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• W2034720263 abstract "The most virulent form of malaria is caused by waves of replication of blood stages of the protozoan pathogen Plasmodium falciparum. The parasite divides within an intraerythrocytic parasitophorous vacuole until rupture of the vacuole and host-cell membranes releases merozoites that invade fresh erythrocytes to repeat the cycle. Despite the importance of merozoite egress for disease progression, none of the molecular factors involved are known. We report that, just prior to egress, an essential serine protease called PfSUB1 is discharged from previously unrecognized parasite organelles (termed exonemes) into the parasitophorous vacuole space. There, PfSUB1 mediates the proteolytic maturation of at least two essential members of another enzyme family called SERA. Pharmacological blockade of PfSUB1 inhibits egress and ablates the invasive capacity of released merozoites. Our findings reveal the presence in the malarial parasitophorous vacuole of a regulated, PfSUB1-mediated proteolytic processing event required for release of viable parasites from the host erythrocyte. The most virulent form of malaria is caused by waves of replication of blood stages of the protozoan pathogen Plasmodium falciparum. The parasite divides within an intraerythrocytic parasitophorous vacuole until rupture of the vacuole and host-cell membranes releases merozoites that invade fresh erythrocytes to repeat the cycle. Despite the importance of merozoite egress for disease progression, none of the molecular factors involved are known. We report that, just prior to egress, an essential serine protease called PfSUB1 is discharged from previously unrecognized parasite organelles (termed exonemes) into the parasitophorous vacuole space. There, PfSUB1 mediates the proteolytic maturation of at least two essential members of another enzyme family called SERA. Pharmacological blockade of PfSUB1 inhibits egress and ablates the invasive capacity of released merozoites. Our findings reveal the presence in the malarial parasitophorous vacuole of a regulated, PfSUB1-mediated proteolytic processing event required for release of viable parasites from the host erythrocyte. Malaria is caused by protozoan parasites of the genus Plasmodium. The parasite life-cycle is divided between a vertebrate host and a mosquito vector. Clinical manifestations of malaria are caused by the asexual blood stage life cycle. The parasite invades erythrocytes to form a ring which undergoes mitotic replication within a parasitophorous vacuole (PV) to become a schizont. This eventually ruptures in a process known as egress, releasing invasive merozoites which invade fresh erythrocytes to repeat the cycle. The molecular events leading to egress are obscure, but studies using broad-spectrum protease inhibitors have strongly implicated parasite protease activity (Delplace et al., 1988Delplace P. Bhatia A. Cagnard M. Camus D. Colombet G. Debrabant A. Dubremetz J.F. Dubreuil N. Prensier G. Fortier B. et al.Protein p126: a parasitophorous vacuole antigen associated with the release of Plasmodium falciparum merozoites.Biol. Cell. 1988; 64: 215-221Crossref PubMed Scopus (73) Google Scholar, Gelhaus et al., 2005Gelhaus C. Vicik R. Schirmeister T. Leippe M. Blocking effect of a biotinylated protease inhibitor on the egress of Plasmodium falciparum merozoites from infected red blood cells.Biol. Chem. 2005; 386: 499-502Crossref PubMed Scopus (24) Google Scholar, Salmon et al., 2001Salmon B.L. Oksman A. Goldberg D.E. Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis.Proc. Natl. Acad. Sci. USA. 2001; 98: 271-276Crossref PubMed Scopus (188) Google Scholar, Soni et al., 2005Soni S. Dhawan S. Rosen K.M. Chafel M. Chishti A.H. Hanspal M. Characterization of events preceding the release of malaria parasite from the host red blood cell.Blood Cells Mol. Dis. 2005; 35: 201-211Crossref PubMed Scopus (33) Google Scholar, Wickham et al., 2003Wickham M.E. Culvenor J.G. Cowman A.F. Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte.J. Biol. Chem. 2003; 278: 37658-37663Crossref PubMed Scopus (133) Google Scholar). Particular interest has focused on members of the serine-rich antigen (SERA) family of malarial papain-like proteins. Disruption of the SERA8 gene prevents release of sporozoites from oocysts in the insect vector (Aly and Matuschewski, 2005Aly A.S. Matuschewski K. A malarial cysteine protease is necessary for Plasmodium sporozoite egress from oocysts.J. Exp. Med. 2005; 202: 225-230Crossref PubMed Scopus (129) Google Scholar). Also, antibodies against the most abundant blood-stage family member, SERA5, interfere with merozoite egress (Pang et al., 1999Pang X.L. Mitamura T. Horii T. Antibodies reactive with the N-terminal domain of Plasmodium falciparum serine repeat antigen inhibit cell proliferation by agglutinating merozoites and schizonts.Infect. Immun. 1999; 67: 1821-1827Crossref PubMed Google Scholar), and both SERA5 and SERA6 appear indispensable for blood-stage parasite growth (Miller et al., 2002Miller S.K. Good R.T. Drew D.R. Delorenzi M. Sanders P.R. Hodder A.N. Speed T.P. Cowman A.F. de Koning-Ward T.F. Crabb B.S. A subset of Plasmodium falciparum SERA genes are expressed and appear to play an important role in the erythrocytic cycle.J. Biol. Chem. 2002; 277: 47524-47532Crossref PubMed Scopus (128) Google Scholar, McCoubrie et al., 2007McCoubrie J.E. Miller S.K. Sargeant T. Good R.T. Hodder A.N. Speed T.P. de Koning-Ward T.F. Crabb B.S. Evidence for a common role for the serine-type Plasmodium falciparum SERA proteases: Implications for vaccine and drug design.Infect. Immun. 2007; (in press Published online September 24, 2007)https://doi.org/10.1128/IAI.00405.07Crossref PubMed Google Scholar). However direct evidence for a role for these or any other known malarial proteases in egress of blood-stage merozoites is lacking, and the cellular mechanisms that govern this essential, highly regulated event are unknown. The genome of P. falciparum, the species causing the most virulent form of malaria, encodes three subtilisin-like serine proteases (subtilases). Of these, PfSUB3 is not essential in asexual blood stages (R.O., unpublished data), while PfSUB2 was recently identified as the ‘sheddase’ responsible for the release of merozoite surface proteins during erythrocyte invasion (Harris et al., 2005Harris P.K. Yeoh S. Dluzewski A.R. O'Donnell R.A. Withers-Martinez C. Hackett F. Bannister L.H. Mitchell G.H. Blackman M.J. Molecular identification of a malaria merozoite surface sheddase.PLoS Pathog. 2005; 1: 241-251Crossref PubMed Scopus (158) Google Scholar). The third P. falciparum subtilase, PfSUB1, is maximally expressed in the final stages of schizont maturation (Blackman et al., 1998Blackman M.J. Fujioka H. Stafford W.H. Sajid M. Clough B. Fleck S.L. Aikawa M. Grainger M. Hackett F. A subtilisin-like protein in secretory organelles of Plasmodium falciparum merozoites.J. Biol. Chem. 1998; 273: 23398-23409Crossref PubMed Scopus (106) Google Scholar). Production of recombinant PfSUB1 has enabled studies of its substrate specificity and the development of a simple activity assay (Blackman et al., 2002Blackman M.J. Corrie J.E. Croney J.C. Kelly G. Eccleston J.F. Jameson D.M. Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate.Biochemistry. 2002; 41: 12244-12252Crossref PubMed Scopus (61) Google Scholar, Withers-Martinez et al., 2002Withers-Martinez C. Saldanha J.W. Ely B. Hackett F. O'Connor T. Blackman M.J. Expression of recombinant Plasmodium falciparum subtilisin-like protease-1 in insect cells: Characterization, comparison with the parasite protease, and homology modelling.J. Biol. Chem. 2002; 277: 29698-29709Crossref PubMed Scopus (42) Google Scholar). Homology modeling of PfSUB1 has highlighted distinctive structural features as well as similarities to bacterial subtilisins (Withers-Martinez et al., 2002Withers-Martinez C. Saldanha J.W. Ely B. Hackett F. O'Connor T. Blackman M.J. Expression of recombinant Plasmodium falciparum subtilisin-like protease-1 in insect cells: Characterization, comparison with the parasite protease, and homology modelling.J. Biol. Chem. 2002; 277: 29698-29709Crossref PubMed Scopus (42) Google Scholar), raising the possibility that PfSUB1 may be a target for the development of selective protease inhibitor-based antimalarial drugs. However, the function of PfSUB1 has remained elusive. Here, we show that that the pfsub1 gene is refractory to