Calcium and Hydrogen Ion Concentrations in the Parasitophorous Vacuoles of Epithelial Cells Infected with the Microsporidian Encephalitozoon hellem

Abstract

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.

The Nascent Parasitophorous Vacuole Membrane of Encephalitozoon cuniculi Is Formed by Host Cell Lipids and Contains Pores Which Allow Nutrient Uptake

“…Speedy DiO was accessible to detergent extraction, which is typical for the integration into nonraft membrane subdomains. In contrast, Triton X-100 extraction in DiIC 16 -labeled cells resulted in a "Swiss cheese" pattern, which is characteristic for an association with lipid rafts after extraction of the nonraft subdomains (10).…”

“…Speedy DiO treatment resulted in a weak fluorescence of spores and was thus not further used for the ongoing experiments. DiIC 16 and Bodipy-PC labeling resulted in a heterogeneous staining pattern of the spore, in which internal structures appeared to be labeled (Fig. 1).…”

“…While Bodipy-PC treatment resulted in the labeling of all spores, a fraction of spores was unstained after DiIC 16 treatment. When these spores were used to infect a new host cell monolayer, the meronts, which are derived from DiIC 16 -labeled spores, did not show any fluorescence. In con-trast, we obtained fluorescent meronts with Bodipy-PC-labeled spores, indicating that this lipid can be used to trace the distribution and the fate of sporoplasma membrane compounds which are injected during infection into the host cell (Fig.…”

“…Two different fluorescent lipid probes were used in order to discriminate between lipid raft and nonraft microdomains. DiIC 16 is a compound that is specifically integrated into packed, rigid (raft) membrane subdomains, while Speedy DiO integrates into disordered, fluid (nonraft) membrane subdomains (6,19,24,27). To confirm the specificity of these lipid probes for raft and nonraft microdomains in our experimental setup, we applied cold Triton X-100 extraction after labeling of BHK cells with these compounds (Fig.…”

Identification and Characterization of Two Subpopulations of Encephalitozoon intestinalis

“…spores, with purification by Percoll gradient separation, has provided researchers with large numbers of highly purified spores (22). Spores purified by these methods have been used to test disinfectant (13,14,19,25) and therapeutic-agent efficacies (6), to define the immunologic responses of microsporidium-infected animals (6a), to determine parasite physiological and biochemical responses (16), to test primers for use in molecular studies and detection methods (1,5), and to develop antibodies (2,18). However, little is known about the comparability of these in vitro-cultured organisms to those occurring naturally.…”