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PubMed1998

On proteins of the microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that can cause opportunistic infections in AIDS patients. Spore invasion of host cells involves extrusion of a polar tube. After immunocytochemical identification of several polar tube proteins (PTPs) in E. cuniculi, a major PTP was isolated from two-dimensional gels and two peptide fragments were sequenced. The complete nucleotide sequence of the corresponding gene was obtained using a combination of PCR amplification and cloning techniques. The gene exists as a single copy per haploid genome and encodes an acidic proline-rich protein, with a deduced molecular mass of 37 kDa, that contains four tandemly arranged 26-amino-acid repeats. An N-terminal region of 22 residues represents a cleaved signal peptide, probably involved in the targeting of the PTP. No similarity with known proteins has been found. The protein was expressed in Escherichia coli, purified and injected into mice. The antisera reacted specifically with the polar tube in indirect immunofluorescence assays and electron microscope immunocytochemistry. Further identification of conserved and variable PTP structural motifs should be useful for diagnostic purposes and new therapeutic strategies.

Amino Acid Sequence↗
PubMed1996

Experimental model for human intestinal microsporidiosis in interferon gamma receptor knockout mice infected by Encephalitozoon intestinalis.

Interferon gamma receptor knockout mice developed a chronic infection when inoculated with spores of Encephalitozoon intestinalis which is a cause of intestinal microsporidiosis in AIDS patients. The infection was evaluated by enumeration of the spores of the parasite shed in the stools, histological examination and follow up over a period of six months. A dose-response was demonstrated since higher numbers of spores were excreted and more infection sites were found in mice which were given an increased quantity of parasites. In infected wild type mice, the number of excreted spores decreased until day 16 post-inoculation, then spores were detected sporadically in low numbers. These data confirmed the role of IFN-gamma in the control of E. intestinalis infection. The infection was not lethal suggesting that other factors are involved in regulation of the parasite infection. This model, with the long survival time of the animals together with the measurable quantity of spores shed in the stools will be useful for testing potential therapeutical agents.

AIDS-Related Opportunistic Infections↗
PubMed2001

Dissemination of Encephalitozoon intestinalis, a causative agent of human microsporidiosis, in IFN-gamma receptor knockout mice.

The dissemination of Encephalitozoon intestinalis, a microsporidium causing intestinal diseases and systemic infection in humans, was investigated in IFN-gamma Ro/o mice. Although lesions were seen in organs of autopsied animals, the parasites were rarely detected using histological examination. Nevertheless, infection of the duodenum, liver, kidneys and lungs was demonstrated by polymerase chain reaction. This method also enabled the detection of the parasite in the brain and the heart. The development of E. intestinalis in RK13 cell cultures to which cell suspensions from liver, kidney, lung or brain of infected IFN-gamma Ro/o mice were added, confirmed the spread of intestinal microsporidiosis to these organs. No dissemination was observed in wild-type mice. These results confirm those of previous studies and emphasize the low morbidity of the infection in IFN-gamma Ro/o mice and confirm the role of IFN-gamma in the control of E. intestinalis infection. These mice infected with E. intestinalis offer important information about this interesting and important parasitic disease of man and animals.

Animals↗
PubMed2002

Phacoemulsification for the management of Encephalitozoon cuniculi-induced phacoclastic uveitis in a rabbit.

Phacoemulsification was performed on a New Zealand White rabbit with slowly progressive unilateral phacoclastic uveitis and cataract formation. The irrigating solution with lenticular contents were centrifuged and examined cytologically using Weber's chromotrope-based stain. Microsporidial spores were observed and positively identified as Encephalitozoon cuniculi via polymerase chain reaction. More than 1 year following surgical therapy, the rabbit is visual and comfortable without medications.

Albendazole↗
PubMed2000

Disseminated infection due to Encephalitozoon cuniculi in a patient with AIDS: case report and review.

OBJECTIVE AND METHODS: Infections due to microsporidia are increasingly recognized as opportunistic infections in patients with AIDS. We describe here a case of disseminated infection due to Encephalitozoon cuniculi and review the literature on this microsporidial infection. RESULTS: All 12 patients reported in the literature had AIDS and nine presented with disseminated infection involving the kidneys, sinuses, lungs, brain and conjunctiva. Asymptomatic infection was seen in three patients. Microsporidia were detected by light microscopy examination of urine samples in all the cases. Species identification was performed by various genotypic methods or transmission electron microscopy. Eight of 12 patients who received albendazole therapy experienced clinical improvement with documented clearance of spores in five of these eight patients. Two patients relapsed. CONCLUSIONS: E. cuniculi infection should be considered in severely immunocompromised HIV-infected patients with multi-organ involvement and fever, especially when renal failure is present. Microsporidial spores are usually seen in urine samples and in the involved organ. Albendazole therapy seems to be effective.