disruption in blood stages, indicating that it performs an essential task. We demonstrate that PfSUB1 is stored in a set of parasite organelles that are distinct from those involved in invasion. Using a high-throughput screen, we isolated a selective inhibitor of PfSUB1 and used it in “chemical knockdown” studies. Our results provide the first insights into the role of PfSUB1 and reveal a hitherto unsuspected regulatory pathway in which discharge of PfSUB1 into the PV in the final stages of schizont maturation triggers a series of proteolytic events that culminate in egress of invasive merozoites. To investigate the function of PfSUB1, we first determined whether it is required for blood-stage growth. Parasites were transfected with a construct designed to disrupt the pfsub1 gene by double homologous recombination (Duraisingh et al., 2002Duraisingh M.T. Triglia T. Cowman A.F. Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination.Int. J. Parasitol. 2002; 32: 81-89Crossref PubMed Scopus (144) Google Scholar). This did not integrate, as determined by Southern blot analysis, but was maintained in episomal form for up to five drug cycles (data not shown). In contrast, in parallel experiments this approach was successfully used to disrupt the pfsub3 gene (R.O., unpublished data). We next attempted to modify the pfsub1 gene by fusion of its 3′ end to green fluorescent protein (GFP) using a single-crossover homologous recombination strategy (Harris et al., 2005Harris P.K. Yeoh S. Dluzewski A.R. O'Donnell R.A. Withers-Martinez C. Hackett F. Bannister L.H. Mitchell G.H. Blackman M.J. Molecular identification of a malaria merozoite surface sheddase.PLoS Pathog. 2005; 1: 241-251Crossref PubMed Scopus (158) Google Scholar). Again, repeated transfections resulted only in long-term maintenance of episomes (data not shown). These results suggested either that modifications of the pfsub1 gene that interfere with its function are deleterious, or that the locus is inaccessible to homologous recombination. To test both possibilities, we attempted to more subtly modify the gene. Parasites were independently transfected with four related constructs, each designed to integrate into the genomic pfsub1 locus by single-crossover homologous recombination with the result of reconstituting a complete open reading frame (ORF) while simultaneously introducing a C-terminal triple haemagglutinin (HA) epitope tag (Figure S1 in the Supplemental Data available online). Constructs pPfSUB1HA3 and pPfSUB1HA3-UTtrun contained targeting sequence comprising the extreme 3′ 943 bp of the authentic pfsub1 ORF. Constructs pPfSUB1chiHA3 and pPfSUB1chiHA3 mut contained a shorter targeting fragment of the authentic pfsub1 gene, fused in frame to synthetic “recodonised” sequence encoding the C-terminal part of PfSUB1. The synthetic gene shares low identity at the nucleotide level with the authentic pfsub1 sequence, so these two constructs were expected to crossover only upstream of the catalytic Ser codon (Ser608), with the predicted result of creating a chimeric modified pfsub1 locus; in the case of pPfSUB1chiHA3 this would encode wild-type, catalytically active PfSUB1, whereas in the case of pPfSUB1chiHA3 mut the chimeric gene product would possess an Ala substitution of Ser608, and so would lack proteolytic activity (Figure S1A). PfSUB1 coding sequences in constructs pPfSUB1HA3, pPfSUB1chiHA3 and pPfSUB1chiHA3 mut were flanked by the 848 bp-long 3′ UTR of the P. berghei dihydrofolate reductase (dhfr) gene to ensure correct transcription termination and polyadenylation of the modified gene, whereas pPfSUB1HA3-UTtrun contained instead a severely truncated form of the same 3′ UTR. Truncation of 3′ UTR sequences in several eukaryotes including Plasmodium can reduce gene expression, so integration of this plasmid was predicted to downregulate or ablate PfSUB1 expression. After two drug cycles, PCR analysis indicated integration of pPfSUB1HA3 and pPfSUB1chiHA3 into the parasite genome (Figure S1B). In contrast, no integration of the other two constructs could be detected, suggesting that integration was detrimental to parasite growth. Since the chimeric constructs were intended solely to test the requirement for catalytically active