A P-glycoprotein homologue of Plasmodium falciparum is localized on the digestive vacuole

Resistance to chloroquine in Plasmodium falciparum bears a striking similarity to the multi-drug resistance (MDR) phenotype of mammalian tumor cells which is mediated by overexpression of P-glycoprotein. We show here that the P. falciparum homologue of the P-glycoprotein (Pgh1) is a 160,000-D protein that is expressed throughout the asexual erythrocytic life cycle of the parasite. Quantitative immunoblotting analysis has shown that the protein is expressed at approximately equal levels in chloroquine resistant and sensitive isolates suggesting that overexpression of Pgh1 is not essential for chloroquine resistance. The chloroquine-resistant cloned line FAC8 however, does express approximately threefold more Pgh1 protein than other isolates which is most likely because of the increased pfmdr1 gene copy number present in this isolate. Immunofluorescence and immunoelectron microscopy has demonstrated that Pgh1 is localized on the membrane of the digestive vacuole of mature parasites. This subcellular localization suggests that Pgh1 may modulate intracellular chloroquine concentrations and has important implications for the normal physiological function of this protein.     

Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function

Knockout mutants of Plasmodium falciparum lacking pfpm1, pfpm2 and pfhap (triple-PM KO), and mutants lacking all four digestive vacuole (DV) plasmepsins (pfpm4, pfpm1, pfpm2 and pfhap; quadruple-PM KO), were prepared by double cross-over integration effecting chromosomal deletions of up to 14.6 kb. The triple-PM KO was similar to the parental line (3D7) in growth rate, morphology and sensitivity to proteinase inhibitors. The quadruple-PM KO showed a significantly slower rate of growth in standard medium, which manifested as delayed schizont maturation accompanied by reduced formation of haemozoin. In amino acid-limited medium, the reduction in growth rate of the quadruple-PM KO was pronounced. The sensitivity of both the triple- and quadruple-PM KOs to six different HIV aspartic proteinase inhibitors was comparable to that of 3D7, thus establishing that the DV plasmepsins were not the primary targets of the antimalarial activity of these clinically important compounds. Electron microscopic analysis revealed the presence of multilamellar bodies resembling ceroid in the DV of the quadruple-PM KO, and intermediates of the autophagic pathway accumulated as determined by Western blot analysis. Thus, the DV plasmepsins, although not essential, contribute significantly to the fitness of the parasite and are required for efficient degradation of endosomal vesicles delivered to the DV.     

Genetic disruption of the Plasmodium falciparum digestive vacuole plasmepsins demonstrates their functional redundancy

The digestive vacuole plasmepsins PfPM1, PfPM2, PfPM4, and PfHAP (a histoaspartic proteinase) are 4 aspartic proteinases among 10 encoded in the Plasmodium falciparum malarial genome. These have been hypothesized to initiate and contribute significantly to hemoglobin degradation, a catabolic function essential to the survival of this intraerythrocytic parasite. Because of their perceived significance, these plasmepsins have been proposed as potential targets for antimalarial drug development. To test their essentiality, knockout constructs were prepared for each corresponding gene such that homologous recombination would result in two partial, nonfunctional gene copies. Disruption of each gene was achieved, as confirmed by PCR, Southern, and Northern blot analyses. Western and two-dimensional gel analyses revealed the absence of mature or even truncated plasmepsins corresponding to the disrupted gene. Reduced growth rates were observed with PfPM1 and PfPM4 knockouts, indicating that although these plasmepsins are not essential, they are important for parasite development. Abnormal mitochondrial morphology also appeared to accompany loss of PfPM2, and an abundant accumulation of electron-dense vesicles in the digestive vacuole was observed upon disruption of PfPM4; however, those phenotypes only manifested in about a third of the disrupted cells. The ability to compensate for loss of individual plasmepsin function may be explained by close similarity in the structure and active site of these four vacuolar enzymes. Our data imply that drug discovery efforts focused on vacuolar plasmepsins must incorporate measures to develop compounds that can inhibit two or more of this enzyme family.     

The role of Plasmodium falciparum food vacuole plasmepsins

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.     

