The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion

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

The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host

Conformational changes influence functional properties of circumsporozoite protein expressed on the surface of Plasmodium sporozoites

Antimalarial Activity of Allicin, a Biologically Active Compound from Garlic Cloves

The incidence of malaria is increasing, and there is an urgent need to identify new drug targets for both prophylaxis and chemotherapy. Potential new drug targets include Plasmodium proteases that play critical roles in the parasite life cycle. We have previously shown that the major surface protein of Plasmodium sporozoites, the circumsporozoite protein (CSP), is proteolytically processed by a parasite-derived cysteine protease, and this processing event is temporally associated with sporozoite invasion of host cells. E-64, a cysteine protease inhibitor, inhibits CSP processing and prevents invasion of host cells in vitro and in vivo. Here we tested allicin, a cysteine protease inhibitor found in garlic extracts, for its ability to inhibit malaria infection. At low concentrations, allicin was not toxic to either sporozoites or mammalian cells. At these concentrations, allicin inhibited CSP processing and prevented sporozoite invasion of host cells in vitro. In vivo, mice injected with allicin had decreased Plasmodium infections compared to controls. When sporozoites were treated with allicin before injection into mice, malaria infection was completely prevented. We also tested allicin on erythrocytic stages and found that a 4-day regimen of allicin administered either orally or intravenously significantly decreased parasitemias and increased the survival of infected mice by 10 days. Together, these experiments demonstrate that the same cysteine protease inhibitor can target two different life cycle stages in the vertebrate host

Shedding of TRAP by a Rhomboid Protease from the Malaria Sporozoite Surface Is Essential for Gliding Motility and Sporozoite Infectivity

Plasmodium sporozoites, the infective stage of the malaria parasite, move by gliding motility, a unique form of locomotion required for tissue migration and host cell invasion. TRAP, a transmembrane protein with extracellular adhesive domains and a cytoplasmic tail linked to the actomyosin motor, is central to this process. Forward movement is achieved when TRAP, bound to matrix or host cell receptors, is translocated posteriorly. It has been hypothesized that these adhesive interactions must ultimately be disengaged for continuous forward movement to occur. TRAP has a canonical rhomboid-cleavage site within its transmembrane domain and mutations were introduced into this sequence to elucidate the function of TRAP cleavage and determine the nature of the responsible protease. Rhomboid cleavage site mutants were defective in TRAP shedding and displayed slow, staccato motility and reduced infectivity. Moreover, they had a more dramatic reduction in infectivity after intradermal inoculation compared to intravenous inoculation, suggesting that robust gliding is critical for dermal exit. The intermediate phenotype of the rhomboid cleavage site mutants suggested residual, albeit inefficient cleavage by another protease. We therefore generated a mutant in which both the rhomboid-cleavage site and the alternate cleavage site were altered. This mutant was non-motile and non-infectious, demonstrating that TRAP removal from the sporozoite surface functions to break adhesive connections between the parasite and extracellular matrix or host cell receptors, which in turn is essential for motility and invasion

The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct

Disruption of the rhomboid motif impairs TRAP cleavage.

<p>(A) Primary structure of TRAP expressed in TRAP-VAL and TRAP-FFF mutants, with the point mutations introduced to disrupt the putative rhomboid substrate motif indicated above each transmembrane domain. (B) Western blot analysis of recombinant control TRAP-rWT (rWT) and rhomboid cleavage mutant salivary gland sporozoites TRAP-VAL and TRAP-FFF (VAL and FFF) probed with TRAP anti-repeat antisera. As a loading control the bottom half of the membrane was probed with mAb 3D11 which recognizes the repeat region of CSP. (C) Pulse-chase metabolic labeling of TRAP-rWT and rhomboid cleavage site mutants. Salivary gland sporozoites were metabolically labeled for 1 hr and either placed on ice for 4 hrs (Time = 0) or chased for 4 hrs (Time = 4). Sporozoites were then centrifuged and TRAP was immunoprecipitated from either the pellet (P) or supernatant (S) using anti-repeat antisera and analyzed by SDS-PAGE and autoradiography. Molecular weight markers shown on left of top panel. Top panel: 6 day exposure. Bottom panel: 14 day exposure, arrows show location of processed VAL and FFF mutant TRAP. (D) Immunofluorescence analysis of surface TRAP staining in TRAP-rWT and rhomboid cleavage site mutants. Shown are representative fluorescence and phase contrast images of the TRAP staining pattern after fixation with paraformaldehyde. Microscope and camera settings were identical for all photographs. (E) Box plot of fluorescence intensity of TRAP surface staining in TRAP-rWT and rhomboid cleavage site mutants. Unpermeabilized sporozoites were stained with anti-TRAP repeat antisera and intensity of staining was measured using NIS Elements software. Identical camera and microscope settings were used for all measurements. Boxes contain 50% of the data around its median (black line in box). Whiskers show the range of data within the 10^th and 90^th percentiles and outliers are shown individually. Results are pooled from 2 to 4 independent experiments. There was a statistically significant difference in staining intensity between TRAP-rWT and TRAP-VAL sporozoites (p<.0001) and between TRAP-rWT and TRAP-FFF sporozoites (p<.0001).</p

In vivo infectivity of TRAP mutant sporozoites as determined by prepatent period.

<p>In vivo infectivity of TRAP mutant sporozoites as determined by prepatent period.</p

TRAP-DMut sporozoites are severely impaired in gliding motility and host cell invasion.

<p>(A) Gliding motility of TRAP-JMD and TRAP-DMut sporozoites. Salivary gland sporozoites were incubated on slides for 1 hr and trails were visualized and counted. The percentage of sporozoites with and without trails is shown in the pie charts. For those sporozoites associated with trails, the number of circles produced by each sporozoite was counted and shown is their distribution for each parasite line. Over 100 sporozoites per well were counted and shown are the means of triplicate wells ± SD. (B) Representative images of the types of trails produced by each mutant. (C) Hepatocyte invasion. Salivary gland sporozoites were incubated with Hepa 1–6 cells for 1 hr, fixed and stained with a double staining assay that distinguishes extracellular and intracellular sporozoites. Percent invasion was determined and shown are the means ± SD of duplicate wells. All experiments were performed at least twice and shown is a representative experiment.</p