Publications (8)37.44 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: The resistance of malaria parasites to current anti-malarial drugs is an issue of major concern globally. Recently we identified a Plasmodium falciparum cell membrane aspartyl protease, which binds to erythrocyte band 3, and is involved in merozoite invasion. Here we report the complete primary structure of P. falciparum signal peptide peptidase (PfSPP), and demonstrate that it is essential for parasite invasion and growth in human erythrocytes. Gene silencing suggests that PfSPP may be essential for parasite survival in human erythrocytes. Remarkably, mammalian signal peptide peptidase inhibitors (Z-LL)(2)-ketone and L-685,458 effectively inhibited malaria parasite invasion as well as growth in human erythrocytes. In contrast, DAPT, an inhibitor of a related gamma-secretase/presenilin-1, was ineffective. Thus, SPP inhibitors specific for PfSPP may function as potent anti-malarial drugs against the blood stage malaria.
- [Show abstract] [Hide abstract] ABSTRACT: Plasmodium falciparum uses multiple host receptors to attach and invade human erythrocytes. Glycophorins have been implicated as receptors for parasite invasion in human erythrocytes. Here, we screened a phage display cDNA library of P. falciparum (FCR3, a sialic acid-dependent strain) using purified glycophorins and erythrocytes as bait. Several phage clones were identified that bound to immobilized glycophorins and contained the same 74 bp insert encoding the 7-amino acids sequence ETTLKSF. A similar screen using intact human erythrocytes in solution identified additional phage clones containing the same 7-amino acids sequence. Using ELISA and immunofluorescence, direct binding of ETTLKSF peptide to glycophorins and erythrocytes was confirmed. Pull-down and protease treatment assays suggest that ETTLKSF peptide specifically interacts with glycophorin C. The synthetic ETTLKSF peptide partially blocks merozoite invasion in human erythrocytes. Further characterization of ETTLKSF peptide could lead to the development of a novel class of inhibitors against the blood stage malaria.
- [Show abstract] [Hide abstract] ABSTRACT: We describe identification of a Plasmodium falciparum microneme protease involved in RBC invasion. From the yeast two-hybrid screening of a P. falciparum cDNA library, we have identified a 47 kDa membrane protein that interacted with the 5ABC domain of human RBC band 3. This protein shared homology with a Presenilin-type aspartyl protease, the signal peptide peptidase (SPP). An antibody raised against a predicted exposed region of this protein reacted specifically to a single band of approximately 47 kDa in the P. falciparum protein extract. Immunofluorescence microscopy suggested that this protein co-localized with the microneme protein EBA-175 in schizonts, and immunoelectron microscopy established that it is primarily localized to micronemes in merozoites. Functional characterization of Plasmodium falciparum signal peptide peptidase (PfSPP), demonstrates that an antibody to PfSPP blocks RBC invasion by P. falciparumin vitro. Native and recombinant PfSPP bound directly to the 5ABC domain of band 3 in solution and the binding of PfSPP to RBCs was chymotrypsin-sensitive, but trypsin and neuraminidase-resistant. Together, these results suggest that host band 3 interacts with PfSPP during RBC invasion presumably following parasite microneme discharge. PfSPP is the first microneme-associated intramembrane aspartyl protease identified in the apicomplexan parasites that interacts with a major transmembrane receptor on host erythrocytes.
- [Show abstract] [Hide abstract] ABSTRACT: Falcipains, the papain-family cysteine proteases of the Plasmodium falciparum, are potential drug targets for malaria parasite. Pharmacological inhibition of falcipains can block the hydrolysis of hemoglobin, parasite development, and egress, suggesting that falcipains play a key role at the blood stage of parasite life cycle. In the present study, we evaluated the anti-malarial effects of BDA-410, a novel cysteine protease inhibitor as a potential anti-malarial drug. Recombinant falcipain (MBP-FP-2B) and P. falciparum trophozoite extract containing native falcipains were used for enzyme inhibition studies in vitro. The effect of BDA-410 on the malaria parasite development in vitro as well as its anti-malarial activity in vivo was evaluated using the Plasmodium chabaudi infection rodent model. The 50% inhibitory concentrations of BDA-410 were determined to be 628 and 534nM for recombinant falcipain-2B and parasite extract, respectively. BDA-410 inhibited the malaria parasite growth in vitro with an IC(50) value of 173nM causing irreversible damage to the intracellular parasite. In vivo, the BDA-410 delayed the progression of malaria infection significantly using a mouse model of malaria pathogenesis. The characterization of BDA-410 as a potent inhibitor of P. falciparum cysteine proteases, and the demonstration of its efficacy in blocking parasite growth both in vitro and in vivo assays identifies BDA-410 is an important lead compound for the development of novel anti-malarial drugs.
