—In this paper, we investigate a microfluidic based sensing device for cell membrane permeability measurements in real time with applications in rapid assessment of red blood cell (RBC) quality at the individual cell level. The microfluidic chip was designed with unique abilities to line up the RBCs in the centerline of the microchannel using positive dielectrophoresis (p-DEP) forces, rapid mixing of RBCs with various media (e.g. containing permeating or nonpermeating solutes) injected from different inlets to achieve high mixing efficiency. The chip detects the impedance values of the RBCs within 0.19 s from the start of mixing with other media, at ten electrodes along the length of the channel and enables time series measurements of volume change of individual cell caused by cell osmosis in anisosmotic fluids over a 0.8 s postmixing timespan. This technique enables estimating water permeability of individual cell accurately. Here we first present confirmation of a linear voltage-diameter relationship in polystyrene bead standards. Next, we show that under equilibrium conditions, the voltage-volume relationship in rat red blood cells (RBCs) is linear, corresponding to previously published Boyle van ‘t Hoff plots. Using rat cells as a model for human, we present the first measurement of water permeability in individual red blood cells and confirm that these data align with previously published population level values for human RBC. Finally, we present preliminary evidence for possible application of our device to identify individual RBCs infected with Plasmodium falciparum malaria parasites. Future developments using this device will address the use of whole blood with non-homogenous cell populations, a task currently performed by clinical Coulter counters.

Erythrocyte Binding Antigen of 175 kDa (EBA-175) has a well-defined role in binding to glycophorin A (GpA) during Plasmodium falciparum invasion of erythrocytes. However, EBA-175 is shed post invasion and a role for this shed protein has not been defined. We show that EBA-175 shed from parasites promotes clustering of RBCs, and EBA-175-dependent clusters occur in parasite culture. Region II of EBA-175 is sufficient for clustering RBCs in a GpA-dependent manner. These clusters are capable of forming under physiological flow conditions and across a range of concentrations. EBA-175-dependent RBC clustering provides daughter merozoites ready access to uninfected RBCs enhancing parasite growth. Clustering provides a general method to protect the invasion machinery from immune recognition and disruption as exemplified by protection from neutralizing antibodies that target AMA-1 and RH5. These findings provide a mechanistic framework for the role of shed proteins in RBC clustering, immune evasion, and malaria.

Proteases of the malaria parasite Plasmodium falciparum have long been investigated as drug targets. The P. falciparum genome encodes 10 aspartic proteases called plasmepsins, which are involved in diverse cellular processes. Most have been studied extensively but the functions of plasmepsins IX and X (PMIX and PMX) were unknown. Here we show that PMIX is essential for erythrocyte invasion, acting on rhoptry secretory organelle biogenesis. In contrast, PMX is essential for both egress and invasion, controlling maturation of the subtilisin-like serine protease SUB1 in exoneme secretory vesicles. We have identified compounds with potent antimalarial activity targeting PMX, including a compound known to have oral efficacy in a mouse model of malaria.

To mediate its survival and virulence, the malaria parasite Plasmodium falciparum exports hundreds of proteins into the host erythrocyte. To enter the host cell, exported proteins must cross the parasitophorous vacuolar membrane (PVM) within which the parasite resides, but the mechanism remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been suggested to be involved for at least one class of exported proteins; however, direct functional evidence for this has been elusive. Here we show that export across the PVM requires heat shock protein 101 (HSP101), a ClpB-like AAA+ ATPase component of PTEX. Using a chaperone auto-inhibition strategy, we achieved rapid, reversible ablation of HSP101 function, resulting in a nearly complete block in export with substrates accumulating in the vacuole in both asexual and sexual parasites. Surprisingly, this block extended to all classes of exported proteins, revealing HSP101-dependent translocation across the PVM as a convergent step in the multi-pathway export process. Under export-blocked conditions, association between HSP101 and other components of the PTEX complex was lost, indicating that the integrity of the complex is required for efficient protein export. Our results demonstrate an essential and universal role for HSP101 in protein export and provide strong evidence for PTEX function in protein translocation into the host cell.

