We expressed the fragments of three Hsp70 family chaperones from the microsporidium Paranosema (Antonospora) locustae in bacteria Escherichia coli. Immunoblotting with proteins of microsporidia intracellular stages and infected host tissue (locust fat bodies) demonstrated that antibodies against recombinant polypeptides may be used to monitor the integrity of parasite cells during homogenization of infected host tissue and subsequent removal of parasites by centrifugation.

Fish microsporidia: immune response, immunomodulation and vaccination.

Immune response to fish microsporidia is still unknown and there are current research trying to elucidate the events involved in the immune response to this parasite. There is evidence suggesting the role of innate immune response and it is clear that adaptive immunity plays an essential part for eliminating and then mounting a solid resistance against subsequent microsporidian infections. This review article discusses the main mechanisms of resistance to fish microsporidia, which are considered under four main headings. 1) Innate immunity: the inflammatory tissue reaction associated with fish microsporidiosis has been studied at the ultrastructural level, providing identification of many of the inflammatory cells and molecules that are actively participating in the spore elimination, such as macrophages, neutrophils, eosinophilic granular cells, soluble factors and MHC molecules. 2) Adaptive immunity: the study of the humoral response is relatively new and controversial. In some cases, the antibody response is well established and it has a protective role, while in other situations, the immune response is not protective or it is depressed. Study of the cellular response against fish microsporidia is still in its infancy. Although the nature of the microsporidian infection suggests participation of cellular mechanisms, few studies have focused on the cellular immune response of infected fish. 3) Immunomodulation: glucans are compounds that can modulate the immune system and potentiate resistance to microorganisms. These compounds have been proposed that can interact with receptors on the surface of leukocytes that result in the stimulation on non-specific immune responses. 4) Vaccination: little is known about a biological product that could be used as a vaccine for preventing this infection in fish. In the Loma salmonae experience, one of the arguments that favor the production of a vaccine is the development in fish of resistance, associated to a cellular immune response. A recently proved spore-based vaccine to prevent microsporidial gill disease in salmon has recently shown its efficacy by considerably reducing the incidence of infection. This recent discovery would be first anti-microsporidian vaccine that is effective against this elusive parasite.