In Vitro Effects of Resveratrol on the Viability and Infectivity of the Microsporidian Encephalitozoon cuniculi

Abstract

Microsporidians of the genus Encephalitozoon are an important cause of disease in immunocompromised patients, and there are currently no completely effective treatments. The present study investigated the viability and infectivity of spores of Encephalitozoon cuniculi that had been exposed to resveratrol (RESV), a natural phytoalexin found in grapes and red wine. RESV at 50 μM showed significant sporicidal activity, and at 10 to 50 μM it reduced the capacity of the spores to infect dog kidney epithelial cells of the MDCK line. At 10 μM RESV also significantly inhibited intracellular development of the parasite, without affecting host cell viability. These results suggest that RESV may be useful in the treatment of Encephalitozoon infections.

Microsporidians are ubiquitous small intracellular spore-forming pathogens that infect a wide range of vertebrate and invertebrate species, including humans, and that are an increasingly significant cause of disease in immunocompromised patients (36). To date, more than 1,200 species within 144 genera have been described (24). Species of the genera Nosema, Vittaforma, Brachiola, Pleistophora, Encephalitozoon, Enterocytozoon, Septata (now reclassified as Encephalitozoon), and Trachipleistophora have been found in human infections (38). Encephalitozoon cuniculi is commonly found in domestic rabbits and rodents, and also occurs in dogs and other canids and primates, including humans (32, 34, 36, 40). Currently, no completely effective treatment exists for human microsporidiosis. The most widely used drugs for the treatment of microsporidiosis in animals and humans are albendazole and fumagillin (11, 25). However, infections show variable responses to these drugs: albendazole appears to show consistent efficacy against Encephalitozoon but is variable in its efficacy against Enterocytozoon, though fumagillin appears to be effective against both species of Encephalitozoon and Enterocytozoon (7, 25). Resveratrol (3,4′,5-trihydroxystilbene) (RESV), a natural phytoalexin produced by certain spermatophytes in response to infection by phytopathogenic microorganisms and found in grapes and grape products such as red wine, has known anticancer and anti-inflammatory effects (13, 15). One probable function of RESV is to protect the plant against fungal infections (16, 29), and some studies indicate that RESV may be useful for combatting human fungal pathogens (5). The microsporidians were initially considered to be protozoa but are currently considered to be parasitic fungi (4, 18, 23, 35). In the present study we evaluated for the first time the effects of RESV on the in vitro viability and infectivity of spores of the microsporidian E. cuniculi and investigated the possible toxicity of this compound to host cells.

MATERIALS AND METHODS

Organism, cell culture, media, drugs, and chemicals.

A total of 10⁷ spores of a canine subtype of E. cuniculi (ATCC 50502) were grown in 75-cm² culture flasks (Costar) containing mycoplasma-free epithelial dog kidney cells of the MDCK line (ATCC CCL-34). The cells were maintained at confluency at 37°C under 5% CO₂ in Iscove's modified Dulbecco's medium (Sigma, Alcobendas, Spain) containing 10% inactivated fetal bovine serum (Serva, Heidelberg, Germany) and gentamicin sulfate (80 μg/ml; Gevramycin; Schering-Plough, Madrid, Spain). Each week for 4 weeks after introduction of spores, the medium was replaced as follows. First, the old medium was removed and immediately replaced with fresh medium; the old medium was then centrifuged at 40 to 500 × g for 10 min at room temperature in 50-ml polypropylene tubes; and the spore-containing supernatant was collected by aspiration and introduced into the original culture flasks containing the original MDCK cells and the fresh medium. Stock solutions (100 mM) of RESV {3,4,5-trihydroxy-trans-stilbene, 5-[(1E)-2-(4-hydroxyphenyl)ethenyl]-1,3-benzenediol; Sigma} and albendazole [benzimidazolecarbamate, methyl 5-(propylthio)-2-benzimidazolecarbamate; Sigma] were prepared in dimethyl sulfoxide (DMSO) (Sigma) and stored in the dark at −80°C until use.

Preparation of spores.

Spores were harvested from the cell monolayers and pelleted as described above. Cell debris was removed by centrifugation at 50 × g for 3 min. The supernatant, containing the spores, was centrifuged at 1,000 × g for 10 min and resuspended in sterile Hank's balanced salt solution (HBSS) (Vitacell; American Type Culture Collection). A hemacytometer (Bürker) was used to count the spores (n = 5), and spore concentration was adjusted to 10⁹ spores/ml. Spores were maintained at 4°C and used within 1 to 2 weeks of harvesting.

