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.
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.
Tumor necrosis factor (TNF) is cytotoxic to certain transformed cells, whereas normal cells are resistant to its effects. The resistance of normal cells can often be overcome by treatment with inhibitors of transcription or translation such as actinomycin D or cycloheximide (CHI), suggesting that normal cells produce a protein(s) that protects them from TNF-induced cytolysis. In this report, we examine the mechanism of cytolysis in a 3T3-like mouse cell line, C3HA, which was sensitized to TNF by treatment with CHI. We found that an early change in TNF/CHI-treated cells was a significant loss of stress fibers in perinuclear areas of the cytoplasm. The disruption of microfilaments, which was observed within 15 min of treatment, was not seen in untreated cells or in cells treated with either TNF or CHI alone. The dissolution of microfilaments spread peripherally over time and preceded other TNF/CHI-induced effects such as cytoplasmic "boiling," decrease in cell volume, and lysis of the plasma membrane. The breakdown of stress fibers occurred without a change in microtubules or intermediate filaments. Cytochalasin E, which disrupts microfilaments, induced cytolysis of TNF-treated cells even in the absence of CHI; however, demecolcine, which depolymerizes microtubules, did not sensitize cells to TNF. We propose that the TNF-induced cytolysis of certain cell types is preceded by a selective disruption of the microfilament lattice.
ABSTRACT. Microsporidia of the genus Encephalitozoon infect mammalian cells and have become a source of morbidity and mortality in immunocompromised humans. Encephalitozoon microsporidia develop and mature within parasitophorous vacuoles, enlarging the vacuole over time until it eventually occupies most of the cytoplasm of the host cell. The ability of the host cell to accommodate such a large burden for several days suggests that the parasite subverts normal host cell processes to ensure optimal environmental conditions for its growth and development. Since this environment would be threatened if cell division of the host cell occurred, we have formulated the hypothesis that infection with Encephalitozoon microsporidia induces an arrest in the cell cycle of the host cell. In support of this hypothesis, we have found that mitotic index and DNA duplication are reduced in infected cells as compared to uninfected cells. The number of host cell nuclei in S phase is increased. The levels of cyclin D1 and the percentage of cells in G1 are reduced; however, the levels of cyclin B1 are elevated even though the percentage of cells in G2/M is decreased. These results suggest that host cells infected with Encephalitozoon microsporidia are blocked at multiple points in the cell cycle.
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