PfSUB1, work with the pPfSUB1chiHA3-transfected line was not progressed further. The pPfSUB1HA3-transfected parasite line (called 3D7SUB1HA3) was cloned by limiting dilution. Analysis of two clones, C10 and F7, confirmed that the input plasmid had integrated through the expected single-crossover homologous recombination event, placing the HA3 tag at the 3′ end of the pfsub1 gene (Figures S1C and S1D). Western blot confirmed expression of epitope-tagged PfSUB1 (Figure S2A). Neither clone displayed any defect in growth rate compared to the parental 3D7 line (data not shown), indicating that neither fusion to the HA3 tag nor replacement of the pfsub1 3′ UTR with the full-length P. berghei dhfr 3′ UTR affected parasite growth. These results prove that the pfsub1 locus is accessible to homologous recombination. Collectively, our ability to epitope-tag the pfsub1 gene together with our inability to disrupt it, fuse it to GFP, substitute its catalytic Ser codon, or replace its 3′ UTR with a truncated 3′ UTR, strongly suggests that PfSUB1 activity is essential for maintenance of the asexual erythrocytic life cycle of the parasite. Previous studies suggested that PfSUB1 accumulates in subcellular organelles that are distinct from micronemes and rhoptries (secretory organelles at the apical end of the merozoite involved in invasion) but that resemble a third class of secretory vesicles called dense granules (Blackman et al., 1998Blackman M.J. Fujioka H. Stafford W.H. Sajid M. Clough B. Fleck S.L. Aikawa M. Grainger M. Hackett F. A subtilisin-like protein in secretory organelles of Plasmodium falciparum merozoites.J. Biol. Chem. 1998; 273: 23398-23409Crossref PubMed Scopus (106) Google Scholar). We exploited the epitope-tagged PfSUB1 (PfSUB1HA3) expressed by the 3D7SUB1HA3 clones to re-examine this localization. Double immunofluorescence analysis (IFA) confirmed that PfSUB1HA3 resides in a set of organelles that are neither rhoptries nor micronemes. Unexpectedly, PfSUB1HA3 also did not colocalize with the dense granule marker, RESA (Figure 1A). Identical results were obtained using a specific rabbit antiserum to detect PfSUB1; importantly, the signal obtained with this colocalized with the anti-HA signal in the 3D7SUB1HA3 clones, validating the rabbit antibodies (Figure S2B). Dual-labeling immunoelectron microscopy (Figures 1B–1G and S3) confirmed the presence of PfSUB1 and RESA in different organelles. Although these shared the electron-dense character typical of dense granules, the PfSUB1-containing structures appeared less numerous than the RESA-containing organelles and were typically larger and elongate (mean dimensions from a sample of 10 was 101 × 60 nm, in contrast to the more spheroidal RESA-containing dense granules with mean dimensions of 72 × 63 nm). A characteristic of dense granules in apicomplexan parasites is that they are discharged predominantly following host cell invasion. Thus, RESA is released from merozoites soon after invasion and translocates to the erythrocyte cytoskeleton (Aikawa et al., 1990Aikawa M. Torii M. Sjolander A. Berzins K. Perlmann P. Miller L.H. Pf155/RESA antigen is localized in dense granules of Plasmodium falciparum merozoites.Exp. Parasitol. 1990; 71: 326-329Crossref PubMed Scopus (86) Google Scholar, Culvenor et al., 1991Culvenor J.G. Day K.P. Anders R.F. Plasmodium falciparum ring-infected erythrocyte surface antigen is released from merozoite dense granules after erythrocyte invasion.Infect. Immun. 1991; 59: 1183-1187Crossref PubMed Google Scholar). A number of rhoptry proteins are also present in the newly-invaded host cell, including RAP2 (Baldi et al., 2000Baldi D.L. Andrews K.T. Waller R.F. Roos D.S. Howard R.F. Crabb B.S. Cowman A.F. RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum.EMBO J. 2000; 19: 2435-2443Crossref PubMed Scopus (100) Google Scholar). As expected, IFA of newly-invaded ring stage parasites of the 3D7SUB1HA3 clones confirmed the presence of RESA at the host erythrocyte membrane; however, PfSUB1HA3 was not detected (Figure 2A), despite the fact that the IFA signals for both proteins were of similar intensity in schizonts. Western blot confirmed this; whereas both RAP2 and RESA (Figures 2B and 2C) were readily detected in ring-stage