INTRODUCTION

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Plasmodium falciparum, the causative agent of the most severe form of human malaria, is an obligate intracellular apicomplexan parasite. The life cycle of the organism includes a number of specialized invasive (zoite) stages. In the vertebrate host, replication of the parasite in circulating erythrocytes is initiated when the cells are invaded by merozoites. The parasite replicates asexually within the infected erythrocyte to produce a number of progeny merozoites. Upon rupture of the host cell, these are released to invade fresh erythrocytes and perpetuate the blood stage cycle. Erythrocyte invasion by the malaria merozoite has been the subject of intensive study, since intervention strategies that prevent invasion would effectively block both replication of the parasite and the associated clinical disease.

Electron microscopic studies have shown that erythrocyte invasion by the malaria merozoite takes place in a number of discrete stages. Initial reversible attachment of the parasite to the red cell surface is rapidly followed by reorientation, the formation of an irreversible junction between the apical prominence of the merozoite and the host cell surface, and finally entry of the parasite into the cell by a mechanism resembling a form of induced endocytosis (1-4). The process is facilitated by the controlled release of the contents of three types of secretory organelles, called rhoptries, micronemes, and dense granules, situated at or toward the apical domain of the merozoite (2, 5, 6). There is extensive evidence indicating an essential role for parasite-derived proteases in invasion. Invasion by P. falciparum merozoites is blocked in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF)¹ (7), and invasion by merozoites of a number of Plasmodium species is prevented by chymostatin (8-13). The inhibitory effect of chymostatin on invasion can be reversed by pretreatment of target erythrocytes with chymotrypsin (10), suggesting that the chymostatin-sensitive step in invasion involves an essential, parasite-induced proteolytic modification of the red cell surface (10-12). A glycosylphosphatidylinositol (GPI)-anchored malarial serine protease activity has been described that may be involved in this modification (14). Treatment of isolated, invasive merozoites of the simian malaria P. knowlesi with N-tosyl phenylalanylchloromethyl ketone or N-tosyl lysylchloromethyl ketone prevents primary attachment of the parasites to host cells, whereas chymostatin blocks a later stage in the invasion pathway, indicating that more than one distinct protease activity may be involved (15). Consistent with this, a P. falciparum serine protease activity that mediates an essential processing and shedding of a major merozoite surface protein (merozoite surface protein-1; MSP-1) at invasion is highly sensitive to inhibition by PMSF but not by chymostatin (16, 17). Parasite proteases involved in invasion are attractive potential targets for new rational approaches to antimalarial chemotherapy.

Here we report the identification of a novel, single copy P. falciparum gene (denoted pfsub-1) encoding a member of the subtilisin-like serine protease superfamily (subtilases). The primary gene product is expressed in the latter stages of intracellular merozoite maturation, and is post-translationally modified during secretory transport in a manner consistent with it being processed to form a mature, enzymatically active product. The putative mature protease is concentrated in a subset of dense granules within the apical domain of free merozoites and then is released in a soluble form during erythrocyte invasion, suggesting that it may play a role in invasion. This is the first molecular characterization of a putative apicomplexan serine protease.

	EXPERIMENTAL PROCEDURES

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Parasites, Materials, and Nucleic Acid Manipulations-- Blood stage cultures of the T9/96 and FCB-1 lines of P. falciparum were maintained in vitro in human A+ erythrocytes and synchronized when required, as described previously (18, 19). Protease inhibitors and fluorogenic or chromogenic peptide and protein substrates were obtained from Sigma and Boehringer Mannheim. Restriction endonucleases and other DNA modifying enzymes were obtained from Boehringer, Amersham Pharmacia Biotech, and New England Biolabs. The majority of the methods used for DNA manipulations were based on those of Sambrook et al. (20). Preparation of malaria parasite genomic DNA and analysis by Southern blotting was by standard procedures (20, 21). DNA probes for Southern analysis and cDNA library screening were radiolabeled with [-³²P]dATP (220 TBq mmol¹, Amersham Pharmacia Biotech) by the random primer method (Prime-It^TM II random primer labeling kit, Stratagene). DNase-treated total parasite RNA from asynchronous blood stage cultures was prepared from saponin-treated parasites (18) using a S.N.A.P.^TM total RNA isolation kit (Invitrogen), following the manufacturers' recommendations. Oligo(dT)-primed single-stranded cDNA synthesis from total parasite RNA was performed using a cDNA Cycle^® kit (Invitrogen) as directed by the manufacturers; "mock cDNA" preparations were prepared by subjecting similar RNA samples to identical processing except that no reverse transcriptase was present. All DNA sequencing was by the dideoxynucleotide (Sequenase) method using custom synthesized primers. Homology searches and alignments were performed with the Basic Local Alignment Search Tool (BLAST) program of the National Center for Biotechnology Information, National Institutes for Health (22), using the European Bioinformatics Institute server.²

Polymerase Chain Reaction (PCR)-- Most PCR was carried out using AmpliTaq^® DNA polymerase (Perkin-Elmer). Degenerate oligonucleotide primers for amplification by PCR and reverse transcription-PCR of malarial subtilase genes were SUB-1 (sense; 5'-CAYGGIACICAYGTIGCIGG-3') and SUB-2 (antisense; 5'-CCIGCIACRTGIGGIGTIGCCAT-3'), based on the amino acid sequences HGTHVAG and MATPHVAG, respectively. Primers for amplification by inverse PCR (23) of genomic sequences flanking the T9/96 pfsub-1 gene were SUB-SEQ11 (sense; 5'-TGCATGGGCAGGATATGCAG-3') and SUB-SEQ12 (antisense; 5'-ATGGGAACCTCAGTATCCGG-3'). For inverse PCR, 5 µg of T9/96 genomic DNA in a total volume of 20 µl was digested for 5 h with 20 units of Sau3A. The restricted DNA was ethanol-precipitated, redissolved at a concentration of 2.5 µg ml¹, and ligated overnight with T4 DNA ligase. The circularized DNA was then used as template for PCR amplification with SUB-SEQ11 and SUB-SEQ12. A major, ~1.4-kilobase pair PCR product was gel-purified and cloned into the T-cloning vector pMOSBlue (Amersham Pharmacia Biotech) to create plasmid PM494-B. This and all other cloned AmpliTaq^®-generated PCR products were maintained in the MOSBlue Escherichia coli strain (Amersham Pharmacia Biotech). PCR amplification of regions of the pfsub-1 gene for subcloning into expression vectors was carried out with Pfu polymerase (Stratagene). Primers used for this purpose were SUB-EX1 (sense; 5'-GAGCTCGGATCCGAAGGAAGTAAGGTCTGAAGAA-3'), SUB-EX2 (sense; 5'-GAGCTCGGATCCGAGTAGACCAGGTAAATATCATTTC-3'), SUB-EX5 (sense; 5'-GAGCTCGAATTCAGTAGACCAGGTAAATATCATTTC-3') and SUB-EX3 (antisense; 5'-AGCCTCGAATTCCTAGTTAATGCAAATATCTAC-3'). DNA fragments amplified with these primers were verified by sequencing on both strands.

