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At least 217 records · Page 12Linked to original sources
PubMed2001

Inter-strain variability of insertion/deletion events in the Encephalitozoon cuniculi genome: a comparative KARD-PFGE analysis.

We applied a two-dimensional pulsed-field gel electrophoresis procedure to the genomes of two karyotype variants assigned to two different strains of the microsporidian Encephalitozoon cuniculi, termed D (strain III) and F (strain II). Data obtained for BssHII and MluI restriction fragment length polymorphisms in each chromosome are compiled and compared to the reference strain I variant A. Six Insertion/Deletion (InDels) are found in subterminal position, some of these being characteristic of either D or F. Like in strain 1, the terminal fragments extending between each telomere and rDNA locus are conserved in length for each chromosome. They are however smaller than in reference variant. This size reduction is estimated to be 2.5 kbp for the strain III isolate and 3.5 kbp for the strain II isolate. We hypothesize that for the three E. cuniculi strains, all chromosome extremities are prone to a constant process of sequence homogenization through mitotic recombination between conserved regions.

Animals↗
PubMed2001

The identification of rRNA maturation sites in the microsporidian Encephalitozoon cuniculi argues against the full excision of presumed ITS1 sequence.

In Encephalitozoon cuniculi like in other microsporidia, the primary transcript for SSU and LSU rRNAs includes only one internal transcribed spacer (ITS1) which separates SSU rRNA from the 5.8S region associated with LSU rRNA. The extraction of total RNA from E. cuniculi-infected MRC5 cells using a hot phenol/chloroform procedure enabled us to perform primer extension and S1 nuclease protection experiments in the aim of identifying rRNA maturation sites. Our data support a simple processing (four cleavage sites) with elimination of only nine nucleotides between SSU and LSU rRNA regions. Most of the presumed ITS1 sequence characterized by strain-dependent polymorphism therefore remains linked to SSU rRNA 3' end. A new secondary structure for the sixth domain of E. cuniculi LSU rRNA is proposed following the identification of its 3' terminus.

Animals↗
PubMedDate not supplied

Molecular characterization of Encephalitozoon intestinalis (Microspora) replication kinetics in a murine intestinal cell line.

Microsporidia are obligate intracellular pathogens of invertebrate and vertebrate animals. Most human infections are caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis, and result in chronic diarrhea. In order to determine the signals involved in microsporidial spore activation and invasion, kinetics of in vitro E. intestinalis replication were defined using real-time quantitative PCR. Segments of small subunit ribosomal RNA and polar tube protein 2 genes of E. intestinalis were used to quantify parasite gene copy number following infection in murine colon carcinoma cells. Parasite DNA was detectable in small but significant amounts within host cells as early as 4 h postinoculation, genome replication was completed by 36 h, and parasite progeny were released into the supernatant beginning 72 h postinoculation. Heat-treating spores did not prevent transfer of parasite DNA into cells, but did inhibit parasite replication. Treating cell cultures with albendazole suppressed but did not completely inhibit parasite replication. These results confirm observations that E. intestinalis completes its life cycle within the turnover time of its target host cells; invasion into susceptible host cells occurs independently of spore viability; and real-time quantitative PCR is a sensitive and reproducible method with which to monitor microsporidial infection under varying treatments or conditions.

Albendazole↗
PubMedDate not supplied

Relationship between the host cell mitochondria and the parasitophorous vacuole in cells infected with Encephalitozoon microsporidia.

Encephalitozoon microsporidia proliferate and differentiate within a parasitophorous vacuole. Using the fluorescent probe, calcein, and the mitochondrial probe, MitoTracker-CMXRos, a vital method was developed that confirmed ultrastructural reports that the host cell mitochondria frequently lie in immediate proximity to the parasitophorous vacuole. Morphometry failed to demonstrate any infection-induced increase in host cell mitochondria as there was no correlation between the mitochondrial volume and the extent of infection as judged by the parasitophorous vacuole volume. The total ATP concentration of infected cells did not differ from that of uninfected cells in spite of the increased metabolic demands of the infection. Treatment with 10(-6) M albendazole, more than ten times the antiparasitic IC50 dose, and demecolcine had no subjective effect on the proximity of mitochondria to the parasitophorous vacuole membrane when studied by either transmission electron microscopy or by confocal microscopy even though these drug concentrations affected microtubule structure. Thus, once the association between mitochondria and the parasitophorous vacuole has been established, host cell microtubule integrity is probably not required for its maintenance. It is unlikely that the antimicrosporidial action of albendazole involves physically uncoupling developing parasite stages from host cell organelle metabolic support.