extracts, no PfSUB1HA3 signal was evident. In contrast, culture supernatants sampled following schizont rupture contained a strong PfSUB1HA3 signal (Figure 2C), suggesting that PfSUB1 is quantitatively released into culture media at schizont rupture. The intraerythrocytic malaria parasite undergoes nuclear division as a syncytium, until cytokinesis (“segmentation”) and budding of individual merozoites in the final stages of schizogony. To establish more precisely the point at which PfSUB1 release takes place, we examined highly mature, segmented 3D7SUB1HA3 schizonts by IFA. This revealed that late in schizogony the PfSUB1HA3 IFA signal became less dot-like, more diffuse and largely located to the periphery of intracellular merozoites. Dual labeling with an antibody to the merozoite plasma membrane marker MSP1 supported this, suggesting that near the end of schizogony PfSUB1 can translocate from its previous organellar location into the PV (Figure 2D). Additionally, where bursting schizonts were visible, the free merozoites were surrounded by a “cloud” of anti-HA signal, suggesting that at the point of egress PfSUB1 discharge had already begun (Figure 2E). Our results show that PfSUB1 defines a set of subcellular vesicles that are distinct from the three previously described types of Plasmodium secretory organelle. The fact that PfSUB1 is released from these vesicles, which we term “exonemes,” just prior to schizont rupture suggested that the role of PfSUB1 is related not to invasion, but to some pre-invasion proteolytic event. We previously described an assay for PfSUB1 activity using a fluorogenic peptide substrate (Blackman et al., 2002Blackman M.J. Corrie J.E. Croney J.C. Kelly G. Eccleston J.F. Jameson D.M. Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate.Biochemistry. 2002; 41: 12244-12252Crossref PubMed Scopus (61) Google Scholar). To identify small molecule inhibitors of PfSUB1 suitable for investigation of its function, we adapted this assay to an automated, 384-well microplate format and used it to screen over 170,000 low molecular weight compounds from a number of commercial and proprietary sources (see Supplemental Data for details of the screen). This resulted in identification of a potent PfSUB1 inhibitor called MRT12113 (Figure 3A). Secondary assays confirmed that MRT12113 was a fast-acting inhibitor of PfSUB1 with an IC50 of 0.3 μM (Figure 3B). In contrast, at concentrations of up to 50 μM MRT12113 displayed no inhibition of any other protease tested, including the related bacterial subtilisins BPN' and Carlsberg, the mammalian serine proteases trypsin, chymotrypsin, and elastase, the cysteine protease papain, human caspase-3, the P. falciparum cysteine protease falcipain 2, the P. falciparum aspartic protease plasmepsin II, and the P. falciparum sheddase PfSUB2 (data not shown; see Supplemental Experimental Procedures for protocols). These data indicated that MRT12113 is a highly selective inhibitor of PfSUB1. Consistent with this, MRT12113 showed no toxicity against mammalian COS-7 cells or against an organism related to the malaria parasite, the ciliate Tetrahymena thermophila, even when present in cultures for extended periods (up to 7 days) at a concentration of 125 μM (data not shown). On the basis of its high selectivity for PfSUB1, MRT12113 was deemed suitable for studies of PfSUB1 function in the parasite. Addition of MRT12113 to synchronous P. falciparum cultures at up to 250 μM had no effect on intracellular parasite growth over the majority of the erythrocytic life cycle (data not shown). However at the end of the cycle partial inhibition of schizont rupture was observed and invasion by those merozoites that were released was reduced, as evidenced by an accumulation of unruptured schizonts, a decrease in ring formation and the presence of numerous free extracellular merozoites (Figure 3C). These dual effects of MRT12113 were dose-dependent, with an ED50 against schizont rupture of ∼180 μM (Figure 3D), and a much lower ED50 against invasion of ∼25 μM (Figure 3E). To dissect the mechanistic basis of these effects, schizonts were cultured in the presence of high concentrations of MRT12113 and culture supernatants analyzed by SDS PAGE. MRT12113 produced an overall