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Antimicrobial Agents and Chemotherapy, June 2005, p. 2362-2366, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2362-2366.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Inhibitory Activity of Human Immunodeficiency Virus Aspartyl Protease Inhibitors against Encephalitozoon intestinalis Evaluated by Cell Culture-Quantitative PCR Assay

Jean Menotti,1,4* Maud Santillana-Hayat,4 Bruno Cassinat,2 Claudine Sarfati,1,4 Francis Derouin,1,4 and Jean-Michel Molina3,4

Laboratory of Parasitology-Mycology,1 Department of Nuclear Medicine,2 Department of Infectious Diseases, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris,3 EA-3520, Faculté de Médecine Lariboisière-Saint-Louis, Université Paris 7, Paris, France4

Received 1 October 2004/ Returned for modification 25 October 2004/ Accepted 11 February 2005


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ABSTRACT
 
Immune reconstitution might not be the only factor contributing to the low prevalence of microsporidiosis in human immunodeficiency virus (HIV)-infected patients treated with protease inhibitors, as these drugs may exert a direct inhibitory effect against fungi and protozoa. In this study, we developed a cell culture-quantitative PCR assay to quantify Encephalitozoon intestinalis growth in U-373-MG human glioblastoma cells and used this assay to evaluate the activities of six HIV aspartyl protease inhibitors against E. intestinalis. A real-time quantitative PCR assay targeted the E. intestinalis small-subunit rRNA gene. HIV aspartyl protease inhibitors were tested over serial concentrations ranging from 0.2 to 10 mg/liter, with albendazole used as a control. Ritonavir, lopinavir, and saquinavir were able to inhibit E. intestinalis growth, with 50% inhibitory concentrations of 1.5, 2.2, and 4.6 mg/liter, respectively, whereas amprenavir, indinavir, and nelfinavir had no inhibitory effect. Pepstatin A, a reference aspartyl protease inhibitor, could also inhibit E. intestinalis growth, suggesting that HIV protease inhibitors may act through the inhibition of an E. intestinalis-encoded aspartyl protease. These results showed that some HIV protease inhibitors can inhibit E. intestinalis growth at concentrations that are achievable in vivo and that the real-time quantitative PCR assay that we used is a valuable tool for the in vitro assessment of the activities of drugs against E. intestinalis.


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INTRODUCTION
 
Microsporidia are ubiquitous protists that can infect a wide range of invertebrate and vertebrate hosts (39). Among them, Enterocytozoon bieneusi and Encephalitozoon intestinalis are opportunistic pathogens responsible for life-threatening intestinal, renal, pulmonary, and disseminated cases of microsporidiosis in severely immunocompromised patients, mainly human immunodeficiency virus (HIV)-infected patients (2, 12, 21, 29, 35, 39).

Treatment of E. bieneusi microsporidiosis is based on administration of fumagillin, whereas albendazole is recommended for the treatment of Encephalitozoon sp. infections (30, 31). Both treatments are efficient but do not eradicate the parasite, as relapses are frequent after the cessation of therapy in patients with persistent immunodeficiency. However, complete remission of intestinal or disseminated microsporidiosis has also been reported in patients treated only with highly active antiretroviral therapy (HAART) and has been found to be associated with the beneficial effect of HAART on patient immunity (14, 22, 26). These data are consistent with the decreased incidence of intestinal opportunistic protozoan infections in HIV-infected patients since the introduction of HAART (4, 27).

However, immune reconstitution might not be the only factor contributing to the low incidence of intestinal opportunistic protozoan infections, since several HIV protease inhibitors (PIs) were found to have inhibitory effects on the growth of fungi and protozoa. This was first evidenced with Candida albicans (3, 20, 33, 36) and was related to an effect of antiviral drugs on yeast adherence. For Pneumocystis jiroveci, the effect was demonstrated in vitro and was related to the presence of an aspartyl protease in P. jiroveci (1). For Toxoplasma gondii (11) and Cryptosporidium parvum (15), significant inhibition by several protease inhibitors at concentrations that can be achieved in humans was noted. Interestingly, all these studies agreed on the inhibitory effects of some PIs, especially ritonavir, which leads to the possibility of conformational similarities between the drug targets in these fungi and protozoa.