Adenosine Triphosphate↗
PubMedDate not supplied

Variability in infection efficiency in vitro of different strains of the microsporidian Encephalitozoon hellem.

The infection efficiency of different strains of Encephalitozoon hellem of human origin was tested in Vero E6 cell cultures, scoring the number of infection foci (NIF) after 9, 14, 20, and 24 days of inoculation. The results revealed a strong interaction of the strain type with time: different strains showed different proliferative dynamics. Number of infection foci was lower on the first sampling day for CDC: V257, EHVS-96, and PV6-96, with a subsequent increase at a higher rate for the first strain and lower for the latter. In contrast, PV7-96 showed the highest NIF at the first sampling, followed by a slight decrease. Since these strains were selected by their genotype for the polar tube protein (PTP)-1A, 1B, 1C, and 2C, respectively, it is tempting to suggest a major role of this protein in the differences detected, although the influence of other genes that hypothetically may also differ among the strains employed cannot be discarded. The different in vitro infection efficiencies raise the possibility that some strains of E. hellem will also produce more aggressive features in infected patients.

Animals↗
PubMed2002

Comparative analysis of sequences encoding ABC systems in the genome of the microsporidian Encephalitozoon cuniculi.

Microsporidia are amitochondriate eukaryotic microbes with fungal affinities and a common status of obligate intracellular parasites. A set of 13 potential genes encoding ATP-binding cassette (ABC) systems was identified in the fully sequenced genome of Encephalitozoon cuniculi. Our analyses of multiple alignments, phylogenetic trees and conserved motifs support a distribution of E. cuniculi ABC systems within only four subfamilies. Six half transporters are homologous to the yeast ATM1 mitochondrial protein, a finding which is in agreement with the hypothesis of a cryptic mitochondrion-derived compartment playing a role in the synthesis and transport of Fe-S clusters. Five half transporters are similar to the human ABCG1 and ABCG2 proteins, involved in regulation of lipid trafficking and anthracyclin resistance respectively. Two proteins with duplicated ABC domains are clearly candidate to non-transport ABC systems: the first is homologous to mammalian RNase L inhibitor and the second to the yeast translation initiation regulator GCN20. An unusual feature of ABC systems in E. cuniculi is the lack of homologs of P-glycoprotein and other ABC transporters which are involved in multiple drug resistance in a large number of eukaryotic microorganisms.

ATP-Binding Cassette Transporters↗
PubMed2006

Antiparasitic activity of flavonoids and isoflavones against Cryptosporidium parvum and Encephalitozoon intestinalis.

Flavonoids, polyphenolic compounds found in plants, have demonstrated activity against several parasites and can augment the efficacy of other drugs by either increasing the uptake or decreasing the efflux of these drugs. We evaluated 11 of these compounds alone or in combination in order to test the hypothesis that flavonoids are effective against Cryptosporidium parvum and Encephalitozoon intestinalis. Using in vitro cell culture assays, HCT-8 cells or E6 cells were infected with C. parvum and E. intestinalis, respectively, and treated with compounds at doses ranging from 1 to 200 microM. We found that six compounds were active against C. parvum. Naringenin and genistein had the greatest activities with EC(50) of 15 and 25 microM, respectively. Two compounds, quercetin and apigenin, had activity against E. intestinalis at EC(50) of 15 and 50 microM, respectively. The EC(50) of trifluralin, a dinitroaniline compound, was decreased significantly when combined with genistein in an in vitro assay, suggesting that compounds may be used alone on in combination with other moderately active drugs to increase efficacy. In addition, induction of apoptosis by these compounds was studied but not observed to be a significant mechanism of action.

