A novel Plasmodium falciparum ring stage protein, REX, is located in Maurer's clefts

The asexual stages of the malaria parasite Plasmodium falciparum develop inside erythrocytes of the human host. Erythrocytes are highly specialized cells lacking organelles and trafficking machinery. The parasite must therefore establish its own transport system to export proteins and waste and import nutrients. A number of parasite-derived structures, implicated in trafficking, appear in the infected red blood cell at the late ring stage. We have identified a novel gene transcribed in ring stage parasites coding for a protein designated the ring exported protein, REX. REX is located in a red cell modification known as the Maurer's clefts, which are parasite induced structures implicated in trafficking of parasite proteins to the red blood cell surface. REX contains predicted coiled-coil regions and a region with similarity to a domain in vesicle-tethering proteins. REX persists in Maurer's clefts throughout the infection of the erythrocyte, where it may play a role in the biogenesis and/or function of this organelle.     

A Plasmodium falciparum protein located in Maurer's clefts underneath knobs and protein localization in association with Rhop-3 and SERA in the intracellular network of infected erythrocytes

We report on the characterization of monoclonal antibodies against Plasmodium falciparum schizonts, which recognize parasite proteins of 130 kDa and 20 kDa. The 130-kDa protein was released by alkaline sodium carbonate treatment, suggesting that the protein is a peripheral membrane protein, while the 20-kDa protein remained associated with the membranes following alkali treatment, suggesting it may be an integral membrane protein. Both proteins were localized to large cytoplasmic vesicles within the cytoplasm of trophozoite and schizont-infected erythrocytes by immunofluorescence assay and confocal microscopy. Both proteins colocalized with Bodipy-ceramide in trophozoite and immature schizont-infected erythrocytes, but not in segmenters. The 130-kDa protein was localized by immunoelectron microscopy (IEM) to Maurer's clefts underneath knobs in a knobby and cytoadherent (K⁺/C⁺) P. falciparum strain. No IEM reactivity was obtained in a knobless and non-cytoadherent (K⁻/C⁻) parasite strain. We investigated stage-specific protein expression and protein localization by indirect immunofluorescence assay. Bodipy-ceramide colocalization assays with Rhop-3 and serine-rich antigen (SERA)-specific antibodies were performed. A similar colocalization in trophozoites and schizonts was obtained using the rhoptry-specific antibody 1B9 reactive with the 110-kDa Rhop-3 protein. In segmenters, unlike trophozoites and immature schizonts, there was no Bodipy-ceramide colocalization with antibody 1B9. A difference in protein colocalization was seen using specific antibody 152.3F7.1.1, reactive with SERA. Antibodies to SERA colocalized with Bodipy-ceramide in schizonts, including segmenters. Collectively the data suggest that Rhop-3 transits through the intracellular network en route to the rhoptries and both vesicle-specific proteins may function in the intracellular network. Received: 4 January 2000 / Accepted: 9 August 2000     

LANCL1, an erythrocyte protein recruited to the Maurer's clefts during Plasmodium falciparum development

As the malarial parasite Plasmodium falciparum develops inside the erythrocyte, parasite-derived membrane structures, referred to as Maurer's clefts, play an important role in parasite development by delivering parasite proteins to the host cell surface, and participating in the assembly of the cytoadherence complex, essential for the pathogenesis of cerebral malaria. PfSBP1 is an integral membrane protein of the clefts, interacting with an erythrocyte cytosolic protein, identified here as the human Lantibiotic synthetase component C-like protein LANCL1. LANCL1 is specifically recruited to the surface of Maurer's clefts in P. falciparum mature blood stages. We propose that the interaction between PfSBP1 and LANCL1 is central for late steps of the parasite development to prevent premature rupture of the red blood cell membrane.     

