Irreversible effect of cysteine protease inhibitors on the release of malaria parasites from infected erythrocytes.

Culture of Plasmodium falciparum and a synchronization procedure

Plasmodium falciparum strain 3D7 (ATCC, Manassas, VA) was cultured according to the Trager-Jensen method ( Trager and Jensen, 1976 ) in human erythrocytes in RPMI 1640 medium (Gibco) supplemented with 25 mM Hepes (Gibco), 4.5 mg/mL glucose (Sigma), 0.1 mM hypoxanthine (Gibco), 25 μg/mL gentamicin (Gibco), and 0.5% AlbuMax (Gibco). A combination of Percoll-enrichment and sorbitol-treatment procedures were used to obtain a culture of P. falciparum with a 3- to 4-h span of synchronization (detailed description in ( Lambros and Vanderberd, 1979 ; Druzewski et al., 1984 ). Only freshly drawn human erythrocytes or those dated no more than 48 hours after phlebotomy were used to initiate synchronized cultures. While erythrocytes that were stored longer than 48 hours after phlebotomy supported parasite replication, cultures using them had a significant fraction of defective schizonts, and many of the drug-induced parasite clusters were structurally damaged.

Parasite replication assay

To test the effect of E-64 on parasite replication, a synchronized parasite culture was maintained according to the Trager-Jensen method ( Trager and Jensen, 1976 ) at 0.5% hematocrit. To avoid aspiration of parasite clusters formed in drug-containing cultures, we started cultures at low parasitemia and maintained cultures in the normal medium or medium supplemented with drug for the first 3 days without medium change. After 72 h, cells were washed by centrifugation (1,900 × g, 5 min at room temperature), resuspended in the normal medium, and returned to culture for 3 more days with a daily medium change. Daily aliquots of infected cells were taken in order to assess parasite replication by staining parasitized cells with acridine orange (Molecular Probes, Eugene, OR).

Parasite Release Assay

To quantify the parasite release process, we used our recently developed method ( Glushakova et al., 2007 ). Briefly, a highly synchronized parasite culture was used for drug treatment, starting 42–50 h after culture initiation, depending on the length of the drug treatment (as much as 8 h or as little as 30 min). The end of the drug treatment time always coincided with the end of the parasite erythrocyte cycle. Treatment was performed in medium supplemented with Albumax at 37°C. The treatment time included the last 2-h period in chambers for microscopy (for accumulation of release sites from the release events) before the quantification of parasite release. Cells were treated with drug in chambers for microscopy only in short-time experiments. To analyze the reversibility of drug effect on parasite release we diluted drug-treated cultures at least 200 × with the normal medium. This “drug-removal” procedure was used to avoid cell centrifugation and resuspension, which affected the morphology of parasite clusters. After this procedure, cells were immediately injected into chambers for microscopy and incubated for 1 to 3 h at 37°C to assess parasite release. Control cultures were treated the same way but in the normal medium. We analyzed a live culture of P.falciparum using differential interference contrast (DIC) microscopy (confocal microscope LSM 510, Zeiss, 100× or 63× oil 1.4 NA objectives). We tested the cysteine protease inhibitor E-64 (Sigma, St. Louis, MO) at 10 μM concentration, the cysteine/serine protease inhibitors leupeptin (Sigma, St. Louis, MO) at 10 μg/ml concentration and calpeptin (Calbiochem, San Diego, CA) at 0.1 – 1 μM concentrations, the caspase inhibitors Z-VAD-FMK, Z-DEVD-FMK, and Z-IETD-FMK (Calbiochem, San Diego, CA) each at a 100 μM concentration, and calpastatin peptide (CS Peptide; Calbiochem, San Diego, CA) at 5 μM concentration.

Confocal recording of live infected erythrocytes

Confocal microscopy was performed at 37°C using a laser scanning confocal microscope (LSM 510, Zeiss) with a 100× or 63× 1.4 NA oil objective. Laser excitation at 633 nm with an intensity below 10 μW was used to avoid photo-induced cellular damage. Assessment of the food vacuole size in parasites in live infected erythrocytes We performed DIC microscopy of infected cells as described above and analyzed digital images using a software package (Image Browser, Zeiss). Specifically, the area of the spot that represents the food vacuole was measured in 10 randomly selected cells. This value is presented as mean ± s.e (μm 2 ).