Protein targeting to the parasitophorous vacuole membrane of Plasmodium falciparum

Red blood cell (RBC) invasion and parasitophorous vacuole (PV) formation by Plasmodium falciparum are critical for the development and pathogenesis of malaria, a continuing global health problem. Expansion of the PV membrane (PVM) during growth is orchestrated by the parasite. This is particularly important in mature RBCs, which lack internal organelles and no longer actively synthesize membranes. Pfs16, a 16-kDa integral PVM protein expressed by gametocytes, was chosen as a model for studying the trafficking of material from the parasite across the PV space to the PVM. The locations of Pfs16-green fluorescent protein (GFP) reporter proteins containing distinct regions of Pfs16 were tracked from RBC invasion to emergence. Inclusion of the 53 C-terminal amino acids (aa) of Pfs16 to a GFP reporter construct already containing the N-terminal secretory signal sequence was sufficient for targeting to and retention on the PVM. An amino acid motif identified in this region was also found in seven other known PVM proteins. Removal of the 11 C-terminal aa did not affect PVM targeting, but membrane retention was decreased. Additionally, during emergence from the PVM and RBC, native Pfs16 and the full-length Pfs16-GFP reporter protein were found to concentrate on the ends of the gametocyte. Capping was not observed in constructs lacking the amino acids between the N-terminal secretory signal sequence and the transmembrane domain, suggesting that this region, which is not required for PVM targeting, is involved in capping. This is the first report to define the amino acid domains required for targeting to the P. falciparum PVM.     

A contractile vacuole complex is involved in osmoregulation in Trypanosoma cruzi

Acidocalcisomes are dense, acidic organelles with a high concentration of phosphorus present as pyrophosphate and polyphosphate complexed with calcium and other cations. Acidocalcisomes have been linked to the contractile vacuole complex in Chlamydomonas reinhardtii, Dictyostelium discoideum, and Trypanosoma cruzi. A microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex in T. cruzi results in translocation of aquaporin and the resulting water movement which, in addition to swelling of acidocalcisomes, is responsible for the volume reversal not accounted for by efflux of osmolytes. Polyphosphate hydrolysis occurs during hyposmotic stress, probably increasing the osmotic pressure of the contractile vacuole and facilitating water movement.     

Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.     

Raman imaging of hemozoin within the food vacuole of Plasmodium falciparum trophozoites

Micro-Raman spectra of hemozoin encapsulated within the food vacuole of a Plasmodium falciparum-infected erythrocyte are presented. The spectrum of hemozoin is identical to the spectrum of beta-hematin at all applied excitation wavelengths. The unexpected observation of dramatic band enhancement of A(1g) modes including nu(4) (1374 cm(-1)) observed when applying 780 nm excitation enabled Raman imaging of hemozoin in the food vacuole. This unusual enhancement, resulting from excitonic coupling between linked porphyrin moieties in the extended porphyrin array, enables the investigation of hemozoin within its natural environment for the first time.     

Rab14 is involved in membrane trafficking between the Golgi complex and endosomes

Rab GTPases are localized to various intracellular compartments and are known to play important regulatory roles in membrane trafficking. Here, we report the subcellular distribution and function of Rab14. By immunofluorescence and immunoelectron microscopy, both endogenous as well as overexpressed Rab14 were localized to biosynthetic (rough endoplasmic reticulum, Golgi, and trans-Golgi network) and endosomal compartments (early endosomal vacuoles and associated vesicles). Notably overexpression of Rab14Q70L shifted the distribution toward the early endosome associated vesicles, whereas the S25N and N124I mutants induced a shift toward the Golgi region. A similar, although less pronounced, redistribution of the transferrin receptor was also observed in cells overexpressing Rab14 mutants. Impairment of Rab14 function did not however affect transferrin uptake or recycling kinetics. Together, these findings suggest that Rab14 is involved in the biosynthetic/recycling pathway between the Golgi and endosomal compartments.     

Genetic ablation of a Maurer's cleft protein prevents assembly of the Plasmodium falciparum virulence complex

The malaria parasite Plasmodium falciparum assembles knob structures underneath the erythrocyte membrane that help present the major virulence protein, P. falciparum erythrocyte membrane protein-1 (PfEMP1). Membranous structures called Maurer's clefts are established in the erythrocyte cytoplasm and function as sorting compartments for proteins en route to the RBC membrane, including the knob-associated histidine-rich protein (KAHRP), and PfEMP1. We have generated mutants in which the Maurer's cleft protein, the ring exported protein-1 (REX1) is truncated or deleted. Removal of the C-terminal domain of REX1 compromises Maurer's cleft architecture and PfEMP1-mediated cytoadherance but permits some trafficking of PfEMP1 to the erythrocyte surface. Deletion of the coiled-coil region of REX1 ablates PfEMP1 surface display, trapping PfEMP1 at the Maurer's clefts. Complementation of mutants with REX1 partly restores PfEMP1-mediated binding to the endothelial cell ligand, CD36. Deletion of the coiled-coil region or complete deletion of REX1 is tightly associated with the loss of a subtelomeric region of chromosome 2, encoding KAHRP and other proteins. A KAHRP-green fluorescent protein (GFP) fusion expressed in the REX1-deletion parasites shows defective trafficking. Thus, loss of functional REX1 directly or indirectly ablates the assembly of the P. falciparum virulence complex at the surface of host erythrocytes.     

