Microsporidia (Encephalitozoon cuniculi) in Patients with Degenerative Hip and Knee Disease, Czech Republic.

Total joint arthroplasty is a commonly used surgical procedure in orthopedics. Revision surgeries are required in >10% of patients mainly because of prosthetic joint infection caused by bacteria or aseptic implant loosening caused by chronic inflammation. Encephalitozoon cuniculi is a microsporidium, an obligate intracellular parasite, capable of exploiting migrating proinflammatory immune cells for dissemination within the host. We used molecular detection methods to evaluate the incidence of E. cuniculi among patients who had total hip or knee arthroplasty revision. Out of 49 patients, E. cuniculi genotypes I, II, or III were confirmed in joint samples from 3 men and 2 women who had implant loosening. Understanding the risks associated with the presence of microsporidia in periprosthetic joint infections is essential for proper management of arthroplasty. Furthermore, E. cuniculi should be considered a potential contributing cause of joint inflammation and arthrosis.

Encephalitozoon cuniculi Microsporidia in Cerebrospinal Fluid from Immunocompetent Patients, Czech Republic.

We retrospectively analyzed of 211 frozen cerebrospinal fluid samples from immunocompetent persons in the Czech Republic and detected 6 Encephalitozoon cuniculi-positive samples. Microsporidiosis is generally underestimated and patients are not usually tested for microsporidia, but latent infection in immunodeficient and immunocompetent patients can cause serious complications if not detected and treated.

Encephalitozoon cuniculi and Extraintestinal Microsporidiosis in Bird Owners.

We identified Encephalitozoon cuniculi genotype II parasites as a cause of extraintestinal microsporidiosis in 2 owners of birds also infected with E. cuniculi. Patients experienced long-lasting nonspecific symptoms; the disease course was more progressive in a patient with diabetes. Our findings suggest direct bird-to-human transmission of this pathogen.

A multidisciplinary review about Encephalitozoon cuniculi in a One Health perspective.

Encephalitozoon cuniculi is a microsporidian parasite mostly associated with its natural host, the rabbit (Oryctolagus cuniculus). However, other animals can be infected, like other mammals, birds, and even humans. Although it usually causes subclinical infection, it can also lead to encephalitozoonosis, a clinical disease characterized by neurological, ocular, and/or renal signs that can be even fatal, especially in immunocompromised individuals. Therefore, this multidisciplinary review contributes with updated information about the E. cuniculi, deepening in its molecular and genetic characterization, its mechanisms of infection and transmission, and its prevalence among different species and geographic locations, in a One Health perspective. Recent information about the diagnostic and therapeutic approach in the main host species and the prophylaxis and infection control measures currently suggested are also discussed.

The course of infection of Encephalitozoon cuniculi genotype I in mice possess combination of features reported in genotypes II and III.

Out of three genotypes of Encephalitozoon cuniculi (I-III) available for experimental studies, E. cuniculi genotype I remains the less characterized. This study describes for the first time individual phases of microsporidiosis caused by E. cuniculi genotype I and efficacy of albendazole treatment in immunocompetent BALB/c and C57Bl/6 mice and immunodeficient SCID, CD4-/- and CD8-/- mice using molecular detection and quantification methods. We demonstrate asymptomatic infection despite an intense dissemination of microsporidia into most organs within the first weeks post infection, followed by a chronic infection characterized by significant microsporidia persistence in immunocompetent, CD4-/- and CD8-/- mice and a lethal outcome for SCID mice. Albendazole application led to loss E. cuniculi genotype I infection in immunocompetent mouse strains, decreased spore burden by half in CD4-/- and CD8-/- mice, and prolongation of survival of SCID mice. These results showed Encephalitozoon cuniculi genotype I infection extend and albendazole sensitivity was comparable to E. cuniculi genotype II, but the infection onset speed and mortality rate was similar to E. cuniculi genotype III. These imply that differences in the course of infection and the response to treatment depend not only on immunological status of the host, but also on the genotype causing the infection.