Labeling of erythrocyte membrane with fluorescent markers

Two different labeling procedures were used to determine the origin of the clusters' limiting membrane. Neither approach required cell washing before microscopic analysis. Late-infected schizonts were not subjected either to centrifugation or to any other procedure of isolation or purification. To detect multiple proteins on the erythrocyte surface, cells were biotinylated in the early ring stages of infected cultures. Biotinylated cells were labeled with 20 nM Quantum-Dots 525 conjugated with streptavidin (Quantum Dots, Hayward, CA) at the end of the cycle just before microscopy (for details of procedure see reference ( Glushakova et al., 2005 ). Quantum Dots 525 were excited with a 488-nm laser. To detect the most abundant glycoprotein of erythrocyte membranes, glycophorin A, we labeled cells with 2 μg/ml Allophycocyanin- (APC-) antibody to glycophorin A (anti-CD235a) (BD Biosciences, San Jose, CA) just before microscopic analysis. APC was excited with a 514-nm laser. Because of high concentrations of detected antigens on the cell surface and because of the use of the primary-labeled antibodies to glycophorin A or Quantum Dots-conjugated streptavidin, the signal/noise ratio was high enough to avoid cell washing before microscopy. In control experiments (labeling of parasitophorus vacuoles extruded from erythrocytes by hypotonic shock), we confirmed that the membrane of the parasitophorus vacuole does not have glycophorin A. In addition, we also confirmed that it is not biotinylated and that it could not be labeled with Quantum Dots-conjugated streptavidin. Labeled cells were injected into chambers (HybriWell ™ HBW20; Grace Bio-Labs, Bend, OR) at hematocrit ~ 0.2%, sealed, and used for microscopy for all methods described above.

Electron microscopy

Cells were pre-fixed with 0.2% paraformaldehyde to render the sample non-infectious for subsequent handling and then pelleted at 1,900 × g for 5 min. Both high-pressure-frozen and chemically-fixed samples were prepared. For high-pressure freezing, the sample was placed in the cavity of a slot grid that was sandwiched between two flat-bottom planchettes immediately before freezing using an HPM10 (Bal-Tec, Balzers, Liechtenstein). Freeze-substitution occurred in acetone containing 2% osmium tetroxide for 72 h at −90°C, followed by linear warming to room temperature over 24 h in an EM-AFS freeze-substitution system (Leica Microsystems, Wetzlar, Germany). For chemical fixation, the sample were treated with 1.5% glutaraldehyde and 1.5% osmium tetroxide for 45 min at room temperature. The cells were then transferred to acetone in a succession of PBS-acetone solutions (25%,50%,75%, and 100% acetate) for 15–20 minutes each. High pressure-frozen and chemically-fixed samples were washed 3 times in acetone for 10–15 minutes and then infiltrated in 25%, 50%, 75%, and 100% Spurr's resin ( Spurr, 1969 ) for 8–12 h each. A final infiltration at 100% for 12h was followed by the polymerization of the samples at 65°C for 48 h. We sectioned polymerized blocks to 60–70 nm using a diamond knife on a Ultracut microtome (Leica, Microsystems, Wetzlar, Germany) and picked them up on Formvar-coated slot grids. These sections were then stained with 1% uranyl acetate ( Stempak and Ward, 1964 ) for 5 min and lead citrate ( Reynolds, 1963 ) for 5 min. Images were collected with a 2k × 2k UltraScan (Gatan, Pleasanton, CA) digital camera on a Technai F30 (FEI, Hillsboro, OR) microscope operated at 300 kV at the indicated magnification.

Parasite viability assay

We detected apoptotic/necrotic parasites using labeling with a combination of fluorescent dyes, YO-PRO-1 (100 nM) and PI (1 μg/ml) (Molecular Probes, Eugene, OR). Dyes were added into the culture medium, and individual infected cells were monitored for the appearance of parasites with labeled nuclei. Alternatively, 30 min after addition of dyes clusters with labeled parasites were counted. No labeling was detected in morphologically normal schizonts during several hours of cell observation in the presence of these dyes.