Spore treatments.

One milliliter of a spore suspension containing 10⁷ spores/ml in HBSS was incubated with RESV (10 or 50 μM) for 7 days at 4°C. Control spores were incubated over the same period in HBSS containing DMSO at 64 mM (i.e., concentration of DMSO in the 50 μM RESV test medium). After treatments, all spores were washed three times in HBSS, resuspended in 1 ml of HBSS, and maintained at 4°C until use. For experiments in which dead spores were required, spores were killed by incubation in 75% ethanol at 4°C (27).

Assessment of spore viability.

Spore viability was assessed on the basis of staining with the fluorescent compound propidium iodide (PI). PI is membrane impermeant, so it only stains dead cells. Ten microliters of a freshly prepared solution of PI at 10 mg/liter in filtered (pore size, 0.2 μm) HBSS was added to each suspension of treated or control spores (10⁷ spores/ml), and the suspension was incubated at 37°C for 30 min in the dark. A total of 10,000 spores in each suspension were then counted with a Coulter Epics XL flow cytometer (Beckman-Coulter, Fullerton, Calif.), with simultaneous monitoring of fluorescence and light scatter. Fluorescent spores were assumed to be dead, and nonfluorescent spores were assumed to be viable (27).

Assessment of spore infectivity.

Low-passage-number MDCK cells were grown in 24-well tissue culture plates (Corning), then released by incubation with trypsin-EDTA (Sigma) at 37°C, and then diluted in culture medium to a concentration of 2 × 10⁵ cells/ml. To produce confluent monolayers, the 24-well plates were incubated at 37°C in the presence of 5% CO₂ and 95% humidity for 3 days and the culture medium was changed twice weekly. To determine whether the RESV can inhibit microsporidial growth by interference with the spore germination, 1 ml of a spore suspension containing 10⁷ spores/ml in HBSS was incubated with RESV (10 or 50 μM) for 7 days at 4°C (see above), and 10 μl of treated spores was added to each well containing a confluent MDCK cell monolayers. After 7 days of incubation, spores were counted in the culture supernatant using a hemacytometer. To determine whether RESV can inhibit microsporidial growth after the establishment of infections, an in vitro assay was used as described by Didier (8). Briefly, confluent MDCK cell monolayers in 24-well plates were infected with 10 μl of a suspension containing 10⁷ of untreated spores/ml in HBSS. Three hours later, noninternalized parasites were washed off and fresh medium, with or without RESV at 2, 10 or 50 μM, was added; control cultures without RESV received medium with the same DMSO concentrations as in the 50 μM RESV medium. The media were replaced with fresh RESV-containing media every 3 to 4 days. On day 7, 100 μl of 10% sodium dodecyl sulfate (SDS) was added to each of the wells to release microsporidian spores from host cells, and the total number of spores in each wells was counted with a hemacytometer.

To assess the in vitro effects of RESV and the reference drug albendazole, confluent MDCK cell monolayers in 24-well plates were infected with 10 μl of a suspension of untreated spores. Two days postinfection, the culture medium was removed and replaced by new medium containing the drugs at the test concentrations (10 and 50 μM for RESV or 20 and 50 μM for albendazole). Twice weekly, the culture medium was replaced by medium containing the drugs, and 14 days postinfection spores were counted in the culture supernatant using a hemacytometer.

Assay of viability of infected cells.

The viability of MDCK cells in infected and noninfected MDCK monolayers was assayed by PI staining, as for spores. A fresh working solution of PI was diluted 1:100 times in commercial stock solution (1 ng/ml in water; Sigma) in HBSS. The 24-well plates containing cell monolayers were washed three times with HBSS, and after washing 1 ml of HBSS was added. Twenty microliters of the working solution of PI was added to each well, and the plates were then incubated in the dark at 37°C for 15 min. After incubation, fluorescence was read with a microplate fluorescence reader (Bio-Tek Instruments, Winooski, Vt.) with excitation at 535 nm and measurement at 617 nm.

Statistical analyses.

Data are shown in the figures as means ± standard errors (SEM). The inhibitory effects of RESV are expressed as 50% inhibitory concentration, calculated by least-squares linear regression, using a curve-fitting program (Origin 5.0; Microcal Software, Inc., Northampton, Mass.) of log molar concentration of the test compound on percentage of maximal pharmacological response obtained with each concentration. Statistical significances (α = 0.05) were assessed by one-way analysis of variance followed by the Tukey-Kramer test for multiple comparisons.

RESULTS

Effects of RESV on the viability of E. cuniculi spores.