- [Show abstract] [Hide abstract] ABSTRACT: Dematin and adducin are actin-binding proteins of the erythrocyte "junctional complex." Individually, they exert modest effects on erythrocyte shape and membrane stability, and their homologues are expressed widely in non-erythroid cells. Here we report generation and characterization of double knock-out mice lacking beta-adducin and the headpiece domain of dematin. The combined mutations result in altered erythrocyte morphology, increased membrane instability, and severe hemolysis. Peripheral blood analysis shows evidence of severe hemolytic anemia with reduced number of erythrocytes/hematocrit/hemoglobin and an approximately 12-fold increase in the number of circulating reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes correlates with increased osmotic fragility and reduced in vivo life span. Despite the apparently normal protein composition of the mutant erythrocyte membrane, the retention of the spectrin-actin complex in the membrane under low ionic strength conditions is significantly reduced by the double mutation. Atomic force microscopy reveals an increase in grain size and a decrease in filament number of the mutant membrane cytoskeleton, although the volume parameter is similar to wild type erythrocytes. Aggregated, disassembled, and irregular features are visualized in the mutant membrane, consistent with the presence of large protein aggregates. Importantly, purified dematin binds to the stripped inside-out vesicles in a saturable manner, and dematin-membrane binding is abolished upon pretreatment of membrane vesicles with trypsin. Together, these results reveal an essential role of dematin and adducin in the maintenance of erythrocyte shape and membrane stability, and they suggest that the dematin-membrane interaction could link the junctional complex to the plasma membrane in erythroid cells.
- [Show abstract] [Hide abstract] ABSTRACT: In Plasmodium falciparum malaria, erythrocyte invasion by circulating merozoites may occur via two distinct pathways involving either a sialic acid-dependent or -independent mechanism. Earlier, we identified two nonglycosylated exofacial regions of erythrocyte band 3 termed 5ABC and 6A as an important host receptor in the sialic acid-independent invasion pathway. 5ABC, a major segment of this receptor, interacts with the 42-kDa processing product of merozoite surface protein 1 (MSP142) through its 19-kDa C-terminal domain. Here, we show that two regions of merozoite surface protein 9 (MSP9), also known as acidic basic repeat antigen, interact directly with 5ABC during erythrocyte invasion by P. falciparum. Native MSP9 as well as recombinant polypeptides derived from two regions of MSP9 (MSP9/Δ1 and MSP9/Δ2) interacted with both 5ABC and intact erythrocytes. Soluble 5ABC added to the assay mixture drastically diminished the binding of MSP9 to erythrocytes. Recombinant MSP9/Δ1 and MSP9/Δ2 present in the culture medium blocked P. falciparum reinvasion into erythrocytes in vitro. Native MSP9 and MSP142, the two ligands binding to the 5ABC receptor, existed as a stable complex. Our results establish a novel concept wherein the merozoite exploits a specific complex of co-ligands on its surface to target a single erythrocyte receptor during invasion. This new paradigm poses a new challenge in the development of a vaccine for blood stage malaria.
- [Show abstract] [Hide abstract] ABSTRACT: We report the molecular identification of a sialic acid-independent host-parasite interaction in the Plasmodium falciparum malaria parasite invasion of RBCs. Two nonglycosylated exofacial regions of human band 3 in the RBC membrane were identified as a crucial host receptor binding the C-terminal processing products of merozoite surface protein 1 (MSP1). Peptides derived from the receptor region of band 3 inhibited the invasion of RBCs by P. falciparum. A major segment of the band 3 receptor (5ABC) bound to native MSP1(42) and blocked the interaction of native MSP1(42) with intact RBCs in vitro. Recombinant MSP1(19) (the C-terminal domain of MSP1(42)) bound to 5ABC as well as RBCs. The binding of both native MSP1(42) and recombinant MSP1(19) was not affected by the neuraminidase treatment of RBCs, but sensitive to chymotrypsin treatment. In addition, recombinant MSP1(38) showed similar interactions with the band 3 receptor and RBCs, although the interaction was relatively weak. These findings suggest that the chymotrypsin-sensitive MSP1-band 3 interaction plays a role in a sialic acid-independent invasion pathway and reveal the function of MSP1 in the Plasmodium invasion of RBCs.