Background: Kae1 proteins are universally conserved and are regulated by a variety of protein-protein interactions. Results: P. falciparum expresses two Kae1 proteins with distinct localizations and atypical binding partners. Conclusion: Kae1api is essential and a potential regulator of ribosome activity. Significance: The Kae1 protein family likely has an expanded role outside of its known tRNA-modifying activity. The universally conserved kinase-associated endopeptidase 1 (Kae1) protein family has established roles in N6-threonylcarbamoyl adenosine tRNA modification, transcriptional regulation, and telomere homeostasis. These functions are performed in complex with a conserved core of protein binding partners. Herein we describe the localization, essentiality, and protein-protein interactions for Kae1 in the human malaria parasite Plasmodium falciparum. We found that the parasite expresses one Kae1 protein in the cytoplasm and a second protein in the apicoplast, a chloroplast remnant organelle involved in fatty acid, heme, and isoprenoid biosynthesis. To analyze the protein interaction networks for both Kae1 pathways, we developed a new proteomic cross-validation approach. This strategy compares immunoprecipitation-MS data sets across different cellular compartments to enrich for biologically relevant protein interactions. Our results show that cytoplasmic Kae1 forms a complex with Bud32 and Cgi121 as in other organisms, whereas apicoplast Kae1 makes novel interactions with multiple proteins in the apicoplast. Quantitative RT-PCR and immunoprecipitation studies indicate that apicoplast Kae1 and its partners interact specifically with the apicoplast ribosomes and with proteins involved in ribosome function. Together, these data indicate an expanded, apicoplast-specific role for Kae1 in the parasite.

One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. Here we report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of asparagine. PfHsp110c gene-knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes.

The newly synthesized benzimidazole compounds were suggested to be inhibitors of Plasmodium falciparum plasmepsin II and human cathepsin D by virtual screening of an internal library of synthetic compounds. This was confirmed by enzyme inhibition studies that gave IC50 values in the low micromolar range (2–48 μM). Ligand docking studies with plasmepsin II predicted binding of benzimidazole compounds at the center of the extended substrate-binding cleft. According to the plausible mode of binding, the pyridine ring of benzimidazole compounds interacted with S1′ subsite residues whereas the acetophenone moiety was in contact with S1–S3 subsites of plasmepsin II active center. The benzimidazole derivatives were evaluated for capacity to inhibit the growth of intraerythrocytic P. falciparum in culture. Four benzimidazole compounds inhibited parasite growth at ⩽3 μM. The most active compound 10, 1-(4-phenylphenyl)-2[2-(pyridinyl-2-yl)-1,3-benzdiazol-1-yl]ethanone showed an IC50 of 160 nM. The substitution of a phenyl group and a chlorine atom at the para position of the acetophenone moiety were shown to be crucial for antiplasmodial activity.

One in four proteins in Plasmodium falciparum contains asparagine repeats. We probed the function of one such 28-residue asparagine repeat present in the P. falciparum proteasome lid subunit 6, Rpn6. To aid our efforts, we developed a regulatable, fluorescent affinity (RFA) tag that allows cellular localization, manipulation of cellular levels, and affinity isolation of a chosen protein in P. falciparum. The tag comprises a degradation domain derived from Escherichia coli dihydrofolate reductase together with GFP. The expression of RFA-tagged proteins is regulated by the simple folate analog trimethoprim (TMP). Parasite lines were generated in which full-length Rpn6 and an asparagine repeat-deletion mutant of Rpn6 were fused to the RFA tag. The knockdown of Rpn6 upon removal of TMP revealed that this protein is essential for ubiquitinated protein degradation and for parasite survival, but the asparagine repeat is dispensable for protein expression, stability, and function. The data point to a genomic mechanism for repeat perpetuation rather than a positive cellular role. The RFA tag should facilitate study of the role of essential genes in parasite biology.

The Plasmodium falciparum genome encodes a single calpain. By generating P. falciparum clones expressing C-terminally tagged calpain, we localized this protein to the nucleolus. Pf_calpain possesses an unusual and long N-terminal domain in which we identified three subregions that are highly conserved among Plasmodium species. Two have putative targeting signals: a myristoylation motif and a nuclear localization sequence. We assessed their functionality. Our data show that the nuclear localization sequence is an active nuclear import motif that contains an embedded signal conferring nucleolar localization on various chimeras. The N-terminus is myristoylated at Gly2 and palmitoylated at Cys3 and Cys22. Palmitoylation status has an important role in dictating P. falciparum calpain localization. The targeting signals function in mammalian cells as well as in the parasite. P. falciparum calpain is a unique nucleolar protein with an interesting mechanism of targeting.