Animals↗
PubMed1997

Microsporidia (Encephalitozoon cuniculi) in wild rabbits in Australia.

OBJECTIVE: To determine the prevalence of infection with Encephalitozoon cuniculi in wild rabbit populations in Western Australia, and to isolate the organism from seropositive rabbits. DESIGN: Serological screening of wild and clinically affected domestic rabbit populations. SAMPLE POPULATION: Eighty-one wild rabbits from south-western Western Australia and 29 laboratory rabbits. PROCEDURE: Indirect immunofluorescence antibody technique and in-vitro amplification of parasite isolates in fibroblast cultures. RESULTS: Of the 81 wild rabbits and 29 laboratory rabbits, 20 and 22 respectively, had antibodies to E cuniculi. E cuniculi from the urine of one seropositive laboratory rabbit and from brain and kidney tissues of eight and five seropositive laboratory and wild rabbits respectively were isolated in fibroblast cultures. CONCLUSION: E cuniculi infection has been shown for the first time to be prevalent in wild rabbits in Australia. Techniques have been developed for the isolation and culture of the causative agent. Comparative studies can now be undertaken to determine risk factors for clinical disease in domestic rabbits and the relationship among E cuniculi isolates from wild and domestic rabbits.

Animals↗
PubMed1997

In vitro susceptibilities of the AIDS-associated microsporidian Encephalitozoon intestinalis to albendazole, its sulfoxide metabolite, and 12 additional benzimidazole derivatives.

Recent reports have described the successful treatment of Encephalitozoon intestinalis infection in AIDS patients with albendazole. However, this compound is rapidly metabolized in vivo to albendazole sulfoxide, and furthermore it is only 1 of about 15 commercially developed benzimidazole derivatives. To compare the activities of albendazole, albendazole sulfoxide, and other benzimidazoles, an in vitro system involving infection of green monkey kidney cell (E6) monolayers with E. intestinalis spores was developed. After 14 days, the effects of benzimidazoles on spore production were determined. Ten of fourteen derivatives tested, including albendazole, were inhibitory at concentrations of 1 to 10 ng/ml. Derivatives modified at the 1 or 2 position were less active. Albendazole sulfoxide was 1.7-fold more inhibitory than albendazole but significantly less toxic to E6 cells, a finding that explains the clinical efficacy of this compound. Potential alternatives to albendazole are discussed. No albendazole-resistant E. intestinalis mutants were obtained following in vitro selection.

AIDS-Related Opportunistic Infections↗
PubMed2000

In vitro efficacy of nikkomycin Z against the human isolate of the microsporidian species Encephalitozoon hellem.

Since 1985 microsporidia have been recognized as a cause of emerging infections in humans, mainly in immunocompromised human immunodeficiency virus-positive subjects. As chitin is a basic component of the microsporidian infective stage, the spore, we evaluated in vitro the susceptibility of a human-derived strain of Encephalitozoon hellem to nikkomycin Z, a peptide-nucleoside antibiotic known as a competitive inhibitor of chitin synthase enzymes. Transmission electron microscopy showed that this drug, at 25 microgram/ml, reduced the number of parasitic foci by about 35% +/- standard deviation after 7 days of culture (P < 0.0001) and induced cell damage of both mature and immature spores and also other sporogonic and merogonic stages. In particular, an irregular outline of the cell shape and an abnormally condensed cytoplasm in meronts and sporonts were documented. Also, the polar tubule and the polaroplast membranes appeared disarrayed in the sporoblast stage. The spore wall showed an enlarged endospore and delaminated exospore. Mature spores had a complete cytoplasmic disorganization and a swollen and delaminated cell wall. No ultrastructural cell damage was observed in uninfected control cultures treated with the drug.

Aminoglycosides↗
PubMed2001

In vitro activities of two antimitotic compounds, pancratistatin and 7-deoxynarciclasine, against Encephalitozoon intestinalis, a microsporidium causing infections in humans.