Production and Screening of a P. falciparum Blood Stage cDNA Library-- Poly(A⁺) RNA was isolated from total T9/96 parasite RNA using oligo(dT)_12-18-cellulose (Amersham Pharmacia Biotech) column chromatography, using two cycles of binding and elution according to the manufacturer's instructions. Two µg of poly(A+) RNA was reverse transcribed into cDNA using 0.5 µg of a random unidirectional XhoI linker primer (Stratagene) according to the method of Gubler and Hoffman (24). First and second strand synthesis was monitored by incorporation of [-³²P]dCTP (220 Tbq mmol¹; Amersham Pharmacia Biotech) and subsequent agarose-formaldehyde electrophoresis, followed by autoradiography (20). The cDNA was purified by spun column chromatography (Sephacryl S-300, Amersham Pharmacia Biotech), ligated to EcoRI/NotI cohesive end adaptors (Amersham Pharmacia Biotech), and treated with T4 polynucleotide kinase according to standard procedures (20). Following phenol extraction and ethanol precipitation, the cDNA was digested with 100 units of XhoI and purified using Sepharose CL-4B spun column chromatography (SizeSep 400^TM, Amersham Pharmacia Biotech). The cDNA was ligated into EcoRI-XhoI-digested -ZAP II vector (Stratagene) and then packaged (Gigapack II Gold, Stratagene), and the packaged phage was used to infect E. coli SURE^TM host cells (Stratagene). Screening, selection, and in vivo rescue of pBluescript^® phagemid from plaque-purified phage by co-infection with Exassist^TM helper phage was according to the manufacturer's instructions (Stratagene).

RNase Protection Assay-- Recombinant pMOSBlue plasmid PM494-B, containing the ~1.4-kilobase pair DNA fragment obtained by inverse PCR amplification from genomic T9/96 DNA with oligonucleotides SUB-SEQ11 and SUB-SEQ12, was linearized with AvrII and used as a template for production of an [-³²P]UTP-radiolabeled, 422-nucleotide antisense RNA probe using the T7 promoter region of the plasmid (20). Protection of this probe from RNase activity following hybridization to blood stage parasite mRNA was assessed using an RPA II^TM Ribonuclease Protection assay kit (Ambion), following the manufacturer's recommendations. Briefly, aliquots of 2 × 10⁴ cpm of gel-purified probe were hybridized overnight at 44 °C with varying amounts (up to 12 µg) of DNase-treated total parasite or yeast control RNA. The reactions were then treated with an empirically determined dilution of a mixture of RNase A and RNase T1 for 30 min at 30 °C, followed by RNase inactivation and precipitation of the remaining RNA. Samples were subjected to electrophoresis on 6% acrylamide gels in the presence of 8 M urea and analyzed by autoradiography. Markers for electrophoresis were end-radiolabeled HinfI-digested dephosphorylated ØX174 DNA markers (Promega).

Expression, Metabolic Radiolabeling, and Purification of PfSUB-1 Recombinant Fusion Proteins and Antibody Production-- DNA fragments amplified by PCR with primers SUB-EX1 and SUB-EX3, SUB-EX2 and SUB-EX3, or SUB-EX5 and SUB-EX3, were digested with BamHI and/or EcoRI and then ligated into either BamHI- and EcoRI-digested pTrcHisB (Invitrogen), or EcoRI-digested pGEX-1T (Amersham Pharmacia Biotech). E. coli strain DH5 (Stratagene) was transformed to ampicillin resistance, and recombinant clones were selected. In each case, the identity and orientation of the insert in recombinant clones was confirmed by complete sequence analysis on both strands.

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P. falciparum Radiolabeling and Merozoite Purification-- Mature schizonts from highly synchronous P. falciparum cultures were enriched to a parasitemia of 85-90%, and naturally released merozoites were purified by filtration through 3-µm and 1.2-µm pore size acrylic membrane filters as described previously (19). Some merozoites were resuspended in fetal calf serum and used to prepare thin films, which were air-dried and stored desiccated at 70 °C. Merozoite preparations were consistently free of schizont contamination, as determined by microscopic analysis of Giemsa-stained samples. For labeling with tritiated diisopropyl fluorophosphate ([³H]DFP), 10 µl (10 µCi) of [³H]DFP (NEN Life Science Products) in propylene glycol was added with mixing to approximately 5 × 10⁹ merozoites in 200 µl of PBS. Following incubation for 45 min at 37 °C, the merozoites were pelleted, washed three times in PBS, and analyzed directly by SDS-PAGE or by immunoprecipitation with anti-PfSUB-1_m antibodies.

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Pulse-Chase Experiments-- Mature T9/96 schizonts enriched from highly synchronous cultures were washed twice in warm methionine/cysteine-free medium, then cultured for 30 min in the same medium. The culture was then pelleted, resuspended to a hematocrit of 20% in 900 µl of fresh warm methionine/cysteine-free medium, and labeling was initiated by adding [³⁵S]methionine/cysteine to 300 µCi ml¹. After a 9-min incubation at 37 °C, the chase was initiated by adding 100 µl of a nonradioactive methionine and cysteine stock solution in RPMI to a final concentration of 3 and 1 mM, respectively. The culture was then immediately pelleted and resuspended in fresh complete medium at 37 °C supplemented with methionine and cysteine to 3 and 1 mM, respectively, and two 1-ml "zero time" samples were removed to chilled 1.5-ml Eppendorf tubes containing 10 µl of 10% (w/v) sodium azide. The sampled parasites were rapidly pelleted by centrifugation, washed once in ice-cold PBS containing 0.1% sodium azide, and then snap-frozen in a dry ice-ethanol bath. Samples taken from the bulk culture at 5, 15, 30, and 60 min following commencement of the chase were similarly processed.