The aim of this study was to examine the in vitro activities of HIV PIs against E. intestinalis. To reach this goal, we developed a real-time quantitative PCR method for the quantification of E. intestinalis growth in vitro. We then characterized the dose-effect relationships and inhibitory concentrations of six HIV PIs on E. intestinalis.

(This work was presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 30 October to 2 November 2004.)


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MATERIALS AND METHODS
 
Parasites and cultures. The strain of E. intestinalis used in this study, kindly provided by T. Van Gool (Amsterdam, The Netherlands), was obtained from an HIV-infected patient (38). It was maintained in U-373-MG human glioblastoma cells (ATCC-HTB 17) in 75-cm2 culture flasks (37). Every other day from day 10 postinfection, spores were harvested from the supernatant and were stored at 4°C until use. For the drug studies, 24-well tissue culture plates were seeded with U-373-MG cells in RPMI medium and inoculated with E. intestinalis spores.

In order to define the optimal conditions for drug testing, various infection conditions were tested. E. intestinalis spores were added to three replicate wells at infection rates ranging between one spore per five cells and three spores per one cell. The cultures were examined microscopically and by real-time PCR at day 0 and day 5 postinfection. After selection of the optimal spore/cell ratio (see Results section), growth kinetics were assessed for this ratio from day 0 to day 8. In each set of experiments, three replicate culture wells with noninfected cells were used as negative controls.

Experimental design for assessment of drug activity against E. intestinalis spore growth. Albendazole (Sigma, Saint-Quentin-Fallavier, France) was used as the reference drug active against E. intestinalis, and pepstatin A (Sigma) was used as the reference aspartyl protease inhibitor. The following HIV protease inhibitors were studied: amprenavir (GlaxoSmithKline, Greenford, United Kingdom), indinavir (MSD-Chibret, Paris, France), lopinavir and ritonavir (Abbott Laboratories, Abbott Park, Ill.), and nelfinavir and saquinavir (Roche, Basel, Switzerland). Fresh stock solutions of all drugs except ritonavir and saquinavir were prepared in distilled water at 10 mg/ml; ritonavir was dissolved in methanol, and saquinavir was dissolved in 50% methanol-50% acetone. Serial dilutions were then prepared in culture medium.

Twenty-four-well tissue culture plates seeded with U-373-MG cells in RPMI medium were inoculated with one E. intestinalis spore per five cells. Four hours after inoculation, various drug dilutions were added into triplicate culture wells. Pepstatin A was tested over seven concentrations ranging from 0.2 to 20 mg/liter. Albendazole was tested over six 10-fold dilutions ranging from 10–5 to 1 mg/liter. The cytotoxic concentrations of PIs, as assessed under an inverted microscope, ranged from 30 to 40 mg/liter. Amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, or saquinavir was then tested at a concentration of 10 mg/liter, close to the highest nontoxic concentration achievable in plasma in vivo. Drugs which demonstrated some inhibitory activity were retested in triplicate cultures at serial concentrations ranging from 0.2 to 10 mg/liter (0.2 to 15 mg/liter for saquinavir). Each culture plate comprised three replicate E. intestinalis culture wells without drug (positive controls) and three replicate uninfected culture wells (negative controls). The culture plates were incubated at 37°C for 8 days without a change of medium and were microscopically examined for cytopathic effects every 2 days.

The contents of three replicate positive control wells (without drug) and three negative control wells were collected on day 0 and were centrifuged at 3,000 x g for 5 min. The pellet was collected and frozen at –20°C until use as the baseline control for PCR analysis. On day 8 postinfection, the plates were examined microscopically for cytopathic effects by using an inverted microscope at a magnification of x250, and the contents of all culture wells were collected and centrifuged. The pellets were collected for PCR analysis.

DNA extraction. The collected cell pellets were incubated with 10 units of lyticase (Sigma, Saint Quentin Fallavier, France). The DNA was then extracted by using the High Pure PCR template preparation kit (Roche Applied Science, Meylan, France), according to the manufacturer's instructions. The extracted DNA was resuspended in 200 µl of 10 mM Tris buffer (pH 8.5). Five microliters of this suspension was used for PCR.