AIDS-Related Opportunistic Infections↗
PubMed2003

[Disseminated microspiridiosis (Encephalitozoon intestinalis) in a patient with HIV infection].

HISTORY AND CLINICAL FINDINGS: A 39-year-old patient with advanced HIV infection was admitted to our hospital with a 6-month history of diarrhoea, abdominal pain and pansinusitis. INVESTIGATIONS: Ultrasound and endoscopic retrograde cholangiography revealed cholangitis of the larger bile ducts. Stool examinations and coloscopy were unremarkable. No pathogenic organisms were identified by routine investigations. Finally, microsporidia of the genus encephalitozoon were diagnosed by electron microscopy in biopsies from the bile duct and the nasal mucous membrane and in stool samples by polymerase chain reaction (PCR). TREATMENT AND COURSE: Albendazole treatment was successful. The cholestatic liver tests and the ultrasound findings normalized. Control tests of stool, bile and nasal secretions by light microscopy, electron microscopy, and PCR were negative. CONCLUSION: Microsporidia, along with human cytomegalovirus, cryptosporida and mycobacteria other than tuberculosis are increasingly recognized as causing opportunistic infections in immunodeficient patients, especially in AIDS-related cholangitis. Some species can cause systemic infection. Therefore microsporidia infection should be considered in the differential diagnosis of all patients with immunodeficiency.

AIDS-Related Opportunistic Infections↗
PubMed2001

Characterization of the mRNA capping apparatus of the microsporidian parasite Encephalitozoon cuniculi.

A scheme of eukaryotic phylogeny has been suggested based on the structure and physical linkage of the enzymes that catalyze mRNA cap formation. Here we show that the intracellular parasite Encephalitozoon cuniculi encodes a complete mRNA capping apparatus consisting of separate triphosphatase (EcCet1), guanylyltransferase (EcCeg1), and methyltransferase (Ecm1) enzymes, which we characterize biochemically and genetically. The triphosphatase EcCet1 belongs to a metal-dependent phosphohydrolase family that includes the triphosphatase components of the capping apparatus of fungi, DNA viruses, and the malaria parasite Plasmodium falciparum. These enzymes are structurally and mechanistically unrelated to the metal-independent cysteine phosphatase-type RNA triphosphatases found in metazoans and plants. Our findings support the proposed evolutionary connection between microsporidia and fungi, and they place fungi and protozoa in a common lineage distinct from that of metazoans and plants. RNA triphosphatase presents an attractive target for antiprotozoal/antifungal drug development.

Acid Anhydride Hydrolases↗
PubMed2005

Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis.

The Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase Ecm1 has been characterized structurally but not biochemically. Here we show that purified Ecm1 is a monomeric protein that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to GTP. The reaction is cofactor-independent and optimal at pH 7.5. Ecm1 also methylates GpppA, GDP, and dGTP but not ATP, CTP, UTP, ITP, or m(7)GTP. The affinity of Ecm1 for the cap dinucleotide GpppA (K 0.1 mm) is higher than that for GTP (K(m) 1 mm) or GDP (K(m) 2.4 mm). Methylation of GTP by Ecm1 in the presence of 5 microm AdoMet is inhibited by the reaction product AdoHcy (IC(50) 4 microm) and by substrate analogs sinefungin (IC(50) 1.5 microm), aza-AdoMet (IC(50) 100 microm), and carbocyclic aza-AdoMet (IC(50) 35 microm). The crystal structure of an Ecm1.aza-AdoMet binary complex reveals that the inhibitor occupies the same site as AdoMet. Structure-function analysis of Ecm1 by alanine scanning and conservative substitutions identified functional groups necessary for methyltransferase activity in vivo. Amino acids Lys-54, Asp-70, Asp-78, and Asp-94, which comprise the AdoMet-binding site, and Phe-141, which contacts the cap guanosine, are essential for cap methyltransferase activity in vitro.

Adenosine↗
PubMed2006

Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin's antifungal activity.

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo,14 reduced activity in vitro to < or = 2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.

Adenosine↗
PubMed2002

Disseminated infection with encephalitozoon intestinalis in AIDS patients: report of 2 cases.