Encephalitozoon cuniculi Genotype III Evinces a Resistance to Albendazole Treatment in both Immunodeficient and Immunocompetent Mice.

Of four genotypes of Encephalitozoon cuniculi, E. cuniculi genotype II is considered to represent a parasite that occurs in many host species in a latent asymptomatic form, whereas E. cuniculi genotype III seems to be more aggressive, and infections caused by this strain can lead to the death of even immunocompetent hosts. Although albendazole has been considered suitable for treatment of Encephalitozoon species, its failure in control of E. cuniculi genotype III infection has been reported. This study determined the effect of a 100× recommended daily dose of albendazole on an Encephalitozoon cuniculi genotype III course of infection in immunocompetent and immunodeficient mice and compared the results with those from experiments performed with a lower dose of albendazole and E. cuniculi genotype II. The administration of the regular dose of abendazole during the acute phase of infection reduced the number of affected organs in all strains of mice and absolute counts of spores in screened organs. However, the effect on genotype III was minor. Surprisingly, no substantial effect was recorded after the use of a 100× dose of albendazole, with larger reductions seen only in the number of affected organs and absolute counts of spores in all strains of mice, implying variations in albendazole resistance between these Encephalitozoon cuniculi genotypes. These results imply that differences in the course of infection and the response to treatment depend not only on the immunological status of the host but also on the genotype causing the infection. Understanding how microsporidia survive in hosts despite targeted antimicrosporidial treatment could significantly contribute to research related to human health.

The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism.

The eukaryotic helicase is an 11-subunit machine containing an Mcm2-7 motor ring that encircles DNA, Cdc45 and the GINS tetramer, referred to as CMG (Cdc45, Mcm2-7, GINS). CMG is "built" on DNA at origins in two steps. First, two Mcm2-7 rings are assembled around duplex DNA at origins in G1 phase, forming the Mcm2-7 "double hexamer." In a second step, in S phase Cdc45 and GINS are assembled onto each Mcm2-7 ring, hence producing two CMGs that ultimately form two replication forks that travel in opposite directions. Here, we review recent findings about CMG structure and function. The CMG unwinds the parental duplex and is also the organizing center of the replisome: it binds DNA polymerases and other factors. EM studies reveal a 20-subunit core replisome with the leading Pol ϵ and lagging Pol α-primase on opposite faces of CMG, forming a fundamentally asymmetric architecture. Structural studies of CMG at a replication fork reveal unexpected details of how CMG engages the DNA fork. The structures of CMG and the Mcm2-7 double hexamer on DNA suggest a completely unanticipated process for formation of bidirectional replication forks at origins.

Disseminated Infection of Encephalitozoon cuniculi Associated With Osteolysis of Hip Periprosthetic Tissue.

Background: Among patients with hip joint endoprosthesis, periprosthetic osteolysis is the most common complication following primary arthroplasty, and subsequent implant loosening is the leading cause of arthroplasty revision. Causes of stability loss, though not always evident, can be mechanical, allergic, or infectious (bacterial and fungal agents) in nature. Microsporidia, widespread opportunistic fungal pathogens that infect most human tissues, are a potential infectious cause of stability loss. Infections caused by Encephalitozoon species-one of the most common microsporidial pathogens in humans-primarily localize to intestinal and respiratory tracts, but can disseminate to tissues throughout the body. Methods: We examined 53 immunocompetent patients, 23 after revision and 30 after primary hip arthroplasty, for infection by Encephalitozoon species. Periprosthetic tissue, urine sediments, and stool samples were tested by microscopic examination and genus-specific nested polymerase chain reaction followed by genotyping. Results: Ten patients had Encephalitozoon-positive periprosthetic tissues, 9 (39%) after revision and 1 (3.3%) after primary hip arthroplasty. Among the tissue-positive postrevision patients, 7 had a positive urine sample and 1 had a positive stool sample. Encephalitozoon cuniculi genotype II was identified in 88.8% (16/18) of samples. Two urine samples were positive for a novel Encephalitozoon species. Conclusions: Encephalitozoon cuniculi should be considered as a cause of osteolysis in hip periprosthetic tissue, leading to a loss of implant stability.