The antiparasitic effect of a collection of compounds with antimitotic activity has been tested on a mammalian cell line infected with Encephalitozoon intestinalis, a microsporidian causing intestinal and systemic infection in immunocompromised patients. The antiparasitic effect was evaluated by counting the number of parasitophorous vacuoles detected by immunofluorescence. Out of 526 compounds tested, 2 (pancratistatin and 7-deoxynarciclasine) inhibited the infection without affecting the host cell. The 50% inhibitory concentrations (IC(50)s) of pancratistatin and 7-deoxynarciclasine for E. intestinalis were 0.18 microM and 0.2 microM, respectively, approximately eightfold lower than the IC(50)s of these same compounds against the host cells. Electron microscopy confirmed the gradual decrease in the number of parasitophorous vacuoles and showed that of the two life cycle phases, sporogony was more sensitive to the inhibitors than merogony. Furthermore, the persistence of meronts in some cells apparently devoid of sporonts and spores indicated that the inhibitors block development rather than entry of the parasite into the host cell. The occurrence of binucleate sporoblasts and spores suggests that these inhibitors blocked a specific phase of cell division.

Amaryllidaceae Alkaloids↗
PubMed2002

Effects of chemical and physical agents on viability and infectivity of Encephalitozoon intestinalis determined by cell culture and flow cytometry.

We combined tissue culture and flow cytometry to assess the activities of various temperatures, chemicals, and disinfectants on the viability and infectivity of spores of Encephalitozoon intestinalis. Surfanios and benzalkonium chloride, disinfectants currently used in the hospital, were remarkably efficient in destroying spore viability and infectivity.

Animals↗
PubMed2005

Inhibitory activity of human immunodeficiency virus aspartyl protease inhibitors against Encephalitozoon intestinalis evaluated by cell culture-quantitative PCR assay.

Immune reconstitution might not be the only factor contributing to the low prevalence of microsporidiosis in human immunodeficiency virus (HIV)-infected patients treated with protease inhibitors, as these drugs may exert a direct inhibitory effect against fungi and protozoa. In this study, we developed a cell culture-quantitative PCR assay to quantify Encephalitozoon intestinalis growth in U-373-MG human glioblastoma cells and used this assay to evaluate the activities of six HIV aspartyl protease inhibitors against E. intestinalis. A real-time quantitative PCR assay targeted the E. intestinalis small-subunit rRNA gene. HIV aspartyl protease inhibitors were tested over serial concentrations ranging from 0.2 to 10 mg/liter, with albendazole used as a control. Ritonavir, lopinavir, and saquinavir were able to inhibit E. intestinalis growth, with 50% inhibitory concentrations of 1.5, 2.2, and 4.6 mg/liter, respectively, whereas amprenavir, indinavir, and nelfinavir had no inhibitory effect. Pepstatin A, a reference aspartyl protease inhibitor, could also inhibit E. intestinalis growth, suggesting that HIV protease inhibitors may act through the inhibition of an E. intestinalis-encoded aspartyl protease. These results showed that some HIV protease inhibitors can inhibit E. intestinalis growth at concentrations that are achievable in vivo and that the real-time quantitative PCR assay that we used is a valuable tool for the in vitro assessment of the activities of drugs against E. intestinalis.

Animals↗
PubMed2000

A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis.

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.

Animals↗
PubMed2006

Detecting immunoglobulin M antibodies against microsporidian Encephalitozoon cuniculi polar tubes in sera from healthy and human immunodeficiency virus-infected persons in Japan.

Encephalitozoon cuniculi, a spore-forming obligate intracellular parasitic pathogen belonging to the phylum Microsporidia, has a unique and highly specialized organelle called the polar tube. Using an enzyme immunostaining assay in which germinated E. cuniculi spores were coated onto plastic surfaces, we tested healthy and human immunodeficiency virus (HIV)-infected individuals in Japan for anti-polar tube antibodies of each immunoglobulin (Ig) class. Anti-polar tube IgG was detected in just 4 of 380 healthy individuals; no anti-polar tube IgA was detected in any individuals; however, unexpectedly, anti-polar tube IgM antibodies were detected in 138 individuals (36%). When the healthy individuals were grouped by age, the highest rate of positivity to anti-polar tube IgM antibodies was seen in individuals aged 20 years old or younger. Fifty-nine percent (24/41) of the individuals aged 20 years or younger were anti-polar tube IgM antibody positive. This rate tended to decrease among individuals in older age groups. However, no anti-polar tube IgM antibodies were detected in 21 HIV-infected persons who were younger than 30 years of age and who had CD4 cell levels below 250/mul. These seroepidemiological results clearly indicate that circulating anti-polar tube IgM antibodies that are capable of strongly reacting with filaments extruded from geminated spores exist and suggest that such antibodies may play a part in protective immunity.