Western Blotting, Immunoprecipitation, and Peptide Mapping-- Analysis of proteins by SDS-PAGE and Western blotting was as described previously (19, 27) except that in most cases antigen detection following incubation of blots with a horseradish peroxidase-conjugated second antibody was by ECL using a commercially obtained substrate (SuperSignal^® Substrate, Pierce). For immunoprecipitation analysis, radiolabeled parasite or bacterial pellets were solubilized into five volumes of a denaturing solubilization buffer (1% (w/w) SDS in 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM PMSF) with sonication. Samples were immediately boiled for 5 min and clarified by centrifugation at 12,000 × g for 30 min, and then the clarified supernatants were diluted 10-fold into immunoprecipitation buffer (1% (v/v) Triton X-100 in 50 mM Tris-HCl, pH 8.2, 5 mM EGTA, 5 mM EDTA, 150 mM NaCl, supplemented with leupeptin, antipain, pepstatin A, and aprotinin, all at 10 µg ml¹, and N-tosyl lysylchloromethyl ketone at 10 µM). Aliquots of the extract were then incubated overnight on ice with 2-8 µl of anti-PfSUB-1_m antiserum. Immune complexes were bound to protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech) and then washed twice with wash buffer I (0.5% (v/v) Triton X-100 in 50 mM Tris-HCl, pH 8.2, 5 mM EDTA, 1 mg ml¹ bovine serum albumin, 0.5 M NaCl), and four times in wash buffer II (wash buffer I minus bovine serum albumin or NaCl), before elution into SDS sample buffer. Immunoprecipitates were subjected to SDS-PAGE in the presence or absence of 100 mM dithiothreitol as a reducing agent, and radiolabeled proteins were detected by autoradiography or fluorography.

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Indirect Immunofluorescence (IFA), Confocal Microscopy, and Immunoelectron Microscopy-- Analysis of acetone-fixed thin smears of malaria parasite cultures or purified merozoites by IFA and confocal microscopy was essentially as described previously (29). Briefly, samples were fixed in cold acetone and then probed with a primary mouse anti-PfSUB-1_m serum or a mouse antiserum against Schistosoma japonicum glutathione S-transferase (mouse anti-GST), typically at a dilution of 1:100, followed by a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Seralab) or a Texas Red-conjugated goat anti-mouse IgG as second antibody, also diluted 1:100. Evans blue (0.01% (w/v) in PBS) was used as a counterstain, and 4,6-diamidino-2-phenylindole was used at 1 µg ml¹ in PBS as a nuclear stain. Direct conjugation of FITC to MSP-1-specific monoclonal antibody 2F10 (30) was as described previously (29). Slides were mounted in Citifluor (Citifluor UKC Chemical Laboratories, Canterbury, UK) and examined using a Zeiss III Photomicroscope fitted with an epifluorescence III RS condenser. For confocal analysis, samples were examined on a Bio-Rad MRC 600 confocal microscope using an argon-krypton laser for two-channel recording. Images were taken at 400-nm intervals along the z axis. For immunoelectron microscopy, preparations of mature segmented schizonts containing some free merozoites were fixed for 30 min at 4 °C with 1% formaldehyde, 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Fixed samples were washed, dehydrated, and embedded in LR White resin as described by Aikawa and Atkinson (31). Thin sections were blocked in PBS containing 5% (w/v) nonfat milk powder and 0.01% (v/v) Tween 20. Grids were then incubated for 2 h at room temperature with primary antibodies (mouse anti-PfSUB-1_m serum, mouse anti-GST serum, or normal mouse serum) diluted in PBS containing 1% (w/v) bovine serum albumin and 0.01% (v/v) Tween 20 (PBT). After washing, grids were incubated for 1.5 h in 15-nm gold-conjugated goat anti-mouse IgG (Amersham Pharmacia Biotech) diluted 1:20 in PBT, rinsed with PBT, and fixed with glutaraldehyde to stabilize the gold particles. Samples were finally stained with uranyl acetate and lead citrate and examined using a Zeiss CEM902 electron microscope.

	RESULTS

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PCR Amplification and Molecular Cloning of the pfsub-1 Gene-- Oligonucleotide primers were designed based on conserved motifs flanking the catalytic histidine and serine residues of the subtilisin-like class of serine proteases. PCR primers SUB-1 (sense), and SUB-2 (antisense) were based on sequences His¹⁷⁰ to Gly¹⁷⁶ and Met³²⁸ to Gly³³⁵ of the Bacillus subtilis subtilisin E gene product (Genbank^TM/EMBL accession no. K01988). Primers were completely redundant, no account being taken of the bias toward A + T-rich codon usage in the P. falciparum genome. Inosine was used in positions of more than 2-fold degeneracy in order to minimize overall primer degeneracy and maximize promiscuity. Reverse primer SUB-2 was designed to take advantage of the nonredundant methionine codon at the 3'-end of the oligonucleotide. In PCR reactions using as template single-stranded cDNA prepared from DNA-free total blood stage parasite RNA, a single DNA fragment of approximately 560 bp was amplified. No product was obtained from mock cDNA preparations produced in the absence of reverse transcriptase. A product of identical size was also obtained by PCR amplification from T9/96 or FCB-1 genomic DNA (not shown). Products from all three PCR reactions were cloned into the T-vector pMOSBlue, and 2-5 representative clones from each transformation were sequenced on both strands. All of the cloned sequences were identical. The cloned DNA was 561 bp long and contained a single open reading frame (ORF); a comparison of the deduced amino acid sequence with the protein data bases indicated strong homology with a number of bacterial subtilisins. The 561-bp fragment was then used to probe a T9/96 -ZAP asexual blood stage cDNA library, and a total of four strongly hybridizing clones were obtained (Fig. 1, top). Complete sequence analysis of the cDNA clones showed that they represented overlapping fragments of a single contiguous sequence containing an uninterrupted ORF of 2073 bp, encoding a protein of 690 amino acids with an estimated molecular mass of 77,874 daltons (Fig. 1, bottom). Consistent with the A + T-rich P. falciparum genome, the A + T content of the ORF is 72%.