We first investigated whether RESV, at 10 or 50 μM, affects the viability of E. cuniculi spores. To this end, we incubated spores at 4°C for 7 days with RESV; as negative (i.e., maximal viability) controls we used spores incubated in the presence of 64 mM DMSO only, and as positive (i.e., minimal viability) controls we used spores maintained in 70% ethanol for 10 min. Spore viabilities as evaluated by flow cytometry are summarized in Fig. 1. These results suggest that the negative controls comprise two populations, a nonfluorescent (viable) population making up on average about 57% of the total, and a fluorescent (nonviable) population making up on average about 43% of the total (Fig. 1). Incubation with RESV at 10 to 50 μM, or ethanol treatment, increases the proportion of nonviable spores, and these increases were marked and statistically significant (P < 0.01) in the cases of 50 μM RESV and ethanol (Fig. 1).

FIG. 1.

Viability of spores of E. cuniculi, assessed on the basis of PI staining. Spores were treated for 7 days with RESV (10 or 50 μM) or for 10 min with 70% ethanol and then were analyzed by flow cytometry. Bars represent mean percentages of fluorescent (dead) spores in a sample of 10,000 spores ± SEMs (error bars) for five analyses. *, P < 0.01 with respect to untreated control spores.

In vitro infectivity of E. cuniculi spores treated with RESV.

This experiment investigated whether treatment of spores with RESV affects their capacity to infect microcultures of MDCK cells. The protocol used was the same as that for the previous experiment. A week after addition of spores to microcultures, spores were counted (Fig. 2). As can be seen, the mean number of spores per ml in MDCK cultures infected with RESV-treated spores (RESV at 10 or 50 μm) was significantly lower than in cultures infected with untreated spores.

FIG. 2.

Infectivity of E. cuniculi spores treated for 7 days with RESV at 10 or 50 μM. MDCK cell cultures were inoculated with treated or untreated spores, and 7 days later the number of spores per milliliter of medium was evaluated using a hemacytometer. Bars represent means ± SEM (error bars) for five replicate assays. *, P < 0.01 with respect to untreated control spores.

Effects of RESV on in vitro development of E. cuniculi.

This experiment was designed to investigate whether in vitro treatment of E. cuniculi-infected MDCK cells with RESV affects development of the parasite. MDCK cells were first infected with untreated E. cuniculi spores for 3 h, and then 2, 10, or 50 μM RESV was added to the medium. Control cultures without RESV received medium containing 64 mM DMSO (i.e., the same DMSO concentration as in the 50 μM RESV culture). Seven days postinfection the cells were lysed and spores present in the medium were counted. Cultures treated with RESV at 10 μM or more showed a significantly lower number of spores than untreated cultures, with a 50% inhibitory concentration of 8.7 ± 0.7 μM (n = 5; Fig. 3). In a second experiment, MDCK cells were first infected with untreated E. cuniculi spores; 2 days later the medium was replaced with medium containing 10 or 50 μM RESV or 20 or 50 μM albendazole; spore production was determined 14 days later. As shown in Fig. 4, the mean number of spores per milliliter in MDCK cultures treated with RESV (at 10 or 50 μm) or albendazole (20 to 50 μM) was significantly lower than that in untreated cultures.

FIG. 3.

Dose-response effect of RESV on infectivity of E. cuniculi-infected MDCK cells. RESV (2 to 50 μM) were added to MDCK cells cultures 3 h after addition of infective spores. The media were replaced with fresh RESV-containing media every 3or 4 days. Bars represent the mean ± SEM (error bars) (n = 5) number of spores per milliliter released by lysed cells on day 7 postinfection. *, P < 0.01 with respect to untreated control cultures.

FIG. 4.

Effects of RESV on spore production from E. cuniculi-infected MDCK cells. From 2 days postinfection onward, the medium included RESV (10 or 50 μM) or albendazole (20 or 50 μM). The number of spores per milliliter of medium was evaluated 14 days postinfection using a hemacytometer. Bars represent means ± SEM (error bars)for five replicate assays. *, P < 0.01 with respect to untreated control cultures.

Effects of RESV on cell viability.

This experiment was designed to assess whether the viability of MDCK cells is affected by E. cuniculi infection and/or RESV treatment following the protocols described in the previous section. The results obtained (Fig. 5) indicate that infection by E. cuniculi significantly reduces cell viability, as does treatment with 50 μM albendazole. By contrast, treatment with RESV, or infection by E. cuniculi followed by treatment with RESV, did not significantly affect viability by comparison with untreated uninfected control cells.