During their intraerythrocytic development, malaria parasites export hundreds of proteins to remodel their host cell. Nutrient acquisition, cytoadherence and antigenic variation are among the key virulence functions effected by this erythrocyte takeover. Proteins destined for export are synthesized in the endoplasmic reticulum (ER) and cleaved at a conserved (PEXEL) motif, which allows translocation into the host cell via an ATP-driven translocon called the PTEX complex. We report that plasmepsin V, an ER aspartic protease with distant homology to the mammalian processing enzyme BACE, recognizes the PEXEL motif and cleaves it at the correct site. This enzyme is essential for parasite viability and ER residence is essential for its function. We propose that plasmepsin V is the PEXEL protease and is an attractive enzyme for antimalarial drug development.

Plasmodium falciparum encodes a single calpain that has a distinct domain composition restricted to alveolates. To evaluate the potential of this protein as a drug target, we assessed its essentiality. Both gene disruption by double cross-over and gene truncation by single cross-over recombination failed. We were also unable to achieve allelic replacement by using a missense mutation at the catalytic cysteine codon, although we could obtain synonymous allelic replacement parasites. These results suggested that the calpain gene and its proteolytic activity are important for optimal parasite growth. To gain further insight into its biological role, we used the FKBP degradation domain system to generate a fusion protein whose stability in transfected parasites could be modulated by a small FKBP ligand, Shield1 (Shld1). We made a calpain-GFP-FKBP fusion through single cross-over integration at the endogenous calpain locus. Calpain levels were knocked down and parasite growth was greatly impaired in the absence of Shld1. Parasites were delayed in their ability to transition out of the ring stage and in their ability to progress to the S phase. Calpain is required for cell cycle progression in Plasmodium parasites and appears to be an attractive drug target. We have shown that regulated knockdowns are possible in P. falciparum and can be useful for evaluating essentiality and function.

The synthesis, characterization, and antimalarial potency of an amine-phenol complex of gallium(III), [{1,12-bis(2-hydroxy-3-methoxy-5-(quinolin-3-yl)-benzyl)-1,5,8,12-tetraazadodecane}-gallium(III)]+, [Ga-3-M-5-Quadd]+ (7) is described; a novel agent that targets Plasmodium falciparum strains.

Extracts and isolated compounds from seedlings of Ailanthus altissima, were assessed for antiplasmodial activity in vitro. Two quassinoids, ailanthone and 6alpha-tigloyloxychaparrinone, isolated from the active extracts showed activity against both chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum in vitro. Only ailanthone demonstrated low toxicity against the Vero cell line (kidney cells from the African green monkey). This is the first report of the isolation and antiplasmodial activity of 6alpha-tigloyloxychaparrinone from this species.

Emergence of chloroquine (CQ)-resistant Plasmodium falciparum strains necessitates discovery of potent and inexpensive antimalarial drugs. The high cost of new drugs negatively impacts their access and distribution in the regions of the world with scarce economic resources. While exploring structure-activity relationships, using gallium(III) as a surrogate marker for iron(III), we found cationic, and moderately hydrophobic, compounds, [[1,12-bis(2-hydroxy-3-ethyl-benzyl)-1,5,8,12-tetraazadodecane]metal(III)](+) (metal = Fe(III) and Ga(III); [Fe-3-Eadd](+), 3; [Ga-3-Eadd](+), 4), that possessed antimalarial activity. Crystal structure analyses revealed octahedral geometry for these complexes. The RP-HPLC analysis, after incubation in PBS or HEPES buffer (pH 7.4) at 37 degrees C for 3 days, detected only parental compounds thereby providing evidence for stability under physiological conditions. Both 3 and 4 demonstrated promising half-maximum inhibitory concentration (IC(50)) values of approximately 80 and 86 nM in the CQ-sensitive HB-3 line, respectively. However, both 3 and 4 were found to possess elevated IC(50) values of 2.5 and 0.8 microM, respectively, in the CQ-resistant Dd2 line, thus displaying preferential cytotoxicity toward the CQ-sensitive HB3 line. In cultured parasites, 3 and 4 targeted hemozoin formation. Thus, these compounds acted similarly to chloroquine with regard to action and resistance, despite the lack of structural similarity to quinolines. Finally, similarity in coordination chemistry, stability, and antimalarial cytotoxicity profiles indicated that gallium(III) ion can serve as a template for iron(III) in structure elucidation of active molecules in solution.