View larger version (107K): [in this window] [in a new window]   Fig. 1.   Molecular cloning of the pfsub-1 gene. Top, cloned pfsub-1 PCR products and cDNAs. The schematic depicts the relative positions and sizes of the 561-bp PCR product (PM443-7) amplified with degenerate oligonucleotide primers SUB-1 and SUB-2; the four partial cDNA clones (8-1, 9-1, 7-2, and 10-1) obtained by using PM443-7 to probe a P. falciparum cDNA library; the composite cDNA clone (PM481-1) constructed from partial cDNA clones 8-1 and 10-1; and the pfsub-1 ORF (shaded box) relative to flanking genomic sequence obtained by inverse PCR amplification of a 3183-bp Sau3A genomic fragment. The positions and orientations of primers SUB-SEQ11 and SUB-SEQ12 used for the inverse PCR are also shown (small arrows). Restriction endonuclease sites shown are as follows: Sau3A (S); AvrII (A); HindIII (H). Bottom, nucleotide and deduced amino acid sequence of the pfsub-1 cDNA (clone PM481-1). Noncoding and coding regions of the nucleotide sequence are shown in lowercase and uppercase letters, respectively. The TAA translational stop codon is marked with an asterisk. The putative amino-terminal signal peptide and the catalytic triad residues Asp374, His430, and Ser608 are shown in boldface type and underlined. The putative C-terminal catalytic domain (PfSUB-1m) is shown shaded.

The pfsub-1 Gene Encodes a Subtilase-- The deduced pfsub-1 gene product (designated PfSUB-1) clearly belongs to the subtilisin-like (subtilase, S8) protease clan or superfamily as defined by Rawlings and Barrett (32). The C-terminal 361 residues of the deduced sequence (Ser³³⁰-His⁶⁹⁰) was aligned with the catalytic domains (as defined by Siezen et al.; Refs. 33 and 34) of a number of bacterial and eukaryotic subtilases (not shown). A number of points can be made concerning this comparison. First, within the putative catalytic core domain of the protein (see below), the PfSUB-1 sequence exhibits a number of residues that are highly or completely conserved among all known subtilases (33, 34); these include Asp³⁷⁴, His⁴³⁰, and Ser⁶⁰⁸ (the catalytic site residues); the oxyanion hole residue Asn⁵²²; and residues Gly⁶⁰⁶, Thr⁶⁰⁷, and Pro⁶¹². Furthermore, PfSUB-1 clearly does not belong to the proteinase K or lantibiotic peptidase families (subtilase families C and D; Ref. 34), both of which lack the 6-residue-long Ca1 Ca²⁺-binding loop sequence (Asn⁴⁴³-Val⁴⁴⁸ in the deduced PfSUB-1 sequence) just C-terminal to the active site histidine residue. Additionally, PfSUB-1 clearly does not belong to the kexin family (family E; Ref. 34) of prohormone-processing proteases of yeasts and higher eukaryotes. One characteristic of this group, for example, is the DDG motif present at the active site Asp residue; PfSUB-1 possesses the more common DSG motif at this position (Asp³⁷⁴-Gly³⁷⁶ of the PfSUB-1 sequence). It is thus unlikely that PfSUB-1, if expressed in the parasite as a catalytically active protease, has a dibasic cleavage specificity (35).

The pfsub-1 Gene Is Single Copy, Contains No Introns, and Is Transcribed in Blood Stages of the Parasite-- Southern blot analysis of T9/96 genomic DNA was performed using as a probe the cloned 561-bp PCR product. Fig. 2A shows that following digestion of genomic DNA with any of six different restriction enzymes (none of which cleaves within the fragment used as a probe), the probe hybridized strongly to only one DNA fragment in each case. However, even under highly stringent washing conditions, weak hybridization was also reproducibly evident with at least one other DNA fragment in some digests. This result was interpreted as indicating that pfsub-1 is a single copy gene, but that the genome also contains at least one other locus with significant homology to the probe. The 561-bp pfsub-1 fragment was also used to probe blots of P. falciparum chromosomes separated by pulse-field gel electrophoresis (a kind gift of D. Williamson and P. Moore, Division of Parasitology, National Institute for Medical Research); the probe hybridized strongly to a single chromosome, further supporting the existence of a single genomic copy of the pfsub-1 gene (not shown).

View larger version (53K): [in this window] [in a new window]   Fig. 2.   The pfsub-1 gene is single copy and is transcribed in blood stage parasites. A, Southern hybridization analysis. P. falciparum (clone T9/96) genomic DNA was digested with restriction enzymes EcoRI (lane 1), BglII (lane 2), SpeI (lane 3), Sau3A (lane 4), ClaI (lane 5), and DdeI (lane 6), and then 10 µg of each digest was electrophoresed on a 0.7% agarose gel and transferred to nylon membrane. The blot was probed with the cloned 561-bp fragment of the gene (PM443-7; see Fig. 1 top). Following hybridization for 18 h in 6× SSC at 65 °C, the blot was washed twice for 15 min each in 2× SSC and finally for 30 min in 0.2× SSC at 65 °C prior to analysis by autoradiography. B, RNase protection analysis. A radiolabeled 422-nucleotide antisense RNA probe was produced using T7 RNA polymerase and the bacteriophage T7 promoter region of AvrII-linearized plasmid PM494-B. Equal amounts (2 × 104 cpm) of the probe were hybridized with varying amounts of DNase I-treated total parasite RNA (sample RNA) isolated from asynchronous asexual blood stage parasites (lanes 4-8 correspond, respectively, to hybridization with 12, 6, 3, 1.5, and 0.75 µg of sample RNA) before the addition of an empirically determined amount of RNase. A major protected RNA fragment (arrow) of about 366 nucleotides was observed, the amount of protection being dependent of the amount of sample RNA present in each hybridization reaction. The bands of approximately 170, 175, and 240 nucleotides in lanes 4-8 probably represent partially protected RNA species derived from internal nicking of the major 366-nucleotide protected fragment. This is a frequently observed feature of RNase protection experiments, particularly in the analysis of AU-rich sequences due to "breathing" (local denaturation) of the hybrid formed between the RNA probe and the target mRNA. No protection from RNase digestion was observed following hybridization of probe with 12 µg of torulla yeast RNA (lane 3). Lanes 1 and 2 were loaded with 1 × 103 cpm and 2 × 102 cpm, respectively, of a sample of probe hybridized with 12 µg of yeast RNA only, without subsequent RNase digestion. No protection was observed when sample parasite RNA was RNase-treated prior to incubation with the probe (not shown).

