Simultaneous multiple allelic replacement in the malaria parasite enables dissection of PKG function.

Over recent years, a plethora of new genetic tools has transformed conditional engineering of the malaria parasite genome, allowing functional dissection of essential genes in the asexual and sexual blood stages that cause pathology or are required for disease transmission, respectively. Important challenges remain, including the desirability to complement conditional mutants with a correctly regulated second gene copy to confirm that observed phenotypes are due solely to loss of gene function and to analyse structure-function relationships. To meet this challenge, here we combine the dimerisable Cre (DiCre) system with the use of multiple lox sites to simultaneously generate multiple recombination events of the same gene. We focused on the Plasmodium falciparum cGMP-dependent protein kinase (PKG), creating in parallel conditional disruption of the gene plus up to two allelic replacements. We use the approach to demonstrate that PKG has no scaffolding or adaptor role in intraerythrocytic development, acting solely at merozoite egress. We also show that a phosphorylation-deficient PKG is functionally incompetent. Our method provides valuable new tools for analysis of gene function in the malaria parasite

Identification of a Plasmodium falciparum phospholipid transfer protein.

Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte

Essentiality of Plasmodium falciparum plasmepsin V.

The malaria parasite replicates within erythrocytes. The pathogenesis of clinical malaria is in large part due to the capacity of the parasite to remodel its host cell. To do this, intraerythrocytic stages of Plasmodium falciparum export more than 300 proteins that dramatically alter the morphology of the infected erythrocyte as well as its mechanical and adhesive properties. P. falciparum plasmepsin V (PfPMV) is an aspartic protease that processes proteins for export into the host erythrocyte and is thought to play a key role in parasite virulence and survival. However, although standard techniques for gene disruption as well as conditional protein knockdown have been previously attempted with the pfpmv gene, complete gene removal or knockdown was not achieved so direct genetic proof that PMV is an essential protein has not been established. Here we have used a conditional gene excision approach combining CRISPR-Cas9 gene editing and DiCre-mediated recombination to functionally inactivate the pfpmv gene. The resulting mutant parasites displayed a severe growth defect. Detailed phenotypic analysis showed that development of the mutant parasites was arrested early in the ring-to-trophozoite transition in the erythrocytic cycle following gene excision. Our findings are the first to elucidate the effects of PMV gene disruption, showing that it is essential for parasite viability in asexual blood stages. The mutant parasites can now be used as a platform to further dissect the Plasmodium protein export pathway

Function and essentiality of <i>Plasmodium falciparum</i> plasmepsin V

AbstractThe malaria parasite replicates within erythrocytes. The pathogenesis of clinical malaria is in large part due to the capacity of the parasite to remodel its host cell. To do this, intraerythrocytic stages of Plasmodium falciparum export more than 300 proteins that dramatically alter the morphology of the infected erythrocyte as well as its mechanical and adhesive properties. P. falciparum plasmepsin V (PfPMV) is an aspartic protease that processes proteins for export into the host erythrocyte and is thought to play a key role in parasite virulence and survival. However, although standard techniques for gene disruption as well as conditional protein knockdown have been previously attempted with the pfpmv gene, complete gene removal or knockdown was not achieved so direct genetic proof that PMV is an essential protein has not yet been established. Here we have used a conditional gene excision approach combining CRISPR-Cas9 gene editing and DiCre-mediated recombination to functionally inactivate the pfpmv gene. The resulting mutant parasites displayed a severe growth defect. Detailed phenotypic analysis showed that development of the mutant parasites was arrested at the ring-to-trophozoite transition in the erythrocytic cycle following gene excision, likely due to a defect in protein export. Our findings are the first to elucidate the effects of PMV gene disruption, showing that it is essential for parasite viability in asexual blood stages. The mutant parasites can now be used as a platform to further dissect the Plasmodium protein export pathway.</jats:p

Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite.

