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IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

The sexual phase of the malaria parasite Plasmodium falciparum is accompanied by the coordinated expression of stage-specific adhesive proteins. Among these are six secreted proteins with multiple adhesion domains, termed P. falciparum LCCL domain-containing protein (PfCCp) proteins, which are expressed in the parasitophorous vacuole of the differentiating gametocytes and which are later associated with macrogametes. Although the majority of the PfCCp proteins are implicated in parasite development in the mosquito vector, their functions remain unknown. In the present study we investigated the molecular interactions between the PfCCp proteins during gametocyte development and emergence. Using five different gene-disruptant parasite lines, we show that the lack of one PfCCp protein leads to the loss of other PfCCp family members. Co-immunoprecipitation assays on gametocyte lysates revealed formation of complexes involving all PfCCp proteins, and affinity chromatography co-elution binding assays with recombinant PfCCp domains further indicated direct binding between distinct adhesion domains. PfCCp-coated latex beads bind to newly formed macrogametes but not to gametocytes or older macrogametes 6 or 24 h post-activation. In view of these data, we propose that the PfCCp proteins form multi-protein complexes that are exposed during gametogenesis, thereby mediating cell contacts of macrogametes.

Intraerythrocytic malaria parasites use host hemoglobin as a major nutrient source. Aspartic proteases (plasmepsins) and cysteine proteases (falcipains) function in the early steps of the hemoglobin degradation pathway. There is extensive functional redundancy within and between these protease families. Plasmepsins are synthesized as integral membrane proenzymes that are activated by cleavage from the membrane. This cleavage is mediated by a maturase activity whose identity has been elusive. We have used a combination of cell biology, chemical biology, and enzymology approaches to analyze this processing event. These studies reveal that plasmepsin processing occurs primarily via the falcipains; however, if falcipain activity is blocked, autoprocessing can take place, serving as an alternate activation system. These results establish a further level of redundancy between the protease families involved in Plasmodium hemoglobin degradation.

Non-coding RNAs (ncRNAs) play an important role in a variety of nuclear processes, including genetic imprinting, RNA interference-mediated transcriptional repression, and dosage compensation. These transcripts are thought to influence chromosome organization and, in some cases, gene expression by directing the assembly of specific chromatin modifications to targeted regions of the genome. In the malaria parasite Plasmodium falciparum, little is known about the regulation of nuclear organization or gene expression, although a notable scarcity of identifiable transcription factors encoded in its genome has led to speculation that this organism may be unusually reliant on chromatin modifications as a mechanism for regulating gene expression. To study the mechanisms that regulate chromatin structure in malaria parasites, we examined the role of ncRNAs in the assembly of chromatin at the centromeres of P. falciparum. We show that centromeric regions within the Plasmodium genome contain bidirectional promoter activity driving the expression of short ncRNAs that are localized within the nucleus and appear to associate with the centromeres themselves, strongly suggesting that they are central characters in the maintenance and function of centromeric chromatin. These observations support the hypothesis that ncRNAs play an important role in the proper organizational assembly of chromatin in P. falciparum, perhaps compensating for a lack of both regulatory transcription factors and RNA interference machinery.

Phosphatidylinositol 3-phosphate (PI3P) is a key ligand for recruitment of endosomal regulatory proteins in higher eukaryotes. Subsets of these endosomal proteins possess a highly selective PI3P binding zinc finger motif belonging to the FYVE domain family. We have identified a single FYVE domain-containing protein in Plasmodium falciparum which we term FCP. Expression and mutagenesis studies demonstrate that key residues are involved in specific binding to PI3P. In contrast to FYVE proteins in other organisms, endogenous FCP localizes to a lysosomal compartment, the malaria parasite food vacuole (FV), rather than to cytoplasmic endocytic organelles. Transfections of deletion mutants further indicate that FCP is essential for trophozoite and FV maturation and that it traffics to the FV via a novel constitutive cytoplasmic to vacuole targeting pathway. This newly discovered pathway excludes the secretory pathway and is directed by a C-terminal 44-amino acid peptide domain. We conclude that an FYVE protein that might be expected to participate in vesicle targeting in the parasite cytosol instead has a vital and functional role in the malaria parasite FV.

Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions.

The molecular mechanisms regulating cell proliferation and development during the life cycle of malaria parasites remain to be elucidated. The peculiarities of the cell cycle organization during Plasmodium falciparum schizogony suggest that the modalities of cell cycle control in this organism may differ from those in other eukaryotes. Indeed, existing data concerning Plasmodium cell cycle regulators such as cyclin-dependent kinases reveal structural and functional properties that are divergent from those of their homologues in other systems. The work presented here lies in the context of the exploitation of the recently available P. falciparum genome sequence toward the characterization of putative cell cycle regulators. We describe the in silico identification of three open reading frames encoding proteins with maximal homology to various members of the cyclin family and demonstrate that the corresponding polypeptides are expressed in the erythrocytic stages of the infection. We present evidence that these proteins possess cyclin activity by demonstrating either their association with histone H1 kinase activity in parasite extracts or their ability to activate PfPK5, a P. falciparum cyclin-dependent kinase homologue, in vitro. Furthermore, we show that RINGO, a protein with no sequence homology to cyclins but that is nevertheless a strong activator of mammalian CDK1/2, is also a strong activator of PfPK5 in vitro. This raises the possibility that "cryptic" cell cycle regulators may be found among the 50% of the open reading frames in the P. falciparum genome that display no homology to any known proteins.