Expression of PfSUB-1 in E. coli and Production of Monospecific Antibodies-- pfsub-1 sequences encoding Lys²⁶ to the final His⁶⁹⁰ residue (the putative proprotease sequence, denoted PfSUB-1_pm), or Ser³³⁰ to His⁶⁹⁰ (the predicted mature protease domain, PfSUB-1_m), were amplified by PCR and cloned into the expression vector pTrcHisB to produce constructs encoding fusion proteins with an N-terminal hexahistidine domain, separated from the insert sequence by an enterokinase cleavage site (Fig. 3). The region encompassing the cleavage site also contains an 8-amino acid residue epitope recognized by a commercially available monoclonal antibody (Anti-Xpress^TM). When clones PM500-6E and PM500-7A, containing the proprotease and mature protease sequences, respectively, of PfSUB-1, were assessed for fusion protein expression following induction by Western blot, using the Anti-Xpress^TM monoclonal antibody, fusion proteins of the expected size were detected (not shown, but see Fig. 4); the calculated size of the fusion partner sequence encoded by these constructs is 3659 daltons, so the predicted M_r of the proprotease PfSUB-1 fusion protein (denoted His₆-PfSUB-1_pm) is 78,707, and that of the mature PfSUB-1 fusion protein (denoted His₆-PfSUB-1_m; Fig. 3) is 44,073. The latter fusion protein was partially purified by metal chelate chromatography for use in experiments described below. DNA encoding Ser³³⁰ to His⁶⁹⁰ of pfsub-1 was similarly amplified and cloned into the EcoRI site of pGEX-1T to produce clone PM511-3A. The resulting fusion protein, denoted GST-PfSUB-1_m (Fig. 3), was purified to homogeneity and then used to raise anti-PfSUB-1_m antisera in mice; the sera were used to identify and characterize the parasite pfsub-1 gene product.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Primary structure of PfSUB-1 and recombinant fusion proteins expressed in E. coli. Shown is a schematic of the deduced primary sequence of the T9/96 P. falciparum PfSUB-1 subtilase. The protein consists of 690 amino acid residues, with a putative signal peptide sequence (Met1-Gly25), a putative propeptide sequence (Lys26-Arg329), and a putative catalytic, or mature domain within the C-terminal part of the protein (Ser330-His690). The putative PfSUB-1 mature protease domain (PfSUB-1m) was expressed in E. coli as a C-terminal fusion protein in two forms: 1) with a 33-residue-long polypeptide containing a hexahistidine and XpressTM monoclonal antibody epitope tag sequence (denoted His6-PfSUB-1m; middle) and 2) with the S. japonicum GST (denoted GST-PfSUB-1m; bottom). The latter construct was used to raise polyclonal antibodies reactive with the C-terminal domain of PfSUB-1.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Identification of PfSUB-1 in blood stage parasites. A, T9/96 schizonts were biosynthetically radiolabeled with [35S]methionine/cysteine and then detergent-solubilized and analyzed by immunoprecipitation using a mouse anti-PfSUB-1m serum (lane 2), a mouse anti-GST serum (lane 3), or normal mouse serum (lane 4). Immunoprecipitates were subjected to SDS-PAGE under reducing conditions on a 10% gel. Lane 1 contains a sample of the total parasite extract, and the positions of molecular weight markers are shown. The positions of the two proteins (denoted p54 and p47) specifically precipitated by the anti-PfSUB-1m antibodies are also indicated. B, PfSUB-1 is expressed in merozoites, and its migration on SDS-PAGE is reduction-sensitive. Lanes 1-5, proteins immunoprecipitated from extracts of metabolically radiolabeled T9/96 schizonts were subjected to SDS-PAGE on a 10% gel in the presence (+) or absence () of 100 mM dithiothreitol as a reducing agent. Lanes 6-11, purified, naturally released T9/96 merozoites (lanes 6-10) or a sample of fusion protein His6-PfSUB-1m (lane 11) was SDS-solubilized and subjected directly to SDS-PAGE on a 10% gel in the presence (+) or absence () of dithiothreitol. The proteins were transferred electrophoretically to nitrocellulose, and the blot was probed with the anti-PfSUB-1m antibodies. The migration of both p54 and p47 is reduction-sensitive, indicating the presence of intramolecular disulfide bonds. In addition, the His6-PfSUB-1m fusion protein was strongly recognized by the anti-PfSUB-1m antibodies. The positions of migration of molecular mass markers bovine serum albumin (68 kDa) and ovalbumin (44 kDa) are also shown.

Identification of pfsub-1 Gene Products in the Malaria Parasite-- Fig. 4 shows that in both Western blot and immunoprecipitation analysis of P. falciparum merozoite or schizont extracts, the anti-PfSUB-1_m antibodies recognized only two polypeptide species; these migrated at 47 kDa (the major species) and 54 kDa (minor species) on SDS-PAGE under reducing conditions, and the mobility of both proteins was reduction-sensitive. The proteins were denoted p47 and p54, respectively. Efficient immunoprecipitation of these proteins was obtained only after SDS extraction of parasite preparations; solubilization with TX-100 or sodium deoxycholate alone resulted in poor yields (not shown). To investigate the relationship between these two proteins and to seek further evidence that one or both proteins were products of the pfsub-1 gene, p47 and p54 were immunoprecipitated from extracts of biosynthetically radiolabeled T9/96 schizonts, digested with trypsin or chymotrypsin, and then analyzed by two-dimensional peptide mapping in parallel with similar digests of biosynthetically labeled recombinant fusion protein His₆-PfSUB-1_m. Peptide maps derived from the parasite-derived proteins and the recombinant His₆-PfSUB-1_m (Fig. 5) showed high relatedness but were not identical. Given the known differences between these polypeptides (i.e. the presence of approximately 3 kDa of nonmalarial sequence at the N terminus of His₆-PfSUB-1_m and the difference in size between the malarial and recombinant proteins), as well as the possibility of differential post-translational modification of the malarial and E. coli-derived proteins, identity was not expected. The maps are, however, sufficiently similar to strongly suggest that p47 and p54 are products of the pfsub-1 gene. Further peptide mapping of radiolabeled in vitro translated pfsub-1 gene products has confirmed this (not shown). Peptide maps of p47 and p54 were virtually indistinguishable (Fig. 5). It was concluded that both proteins are pfsub-1 gene products, probably derived from differential post-translational processing of a full-length precursor protein.