Microsporidiosis must be regarded as a late opportunistic infection when HIV is advanced. In this article we describe 2 cases of disseminated infection with Encephalitozoon intestinalis. The first case had a local intestinal infection for > 1 y before it disseminated and microsporidia were found intracellularly in sputum. In the second case, spores were initially found in conjunctival cells, sinus lavage, sputum and urine. This patient had clinical symptoms and radiological findings from the central nervous system. Signs of cerebral lymphoma developed after treatment of the opportunistic microsporidial infection.

AIDS-Related Opportunistic Infections↗
PubMedDate not supplied

Fatal pulmonary microsporidiosis due to encephalitozoon cuniculi following allogeneic bone marrow transplantation for acute myelogenous leukemia.

Microsporidia are ubiquitous obligate eukaryotic intracellular parasites that are now felt to be more akin to degenerate fungi than to protozoa. Microsporidia can be highly pathogenic, causing a broad range of symptoms in humans, especially individuals who are immunocompromised. The vast majority of human cases of microsporidiosis have been reported during the past 20 years, in patients with HIV/AIDS, while only relatively rare cases have been described in immunocompetent individuals. However, microsporidia infections are being increasingly reported in patients following solid-organ transplanation, where the main symptom has been diarrhea. The authors report the first case of pulmonary microsporidial infection in an allogeneic bone marrow transplant recipient in the United States and only the second case in the world. The patient, with a history of Hodgkin disease followed by acute myelogenous leukemia received a T-cell-depleted graft, but succumbed to respiratory failure 63 days post transplantation. An open lung biopsy, taken just before death, was originally thought to show toxoplasmosis. The correct diagnosis of microsporidiosis was made postmortem by light and electron microscopy. DNA polymerase chain reaction analysis confirmed the diagnosis and furthermore revealed it to be the dog strain of the microsporidia species Encephalitozoon cuniculi. Although to date rarely diagnosed, microsporidial infection should also be considered in the differential diagnosis of, e.g., unexplained pulmonary infection in bone marrow transplant patients.

Acute Disease↗
PubMed2003

Removal of Encephalitozoon intestinalis, calicivirus, and coliphages by conventional drinking water treatment.

The removal of the Microsporidia, Encephalitozoon intestinalis, feline calicivirus and coliphages MS-2, PRD-1, and Fr were evaluated during conventional drinking water treatment in a pilot plant. The treatment consisted of coagulation, sedimentation, and mixed media filtration. Fr coliphage was removed the most (3.21 log), followed by feline calicivirus (3.05 log), E. coli (2.67 log), E. intestinalis (2.47 log), MS-2 (2.51 log). and PRD-1 (1.85 log). With the exception of PRD-1 the greatest removal of the viruses occurred during the flocculation step of the water treatment process.

Animals↗
PubMed1999

Encephalitozoon cuniculi strain III is a cause of encephalitozoonosis in both humans and dogs.

Microsporidia are obligate intracellular eukaryotic organisms found in a wide range of vertebrate and invertebrate hosts. Encephalitozoon cuniculi is commonly found in domestic rabbits and rodents and also occurs in dogs, other canids, and primates, including humans. DNA sequencing of the ribosomal RNA genes has been used to identify these parasites to a species level and to define E. cuniculi strains I, II, and III. Eight new dog isolates were characterized as E. cuniculi strain III by use of molecular methods. This strain has also been identified in isolates from immunocompromised humans, suggesting the zoonotic potential of this parasite species. Prolonged microsporidial spore shedding from asymptomatic dogs is also reported.

Animals↗
PubMed1998

Dual microsporidial infection due to Vittaforma corneae and Encephalitozoon hellem in a patient with AIDS.

A 46-year-old human immunodeficiency virus-infected Swiss citizen living in Tanzania presented with respiratory, abdominal, and urogenital complaints. Microsporidial spores were isolated from urine and a sinunasal aspirate and were propagated in MRC-5 cell cultures. Western blot analysis and riboprinting identified the sinunasal isolate as Encephalitozoon hellem. Electron microscopic investigation of the urine isolate revealed spores with diplokaryotic nuclei and five to six isofilar coils of the polar tube and sporonts with two or three diplokarya. All stages were enveloped by two membranes, corresponding to a cisterna of host endoplasmic reticulum studded with ribosomes. These characteristics have been described for the genus Vittaforma. Western blot analysis of this isolate revealed a banding pattern identical to that of the Vittaforma corneae reference isolate. Part of the small subunit rRNA gene was amplified, sequenced (239 base pairs), and found to be identical to that of V. corneae. This is the second isolation of V. corneae and the first description of urinary tract infection due to V. corneae in a patient with AIDS.