Evidence of transplacental transmission of Encephalitozoon cuniculi genotype II in murine model.

Microsporidia are obligate intracellurar unicellular parasite of wide range of vertebrates. Although ingestion or inhalation of microsporidian spores is the main route of infection, assumed vertical transmission was described in some mammals. The present study was focused on proof of vertical transmission in mice under experimental conditions. Mice were infected with E. cuniculi genotype II intraperitoneally after mating, or perorally followed by mating in acute or chronic phase of infection. Fetuses were delivered by Caesarean section or mice were kept up to the parturition. Some of cubs were immediately after birth transferred to uninfected surrogate mothers. Group of cubs was immunosuppressed. All cubs were examined using polymerase chain reaction for the presence of Encephalitozoon after birth or in their age of 3 or 6 weeks, respectively. All fetuses delivered by Caesarean section, which were intraperitoneally or perorally infected were negative as well as all neonatal mice and youngsters tested in age of 6 weeks. Only immunosuppressed cubs and cubs of immunodeficient mice in age of 21 days were positive for Encephalitozoon cuniculi genotype II. Present results provided the evidence that transplacental transmission of Encephalitozoon cuniculi in mice occurs, but the mechanism of these transport is still unknown.

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.

[The Common Partner of Several Methyltransferases Modifying the Components of The Eukaryotic Translation Apparatus].

TRM112 is necessary for the activation and stability of several methyltransferases involved in the modification of various components of the translation apparatus. This unique protein is a partner for enzymes that methylate tRNA, rRNA, and the translation termination factor. Here we review the structural and functional features of the TRM112 complexes with methyltransferases and provide, where possible, information on their significance.

Dynamics of parasitophorous vacuoles formed by the microsporidian pathogen Encephalitozoon cuniculi.

It has been a long-standing debate if sexual development occurs in the microsporidian lineages. Previous studies, including morphological observations, ploidy analysis, and the presence of a sex-related locus, provided evidence of possible extant of sexual development. This study presents another line of evidence by monitoring the parasitophorous vacuoles (PVs) formed by Encephalitozoon cuniculi. Time lapse observations of infection cycles of E. cuniculi revealed that multiple PVs can be formed in a single host cell and the PVs in the single cell can merge (fusion) or split (fission). The dynamics of PVs may provide a route for interactions between genetically distinct microsporidian isolates during host infections.

The course of infection caused by Encephalitozoon cuniculi genotype III in immunocompetent and immunodeficient mice.

Encephalitozoon cuniculi is probably the most common microsporidia which infects a wide range of vertebrates, including human. So far, four genotypes of this parasite have been identified based on the rRNA internal transcribed spacer variations. The course of infection caused by E. cuniculi III had very massive onset in immunocompetent host characterized by the presence of this parasite in all organs and tissues within one week after peroral infection. Encephalitozoonosis caused by E. cuniculi III had very progressive spreading into all organs within first week post inoculation in immunocompromised SCID mice and led to the death of the host. The experimental treatment with albendazole of immunocompetent BALB/c mice infected with E. cuniculi III have shown very weak effect. Our findings clearly showed that the different course of infection and response to treatment depends not only on the immunological status of the host, but also on the genotype of microsporidia. It could be very important especially for individuals under chemotherapy and transplant recipients of organs originating from infected donors.

Subtelomere organization in the genome of the microsporidian Encephalitozoon cuniculi: patterns of repeated sequences and physicochemical signatures.