Real-time quantitative PCR. Real-time PCR experiments were performed on an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, Calif.) by using the primer set FEI1 and REI1 and a TaqMan fluorescent probe, as described previously (24). This method results in the amplification of a 127-bp fragment of the E. intestinalis small-subunit rRNA gene, with a detection limit of 20 spores/ml (24). In the present study, each set of real-time PCR assays was run for 45 cycles, and the reaction mixtures comprised serial dilutions of E. intestinalis spores as external standards. Each spore dilution was tested in triplicate. A standard curve between the threshold cycle (CT) values and the spore numbers was established by using the Applied Biosystems sequence detection system, version 1.9.1, as described previously (24). The spore number in each culture well, tested in duplicate, was estimated by interpolation of the CT value obtained by real-time PCR on the standard curve. The results were expressed as equivalent spore numbers per culture well.

Statistical analysis. Student's t test was used to compare the spore numbers in the treated and the untreated cultures. The effect of each drug at various concentrations was then described by plotting the number of E. intestinalis spores as a function of the logarithm of the concentration. A linear regression model was used to summarize the concentration-effect relationship and to determine the 50% inhibitory concentration (IC50) (10). All statistical analyses were performed by using Statview software (SAS Institute Inc., Cary, N.C.) and GraphPad Prism, version 4.00, for Windows (GraphPad Software, San Diego, Calif.).


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RESULTS
 
Determination of optimal culture conditions for real-time PCR assessment of the growth kinetics of E. intestinalis and drug testing. Real-time PCR with noninfected cells yielded undetectable levels of E. intestinalis DNA (CT > 45). The kinetics of growth was then assessed according to the inoculum size and spore/cell ratios. For all infecting ratios, a marked increase in the amount of parasitic DNA in culture between day 0 and day 5 postinfection was observed. The best results were obtained with an inoculum of one spore for five cells (1:5 spore/cell ratio), with an increase in the parasitic load, as assessed by real-time PCR, of from (3.9 ± 1.6) x 103 spores per well at day 0 to (6.5 ± 2.1) x 104 spores per well at day 5. Prolongation of these cultures until day 8 resulted in a significant increase in the spore count compared to that on day 5 (1.5 x 105 ± 0.4 x 105 spores per well, as assessed by real-time PCR [P < 0.0001 versus that on day 5]) without alteration of the monolayers. Thus, this ratio and an 8-day incubation time were selected, as they provided the lowest background at day 0 (at least 10-fold lower than that achieved with other spore/cell ratios) and resulted in marked parasitic growth for 8 days. In order to assess if these conditions were well adapted for drug testing, serial 10-fold dilutions of albendazole were tested. As expected, a marked decrease in spore number was observed with concentrations of albendazole ≥10–2 mg/liter (Fig. 1A), which were associated with the inhibition of the cytopathic effect, confirming the inhibitory activity of albendazole on E. intestinalis. By the use of linear regression models, the IC50 was assessed to be 4.8 µg/liter, with a 95% confidence interval ranging from 4.7 to 4.9 µg/liter.



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  		FIG. 1. Inhibitory effects of albendazole (A), ritonavir (B), lopinavir (C), and saquinavir (D) on E. intestinalis growth in U-373-MG human glioblastoma cells. Number of E. intestinalis spores per culture well as assessed by real-time PCR (y axis) versus drug concentration in mg/liter (x axis). +, presence of parasitic foci at microscopic observation. –, absence of parasitic foci at microscopic observation.

Assessment of the inhibitory activities of HIV PIs. In a first experiment, HIV PIs were tested at a concentration of 10 mg/liter. At day 8 postinfection, no cell toxicity was recorded by microscopic examination of the monolayers.