FIG. 5.

Effects of RESV on the viability of MDCK cells. E. cuniculi-infected (I) or uninfected (UI) cells were treated for 14 days with RESV (10 or 50 μM) or albendazole (20 or 50 μM). Cell viability was determined by fluorimetry following propidium iodide staining. Bars show mean fluorescence (indicating mean proportion of dead cells) ± SEM (error bars)for five replicate cultures. *, P < 0.05; **, P < 0.01 with respect to untreated control cultures.

DISCUSSION

There is currently a broad consensus that diverse plant polyphenols have positive effects on many diseases related to oxidative stress (33). However, there have been very few studies of the antimicrobial activity of these compounds, and still less is known about their antiparasitic activity (19). Of the widely studied plant polyphenols, perhaps the water-soluble tannins (commonly referred to as tannic acid) are those for which antimicrobial activity has been most comprehensively documented (6, 31). The polyphenol considered in the present study (RESV, found in red wine) has been shown to have antiviral activity (30), as well as antibacterial and antifungal activity (5); to date, however, there have been no studies of its effects on microsporidians, opportunist intracellular parasites that are an important cause of disease, particularly in human immunodeficiency virus-infected individuals (20).

Our results indicate that the population of spores obtained by in vitro culture in MDCK cells can be divided into two subpopulations: viable spores not stained by PI (about 57% of the total) and nonviable spores stained by PI (about 43% of the total). The nonviable spores are probably empty spores that have germinated and that thus no longer contain either sporoplasm or nucleus (9). Our results also indicate that RESV, at micromolar concentrations, significantly reduces the viability of E. cuniculi spores. This sporicide capacity may explain the reduced infection rates observed after infection of MDCK cells with RESV-treated spores. However, the observed inhibition was much greater when infected cells were treated directly with RESV: at concentrations above 50 μM, the inhibitions observed were even higher than those obtained with albendazole, a well-known reference drug in the treatment of human E. cuniculi infections (11). These results indicate that RESV shows sporicidal activity but that its effects on E. cuniculi development in host cells are even more marked. A similar difference in the capacity of polyphenols to kill intracellular stages was observed in a study of the intracellular flagellate Leishmania (19): specifically, this study found that some polyphenols significantly inhibited the intracellular survival of L. donovani amastigotes; in contrast, none of the polyphenols analyzed were directly toxic for the extracellular promastigote form of the parasite. One possible explanation for the sporicidal capacity of RESV is that it interferes in the polyamine metabolism pathways that are essential for microsporidian development (1); indeed, polyamine inhibitors have been demonstrated to be effective for the treatment of microporidiosis (2). It is well known that polyphenols provoke changes in polyamine levels in various cell types (21). Specifically, it has been shown that RESV inhibits polyamine synthesis and increases polyamine catabolism (39). This effect might explain the apparent difference in the capacity of RESV to kill mature spores and intracellular stages, since preemergent spores show active polyamine synthesis and interconversion, while intact mature spores show little metabolic activity (1). Other possible explanations for the observed inhibitory effect of RESV on the development of this parasite are related to its known antimitotic activity, due to inhibition of tubulin polymerization (28), which may block the proliferative phase of development (merogony). Another possibility is that RESV inhibits the germination of mature spores in host cells. Microsporidian germination occurs after discharge of the polar filament through the host cell membrane, allowing “inoculation” of sporoplasm into the cell (37). Ca²⁺ plays a key role in this process (22). Polyphenols are reducing agents that function as antioxidants by virtue of the hydrogen-donating properties of their phenolic hydroxyl groups, and also because of their transition-metal-chelating ability (3, 26). It is well known that RESV is an inhibitor of store-operated Ca²⁺ channels (10), and Ca²⁺ channel blockers inhibit Encephalitozoon polar filament discharge (14, 22).

In the present study, albendazole at 5 μg/ml had inhibitory effects on E. cuniculi, inhibiting spore production by on average 63%; however, at 5 to 50 μg/ml this drug had toxic effects on 1-week-old monolayer RK13 cells (12). Likewise, in the present study RESV at 50 μM had no detectable toxic effects on MDCK cells and, at the same time, inhibited spore production from infected cells by about the same amount as albendazole. On the other hand, in vivo studies have demonstrated that long-term high-dose (20 mg/kg/day) administration of RESV had no harmful effects in rats (17).

In conclusion, the results of the present study suggest that RESV might potentially have therapeutic value in the treatment of human E. cuniculi infections, though of course in vivo trials will be required to confirm this possibility.