First characterization of a microsporidial triosephosphate isomerase and the biochemical mechanisms of its inactivation to propose a new druggable target.

The microsporidia are a large group of intracellular parasites with a broad range of hosts, including humans. Encephalitozoon intestinalis is the second microsporidia species most frequently associated with gastrointestinal disease in humans, especially immunocompromised or immunosuppressed individuals, including children and the elderly. The prevalence reported worldwide in these groups ranges from 0 to 60%. Currently, albendazole is most commonly used to treat microsporidiosis caused by Encephalitozoon species. However, the results of treatment are variable, and relapse can occur. Consequently, efforts are being directed toward identifying more effective drugs for treating microsporidiosis, and the study of new molecular targets appears promising. These parasites lack mitochondria, and oxidative phosphorylation therefore does not occur, which suggests the enzymes involved in glycolysis as potential drug targets. Here, we have for the first time characterized the glycolytic enzyme triosephosphate isomerase of E. intestinalis at the functional and structural levels. Our results demonstrate the mechanisms of inactivation of this enzyme by thiol-reactive compounds. The most striking result of this study is the demonstration that established safe drugs such as omeprazole, rabeprazole and sulbutiamine can effectively inactivate this microsporidial enzyme and might be considered as potential drugs for treating this important disease.

A new Encephalitozoon species (Microsporidia) isolated from the lubber grasshopper, Romalea microptera (Beauvois) (Orthoptera: Romaleidae).

We describe a new microsporidian species, Encephalitozoon romaleae n. sp., isolated from an invertebrate host, the grasshopper Romalea microptera, collected near Weeks Island, Louisiana, and Jacksonville, Florida. This microsporidian is characterized by specificity to the gastric caecae and midgut tissues of the host and a life cycle that is nearly identical to that of Encephalitozoon hellem and Encephalitozoon cuniculi. Mature spores are larger (3.97 x 1.95 microm) than those of other Encephalitozoon species. Polar filament coils number 7 to 8 in a single row. Analysis of the small subunit (SSU) rDNA shows that E. romaleae fits well into the Encephalitozoon group and is a sister taxon to E. hellem. This is the first Encephalitozoon species that has been shown to complete its life cycle in an invertebrate host.

Confirmed detection of Cyclospora cayetanesis, Encephalitozoon intestinalis and Cryptosporidium parvum in water used for drinking.

Human enteropathogenic microsporidia (HEM), Cryptosporidium parvum, Cyclospora cayetanesis, and Giardia lamblia are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of HEM (Encephalitozoon intestinalis and Enterocytozoon bieneusi) and Cyclospora cayetanesis have not been fully elucidated due to lack of sensitive and specific environmental screening methods. The present study was undertaken with recently developed methods, to screen various water sources used for public consumption in rural areas around the city of Guatemala. Water concentrates collected in these areas were subjected to community DNA extraction followed by PCR amplification, PCR sequencing and computer database homology comparison (CDHC). All water samples screened in this study had been previously confirmed positive for Giardia spp. by immunofluorescent assay (IFA). Of the 12 water concentrates screened, 6 showed amplification of microsporidial SSU-rDNA and were subsequently confirmed to be Encephalitozoon intestinalis. Five of the samples allowed for amplification of Cyclospora 18S-rDNA; three of these were confirmed to be Cyclospora cayetanesis while two could not be identified because of inadequate sequence information. Thus, this study represents the first confirmed identification of Cyclospora cayetanesis and Encephalitozoon intestinalis in source water used for consumption. The fact that the waters tested may be used for human consumption indicates that these emerging protozoa may be transmitted by ingestion of contaminated water.

In situ hybridization: a molecular approach for the diagnosis of the microsporidian parasite Enterocytozoon bieneusi.

Microsporidia are emerging as opportunistic pathogens in patients with acquired immunodeficiency syndrome (AIDS). Enterocytozoon bieneusi is the most commonly reported microsporidium that is detected in gastrointestinal specimens. This report describes an in situ hybridization technique with a 30-base specific synthetic DNA probe for detection of E bieneusi by light microscopy. Formalin-fixed paraffin-embedded duodenal biopsy specimens from three patients with AIDS, chronic diarrhea, and E bieneusi infection confirmed by electron microscopy were used in this study. Light microscopic examination after colorimetric detection allowed the identification of different stages of the pathogen's life cycle in the cytoplasm of enterocytes. No cross-reactivity was noted between the probe and human DNA. Our study underscores the applicability of a synthetic-labeled oligonucleotide for the detection and identification of E bieneusi in clinical samples.