decrease in protein content compared to control supernatants, due to the inhibition of schizont rupture. However, close examination of the gels revealed changes in the relative abundance of two prominent species; a ∼120 kDa protein, levels of which were increased, and a ∼50 kDa protein, which almost disappeared in the presence of MRT12113 (Figure 4A). Tryptic peptide mapping (Tables S1 and S2) identified both polypeptides as being derived from the P. falciparum protein SERA5. SERA5 is an abundant, soluble protein of the P. falciparum PV. Expressed as a precursor of ∼126 kDa (SERA5 P126), it undergoes extensive proteolytic processing (Figure 4B) (Debrabant et al., 1992Debrabant A. Maes P. Delplace P. Dubremetz J.F. Tartar A. Camus D. Intramolecular mapping of Plasmodium falciparum P126 proteolytic fragments by N-terminal amino acid sequencing.Mol. Biochem. Parasitol. 1992; 53: 89-95Crossref PubMed Scopus (55) Google Scholar, 1988, 1985; Li et al., 2002aLi J. Matsuoka H. Mitamura T. Horii T. Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA).Mol. Biochem. Parasitol. 2002; 120: 177-186Crossref PubMed Scopus (44) Google Scholar). SERA5 P126 is initially converted to a ∼47 kDa N-terminal and an ∼73 kDa C-terminal fragment (P47 and P73). P73 is further cleaved to produce P56, which derives from the central region of the precursor, plus P18. P47 can also undergo further cleavage, although this only occurs in some allelic forms of SERA5 (Li et al., 2002bLi J. Mitamura T. Fox B.A. Bzik D.J. Horii T. Differential localization of processed fragments of Plasmodium falciparum serine repeat antigen and further processing of its N-terminal 47 kDa fragment.Parasitol. Int. 2002; 51: 343-352Crossref PubMed Scopus (46) Google Scholar). P56 is finally truncated at its C terminus to produce P50. Importantly, P50 is abundant in culture supernatants following schizont rupture, but processed forms of SERA5 are not prominent in schizonts, suggesting that processing occurs rapidly just prior to or at the point of egress. Previous studies have indicated that a parasite serine protease plus at least one other enzyme are involved in SERA5 processing, but their identities are unknown (Debrabant and Delplace, 1989Debrabant A. Delplace P. Leupeptin alters the proteolytic processing of P126, the major parasitophorous vacuole antigen of Plasmodium falciparum.Mol. Biochem. Parasitol. 1989; 33: 151-158Crossref PubMed Scopus (36) Google Scholar, Li et al., 2002aLi J. Matsuoka H. Mitamura T. Horii T. Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA).Mol. Biochem. Parasitol. 2002; 120: 177-186Crossref PubMed Scopus (44) Google Scholar). Interest has focused on SERA5 for a number of reasons, including the presence of a papain-like central domain containing a Ser residue in place of the putative catalytic Cys. SERA5 therefore resembles a protease, and indeed a recombinant form of SERA5 displays chymotrypsin-like activity (Hodder et al., 2003Hodder A.N. Drew D.R. Epa V.C. Delorenzi M. Bourgon R. Miller S.K. Moritz R.L. Frecklington D.F. Simpson R.J. Speed T.P. et al.Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.J. Biol. Chem. 2003; 278: 48169-48177Crossref PubMed Scopus (80) Google Scholar). Attempts to disrupt the SERA5 gene have been unsuccessful, suggesting it is important for blood-stage growth (Miller et al., 2002Miller S.K. Good R.T. Drew D.R. Delorenzi M. Sanders P.R. Hodder A.N. Speed T.P. Cowman A.F. de Koning-Ward T.F. Crabb B.S. A subset of Plasmodium falciparum SERA genes are expressed and appear to play an important role in the erythrocytic cycle.J. Biol. Chem. 2002; 277: 47524-47532Crossref PubMed Scopus (128) Google Scholar, McCoubrie et al., 2007McCoubrie J.E. Miller S.K. Sargeant T. Good R.T. Hodder A.N. Speed T.P. de Koning-Ward T.F. Crabb B.S. Evidence for a common role for the serine-type Plasmodium falciparum SERA proteases: Implications for vaccine and drug design.Infect. Immun. 