INTRODUCTION

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Microsporidia are widespread obligatory intracellular parasites, apparently able to invade any cell in animals and humans (4, 16). These unicellular parasites have been increasingly recognized as opportunistic pathogens of immunodeficient patients (24). Two species cause diarrhea, malabsorption, and weight loss in AIDS patients (6). Enterocytozoon bieneusi is the most prevalent cause of these symptoms and is occasionally associated with hepatobiliary disease or infection of the upper respiratory tract (24). The second prevalent species, Encephalitozoon (Septata) intestinalis, is responsible for nephritis, bronchitis, and lytic mandibular lesions (11, 16). Rational strategies for the development of chemotherapeutic agents against microsporidia require a better understanding of the mechanisms controlling the proliferation of these parasites.

The life cycle of Encephalitozoon intestinalis (Fig. 1) consists of two successive developmental sequences, merogony and sporogony, both of which occur in a parasitophorous vacuole (PV) within host cells. During merogony, proliferative stages known as meronts are produced. After multiple divisions, meronts are transformed into sporonts. During sporogony, each sporont divides into two sporoblasts, which mature into spores which are approximately 2.0 by 1.2 µm. They contain a complex extrusion apparatus which ensures inoculation of the infective sporoplasm into a host cell. Meronts and sporonts are mononucleate cells which replicate by binary fission. Sometimes, the karyokinetic process is repeated before cytokinesis occurs, resulting in a ribbon-like cell containing two to four nuclei. The production of tetranucleate meronts and sporonts suggests some variability in the timing of cytokinetic cycles in E. intestinalis (3). Sporoblasts and spores are exclusively mononucleate, an indication that the regulation of development must be linked to the control of the cell cycle. Although very little is known about cell cycle control in microsporidia, a gene encoding a putative homologue of the cyclin-dependent kinase 1 (CDK1) has been recently identified in Encephalitozoon cuniculi (20), and a similar gene from E. intestinalis is being characterized in our laboratory (unpublished data). CDKs are major players in the progression of the eukaryotic cell cycle. Their activity is regulated by their phosphorylation status and by the association with negative (cyclin kinase inhibitors) or positive (cyclins) regulators, and by intracellular translocations. The temporary association of kinase subunits with different cyclins define time windows during which kinase activity is directed at distinct sets of substrates at the appropriate phase of the cell cycle.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Life cycle of E. intestinalis. Only the spores survive in the extracellular medium. The inoculation of the sporoplasm into the host cell is the initial step of the intracellular development.

Because of the importance of CDKs and their regulators in the multiplication and development of eukaryotes, these enzymes represent attractive potential targets for antiparasitic chemotherapy. The phylogenetic divergence between the parasite and its host is likely to result in divergences in the structure of their regulatory genes, as has been shown in other parasite-host systems (9, 10, 15); such divergences might confer to parasite and host differential susceptibilities to a given inhibitor. Therefore, we decided to evaluate the effect of a collection of antimitotic compounds (many but not all of which are CDK inhibitors) on the course of cellular infection by E. intestinalis.

	MATERIALS AND METHODS

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Inhibitors. Pancratistatin and 7-deoxynarciclasine were extracted and purified from Pancratium (Hymenocallis) littorale in Hawaii (17).

Parasites. E. intestinalis spores were collected from monolayers of rabbit kidney cells (RK13) as described by Van Gool et al. (22). Spores were harvested every 3 days, and suspensions of parasites were centrifuged at 350 × g for 5 min to eliminate cellular fragments. Spores were then pelleted by centrifugation at 2,000 × g for 20 min and washed twice. Spores were counted with a Malassez slide and used immediately for infection of cultured cells.

Culturing of parasites and treatment. RK13 culture cells were cultivated in Lab-Tek slides, in RPMI 1640 medium (Gibco BRL, Cergy Potoise, France) supplemented with 8% heat-inactivated fetal calf serum (56°C for 30 min) (Sigma, St. Quentin-Fallavier, France), streptomycin (100 µg/ml), penicillin (100 U/ml) and L-glutamine (2 mM). Cells were adjusted to 10⁵ cells/well and spores from E. intestinalis were added to cultured cells at a ratio of 1 spore/10 cells. The compounds were added to cultured cells at infection time. The effects of the compounds were determined by counting the PVs 48 h after infection and treatment. The detection of PVs was performed by immunofluorescence assay (IFA).