Intravacuolar proteolysis in Plasmodium falciparum digestive vacuoles is similar to intralysosomal proteolysis in mammalian cells

Hemoglobin of intact human erythrocytes was labelled with [125I]iodide and these cells were inoculated with late-stage cultures of Plasmodium falciparum. Subcellular parasite particulate material containing intact digestive vacuoles filled with 125I-labelled hemoglobin was incubated in sucrose-buffer medium and degradation of labelled intravacuolar hemoglobin was measured by precipitation with trichloroacetic acid. Proteolysis was maximal at pH 5.0 or in the presence of MgATP at pH 8.0. The stimulatory effect of MgATP was probably due to energization of a proton pump activity as reported by others (Krogstad, D.J., Schlesinger, P.H. and Gluzman, I.Y. (1984) J. Cell Biol. 101, 2302-2309). Proteolysis was also inhibited by ionophores and antimalarials. These results suggest that P. falciparum digestive vacuoles have an ATP-dependent acidification mechanism similar to mammalian lysosomes but with some exceptions. The properties of this intravacuolar proteolysis were remarkably similar to intralysosomal proteolysis in mouse liver or kidney preparations.     

Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum

A family of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole. To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II-GFP chimera. By taking advantage of green fluorescent protein fluorescence in live parasites, the ultrastructural resolution of immunoelectron microscopy, and inhibitors of trafficking and PM maturation, we have investigated the biosynthetic path leading to mature PM II in the food vacuole. Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.     

2-Cys peroxiredoxin of Plasmodium falciparum is involved in resistance to heat stress of the parasite

In the cytoplasm of Plasmodium falciparum, two peroxiredoxins: PfTPx-1 and Pf1-Cys-Prx, are expressed at different time-points of the parasite cell cycle during the intraerythrocytic stage. In the present study, to gain insight into the functions of Prxs in the cytoplasm of P. falciparum, we investigated the heat stress sensitivity of the previously established PfTPx-1 KO line and found that PfTPx-1 disruption renders the parasite hypersensitive to heat stress. In addition, we established Pf1-Cys-Prx knockout (KO) parasite lines. The phenotypes of Pf1-Cys-Prx KO lines were different to those of the PfTPx-1 KO line and did not show hypersensitivity to reactive oxygen species, reactive nitrogen species, chloroquine or heat stress. These results suggest that the function of Pf1-Cys-Prx in the parasite cytoplasm is independent from that of PfTPx-1. The hyperthermal protective function of the PfTPx-1 is obviously important for the parasite physiology in the human patient body, in which it must survive repeated incidences of fever.     

Effects on growth, hemoglobin metabolism and paralogous gene expression resulting from disruption of genes encoding the digestive vacuole plasmepsins of Plasmodium falciparum

Four of the plasmepsins of Plasmodium falciparum are localised in the digestive vacuole (DV) of the asexual blood stage parasite (PfPM1, PfPM2, PfPM4 and PfHAP), and each of these aspartic proteinases has been successfully targeted by gene disruption. This study describes further characterisation of the single-plasmepsin knockout mutants, and the creation and characterisation of double-plasmepsin knockout mutants lacking complete copies of pfpm2 and pfpm1 or pfhap and pfpm2. Double-plasmepsin knockout mutants were created by transfecting pre-existing knockout mutants with a second plasmid knockout construct. PCR and Southern blot analysis demonstrate the integration of a large concatamer of each plasmid construct into the targeted gene. All mutants have been characterised to assess the involvement of the DV plasmepsins in sustaining growth during the asexual blood stage. Analyses reaffirmed that knockout mutants Deltapfpm1 and Deltapfpm4 had lower replication rates in the asexual erythrocytic stage than the parental line (Dd2), but double-plasmepsin knockout mutants lacking intact copies of either pfpm2 and pfpm1, or pfpm2 and pfhap, had normal growth rates compared with Dd2. The amount of crystalline hemozoin produced per parasite during the asexual cycle was measured in each single-plasmepsin knockout to estimate the effect of each DV plasmepsin on hemoglobin digestion. Only Deltapfpm4 had a statistically significant reduction in hemozoin accumulation, indicating that hemoglobin digestion was impaired in this mutant. In the single-plasmepsin knockouts, no statistically significant differences were found in the steady state levels of mRNA from the remaining intact DV plasmepsin genes. Disruption of a DV plasmepsin gene does not affect the accumulation of mRNA encoding the remaining paralogous plasmepsins, and Western blot analysis confirmed that the accumulation of the paralogous plasmepsins in each knockout mutant was similar among all clones examined.     