View larger version (78K): [in this window] [in a new window]   Fig. 5.   Two-dimensional peptide mapping indicates structural similarity between p54/p47 and a recombinant pfsub-1 gene product. Biosynthetically radiolabeled E. coli-derived recombinant His6-PfSUB-1m or parasite-derived p54 and p47 proteins were purified by immunoprecipitation and then subjected to digestion with trypsin (T) or chymotrypsin (C). Digests were analyzed by two-dimensional thin layer chromatography using electrophoresis at pH 4.4 in the first dimension (A), followed by ascending chromatography in the second dimension (B). Labeled peptides were detected by fluorography.

PfSUB-1 Undergoes Post-translational Processing during Secretory Transport-- This indication was confirmed by further immunoprecipitation analysis of parasites biosynthetically radiolabeled in the presence of the drug BFA and by pulse-chase experiments. Fig. 6 shows that radiolabeling in the presence of BFA, which blocks secretory transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus, resulted in a complete absence of detectable labeled p47 and a clear relative increase in levels of labeled p54. This is consistent with p47 being derived from p54 via a proteolytic processing step taking place during or following transport from the ER to the Golgi; blockade of this step with BFA would be expected to result in an accumulation of p54 in the ER. Pulse-chase experiments further confirmed the derivation of p47 (Fig. 7). Highly synchronous cultures of mature T9/96 schizonts were metabolically pulse-radiolabeled with [³⁵S]methionine/cysteine and then chased in the presence of excess nonradioactive methionine and cysteine. Samples of the culture taken at 0, 5, 15, 30, and 60 min following commencement of the chase were again analyzed by immunoprecipitation with the anti-PfSUB-1_m antibodies. Fig. 7 shows that at zero chase time following the pulse labeling (which was extended for a 9-min period in order to obtain detectable amounts of labeled protein) a number of immunoreactive proteins were evident; as well as p54, a clear doublet was evident at about 60 and 61 kDa, in addition to a larger band at 82 kDa. By 15 min of chase, the 82-kDa species and the 60/61-kDa doublet had virtually disappeared, concomitant with an increase in the intensity of p54 and a just detectable appearance of p47. By 30 min of chase, all bands other than p54 and p47 had disappeared, and between 30 and 60 min of chase, clear conversion of p54 to p47 was evident. These results are consistent with the following processing scheme. PfSUB-1 is initially detected as a large precursor protein with an approximate molecular mass of 82 kDa. Since signal peptide cleavage is generally cotranslational, it is likely that this largest species detectable in the pulse-chase experiments represents the complete PfSUB-1 sequence minus the predicted 25-residue-long signal sequence. This product is then rapidly processed (estimated t1/2 < 15 min) in an early secretory compartment, probably the ER, possibly via the 60/61-kDa forms, to p54, which is subsequently converted to p47 upon transport to the Golgi. Note that since the antibodies used in these analyses were raised against a fusion protein containing only the C-terminal 361 residues of the PfSUB-1 sequence (PfSUB-1_m), only those proteins corresponding to, or significantly overlapping, this putative catalytic domain sequence would be detectable in these experiments. Species derived from the N-terminal portion of PfSUB-1 (such as, for example, the putative pro-domain) would not be detected.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   p47 is derived from p54 via a brefeldin A-sensitive step. Cultures containing mature T9/96 schizonts were resuspended in medium and then divided into two identical 2-ml cultures. To one culture was added 10 µl of a 1 mg ml1 solution of BFA in methanol. To the other culture was added 10 µl of methanol only. The cultures were incubated at 37 °C for 15 min, and then both were metabolically radiolabeled with [35S]methionine/cysteine for a period of 2 h. The labeled parasites were then analyzed by immunoprecipitation using the anti-PfSUB-1m antibodies (lanes 1 and 2) or an anti-GST serum as a control (lanes 3 and 4). In BFA-treated parasites, the appearance of labeled p47 is completely blocked, with a concomitant increase in levels of p54. The fluorograph is deliberately overexposed, and the arrows indicate the positions of minor labeled species immunoreactive with the anti-PfSUB-1m but not the anti-GST serum. Positions of molecular weight markers are shown.

View larger version (73K): [in this window] [in a new window]   Fig. 7.   Pulse-chase analysis shows that p54 and p47 are derived from higher molecular weight precursors. Highly synchronous cultures of mature T9/96 schizonts were metabolically pulse-radiolabeled for 9 min with [35S]methionine/cysteine. A chase was then initiated, and cells were harvested at the indicated times and analyzed by immunoprecipitation. The positions of p54 and p47 are indicated, and positions of larger precursor proteins labeled with arrows. The bands at about 45 and 42 kDa present in all lanes are likely to be non-PfSUB-1-derived proteins co-precipitated or cross-reactive with the anti-PfSUB-1m serum used.

Immunolocalization of PfSUB-1 to a Subset of Dense Granules of the P. falciparum Merozoite-- The above experiments showed convincingly that antibodies raised against the GST-PfSUB-1_m fusion protein recognize exclusively products of the pfsub-1 gene in merozoite or schizont extracts. The same antibodies were therefore used to investigate the subcellular localization of these proteins. T9/96 schizonts, or naturally released T9/96 or FCB-1 merozoites were acetone permeabilized and analyzed by IFA. The anti-PfSUB-1_m antibodies reacted exclusively and strongly with a highly localized domain within the apical tip of free merozoites (Fig. 8, top). Similar IFA analysis of mature segmented schizonts (not shown) produced a weak punctate fluorescence similar to that previously observed with antibodies reactive with components of secretory apical organelles of the merozoite. Absolutely no reactivity was observed with intracellular trophozoite or young "ring-stage" forms of the parasite (not shown). To more stringently define the location of PfSUB-1, the antibodies were used in immunoelectron microscopy examination of thin sections of schizonts and free merozoites. Discrete antibody reactivity was observed with circular, electron-dense organelles with the morphological characteristics of merozoite dense granules (Fig. 8, bottom). Interestingly, although these organelles are dispersed throughout the merozoite cytoplasm (2), immunoreactivity was observed only with a subset of granules situated toward the apical end of the parasite. This result was entirely consistent with the IFA data showing a predominantly apical reactivity. Since p47 is the major pfsub-1 gene product detectable in merozoite extracts by Western blot and appears to be the terminal processing product in the parasite, it is likely that this is the species that concentrates in merozoite dense granules.