IFA. The Lab-Tek slides were fixed in ethanol at 20°C for 10 min and then incubated for 1 h at 37°C with the monoclonal antibody (MAb) M₁.6C₁.2C₁₁ (1) at a dilution of 1/500. This MAb is directed against a coat protein of sporogonic stages generated in the PVs. The slides were then washed in phosphate-buffered saline (PBS) and incubated with fluorescein-conjugated anti-mouse immunoglobulin G, diluted to 1/100 in Evans blue (1/1,000). After several washes with PBS, the slides were mounted in PBS-glycerol (50:50, vol/vol), and PVs were counted using an epifluorescence microscope. The entire surface of each well was examined at a magnification of ×500.

Determination of IC₅₀s on parasite. The concentration of inhibitor required to inhibit parasite growth by 50% (IC₅₀) was determined by IFA. The data were plotted and the IC₅₀s were determined using Cricket Graph software.

Determination of IC₅₀s on host cells. Microculture plates (96-well flat-bottom plates; Falcon) were prepared with RK13 cells. Six concentrations of each inhibitor were tested in triplicate. One microcurie of [³H] thymidine (5 Ci/mmol) was added to each well. Plates were incubated for 48 h and [³H]thymidine incorporation was measured with a scintillation counter (Beckman). The data were plotted and the IC₅₀s were determined using Cricket Graph software.

Electron microscopy. The effect of inhibitors on the morphology of E. intestinalis and RK13 cells was examined by electron microscopy. Infected monolayers were fixed at 48 h postinfection in 2.5% glutaraldehyde in 0.1 M Na cacodylate buffer (pH 7.2) for 1 h. They were rinsed in the same buffer and postfixed in ferriosmium [OsO4 and K₃Fe(CN)₆ (1%, wt/vol) in cacodylate buffer] for 1 h at room temperature. After ethanolic dehydration, the samples were embedded in Spurr's resin. Thin sections were stained with uranyl acetate and lead citrate and then examined with a JEOL TEM 100CX transmission electron microscope.

	RESULTS

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Effect of inhibitors on parasite growth. RK13 cells infected with E. intestinalis spores were incubated for 48 h in the presence of various concentrations of compounds. Since the stocks of inhibitors were dissolved in dimethyl sulfoxide (DMSO), controls were run simultaneously with the highest concentration of DMSO (0.03 µl/ml) without inhibitor. The effect of the inhibitors on parasite multiplication was evaluated from the number of PVs detected by IFA. Microsporidia are fast-growing organisms, and 48 h postinfection, PVs were detectable by IFA due to the occurrence of sporogonic stages cross-reacting with the MAb.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Structures of pancratistatin and 7-deoxynarciclasine.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   (a) In vitro effect of inhibitors on E. intestinalis multiplication. RK13 cells infected with E. intestinalis spores were incubated for 48 h, in the presence of various concentrations of inhibitors. The number of PVs was determined by IFA. The data represent means ± standard deviations (error bars) of triplicate cultures. *, significant difference between the PV number obtained in treated culture and nontreated culture, as determined by Student's test (P < 0.05). (b) In vitro effect of inhibitors on host cells (RK13). Microculture plates prepared with RK13 cells were treated with six concentrations of inhibitors. One microcurie of [3H]thymidine (5 Ci/mmol) was added to each well. Plates were incubated for 48 h, and [3H]thymidine incorporation was measured. The data represent means ± standard deviations (error bars) of triplicate cultures. *, significant difference between the 3H incorporation by the treated culture and nontreated culture, as determined by Student's test (P < 0.05).

Host cell IC₅₀s. To determine whether any of these two molecules may represent a useful lead compound, we next measured their effect on host cells in a standard [³H]thymidine incorporation assay. RK13 cells were treated with the two inhibitors (concentrations ranging from 0.01 to 10 µM), and incorporated radiolabel was then measured (see Materials and Methods). Again, cells treated with DMSO diluted in RPMI and RPMI alone were used as controls. The IC₅₀s of pancratistatin and 7-deoxynarciclasine obtained from the graphs (Fig. 3b) were both 1.5 µM and thus were 8- and 7.5-fold higher than the parasite IC₅₀s, respectively.