Animals↗
PubMed2005

The parasitophorous vacuole membrane of Encephalitozoon cuniculi lacks host cell membrane proteins immediately after invasion.

Microsporidia of the genus Encephalitozoon develop inside a parasitophorous vacuole (PV) of unknown origin. Using colocalization studies, the PV was found to be absent from the endocytic pathway markers early endosomal autoantigen 1, transferrin receptor, and lysosome-associated membrane protein 1 and for the endoplasmic reticulum marker calnexin. The nonfusiogenic characteristic of the PV appears to be acquired as early as 1 min postinfection and is not reversed by drug treatment with albendazole or fumagillin.

Albendazole↗
PubMed1999

Role of gamma interferon in cellular immune response against murine Encephalitozoon cuniculi infection.

Microsporidia are obligate intracellular protozoan parasites that cause a wide variety of opportunistic infection in patients with AIDS. Because it is able to grow in vitro, Encephalitozoon cuniculi is currently the best-studied microsporidian. T cells mediate protective immunity against this parasite. Splenocytes obtained from infected mice proliferate in vitro in response to irradiated parasites. A transient state of hyporesponsiveness to parasite antigen and mitogen was observed at day 17 postinfection. This downregulatory response could be partially reversed by addition of nitric oxide (NO) antagonist to the culture. Mice infected with E. cuniculi secrete significant levels of gamma interferon (IFN-gamma). Treatment with antibody to IFN-gamma or interleukin-2 (IL-12) was able to neutralize the resistance to the parasite. Mutant animals lacking the IFN-gamma or IL-12 gene were highly susceptible to infection. However, mice unable to secrete NO withstood high doses of parasite challenge, similar to normal wild-type animals. These studies describe an IFN-gamma-mediated protection against E. cuniculi infection that is independent of NO production.

Animals↗
PubMed2000

Lack of CD4(+) T cells does not affect induction of CD8(+) T-cell immunity against Encephalitozoon cuniculi infection.

Cell-mediated immunity has been reported to play an important role in defense against Encephalitozoon cuniculi infection. Previous studies from our laboratory have underlined the importance of cytotoxic CD8(+) T lymphocytes (CTL) in survival of mice infected with E. cuniculi. In the present study, immune response against E. cuniculi infection in CD4(+) T-cell-deficient mice was evaluated. Similar to resistant wild-type animals, CD4(-/-) mice were able to resolve E. cuniculi infection even at a very high challenge dose (5 x 10(7) spores/mouse). Tissues from infected CD4(-/-) mice did not exhibit higher parasite loads in comparison to the parental wild-type mice. Conversely, at day 21 postinfection, susceptible CD8(-/-) mice had 10(14) times more parasites in the liver compared to control wild-type mice. Induction of the CD8(+) T-cell response in CD4(-/-) mice against E. cuniculi infection was studied. Interestingly, a normal antigen-specific CD8(+) T-cell response to E. cuniculi infection was observed in CD4(-/-) mice (precursor proliferation frequency, 1/2.5 x 10(4) versus 1/10(4) in wild-type controls). Lack of CD4(+) T cells did not alter the magnitude of the antigen-specific CTL response (precursor CTL frequency; 1/1.4 x 10(4) in CD4(-/-) mice versus 1/3 x 10(4) in control mice). Adoptive transfer of immune CD8(+) T cells from both CD4(-/-) and wild-type animals prevented the mortality in CD8(-/-) mice. E. cuniculi infection thus offers an example of an intracellular parasitic infection where CD8(+) T-cell immunity can be induced in the absence of CD4(+) T cells.

Adoptive Transfer↗