2007; (in press Published online September 24, 2007)https://doi.org/10.1128/IAI.00405.07Crossref PubMed Google Scholar). Our observations indicated that blockade of PfSUB1 activity with MRT12113 inhibits SERA5 processing. We confirmed this using a SERA5-specific mAb to probe extracts and culture supernatants from schizonts cultured with MRT12113 (Figure 4C); the compound blocked the small amount of SERA5 processing detectable in the parasite, and resulted in accumulation of SERA5 P126 in culture supernatants. These results suggested that PfSUB1 plays a role in SERA5 processing. They also implied that PfSUB1 can access SERA5 intracellularly (presumably in the PV) just prior to its release into culture supernatants at egress. Furthermore, given the inhibitory effects of MRT12113 on schizont rupture and merozoite invasion, they suggested that SERA5 processing by PfSUB1 is important for release of invasive merozoites. To test directly whether SERA5 can be correctly processed by PfSUB1, SERA5 P126 was purified from schizont extracts (Figure S4) and incubated with recombinant PfSUB1 (rPfSUB1). This resulted in rapid conversion to the P56 form via the P73 intermediate (Figure 5A). No conversion to P50 occurred, consistent with previous reports that this last step of SERA5 processing is mediated by a distinct, leupeptin-sensitive activity (Debrabant and Delplace, 1989Debrabant A. Delplace P. Leupeptin alters the proteolytic processing of P126, the major parasitophorous vacuole antigen of Plasmodium falciparum.Mol. Biochem. Parasitol. 1989; 33: 151-158Crossref PubMed Scopus (36) Google Scholar, Li et al., 2002aLi J. Matsuoka H. Mitamura T. Horii T. Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA).Mol. Biochem. Parasitol. 2002; 120: 177-186Crossref PubMed Scopus (44) Google Scholar) and that PfSUB1 is insensitive to leupeptin (Withers-Martinez et al., 2002Withers-Martinez C. Saldanha J.W. Ely B. Hackett F. O'Connor T. Blackman M.J. Expression of recombinant Plasmodium falciparum subtilisin-like protease-1 in insect cells: Characterization, comparison with the parasite protease, and homology modelling.J. Biol. Chem. 2002; 277: 29698-29709Crossref PubMed Scopus (42) Google Scholar). An identical profile of processing was seen using SERA5 P126 that had been treated during purification with a cocktail of protease inhibitors (Figure 5A). Also, processing was completely sensitive to MRT12113 even if added after processing had commenced (Figure 5B), demonstrating that all the observed processing was mediated directly by PfSUB1 and was not the result of autocatalytic maturation of SERA5 triggered by the presence of PfSUB1. To establish whether the cleavage mimicked authentic SERA5 processing, we examined larger amounts of rPfSUB1-processed SERA5 P126 by silver-staining. This allowed us to detect all the products of cleavage (Figure 5C). N-terminal sequencing confirmed correct cleavage at the known sites 1 and 2, and mapped a third, allele-specific processing site (site 3) described by Li et al., 2002bLi J. Mitamura T. Fox B.A. Bzik D.J. Horii T. Differential localization of processed fragments of Plasmodium falciparum serine repeat antigen and further processing of its N-terminal 47 kDa fragment.Parasitol. Int. 2002; 51: 343-352Crossref PubMed Scopus (46) Google Scholar but not previously precisely defined. These results suggested that SERA5 is an authentic physiological substrate for PfSUB1. By analogy with other processing proteases we predicted that peptides based on the SERA5 processing sites would be substrates for PfSUB1. To explore this, peptides incorporating SERA5 processing sites 1, 2, and 3 were assessed for susceptibility to PfSUB1 cleavage. Synthetic decapeptides Ac-TVRGDTEPIS, Ac-EIKAETEDDD and Ac-IIFGQDTAGS—but not similar peptides with a P1 or P2 Leu substitution—were rapidly and specifically cleaved by rPfSUB1 at the expected Asp-Thr, Glu-Thr and Gln-Asp bonds respectively (data not shown). To quantify this, two fluorogenic derivatives were produced by labeling the site 1 and site 2-related peptides Ac-CIKAETEDDC and Ac-CIFGQDTAGC with 6 iodoacetamidotetramethylrhodamine (6-IATR). These peptides, named SERAst1F-6R and SERAst2F-6R respectively, were also cleaved" @default.
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• W2034720263 date "2007-12-01" @default.
• W2034720263 modified "2023-10-10" @default.
• W2034720263 title "Subcellular Discharge of a Serine Protease Mediates Release of Invasive Malaria Parasites from Host Erythrocytes" @default.
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