View larger version (81K): [in this window] [in a new window]   Fig. 8.   The p47 pfsub-1 gene product localizes to dense granules within the apical domain of free P. falciparum merozoites. Top, air-dried smears of naturally released merozoites were fixed with acetone and then probed with anti-PfSUB-1m antibodies. Bound antibody was detected with a Texas Red-conjugated anti-mouse IgG. The preparations were finally incubated briefly with an FITC-conjugated monoclonal antibody (2F10) reactive with MSP-1 and examined by confocal microscopy. The plate consists of an overlaid z series of 12 images recorded at 400-nm intervals, projected as a single image, with Texas Red fluorescence and FITC fluorescence shown overlaid. Bottom, thin sections of resin embedded merozoites (A) or mature schizonts (B and C) were probed with the anti-PfSUB-1m antiserum, and then bound antibodies were detected using a gold-labeled anti-mouse IgG antibody. Dense granules (D), rhoptries (R), and micronemes (Mi) are indicated, as is the cytoplasm of the infected erythrocyte (E). Bar, 0.5 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 9.   The pfsub-1 gene product p47 is shed in the form of a truncated, soluble protein. Synchronous cultures of mature T9/96 schizonts were metabolically radiolabeled with [35S]methionine/cysteine, washed extensively, and then either immediately snap-frozen or placed back into culture with the addition of a 10-fold excess of fresh red blood cells and cultured for 4 h to allow merozoite release and invasion to take place. Culture supernatants were harvested, and schizont and supernatant samples were analyzed by immunoprecipitation. Whereas both p54 and p47 are detected in immunoprecipitates from the schizont preparations (lane 1), only a 43-kDa immunoreactive species was present in the supernatant sample (lane 2). Positions of molecular weight markers are shown.

PfSUB-1 Does Not React with DFP-- In attempts to further characterize PfSUB-1, we explored its reactivity with DFP. This compound inhibits a wide range of serine proteases by forming a covalent adduct between the nucleophilic oxygen atom of the active site serine residue and the diisopropyl phosphoryl function of DFP. Treatment of purified merozoites with [³H]DFP resulted in the radiolabeling of three major species of approximately 35, 40, and 55 kDa, as well as two minor species of about 70 and 85 kDa (Fig. 10). This labeling profile was completely reproducible in a number of experiments (n = 8) using different batches of intact or sonicated merozoites (not shown). Since DFP is known to be able to react with certain residues other than reactive serine residues (46), cold competition experiments using other well characterized active site serine affinity reagents were performed. Pretreatment of the merozoites with nonradioactive PMSF abolished [³H]DFP reactivity with the 40- and 70-kDa proteins. Similarly, reactivity of [³H]DFP with the 40-kDa moiety was completely abolished by pretreatment with another active site serine affinity reagent, 3,4-dichloroisocoumarin (47). It is therefore highly likely that at least the 40- and 70-kDa species are active site serine enzymes. However, none of the [³H]DFP-reactive merozoite proteins migrated on SDS-PAGE at the position of any of the major PfSUB-1 gene products. Immunoprecipitation from extracts of [³H]DFP-labeled merozoites with anti-PfSUB-1_m sera similarly failed to precipitate radiolabeled p47 or p54 (not shown). Thus, at least in the form in which it is resident in the merozoite, PfSUB-1 does not detectably react with DFP.

View larger version (65K): [in this window] [in a new window]   Fig. 10.   Identification of merozoite proteins reactive with [3H]DFP. Highly purified T9/96 merozoites suspended in PBS were labeled with [3H]DFP after no pretreatment (CON) or after pretreatment for 10 min at room temperature with 2 mM PMSF in 1% (v/v) ethanol (PMSF); 2 mM DFP in 2% (v/v) isopropyl alcohol (DFP); 0.1 mM 3,4-dichloroisocoumarin in 2.5% (v/v) Me2SO (DCI); 1% (v/v) ethanol only (EtOH); 2% (v/v) isopropyl alcohol only (Isopro); or 2.5% (v/v) Me2SO only (DMSO). The merozoites were then analyzed by reducing SDS-PAGE and fluorography to detect [3H]DFP-reactive proteins. Positions of molecular weight markers are shown.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

This report describes the first gene to be identified of any apicomplexan parasite that encodes a serine protease-like protein. We have not directly demonstrated any proteolytic activity associated with any pfsub-1 gene product, but a number of indications lead us to propose that p47 or its truncated, secreted product is likely to be an enzymatically active protease and that it may play a role in erythrocyte invasion.

First, at the primary sequence level PfSUB-1 shows significant homology to known subtilases, and possesses all of the features known to be required of an active subtilase. Only four amino acid residues are completely conserved among known subtilases (34); these are the catalytic triad residues (PfSUB-1 residues Asp³⁷⁴, His⁴³⁰, and Ser⁶⁰⁸) and a glycine residue (Gly⁶⁰⁶ in the PfSUB-1 sequence) two positions N-terminal to the active site serine. PfSUB-1 also possesses an asparagine (Asn⁵²²) at the position of the oxyanion hole residue; the only substitution acceptable at this position is that of an aspartate, found in the PC2 subfamily of kexin-like convertases (34). While many bacterial subtilisins are devoid of cysteine residues, an increasingly large number of subtilases are known to possess up to two intramolecular disulfide bonds within the catalytic domain (34). There are seven cysteine residues within the putative catalytic domain of PfSUB-1, and the reduction-sensitive mobility on SDS-PAGE of p47 is consistent with the presence of intramolecular disulfide bonds. Detailed homology-based molecular modeling of the putative PfSUB-1 catalytic domain³ supports this postulate. Thus, at the structural level, PfSUB-1 resembles a subtilase.