Helicases involved in splicing from malaria parasite Plasmodium falciparum

An interesting element of eukaryotic genomes is the large quantity of non-coding intervening sequences commonly known as introns, which regularly interrupt functional genes and therefore must be removed prior to the use of genetic information by the cell. After splicing, the mature RNA is exported from the nucleus to the cytoplasm. Any error in the process of recognition and removal of introns, or splicing, would lead to change in genetic message and thus has potentially catastrophic consequences. Thus splicing is a highly complex essential step in eukaryotic gene expression. It takes place in spliceosome, which is a dynamic RNA-protein complex made of snRNPs and non-snRNP proteins. The splicing process consists of following sequential steps: spliceosome formation, the first transesterification and second transesterification reactions, release of the mature mRNA and recycling of the snRNPs. The spliceosomal components produce a complex network of RNA-RNA, RNA-protein and protein-protein interactions and spliceosome experience remodeling during each splicing cycle. Helicases are essentially required at almost each step for resolution of RNA-RNA and/or RNA-protein interactions. RNA helicases share a highly conserved helicase domain which includes the motif DExD/H in the single letter amino acid code. This article will focus on members of the DExD/H-box proteins involved specially in splicing in the malaria parasite Plasmodium falciparum.     

Protein transport across the parasitophorous vacuole of Plasmodium falciparum: into the great wide open

The human malaria parasite Plasmodium falciparum resides and multiplies within a membrane-bound vacuole in the cytosol of its host cell, the mature human erythrocyte. To enable the parasite to complete its intraerythrocytic life cycle, a large number of parasite proteins are synthesized and transported from the parasite to the infected cell. To gain access to the erythrocyte, parasite proteins must first cross the membrane of the parasitophorous vacuole (PVM), a process that is not well understood at the mechanistic level. Here, we review past and current literature on this topic, and make tentative predictions about the nature of the transport machinery required for transport of proteins across the PVM, and the molecular factors involved.     

Falcipain cysteine proteases require bipartite motifs for trafficking to the Plasmodium falciparum food vacuole

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 hydrolyze hemoglobin in an acidic food vacuole to provide amino acids for erythrocytic malaria parasites. Trafficking to the food vacuole has not been well characterized. To study trafficking of falcipains, which include large membrane-spanning prodomains, we utilized chimeras with portions of the proteases fused to green fluorescent protein. The prodomains of falcipain-2 and falcipain-3 were sufficient to target green fluorescent protein to the food vacuole. Using serial truncations, deletions, and point mutations, we showed that both a 20-amino acid stretch of the lumenal portion and a 10-amino acid stretch of the cytoplasmic portion of the falcipain-2 prodomain were required for efficient food vacuolar trafficking. Mutants with altered trafficking were arrested at the plasma membrane, implicating trafficking via this structure. Our results indicate that falcipains utilize a previously undescribed bipartite motif-dependent mechanism for targeting to a hydrolytic organelle, suggesting inhibition of this unique mechanism as a new means of antimalarial chemotherapy.     

ATP synthase complex of Plasmodium falciparum: dimeric assembly in mitochondrial membranes and resistance to genetic disruption

The rotary nanomotor ATP synthase is a central player in the bioenergetics of most organisms. Yet the role of ATP synthase in malaria parasites has remained unclear, as blood stages of Plasmodium falciparum appear to derive ATP largely through glycolysis. Also, genes for essential subunits of the F(O) sector of the complex could not be detected in the parasite genomes. Here, we have used molecular genetic and immunological tools to investigate the localization, complex formation, and functional significance of predicted ATP synthase subunits in P. falciparum. We generated transgenic P. falciparum lines expressing seven epitope-tagged canonical ATP synthase subunits, revealing localization of all but one of the subunits to the mitochondrion. Blue native gel electrophoresis of P. falciparum mitochondrial membranes suggested the molecular mass of the ATP synthase complex to be greater than 1 million daltons. This size is consistent with the complex being assembled as a dimer in a manner similar to the complexes observed in other eukaryotic organisms. This observation also suggests the presence of previously unknown subunits in addition to the canonical subunits in P. falciparum ATP synthase complex. Our attempts to disrupt genes encoding β and γ subunits were unsuccessful, suggesting an essential role played by the ATP synthase complex in blood stages of P. falciparum. These studies suggest that, despite some unconventional features and its minimal contribution to ATP synthesis, P. falciparum ATP synthase is localized to the parasite mitochondrion, assembled as a large dimeric complex, and is likely essential for parasite survival.