The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion

Abstract

The circumsporozoite protein (CSP) is the major surface protein of Plasmodium sporozoites, the infective stage of malaria. Although CSP has been extensively studied as a malaria vaccine candidate, little is known about its structure. Here, we show that CSP is proteolytically cleaved by a papain family cysteine protease of parasite origin. Our data suggest that the highly conserved region I, found just before the repeat region, contains the cleavage site. Cleavage occurs on the sporozoite surface when parasites contact target cells. Inhibitors of CSP processing inhibit cell invasion in vitro, and treatment of mice with E-64, a highly specific cysteine protease inhibitor, completely inhibits sporozoite infectivity in vivo.

Figure 1.

CSP is proteolytically cleaved by a cysteine protease. (A) CS proteins from all species of Plasmodium have several conserved features: a central repeat region (gray box) and two conserved regions (black boxes, region I and the cell-adhesive TSR). The first 20 residues of CSP have the features of a eukaryotic signal sequence (reference 28) and the COOH- terminal sequence contains a canonical GPI-anchor addition site. Bars show the location of peptides used for the generation of antisera. (B) Western blot of a P. berghei sporozoite lysate probed with polyclonal antisera generated against the NH₂- and COOH-terminal peptides shown in A; mAb 3D11 was used as a control. The membrane was cut where indicated so that it could be probed with the three different antisera, and the membrane strips were developed together. (C) P. berghei sporozoites were metabolically labeled and kept on ice (lane 1) or chased for 2 h in the absence (lane 2) or presence of the indicated protease inhibitors (lanes 3–11). CSP was immunoprecipitated from lysates of sporozoites and analyzed by SDS-PAGE and autoradiography. The following inhibitors were used: 10 μM E-64, 1 mM PMSF, 0.3 μM aprotinin, 100 μM 3,4 DCI, 75 μM leupeptin, 100 μM TLCK, 1 μM pepstatin, 1 mM 1,10 phenanthroline, and 5 mM EDTA. All samples are from the same experiment but were run on two gels and grouped as indicated. (D) P. falciparum sporozoites were metabolically labeled and kept on ice (lane 1) or chased with cold medium for 90 min in the absence (lane 2) or presence of E-64 (lane 3). Samples were processed as outlined before.

Figure 2.

The conserved region I likely contains the proteolytic cleavage site. (A) The epitopes recognized by the NH₂-terminal antiserum were determined by ELISA by testing its reactivity to overlapping peptides encompassing the NH₂-terminal third of CSP. Pep 1, GYGQNKSIQAQRNLNE; Pep 2, RNLNELCYNEGNDNKL; Pep 3, NDNKLYHVLNSKNGKI; Pep 4, KNGKIYIRNTVNRLLA; Pep 5, NRLLADAPEGKKNEKK; Pep 6, KNEKKNKIERNNKLK; and N-term, full-length NH₂-terminal peptide. (B–D) Proteolytic cleavage of hybrid CSP. (B) Structure of CSP from Pf/Pb sporozoites. The portion of P. berghei CSP that has been replaced with the orthologous sequence of P. falciparum CSP is shown in orange and includes region I, which is shown in black. (C) Western blot of Pf/Pb sporozoites probed with mAb 2A10, directed against the P. falciparum CSP repeat region. (D) Pf/Pb sporozoites were metabolically labeled and kept on ice (lane 1) or chased with cold medium for 4 h, in the absence or presence of E-64. Samples were processed as outlined in Fig. 1.

Figure 3.

CSP is processed extracellularly by a parasite protease. (A) Staining of live sporozoites with the NH₂-terminal antiserum. Phase contrast and fluorescence views are shown. Bar, 10 μm. (B and C) P. berghei sporozoites expressing GFP were biotinylated, lysed, and CSP (B) and GFP (C) were immunoprecipitated. A Western blot of the immunoprecipitated material was probed with streptavidin (B and C, lane 1), mAb 3D11 (B, lane 2), or polyclonal antisera to GFP (C, lane 2). The membrane was cut where indicated so that it could be probed with individual antisera and, the membrane strips were developed together. (D) Metabolically labeled P. berghei sporozoites were kept on ice (time = 0) or chased for 1 h, and incubated in medium containing pronase (+) or pronase plus pronase inhibitor cocktail (−). CSP was immunoprecipitated from sporozoite lysates and analyzed by SDS-PAGE and autoradiography. (E) P. berghei sporozoites were dissected and purified in the absence (−) or presence (+) of E-64, washed, metabolically labeled, and either kept on ice (time = 0) or chased for 2 h. Sporozoites were lysed, and CSP was immunoprecipitated and analyzed by SDS-PAGE and autoradiography.

Figure 4.

E-64 inhibits sporozoite infectivity in vitro and in vivo but not sporozoite migration through cells. (A) The effect of protease inhibitors on cell invasion by Plasmodium sporozoites. P. berghei (gray bars), P. yoelii (white bar), or P. falciparum (black bar) sporozoites were preincubated with the indicated protease inhibitors before being added to cells. A control (diagonally striped bar) was performed in which the target cells were preincubated with E-64, the medium was removed, and untreated P. berghei sporozoites were added. The following inhibitors were used: 10 μM E-64, 1 mM PMSF, 75 μM leupeptin, 0.3 μM aprotinin, 100 μM 3,4 DCI, and 1 μM pepstatin. Each point was performed in triplicate, 50 fields/well were counted, and the means ± SD are shown. Inhibition of invasion was calculated based on the invasion rate for sporozoites pretreated with buffer alone, which was 54% for P. berghei, 26% for P. yoelii, and 52% for P. falciparum. (B) Shown are the numbers of extracellular sporozoites when sporozoites are preincubated in the absence (gray bars) or presence (white bars) of E-64. Data are from the invasion assay shown. (C) Shown are the numbers of dextran-positive cells when sporozoites were preincubated ± E-64 and added to cells in the presence of rhodamine–dextran. Each point was performed in triplicate, 50 fields/coverslip were counted, and shown are the means ± SD. (D) E-64 inhibits sporozoite infectivity in vivo. Mice were injected with E-64 or buffer alone before inoculation of P. yoelii sporozoites. 40 h later, the mice were killed, total liver RNA was extracted, and malaria infection was measured by quantitative PCR. Infection is expressed as the number of copies of P. yoelii 18S rRNA. Results from two experiments are shown. There were six mice per group per experiment.