AIDS-Related Opportunistic Infections↗
PubMed1998

Albendazole for treatment and prophylaxis of microsporidiosis due to Encephalitozoon intestinalis in patients with AIDS: a randomized double-blind controlled trial.

A double-blind placebo-controlled trial was conducted to assess the efficacy and safety of albendazole (400 mg twice daily for 3 weeks) for the treatment of Encephalitozoon intestinalis infection in patients with AIDS. Clearance of microsporidia from the intestinal tract was obtained in 4 of 4 patients in the albendazole group versus 0 of 4 in the control group (P = .01, one-sided Fisher's exact test) and was associated with significant clinical benefit. All 4 controls subsequently cleared microsporidia following open-labeled albendazole treatment. To investigate the effect of albendazole in preventing relapse, these 8 patients were then randomly assigned to receive either albendazole (400 mg twice daily) or no treatment for the next 12 months. Albendazole significantly delayed the occurrence of relapse (P = .04, one-sided log-rank test). In human immunodeficiency virus-infected patients with E. intestinalis infection, albendazole has parasitologic and clinical efficacy and reduces the risk of relapse.

AIDS-Related Opportunistic Infections↗
PubMed1998

First report on the systematic sequencing of the small genome of Encephalitozoon cuniculi (Protozoa, Microspora): gene organization of a 4.3 kbp region on chromosome I.

Belonging to a large group of parasitic amitochondrial protozoans (Microspora), Encephalitozoon cuniculi infects humans and other mammals. Because of its medical importance and small genome size (2.9 Mbp), we are systematically sequencing its smallest (217 kbp) chromosome. The shotgun cloning strategy now has produced the sequence of randomly dispersed contigs representing more than 180 kbp of this chromosome. The present report describes analysis of the 4.3 kbp contig, which includes the complete coding regions of dihydrofolate reductase (DHFR), thymidylate synthase (TS), and serine hydroxymethyl transferase (SHMT) genes and the partial coding region of an aminopeptidase (AP) gene. In contrast to the other reported protozoan genes, DHFR and TS are encoded by two different open reading frames (ORFs). The SHMT gene is the first one identified in a protozoan and corresponds to the cytosolic form of the enzyme. No introns were detected, and the intergenic noncoding regions do not exceed 50 bp. The mean GC content is close to 60%, and there is a G or C third-base codon bias. Transcription and translation initiation signals also are analyzed, and a model for the mRNA-ssu rRNA interactions is proposed.

Amino Acid Sequence↗
PubMed1989

A case of Encephalitozoon cuniculi peritonitis in a patient with AIDS.

A 45-year-old man with acquired immune deficiency syndrome (AIDS) presented with an unusual peritonitis resulting from a microsporidian infection. An inflammatory mass formed by the omentum magnum was revealed at partial autopsy. This mass was infested with parasites measuring 2.5-2.6 microns by 1.2-1.5 microns, which appeared gram positive and stained dark blue with Giemsa stain. These organisms were pale pink with Kinyoun stain, and occasionally one of their poles was periodic acid-Schiff positive. Sporoblasts and spores contained in parasitophorous vacuoles were revealed by electron microscopic examination. These spores were mononucleated, without mitochondria, and had a polar filament with four or five coils. They were identified as Encephalitozoon cuniculi. Peritonitis resulting from this parasite has not been reported previously, however, it should be suspected among rare infections resulting from unusual parasites in patients with AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Disseminated microsporidiosis due to Encephalitozoon hellem: pulmonary colonization, microhematuria, and mild conjunctivitis in a patient with AIDS.

Four genera of microsporidia have been associated with disease in humans, which predominantly affects immunocompromised persons. Systemic infection with a newly characterized microsporidian species, Encephalitozoon hellem, was recently reported in a patient with AIDS. This article describes a second patient with AIDS and disseminated E. hellem infection. In this case the parasite was detected in sputum, urine, and conjunctival swab specimens. Apart from recurrent mild conjunctivitis and asymptomatic microhematuria, the patient had no findings or symptoms that could be related to this parasite. Specifically, no microsporidian-associated pulmonary pathology was documented. Detection of E. hellem in the patient's sputum may have epidemiological implications in that this finding suggests transmission of microsporidia by the aerosol route. Because the patient died of unrelated complications, it remains unknown whether he was an asymptomatic carrier of microsporidia or whether microhematuria heralded early microsporidian disease, with the onset of cellular damage in the urinary tract.

AIDS-Related Opportunistic Infections↗