Apicomplexan pathogens are obligate intracellular parasites. To enter cells, they must bind with high affinity to host cell receptors and then uncouple these interactions to complete invasion. Merozoites of Plasmodium falciparum, the parasite responsible for the most dangerous form of malaria, invade erythrocytes using a family of adhesins called Duffy binding ligand-erythrocyte binding proteins (DBL-EBPs). The best-characterized P. falciparum DBL-EBP is erythrocyte binding antigen 175 (EBA-175), which binds erythrocyte surface glycophorin A. We report that EBA-175 is shed from the merozoite at around the point of invasion. Shedding occurs by proteolytic cleavage within the transmembrane domain (TMD) at a site that is conserved across the DBL-EBP family. We show that EBA-175 is cleaved by PfROM4, a rhomboid protease that localizes to the merozoite plasma membrane, but not by other rhomboids tested. Mutations within the EBA-175 TMD that abolish cleavage by PfROM4 prevent parasite growth. Our results identify a crucial role for intramembrane proteolysis in the life cycle of this pathogen

A malaria parasite subtilisin propeptide-like protein is a potent inhibitor of the egress protease SUB1.

Subtilisin-like serine peptidases (subtilases) play important roles in the life cycle of many organisms, including the protozoan parasites that are the causative agent of malaria, Plasmodium spp. As with other peptidases, subtilase proteolytic activity has to be tightly regulated in order to prevent potentially deleterious uncontrolled protein degradation. Maturation of most subtilases requires the presence of an N-terminal propeptide that facilitates folding of the catalytic domain. Following its proteolytic cleavage, the propeptide acts as a transient, tightly bound inhibitor until its eventual complete removal to generate active protease. Here we report the identification of a stand-alone malaria parasite propeptide-like protein, called SUB1-ProM, encoded by a conserved gene that lies in a highly syntenic locus adjacent to three of the four subtilisin-like genes in the Plasmodium genome. Template-based modelling and ab initio structure prediction showed that the SUB1-ProM core structure is most similar to the X-ray crystal structure of the propeptide of SUB1, an essential parasite subtilase that is discharged into the parasitophorous vacuole (PV) to trigger parasite release (egress) from infected host cells. Recombinant Plasmodium falciparum SUB1-ProM was found to be a fast-binding, potent inhibitor of P. falciparum SUB1, but not of the only other essential blood-stage parasite subtilase, SUB2, or of other proteases examined. Mass-spectrometry and immunofluorescence showed that SUB1-ProM is expressed in the PV of blood stage P. falciparum, where it may act as an endogenous inhibitor to regulate SUB1 activity in the parasite

The malaria parasite egress protease SUB1 is a calcium-dependent redox switch subtilisin.

Malaria is caused by a protozoan parasite that replicates within an intraerythrocytic parasitophorous vacuole. Release (egress) of malaria merozoites from the host erythrocyte is a highly regulated and calcium-dependent event that is critical for disease progression. Minutes before egress, an essential parasite serine protease called SUB1 is discharged into the parasitophorous vacuole, where it proteolytically processes a subset of parasite proteins that play indispensable roles in egress and invasion. Here we report the first crystallographic structure of Plasmodium falciparum SUB1 at 2.25 Å, in complex with its cognate prodomain. The structure highlights the basis of the calcium dependence of SUB1, as well as its unusual requirement for interactions with substrate residues on both prime and non-prime sides of the scissile bond. Importantly, the structure also reveals the presence of a solvent-exposed redox-sensitive disulphide bridge, unique among the subtilisin family, that likely acts as a regulator of protease activity in the parasite

Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.

Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1

Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum.

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 microg x l(-1) to >400 microg x l(-1) by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale 'piggyback' inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values 80% at a concentration of 10 microM

Peptidic boronic acids are potent cell-permeable inhibitors of the malaria parasite egress serine protease SUB1.

Malaria is a devastating infectious disease, which causes over 400,000 deaths per annum and impacts the lives of nearly half the world's population. The causative agent, a protozoan parasite, replicates within red blood cells (RBCs), eventually destroying the cells in a lytic process called egress to release a new generation of parasites. These invade fresh RBCs to repeat the cycle. Egress is regulated by an essential parasite subtilisin-like serine protease called SUB1. Here, we describe the development and optimization of substrate-based peptidic boronic acids that inhibit Plasmodium falciparum SUB1 with low nanomolar potency. Structural optimization generated membrane-permeable, slow off-rate inhibitors that prevent P falciparum egress through direct inhibition of SUB1 activity and block parasite replication in vitro at submicromolar concentrations. Our results validate SUB1 as a potential target for a new class of antimalarial drugs designed to prevent parasite replication and disease progression