In cultures containing amprenavir, indinavir, or nelfinavir, spore numbers were not significantly different from those in infected cultures without drug (P > 0.05) (Table 1), and parasitic foci were microscopically observed in all culture wells containing amprenavir, indinavir, and nelfinavir. These drugs were considered noninhibitory against E. intestinalis and were not tested at further concentrations. For ritonavir, lopinavir, and saquinavir, a significant decrease in spore numbers compared to that for the untreated controls (P < 0.0001) was noted (Table 1). Furthermore, testing of serial dilutions of these drugs allowed description of the relationships between their concentrations in the cultures and the inhibitory effect (Fig. 1B to D) and then estimation of the IC50. Ritonavir was found to be the PI that was the most active against E. intestinalis. Significant inhibition was noted for concentrations ≥2 mg/liter (P < 0.0001), and the IC50 of ritonavir was estimated to be 1.5 mg/liter, with a 95% confidence interval ranging from 1.4 to 1.6 mg/liter. For lopinavir and saquinavir, significant inhibition was noted for concentrations ≥2 mg/liter and 5 mg/liter, respectively (P < 0.0001). IC50s were estimated to be 2.2 mg/liter (95% confidence interval, 1.7 to 3.0 mg/liter) for lopinavir and 4.6 mg/liter (95% confidence interval, 3.7 to 5.8 mg/liter) for saquinavir. No cell toxicity was recorded at concentrations observed to inhibit microsporidia, and parasitic foci were microscopically observed only for concentrations ≤1 mg/liter for ritonavir and lopinavir and ≤2 mg/liter for saquinavir, in complete agreement with the PCR results.


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  		TABLE 1. Microscopic observation and real-time PCR assays performed on three replicate culture wells incubated for 8 days with HIV protease inhibitors at 10 mg/liter or pepstatin A at 20 mg/liter

Assessment of inhibitory activity of pepstatin A. Since HIV protease inhibitors were supposed to target an aspartyl protease, we examined the inhibitory effect of pepstatin A, which was used as a reference inhibitor for this enzyme. A significant inhibitory effect on E. intestinalis growth was noted for concentrations ≥10 mg/liter, with an estimated IC50 of 11.0 mg/liter (95% confidence interval, 9.5 to 13.0) (Table 1). No cell toxicity was recorded at concentrations observed to inhibit microsporidia, and parasitic foci were microscopically observed only for concentrations ≤5 mg/liter.


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DISCUSSION
 
The combination of a cell culture system and a specific real-time PCR assay allowed the description of the kinetics of growth of E. intestinalis in vitro and assessment of the inhibitory activities of six HIV protease inhibitors against this parasite. This technique was found to provide an alternative method to classic microscopic examination of cultures and spore counting in order to screen drugs for antimicrosporidial activity. In addition, real-time PCR was sensitive (detection threshold, 20 spores/ml) and reproducible by several repeated experiments without operator-dependent microscopic spore counting. What is more, real-time PCR could be applied to high-throughput drug screening assays performed with automated DNA extraction and 384-well plates. It seems to be a time-effective method, although its cost-effectiveness would need to be assessed.

When used for the assessment of drug activity, the culture-PCR technique allowed the simultaneous testing of a wide range of drug concentrations and description of the relationships between the drug concentrations and the inhibitory effects on E. intestinalis growth. Regression analysis allowed the accurate estimation of the 50% inhibitory concentration of each drug. The results of experiments performed with albendazole, which was used as a reference drug against Encephalitozoon species, confirmed the activity of albendazole against E. intestinalis. By the culture-PCR technique and regression analysis, the IC50 was estimated to be 4.8 µg/liter, a value consistent with those previously reported by Katiyar and Edlind (5 µg/liter) and Didier (8 µg/liter) by "classical" tissue culture and spore-counting methods (13, 18).

Among the six HIV protease inhibitors tested, ritonavir, lopinavir, and saquinavir were found to be inhibitory for E. intestinalis growth, as assessed by a significant and dose-dependent reduction of parasitic DNA excretion in the cultures. This effect was not associated with alteration of the cell monolayer, making unlikely possible nonspecific activity through a toxic effect on cultured cells. The reasons why only three of six protease inhibitors were found to be inhibitory against E. intestinalis are unclear, as all these drugs target the same enzyme (i.e., HIV aspartyl protease). Indeed, similar differences in protease inhibitor activities have already been observed with other parasites and fungi. Pichova et al. (36) found that ritonavir was the most potent inhibitor against Candida sp. aspartic proteases in comparison to saquinavir, whereas indinavir and nelfinavir exhibited almost no activity. Similarly, we previously found that ritonavir and nelfinavir were highly inhibitory for T. gondii growth, whereas indinavir was not (11).