Supplementary Material Movie S1 Movie S1. Confocal recording of parasite release in normal medium . Recording was performed under physiological conditions using low-light-intensity laser scanning microscopy. Movie S2 Movie S2. Confocal recording of the cluster formation in medium supplemented with 10 μM E-64 . Recording was performed under physiological conditions using low-light-intensity laser scanning microscopy. Supl.Fig.1 Figure S1. Reversible protease inhibitors leupeptin and calpeptin block parasite release, produce parasite clusters limited by erythrocyte membrane, and interfere with hemoglobin degradation in food vacuoles upon prolonged drug treatment . A . Synchronized culture of P. falciparum was treated with leupeptin (10 μg/ml) and calpeptin (1 μM) as described in the Fig. 2 legend. The result is presented as mean value ± s.e. of 3–4 independent experiments. B – C . Detection of the cluster-limiting membrane by fluorescent light microscopy. Biotinylated (B) or unmodified (C) RBC membrane was detected in live drug-treated with streptavidin-quantum dots 525 conjugate (green color; B) or APC-antibody to glycophorin A (anti-CD235a, red color; C). Fluorescent images of clusters are supplemented with the DIC images of the same cells. D . Drug-induced clusters with enlarged parasite food vacuoles in the long drug-treated cultures. Scale bars = 5 μM. Supl.Fig.2 Figure S2. Isolated clusters are not infectious in the standard replication assay . Clusters that were treated for 8 h with 10 μM E-64 were isolated according to Salmon et al. ( Salmon et al., 2001 ) and added to uninfected RBC at 0.5% hematocrit to follow the initiation of a new cycle of parasite replication. After 15–18 h in culture, the resulting parasitemia (a fraction of infected erythrocytes) was compared with the parasitemia in control culture that originated with schizonts isolated from the same drug-treated cells. Note that the schizonts but not the clusters initiate a new round of parasite replication. Cl, clusters; T, trophozoites; R, rings. Supl.Fig.3 Figure S3. Isolated clusters harbored dead parasites . Clusters treated for 10 h with 10 μM with E-64 and isolated according Salmon et al. ( Salmon et al., 2001 ) harbored dead parasites , as ascertained with PI. Note that immediately after isolation, clusters harbored multiple PI-labeled dead parasites (red color). Supl.Fig.4 Figure S4. Cysteine protease inhibitor E-64 blocks parasite erythrocyte cycle . Synchronized culture at the ring stage was treated with 10 μM E-64 for 3 days, and then the cycle was followed for 3 more days after the replacement of drug-containing medium with the normal one. The resulting parasitemia was compared with the parasitemia in control cultures not treated with drug. Data are presented as the mean of triplicate values. Supl.Fig.5 Figure S5. Reversible protease inhibitors leupeptin and calpeptin irreversibly block parasite release from drug-induced clusters but not from schizonts upon drug withdrawal . A – B . Evidence that sites of parasite release originate from schizonts upon drug withdrawal from treated cultures. Cultures were treated with 10 μg/ml leupeptin (A) or 1 μM calpeptin (B) (1–3 h for leupeptin and 2 h for calpeptin); after drug withdrawal cells were injected onto the chambers, and the proportion of schizonts, clusters, and sites of release were assessed before and after parasite release recovery (1–3 h for leupeptin and 2 h for calpeptin). Note that the increase in the number of newly ruptured cells upon drug withdrawal is equal to the decrease in the number of schizonts; the number of clusters is slightly increased. Mean ± s.e. (n=5) for leupeptin and a representative experiment for calpeptin. C . Recovery of parasite release after 1–2 h after drug withdrawal in cultures treated for different time intervals with drug (40 min to 1 h for leupeptin and 2 h for calpeptin (mean ± s.e., n=3). Table S1 Table S1. The size of food vacuoles increased with the increased time of drug treatment . The size of food vacuole in the clusters was compared with the size of control food vacuoles released during schizont rupture. The symbol * indicates a significant difference of value from the control.