Using a stage-specific cDNA library from Plasmodium falciparum we have identified a gene coding for a novel histidine-rich protein (MAHRP-1). The gene is exclusively transcribed during early erythrocyte stages and codes for a small transmembrane protein. The C-terminal region contains a polymorphic stretch of histidine-rich repeats. Fluorescence microscopy studies using polyclonal mouse sera revealed that MAHRP-1 is located at the Maurer's clefts, which represent parasite-induced structures within the cytosol of infected erythrocytes. Biochemical studies showed that recombinant MAHRP-1 binds the toxic hemoglobin degradation product, ferriprotoporphyrin (FP) with a submicromolar dissociation constant and a stoichiometry determined by the number of DHGH motifs. The bound FP has increased peroxidase-like activity and is 10-fold more susceptible to H2O2-induced degradation compared with unbound FP. These properties of MAHRP-1 suggest it may play a protective role against oxidative stress, and its location at the Maurer's clefts suggests a function in promoting the correct trafficking of exported proteins, such as P. falciparum erythrocyte membrane protein-1.

The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane.

Live cell fluorescence microscopy has been widely used to study physiological processes in the human malarial parasite Plasmodium falciparum, including pH homeostasis, Ca(2+) signaling and protein targeting. However, the reproducibility of the data is often poor. Controversial statements exist regarding cytosolic and vacuolar baseline pH, as well as regarding the subcellular localization of some of the fluorochromes used. When trying to reproduce published baseline values, we observed an unexpected light sensitivity of P. falciparum, which manifests itself in the form of a strong cytoplasmic acidification. Even short exposure times with moderate to low light intensities caused the parasite cytosol to acidify. We show that this effect arises from the selective disruption of the parasite's acidic food vacuole, brought about by lipid peroxidation initiated by light-induced generation of hydroxyl radicals. Our data suggest that heme serves as a photosensitizer in this process. Our findings have major implications for the use of live cell microscopy in P. falciparum and add a cautionary note to previous studies where live cell fluorometry has been used to determine physiological parameters in P. falciparum.

The malarial "apicoplast" derived from an algal plastid, has stimulated interest for its novel evolutionary biology and potential as a drug target. An endoplasmic reticulum-type signal sequence followed by a plastid targeting sequence are required to target proteins to the apicoplast but the pathway by which proteins are transported to the organelle is unknown. By stage regulating the expression of transgenes we show that early (0-12 h) in the parasite's development in red cells, newly synthesized green fluorescent protein that contains the plastid targeting sequence (plastid targeting sequence-green fluorescent protein (PTS-GFP)) is recruited into the parasite's secretory pathway. PTS-GFP in 0-12-h parasites is found released into the parasitophorous vacuole (PV) and in apposition with the Golgi. However, import into the apicoplast and processing to GFP does not occur until 18-36 h in development. In intermediate, 18-h parasites PTS-GFP resides in the PV. Quantitative exit of PTS-GFP from the PV and its conversion to GFP is seen at 36 h. The data suggest that: (i) import into the apicoplast is stage regulated and (ii) the PTS can signal endomembrane targeting from the PV to the apicoplast.

In the vertebrate host, the malaria parasite invades and replicates asexually within circulating erythrocytes. Parasite proteolytic enzymes play an essential but poorly understood role in erythrocyte invasion. We have identified a Plasmodium falciparum gene, denoted pfsub-1, encoding a member of the subtilisin-like serine protease family (subtilases). The pfsub-1 gene is expressed in asexual blood stages of P. falciparum, and the primary gene product (PfSUB-1) undergoes post-translational processing during secretory transport in a manner consistent with its being converted to a mature, enzymatically active form, as documented for other subtilases. In the invasive merozoite, the putative mature protease (p47) is concentrated in dense granules, which are secretory organelles located toward the apical end of the merozoite. At some point following merozoite release and completion of erythrocyte invasion, p47 is secreted from the parasite in a truncated, soluble form. The subcellular location and timing of secretion of p47 suggest that it is likely to play a role in erythrocyte invasion. PfSUB-1 is a new potential target for antimalarial drug development.

Catalytic RNA (ribozymes) suppressed the growth of the human malarial parasite Plasmodium falciparum in vitro. The phosphorothioated hammerhead ribozymes targeted unique regions of the P. falciparum carbamoyl-phosphate synthetase II gene. The P. falciparum carbamoyl-phosphate synthetase II gene encodes the first and limiting enzyme in the pathway, and its mRNA transcript contains two large insert regions absent in other carbamoyl-phosphate synthetases, including that from humans. These inserts are ideal targets for nucleic acid therapy. Exogenous delivery of ribozymes to cultures reduced malarial viability up to 55% at 0.5 microM ribozyme concentrations, which is significantly greater than control levels (5-15% reduction), suggesting a sequence-specific inhibition. This inhibition was shown to be stage-specific, with optimal inhibitions being detected after 24 h, coincident with maximal production of the carbamoyl-phosphate synthetase enzyme in the course of the life cycle of the parasite. A decrease in total carbamoyl-phosphate synthetase activity was observed only in cultures treated with the ribozymes. The task of developing alternative therapeutic agents against malaria is urgent due to the evolution of drug-resistant strains of P. falciparum, the most virulent of all human malarial parasites. Another critical issue to be addressed is the possibility of eliminating or reducing any systemic toxicity to the host, which can potentially be provided by nucleic acid therapy. This work is the first reported assessment of the ability of ribozymes as antimalarials. Ribozyme inhibition assays can also aid in identifying important antimalarial loci for chemotherapy. The malarial parasite can, in turn, be a useful in vivo host to study the catalysis and function of new ribozyme designs.