For microsporidia as well as for other parasites and fungi that were found to be inhibited by HIV protease inhibitors, the modes of action of these drugs were not clearly determined. The fact that the reference aspartyl protease inhibitor, pepstatin A, was also shown to inhibit E. intestinalis spore growth supports the fact that ritonavir, lopinavir, and saquinavir do target a parasitic aspartyl protease. The presence of such an enzyme has already been demonstrated for P. jiroveci and C. albicans, the growth of which can be inhibited by HIV protease inhibitors (1, 3, 9, 20, 33, 36). Several aspartic protease-encoding genes have also been evidenced in Candida sp. (28, 32, 41) and Plasmodium sp. (7, 40) genomes and are considered potential drug targets (5, 6, 16). For C. parvum and T. gondii, genomic sequencing revealed the presence of aspartic proteinase-coding regions (5, 23). Recently, the complete sequencing of the genome of Encephalitozoon cuniculi has been performed (17), but to our knowledge, an aspartic protease-encoding gene has not yet been identified. Therefore, HIV protease inhibitors might also inhibit E. intestinalis growth through a non-aspartyl protease pathway. However, many genes encode yet unknown proteins in the E. cuniculi genome, and additional studies are needed for their characterization.

The fact that some HIV protease inhibitors can inhibit protozoa or fungi suggests that these drugs are not fully specific for the HIV enzyme and might cross-react with other aspartic proteases. Our results, as well as the fact that aspartic proteases are widely spread in protozoa and fungi, suggest that protease inhibitors might be promising new antiparasitic agents.

The in vivo and clinical relevance in humans of the in vitro activities of HIV protease inhibitors against E. intestinalis is unknown. Indeed, the main activity of all HIV PIs in human microsporidiosis is likely to be through immune reconstitution secondary to suppression of viral replication. However, our study shows that some of them may have an additional antiparasitic effect. The PI concentrations that were found to be inhibitory in vitro were in the range of those that can be achieved in human plasma during HAART, at least for ritonavir and lopinavir. For ritonavir, the reported mean maximal and minimal concentrations at steady state are 11.2 and 3.7 mg/liter, respectively, following oral administration of 600 mg twice daily (8), whereas the IC50 of this drug against E. intestinalis was 1.5 mg/liter. For lopinavir, mean maximal and minimal concentrations at steady state are 9.6 and 5.5 mg/liter, respectively, for a dose of 400 mg twice daily in combination with 100 mg of ritonavir twice daily (34), and the IC50 was assessed to be 2.2 mg/liter. For saquinavir, the IC50 is 4.6 mg/liter, whereas mean maximal and minimal concentrations at steady-state are only 2.2 and 0.2 mg/liter, respectively, with a dose of 1,200 mg three times daily. However, when the treatment was boosted with 300 mg of ritonavir twice daily, which inhibits the cytochrome P450 3A-mediated metabolism of saquinavir (19), the mean maximal concentration with a dose of 600 mg of saquinavir three times daily can be increased to 4.8 mg/liter (25).

From these results, one might expect some clinical benefits of some HIV protease inhibitors against microsporidiosis due to Encephalitozoon. Indeed, although these drugs do not seem to be as efficient as albendazole and cannot reasonably be proposed as an alternative treatment for microsporidiosis, they could contribute to the cure and prevention of microsporidiosis. Whether this hypothesis can be extrapolated to E. bieneusi infection remains hazardous because of the lack of an experimental model for the assessment of drug activity against this species.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Parasitologie-Mycologie, Hôpital Saint Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France. Phone: 33 1 42 49 95 03. Fax: 33 1 42 49 48 03. E-mail: jean.menotti{at}sls.ap-hop-paris.fr. Back


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Antimicrobial Agents and Chemotherapy, June 2005, p. 2362-2366, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2362-2366.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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