BACKGROUND: The microsporidian Encephalitozoon cuniculi is an obligate intracellular eukaryotic pathogen with a small nuclear genome (2.9 Mbp) consisting of 11 chromosomes. Although each chromosome end is known to contain a single rDNA unit, the incomplete assembly of subtelomeric regions following sequencing of the genome identified only 3 of the 22 expected rDNA units. While chromosome end assembly remains a difficult process in most eukaryotic genomes, it is of significant importance for pathogens because these regions encode factors important for virulence and host evasion. RESULTS: Here we report the first complete assembly of E. cuniculi chromosome ends, and describe a novel mosaic structure of segmental duplications (EXT repeats) in these regions. EXT repeats range in size between 3.5 and 23.8 kbp and contain four multigene families encoding membrane associated proteins. Twenty-one recombination sites were identified in the sub-terminal region of E. cuniculi chromosomes. Our analysis suggests that these sites contribute to the diversity of chromosome ends organization through Double Strand Break repair mechanisms. The region containing EXT repeats at chromosome extremities can be differentiated based on gene composition, GC content, recombination sites density and chromosome landscape. CONCLUSION: Together this study provides the complete structure of the chromosome ends of E. cuniculi GB-M1, and identifies important factors, which could play a major role in parasite diversity and host-parasite interactions. Comparison with other eukaryotic genomes suggests that terminal regions could be distinguished precisely based on gene content, genetic instability and base composition biais. The diversity of processes assciated with chromosome extremities and their biological consequences, as they are presented in the present study, emphasize the fact that great effort will be necessary in the future to characterize more carefully these regions during whole genome sequencing efforts.

The genome of an Encephalitozoon cuniculi type III strain reveals insights into the genetic diversity and mode of reproduction of a ubiquitous vertebrate pathogen.

Encephalitozoon cuniculi is a model microsporidian species with a mononucleate nucleus and a genome that has been extensively studied. To date, analyses of genome diversity have revealed the existence of four genotypes in E. cuniculi (EcI, II, III and IV). Genome sequences are available for EcI, II and III, and are all very divergent, possibly diploid and genetically homogeneous. The mechanisms that cause low genetic diversity in E. cuniculi (for example, selfing, inbreeding or a combination of both), as well as the degree of genetic variation in their natural populations, have been hard to assess because genome data have been so far gathered from laboratory-propagated strains. In this study, we aim to tackle this issue by analyzing the complete genome sequence of a natural strain of E. cuniculi isolated in 2013 from a steppe lemming. The strain belongs to the EcIII genotype and has been designated EcIII-L. The EcIII-L genome sequence harbors genomic features intermediate to known genomes of II and III lab strains, and we provide primers that differentiate the three E. cuniculi genotypes using a single PCR. Surprisingly, the EcIII-L genome is also highly homogeneous, harbors signatures of heterozygosity and also one strain-specific single-nucleotide polymorphism (SNP) that introduces a stop codon in a key meiosis gene, Spo11. Functional analyses using a heterologous system demonstrate that this SNP leads to a deficient meiosis in a model fungus. This indicates that EcIII-L meiotic machinery may be presently broken. Overall, our findings reveal previously unsuspected genome diversity in E. cuniculi, some of which appears to affect genes of primary importance for the biology of this pathogen.

Fast Technology Analysis Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians.

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with "state-of-the-art" equipment and well-trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore-concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN-1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies.

The structure of an Iws1/Spt6 complex reveals an interaction domain conserved in TFIIS, Elongin A and Med26.

Binding of elongation factor Spt6 to Iws1 provides an effective means for coupling eukaryotic mRNA synthesis, chromatin remodelling and mRNA export. We show that an N-terminal region of Spt6 (Spt6N) is responsible for interaction with Iws1. The crystallographic structures of Encephalitozoon cuniculi Iws1 and the Iws1/Spt6N complex reveal two conserved binding subdomains in Iws1. The first subdomain (one HEAT repeat; HEAT subdomain) is a putative phosphoprotein-binding site most likely involved in an Spt6-independent function of Iws1. The second subdomain (two ARM repeats; ARM subdomain) specifically recognizes a bipartite N-terminal region of Spt6. Mutations that alter this region of Spt6 cause severe phenotypes in vivo. Importantly, the ARM subdomain of Iws1 is conserved in several transcription factors, including TFIIS, Elongin A and Med26. We show that the homologous region in yeast TFIIS enables this factor to interact with SAGA and the Mediator subunits Spt8 and Med13, suggesting the molecular basis for TFIIS recruitment at promoters. Taken together, our results provide new structural information about the Iws1/Spt6 complex and reveal a novel interaction domain used for the formation of transcription networks.

