Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes.

In Vitro Cultivation of Plasmodiumfalciparum The FCR-3 (Gambia) strain ofP. falciparum was cultured in vitro according to the method of Trager and Jensen (29). P fatciparumwas grown in human (c) The Rockefeller University Press, 0021-9525188105/1507/7 $2.00 The Journal of Cell Biology, Volume 106, May 1988 1507-1513 1507 type A/erythrocytes at 5% hematocrit in RPMI 1640-Hepes medium sup- plemented with 10% human serum and 20 mM glucose. To achieve parasite synchrony, schizont-infectedcultures were fractionated by gelatin flotation (17). Parasites from the same synchronous culture were used in the experi- ments on the stage-dependent synthesis of the rhoptry protein. Schizont- infected erythrocytes concentrated by gelatin flotation, were resuspended with fresh erythrocytesat a parasitemia of 6 % and reinvasionallowed to take place. The time of erythrocyte reinvasion when parasites were 0-6 h was defined as T-3. At each time point, "I"-3(rings), 21 h (trophozoites), 33 h (mid-schizont), 41 h (segmenters, free merozoites) and 48 h (reinvaded rings), parasites were prepared for immunoblotting and IFA as described below. Production ofMonoclonalAntibody 1B9 Hybridomas secreting mAb directed against P. falciparum antigens were produced as described (21). Hybridoma supernatant reacting with rhoptries were selected by IFA. One bybridoma, 1B9, reacting with the rhoptry was selectedand cloned by limiting dilution. Spent medium from in vitro-grown cloned hybridoma IB9 cultures were used as the source of mAb. Culture supernatants were concentrated 100times by ultrafiltration (XM50; Amicon Diaflo, Danvers, MA). lmmunoprecipitation 100 /.tl of mAb 1B9 was incubated with 50 I.tl of goat anti-mouse IgG- sepharose 4B (Cappel Laboratories, Malvern, IL) for 1 h at room tempera- ture, and washed three times in Buffer A (1% BSA, 1% NP40, 1 mM EDTA in PBS). Extracts of schizont-iufectederythrocyte labeled with [35S]methi- onine were prepared (21). The beads were incubated with 100 I.tl of [35S]methionine-labeled parasite extracts for l h at room temperature, and then washed, twice in buffer A, once in buffer B (1% BSA, 1% NP40, l mM EDTA, 0.5 M NaCl in PBS), and once in buffer C (1% NP40, l mM EDTA in PBS). The beads were boiled in 100 I.tlof electrophoresis sample buffer (0.1 M Tris-HCI, pH 6.8, 10% glycerol, 2% SDS and 0.001% bromophenol blue) with or without 100 mM dithiothreitol (DTT). The sam- ples were subjected to electrophoresison a 5-15 % SDS-polyacrylamide gel. The gels were treated with Enhance (New England Nuclear, Boston, MA) before drying for exposure to X-Omat AR5 film.

Immunoblotting

Extracts of parasites collected at T-3 h, 21 h, 33 h, 41 h, and 48 h, were separated on 5-15% gradient SDS-PAGE gels under nonreducing condi- tions and transferred to nitrocellulose paper. The transfer was carried out in 20 mM Tris, 0.15 M glycine and 20% methanol at a constant current of 150 mA for 12 h at 4C. Nitrocellulose paper was blocked in 0.1% Tween 20 in Tris-saline buffer (10 mM Tris, 0.9% NaCI pH 7.4) for I h at room temperature and then incubated with mAb 1B9 in 20% fetal bovine serum in Tris-saline buffer. After washing, the antibody bound to the protein was detected by [125I]rabbitanti-mouse IgG (New England Nuclear) (1 x l& cpm/ml). Indirect Immunofluorescence Assay (IFA)1 Thin smears ofP. falciparum cultures collected at T-3,21, 33, 41, and 48 h were acetone-fixed for 10 rain at 4C and incubated with mAh IB9 for 1 h at room temperature in a humidified atmosphere. After three washes in PBS the slides were incubated with FITC-goat anti-mouse Ig (Boehringer-Mann- helm) diluted 1:20 in PBS for 45 min at room temperature, washed in PBS and then in distilled water. Some slides were counterstained with ethidium bromide (10 mg/ml) for 30 s and then rinsed in distilled water. The slides were mounted with 50% glycerol in PBS, and examined by a Nikon Labo- phot microscope.

Immunoelectron Microscopy with 1B9

Parasite pellets containing mature schizonts and reinvading merozoites were fixed in 0.05 % gluteraldehyde in 0.1 M cacodylate pH 7.4 for 15 min at 4C. The fixed cells were centrifuged at 10,000g for 5 min, dehydrated in graded alcohol, and then embedded in LR White resin (Ernest E Fullam, Inc., Latham, NY). The sections were collected on Formvar carbon-coated 1. Abbreviations used in this paper: DOC, deoxycholate; IFA, immunofluo- rescence assay; NP-40, nonidet P40. nickel grids. Grids with sections were blocked in PBS containing 0.5 % BSA for 10 min followed by incubation with concentrated mAb 1B9. The grids were incubated for 3.5 h at room temperature, washed in PBS, and then in- cubated with protein A bound to 5-nm gold particles for 30-60 min at room temperature. Some sections were stained with uranyl acetate. Samples reacted with mAb IB9 before embedding were prepared by washing parasite pellet in 0.1 M cacodylate buffer after fixation, followed by PBS-0.5% BSA, then reacting pellet with 1:10dilution ofmAb 1B9.The samples were incubated for 2 h at room temperature with manual agitation, washed in PBS and incubated with protein A-gold (20 nm) for 30 min. The pellets were washed in PBS, dehydrated in alcohol, and embedded in LR white resin.