Branching network of proteinaceous filaments within the parasitophorous vacuole of Encephalitozoon cuniculi and Encephalitozoon hellem.

The microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles in Encephalitozoon cuniculi. As a result, a new component of the E. cuniculi developing spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole of Encephalitozoon hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.

Encephalitozoon microsporidia modulates p53-mediated apoptosis in infected cells.

Microsporidia are intracellular obligate parasites which have recently been found to be related to fungi. They have a unique extrusion apparatus that is able to inject the sporoplasm directly into the target cell without using receptors. Encephalitozoon microsporidia are a source of morbidity and mortality in humans. It has been suggested that microsporidia may modulate the host cell cycle and apoptosis. We report here that caspase-3 cleavage is inhibited at different times of Vero cell infection by Encephalitozoon microsporidia and that the phosphorylation and translocation of p53 to the nucleus, previous steps for the activation of this protein, do not occur after infection of Vero cells. Consequently, the transcriptional function of p53 is impaired during the infection cycle as demonstrated by luciferase reporter assays. Thus, to our knowledge, for the first time it is shown that an intracellular parasite may be able to multiply in the host cell without activating the p53 apoptotic pathway of that cell. However, changes in the expression of Bcl-2 or Bax levels were not observed.

Characterisation of benzimidazole binding with recombinant tubulin from Giardia duodenalis, Encephalitozoon intestinalis, and Cryptosporidium parvum.

The binding kinetics of several benzimidazole compounds were determined with recombinant tubulin from benzimidazole-sensitive and -insensitive organisms. This study utilised the naturally occurring high efficacy of the benzimidazoles for the parasitic protozoa Giardia duodenalis and Encephalitozoon intestinalis, and low efficacy with Cryptosporidium parvum. Direct kinetic analysis of the benzimidazole-beta-tubulin interaction was performed using a fluorescence-based quenching method to determine the apparent association (k(on)) and dissociation (k(off)) rate constants from which the affinity constant (K(a)) was calculated. The binding kinetics were determined with recombinant alpha- and beta-tubulin from the parasitic protozoa with several benzimidazole R(2)-carbamate analogues. The affinity constant for the binding of several benzimidazoles with beta-tubulin from benzimidazole-sensitive protozoa was found to be significantly greater than binding to beta-tubulin from benzimidazole-insensitive protozoa. Additionally, the high affinity of several benzimidazole derivatives (albendazole, fenbendazole, mebendazole) for monomeric beta-tubulin and heterodimeric alphabeta-tubulin from benzimidazole-sensitive protozoa was also clearly demonstrated. The affinity constants determined with beta-tubulin from G. duodenalis and E. intestinalis also supported the observed in vitro efficacy of these compounds. The binding characteristics of the benzimidazoles with the highest in vitro efficacy (albendazole, fenbendazole, mebendazole) was reflected in their high association and slow dissociation rates with the beta-tubulin monomer or dimer from benzimidazole-sensitive protozoa compared with insensitive ones. Benzimidazole-bound alphabeta-tubulin heterodimers also had a significantly lower rate of microtubule assembly compared with benzimidazole-free alphabeta-heterodimers. The incorporation of benzimidazole-bound alphabeta-heterodimers into assembling microtubules was shown to arrest polymerisation in vitro although the addition of benzimidazole compounds to assembled microtubules did not result in depolymerisation. These findings indicate that a benzimidazole-beta-tubulin cap may be formed at the growing end of the microtubule and this cap prevents elongation of the microtubule.

Calcium and hydrogen ion concentrations in the parasitophorous vacuoles of epithelial cells infected with the microsporidian Encephalitozoon hellem.

Microsporidia of the genus Encephalitozoon undergo merogony and sporogony in a parasitophorous vacuole within the host cell. Cultured green monkey kidney cells infected with Encephalitozoon hellem were loaded with the fluorescent dyes fura-2 or BCECF in order to measure intracellular concentrations of calcium and hydrogen ions respectively. Both the parasitophorous vacuole calcium concentration and pH values resembled those of the host cell cytoplasm in infected cells. Calcein entered the parasitophorous vacuole but not other host cell vacuoles or parasite stages within the parasitophorous vacuole. The lack of a pH or calcium concentration gradient across the parasitophorous vacuole membrane and the permeability of this membrane to a large anion such as calcein suggest that the vacuole membrane surrounding E. hellem resembles that surrounding some other intracellular parasites such as Toxoplasma gondii. A potential role is discussed for the parasitophorous vacuole calcium concentration in germination in situ.

Antiprotozoal activities of benzimidazoles and correlations with beta-tubulin sequence.

Benzimidazoles have been widely used since the 1960s as anthelmintic agents in veterinary and human medicine and as antifungal agents in agriculture. More recently, selected benzimidazole derivatives were shown to be active in vitro against two protozoan parasites, Trichomonas vaginalis and Giardia lamblia, and clinical studies with AIDS patients have suggested that microsporidia are susceptible as well. Here, we first present in vitro susceptibility data for T. vaginalis and G. lamblia using an expanded set of benzimidazole derivatives. Both parasites were highly susceptible to four derivatives, including mebendazole, flubendazole, and fenbendazole (50% inhibitory concentrations of 0.005 to 0.16 microgram/ml). These derivatives also had lethal activity that was time dependent: 90% of T. vaginalis cells failed to recover following a 20-h exposure to mebendazole at 0.17 microgram/ml. G. lamblia, but not T. vaginalis, was highly susceptible to five additional derivatives. Next, we examined in vitro activity of benzimidazoles against additional protozoan parasites: little or no activity was observed against Entamoeba histolytica, Leishmania major, and Acanthamoeba polyphaga. Since the microtubule protein beta-tubulin has been identified as the benzimidazole target in helminths and fungi, potential correlations between benzimidazole activity and beta-tubulin sequence were examined. This analysis included partial sequences (residues 108 to 259) from the organisms mentioned above, as well as the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi and the sporozoan Cryptosporidium parvum. beta-tubulin residues Glu-198 and, in particular, Phe-200 are strong predictors of benzimidazole susceptibility; both are present in Encephalitozoon spp. but absent in C. parvum.