Morphological effects of the inhibitors. Examination of infected cultures by electron microscopy confirmed that both compounds caused a gradual resorption of the infection. In cultures treated with the lower doses, most cells were infected. Normally fusiform, these cells became rounded due to the presence of a large PV (Fig. 4). Two or three PVs could be seen in some cells. All developmental stages of the parasite were present in these vacuoles, which contained up to 20 spores. At a concentration of 0.5 µM, flat cells containing small PVs with a concomitant decrease in the number of mature spores were observed. In some cells, PVs contained meronts as well as a small number of sporoblasts and spores that were larger (3 to 4 µm) than those produced in the absence of inhibitors. Additionally, some alterations were observed in the polar tube, a major component of the spore extrusion apparatus. Eight to nine coils of the polar tube were numbered in large sporoblasts and spores, instead of the five to six characteristic of the species (data not shown). Most interesting was the occurrence of two nuclei in sporoblasts and in spores (Fig. 5A and B and 6A). In most sections, these nuclei were abutted, thus displaying diplokaryotic arrangement. Used at the same concentration (0.5 µM), 7-deoxynarciclasine exerted a similar effect, although to a lesser extent.

View larger version (138K): [in this window] [in a new window]   FIG. 4.   In nontreated cultures and in those treated with lower doses of inhibitors, the development of the parasite results in the production of numerous spores. The electron-dense stages are sporoblasts maturing into spores. Abbreviations: HN, host cell nucleus; Me, two meronts applied to the PV membrane outlined with glycogen granules (arrowheads). Scale bar, 2 µm.

View larger version (145K): [in this window] [in a new window]   FIG. 5.   Sporoblasts and spores generated in cultures treated with 0.5 µM pancratistatin contain two nuclei (N). (A) Two binucleate sporoblasts. (B) Only one polar tube (arrows) can be seen in this section of a binucleate sporoblast. The nuclei are assembled into a diplokaryon. (C) The mononucleate sporoblast of E. intestinalis in the absence of treatment with pancratistatin or 7-deoxynarciclasine. Scale bars, 0.5 µm.

View larger version (161K): [in this window] [in a new window]   FIG. 6.   Two sporogonic stages in a culture treated with 0.5 µM pancratistatin. (A) An electron dense diplokaryotic sporoblast is transforming into a mature spore. The median constriction (arrow) of the thick walled spore is another indication that this stage originates from a sporont that did not complete its division. (B) A large mononucleate meront in a cell treated with 1 µM pancratistatin. The vesicles (arrows) characteristic of microsporidia mitotic poles indicate that the nucleus is engaged into the division process. Scale bars, 1 µm.

View larger version (132K): [in this window] [in a new window]   FIG. 7.   Alterations of the parasite in cells treated with the inhibitors. Degenerative stages (arrowheads) are scattered in the host cell cytoplasm. Scale bar, 0.5 µm.

	DISCUSSION

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Pancratistatin, 7-deoxynarciclasine, and parasite development. We have tested a small collection of compounds containing a large proportion of kinase inhibitors, especially CDK inhibitors such as purines, paullones, and indirubins. Most compounds proved inefficient in specifically inhibiting parasites while preserving the host cells. Two out of 526 compounds were found to be selective for the parasites, and, interestingly, they appeared to be closely related in their structure (Fig. 2). Pancratistatin and 7-deoxynarciclasin have been originally identified as antitumor agents extracted from P. littorale, a Hawaiian member of the family Amaryllidaceae, and related species (17, 18, 19). As many pancratistatin derivatives have been synthesized, we are now testing this family of compounds, with the purpose of identifying a pancratistatin analogue with improved selectivity for microsporidia.

Binucleate sporoblasts and spores induced by pancratistatin and 7-deoxynarciclasine. These molecules appear to block cell division in microsporidia in a dose-dependent manner. Morphological changes occurred gradually with the concentration. Binucleate sporoblasts and spores were seen in culture treated with 0.5 µM pancratistatin. Although less frequent, they were also observed in those treated with the same dose of 7-deoxynarciclasine. Apparently, the inhibitor caused a block in the cytodieresis of the sporonts, thus generating these binucleate sporoblasts which, however, were still able to differentiate into mature spores (Fig. 5A and B and 6A). Sporogony itself was totally inhibited at the highest dose (1 µM).