Second, the proteolytic processing to which PfSUB-1 is subjected during secretory transport probably represents a process of enzyme activation common among subtilases. In a number of well studied examples, co-translational signal peptide cleavage allows folding of the subtilase catalytic domain, mediated by the intramolecular chaperone activity of the pro-domain (36, 48). This is often (but not always) rapidly followed by autocatalytic cleavage of the pro-domain from the catalytic domain; this cleavage can take place in cis (intramolecular) or in trans (intermolecular) and in some cases simple pro-domain cleavage is sufficient to allow zymogen activation (49, 50). However, there is a growing body of evidence that subtilase activation is often a substantially more complex process, which in eukaryotes may be intimately linked to correct routing of the proenzyme through the secretory pathway and final compartmentalization of the active enzyme (51-54). Here we have shown that the primary pfsub-1 gene product is subjected to at least two major post-translational processing steps. The first of these, conversion of the 82-kDa form, possibly via a 60/61-kDa intermediate, to the p54 form, takes place very rapidly following synthesis and therefore could represent an autocatalytic processing step triggered by signal peptide cleavage and co-translational folding within the lumen of the ER. The p54 form is then quantitatively converted to p47 in a slower, BFA-sensitive process. Further work is required to establish whether either or both of these processing events represent enzyme activation, but certainly they are wholly consistent with a putative activation pathway. If so, the process is clearly more complex than a simple one-step pro-domain removal. BFA sensitivity is a common feature of transport to secretory organelles in apicomplexan parasites (55-59), but little is known about the structural requirements for correct sorting to these organelles (59); it is possible that part of the PfSUB-1 sequence could provide a targeting signal, as has been determined for other subtilases and yeast carboxypeptidase Y (52, 60).

Third, the terminal intracellular PfSUB-1 processing product, p47, localizes to a subpopulation of dense granule-like organelles within the extreme apical domain of the merozoite and appears to be shed in a truncated, soluble form following merozoite release but before completion of erythrocyte invasion; no PfSUB-1-derived proteins were detectable in the newly invaded erythrocyte. These observations are in apparent conflict with the majority of the available data suggesting that dense granule secretion in apicomplexan parasites is a postinvasion event (2, 6, 41-44). However, dense granules in Plasmodium are currently defined essentially on morphological criteria; there is a paucity of specific markers for these organelles, and indeed only two other malarial proteins, RESA/Pf155 and a 14-kDa protein denoted RIMA, have been previously localized to merozoite dense granules (41, 61, 63). It is therefore possible that distinct subpopulations of dense granules may exist in Plasmodium; there is evidence for this in the apicomplexan parasite Cryptosporidium parvum (64), and if so, these subpopulations may be functionally distinct and may undergo exocytosis at different stages of the invasion process. Our evidence that PfSUB-1 is released at invasion suggests that it could play a role in the process of erythrocyte entry. Anti-PfSUB-1_m antibodies had no inhibitory effect on erythrocyte invasion by released merozoites in vitro, but it is unclear whether these antibodies have access to PfSUB-1 in the intact merozoite or whether they can interfere with its function. The demonstration of enzymatic activity in native, merozoite-derived p47 or its truncated soluble product would be a step toward addressing this issue. Consistent with observations of other workers (65), we have not been able to detect activity corresponding to p47 on gelatin substrate SDS-PAGE.⁴ However, the enzyme may be irreversibly denatured by exposure to SDS, and the insolubility of p47 in nonionic detergents precludes ready isolation and analysis of the merozoite-derived protein in a native state. These limitations could be overcome by recombinant expression of enzymatically active PfSUB-1, and this is a major priority of current work.

If PfSUB-1 is involved in erythrocyte invasion, what precise role might the enzyme perform? The best characterized proteolytic event associated with erythrocyte invasion is the processing and shedding of a merozoite surface protein complex derived from the precursor protein MSP-1. A single proteolytic cleavage at a Leu-Asn motif within the membrane-bound component of this complex, known as secondary processing, releases the complex quantitatively from the surface of the merozoite as it enters the host erythrocyte (16-18, 29). MSP-1 secondary processing is thought to be essential for successful erythrocyte invasion, and it is mediated by a parasite-derived, calcium-dependent serine protease (16, 17). Interestingly, this activity is exquisitely sensitive to inhibition by PMSF, but it is only poorly inhibited by even very high concentrations (up to 10 mM) of DFP (19). Given its apparent nonreactivity with DFP, its location in the merozoite, and the timing of its secretion, PfSUB-1 is a good candidate for this activity. This intriguing possibility clearly merits further investigation and is supported by molecular modeling of the proposed catalytic domain of PfSUB-1, which has indicated that a heptapeptide corresponding to the sequence flanking the MSP-1 secondary processing site (i.e. Gln-Gly-Met-Leu-Asn-Ile-Ser) fits well into the active site groove of the model, with the Leu-Asn scissile bond in the appropriate position for cleavage.³ The only other serine protease activity previously localized to P. falciparum merozoites is a DFP-reactive enzyme thought to be activated following cleavage of a GPI anchor (14); it is unlikely that PfSUB-1 corresponds to this activity, since it shows no DFP reactivity and the sequence exhibits no C-terminal hydrophobic domain typical of GPI signal sequences (66).

Of the two major families of active site serine endoproteases, the chymotrypsin-like and the subtilisin-like families (32), chymotrypsin-like proteases are common in higher eukaryotes but appear to be rare in lower eukaryotes and prokaryotic organisms. Sakanari et al. (67) have reported evidence for the existence of a chymotrypsin-like gene in Trypanosoma cruzi, but there is no other previous genetic data on protozoal serine proteases. Extensive attempts in this laboratory to isolate chymotrypsin-like P. falciparum genes using a number of different PCR-based approaches have been unsuccessful.⁴ It is conceivable that, like Saccharomyces cerevisiae (40), the malaria parasite (and perhaps other apicomplexan parasites) does not possess genes of this class. Proteases play a crucial role in the life cycle of the blood stage malaria parasite and are undoubtedly potential targets for the design of protease inhibitor-based drugs. Expression of PfSUB-1 in an enzymatically active form should allow the development and evaluation of such inhibitors.