Emergence of chloroquine-resistant Plasmodium falciparum strains necessitates discovery of novel antimalarial drugs, especially if the agents can be synthesized from commercially available, inexpensive precursors via short synthetic routes. While exploring structure-activity relationships, we found a gallium(III) complex, [(1,12-bis(2-hydroxy-5-methoxybenzyl)-1,5,8,12-tetraazadodecane)-gallium(III)](+) [Ga-5-Madd](+), 1, that possessed antimalarial efficacy. Like previously reported complexes, the crystal structure of 1 revealed gallium(III) in a symmetrical octahedral environment surrounded by four secondary amine nitrogen atoms in equatorial plane and two axial oxygen atoms. In contrast to a previously reported complex, [Ga-3-Madd](+), this novel metallo-antimalarial 1 possessed modest efficacy against chloroquine-sensitive HB3 Plasmodium lines. Thus, slight variation in the positions of methoxy functionalities on the aromatic rings of the organic scaffold dramatically altered specificity thereby suggesting a targeted (e.g., transporter- or receptor-mediated) rather than non-specific (e.g., pH or other gradient-mediated) mechanism of action for these agents.

The heme polymer hemozoin is produced in the food vacuole (fv) of the parasite after hemoglobin proteolysis and is the target of the drug chloroquine. A candidate heme polymerase, the histidine-rich protein II (HRPII), is proposed to be delivered to the fv by ingestion of the infected-red cell cytoplasm. Here we show that 97% of endogenous Plasmodium falciparum (Pf) HRPII (PfHRPII) is secreted as soluble protein in the periphery of the red cell and avoids endocytosis by the parasite, and 3% remains membrane-bound within the parasite. Transfected cells release 90% of a soluble transgene PfHRPIImyc into the red cell periphery and contain 10% membrane bound within the parasite. Yet these cells show a minor reduction in hemozoin production and IC(50) for chloroquine. They also show decreased transport of resident fv enzyme PfPlasmepsin I, the endoplasmic reticulum (ER) marker PfBiP, and parasite-associated HRPII to fvs. Instead, all three proteins accumulate in the ER, although there is no defect in protein export from the parasite. The data suggest that novel mechanisms of sorting (i) soluble antigens like HRPII in the red cell cytoplasm and (ii) fv-bound membrane complexes in the ER regulate parasite digestive processes.

Intraerythrocytic malaria parasites replicate by the process of schizogeny, during which time they copy their genetic material and package it into infective merozoites. These merozoites must then exit the host cell to invade new erythrocytes. To better characterize the events of merozoite escape, erythrocytes containing Plasmodium falciparum schizonts were cultured in the presence of the cysteine protease inhibitor, L-transepoxy-succinyl-leucylamido-(4-guanidino)butane (E64). This treatment resulted in the accumulation of extraerythrocytic merozoites locked within a thin, transparent membrane. Immunomicroscopy demonstrated that the single membrane surrounding the merozoites is not erythrocytic but rather is derived from the parasitophorous vacuolar membrane (PVM). Importantly, structures identical in appearance can be detected in untreated cultures at low frequency. Further studies revealed that (i) merozoites from the PVM-enclosed merozoite structures (PEMS) are invasive, viable, and capable of normal development; (ii) PEMS can be purified easily and efficiently; and (iii) when PEMS are added to uninfected red blood cells, released merozoites can establish a synchronous wave of infection. These observations suggest that L-transepoxy-succinyl-leucylamido-(4-guanidino)butane (E64) causes an accumulation of an intermediate normally present during the process of rupture. We propose a model for the process of rupture: merozoites enclosed within the PVM first exit from the host erythrocyte and then rapidly escape from the PVM by a proteolysis-dependent mechanism.