First characterization in China of Encephalitozoon cuniculi in the blue fox (Alopex lagopus).

Encephalitozoon cuniculi is a microsporidian parasite that infects a wide range of vertebrates, including primates. It has recently emerged as an opportunistic parasite of patients infected with the human immunodeficiency virus. The blue fox (Alopex lagopus; also known as the arctic fox) is one of the most susceptible species for encephalitozoonosis. Here, we report an outbreak of encephalitozoonosis at a fox farm in China. The isolated parasites displayed the typical morphology of E. cuniculi as assessed by Masson's trichrome staining. Analysis of the internal transcribed spacer sequence indicated that the isolated parasite is a genotype III strain of E. cuniculi. Furthermore, phylogenetic analysis of the PTP1 gene verifies classification of this new strain (termed LN-1) with other genotype III E. cuniculi strains, though the PTP3 and SWP1 sequences diverge from the reference strain. This is the first report of encephalitozoonosis in farmed blue foxes in China.

A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi.

Mitochondria use transport proteins of the eukaryotic mitochondrial carrier family (MCF) to mediate the exchange of diverse substrates, including ATP, with the host cell cytosol. According to classical endosymbiosis theory, insertion of a host-nuclear-encoded MCF transporter into the protomitochondrion was the key step that allowed the host cell to harvest ATP from the enslaved endosymbiont. Notably the genome of the microsporidian Encephalitozoon cuniculi has lost all of its genes for MCF proteins. This raises the question of how the recently discovered microsporidian remnant mitochondrion, called a mitosome, acquires ATP to support protein import and other predicted ATP-dependent activities. The E. cuniculi genome does contain four genes for an unrelated type of nucleotide transporter used by plastids and bacterial intracellular parasites, such as Rickettsia and Chlamydia, to import ATP from the cytosol of their eukaryotic host cells. The inference is that E. cuniculi also uses these proteins to steal ATP from its eukaryotic host to sustain its lifestyle as an obligate intracellular parasite. Here we show that, consistent with this hypothesis, all four E. cuniculi transporters can transport ATP, and three of them are expressed on the surface of the parasite when it is living inside host cells. The fourth transporter co-locates with mitochondrial Hsp70 to the E. cuniculi mitosome. Thus, uniquely among eukaryotes, the traditional relationship between mitochondrion and host has been subverted in E. cuniculi, by reductive evolution and analogous gene replacement. Instead of the mitosome providing the parasite cytosol with ATP, the parasite cytosol now seems to provide ATP for the organelle.

Extremely reduced levels of heterozygosity in the vertebrate pathogen Encephalitozoon cuniculi.

The genomes of microsporidia in the genus Encephalitozoon have been extensively studied for their minimalistic features, but they have seldom been used to investigate basic characteristics of the biology of these organisms, such as their ploidy or their mode of reproduction. In the present study, we aimed to tackle this issue by mapping Illumina sequence reads against the genomes of four strains of E. cuniculi. This approach, combined with more conventional molecular biology techniques, resulted in the identification of heterozygosity in all strains investigated, a typical signature of a diploid nuclear state. In sharp contrast with similar studies recently performed on a distant microsporidian lineage (Nematocida spp.), the level of heterozygosity that we identified across the E. cuniculi genomes was found to be extremely low. This reductive intraindividual genetic variation could result from the long-term propagation of these strains under laboratory conditions, but we propose that it could also reflect an intrinsic capacity of these vertebrate pathogens to self-reproduce.