Pfsbp1, a Maurer's cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton

Antibodies from hyperimmune monkey sera, selected by absorption to Plasmodium falciparum-infected erythrocytes, and elution at acidic pH, allowed us to characterize a novel parasite protein, Pfsbp1 (P. falciparum skeleton binding protein 1). Pfsbp1 is an integral membrane protein of parasite-induced membranous structures associated with the erythrocyte plasma membrane and referred to as Maurer's clefts. The carboxy-terminal domain of Pfsbp1, exposed within the cytoplasm of the host cell, interacts with a 35 kDa erythrocyte skeletal protein and might participate in the binding of the Maurer's clefts to the erythrocyte submembrane skeleton. Antibodies to the carboxy- and amino-terminal domains of Pfsbp1 labelled similar vesicular structures in the cytoplasm of Plasmodium chabaudi and Plasmodium berghei-infected murine erythrocytes, suggesting that the protein is conserved among malaria species, consistent with an important role of Maurer's cleft-like structures in the intraerythrocytic development of malaria parasites.     

Identification of putative Plasmodium falciparum mefloquine resistance genes

Mefloquine is an effective antimalarial drug; however, resistant strains of the human malarial pathogen, Plasmodium falciparum, are beginning to arise. The yeast Saccharomyces cerevisiae is sensitive to mefloquine, enabling a screen for P. falciparum genes involved in resistance. Yeast were transformed with a P. falciparum expression library, followed by selection on mefloquine plates and sequencing of plasmids that conferred resistance. We characterized the four genes that conferred the strongest mefloquine-resistant phenotype in yeast. All four (PFD0090c, PFI0195c, PF10_0372 and PF14_0649) are uncharacterized P. falciparum genes from distinct chromosomes (4, 9, 10 and 14, respectively). The mefloquine-resistant phenotype was dependent on induction of the P. falciparum gene and independent of vector context. PFI0195c, which likely encodes a small GTPase activator (GAP), also conferred resistance to cycloheximide and halofantrine in yeast. Immunolocalization of the encoded protein to the Golgi complex in yeast is consistent with potential GAP function. The other three candidate proteins localized to the cytoplasm and plasma membrane (PF14_0649), nuclear envelope/ER (PF10_0372) and Golgi (PFD0090c) of yeast. Analysis of mefloquine-resistant P. falciparum strains and the mefloquine-sensitive strain, W2, by sequencing and semi-quantitative RT-PCR identified no relevant mutations in the resistant strains but showed that PFI0195c was upregulated in two out of three resistant strains and PF14_0649 was upregulated in all resistant strains tested.     

Plasmodium falciparum homologue of the genes for Plasmodium vivax and Plasmodium yoelii adhesive proteins, which is transcribed but not translated

The 235-kDa family of rhoptry proteins in Plasmodium yoelii and the two reticulocyte binding proteins of P. vivax comprise a family of proteins involved in host cell selection and erythrocyte invasion. Here we described a member of the gene family found in P. falciparum (PfRH3) that is transcribed in its entirety, under stage-specific control, with correct splicing of the intron, but appears not to be translated, probably due to two reading frameshifts at the 5' end of the gene.     

Characterisation of a delta-COP homologue in the malaria parasite,Plasmodium falciparum

The mature human erythrocyte is a simple haemoglobin-containing cell with no internal organelles and no protein synthesis machinery. The malaria parasite invades this cell and develops inside a parasitophorous vacuole (PV). The parasite exports proteins into the erythrocyte to bring about extensive remodelling of its adopted cellular home. Plasmodial homologues of two COPII proteins, PfSar1p and PfSec31p, are exported to the erythrocyte cytosol where they appear to play a role in the trafficking of proteins across the erythrocyte cytoplasm [Eur. J. Cell Biol. 78 (1999) 453; J. Cell Sci. 114 (2001) 3377]. We have now characterised a homologue of the COPI protein, delta-COP. A recombinant protein corresponding to 90% of the Pfdelta-COP sequence was used to raise antibodies. The affinity-purified antiserum recognised a protein with an apparent M(r) of 58 x 10(3) on Western blots of malaria parasite-infected erythrocytes but not on blots of uninfected erythrocytes. Pfdelta-COP was shown to be largely insoluble in non-ionic detergent, possibly suggesting cytoskeletal attachment. Confocal immunofluorescence microscopy of parasitised erythrocytes was used to show that, in contrast to the COPII proteins, Pfdelta-COP is located entirely within the parasite. The location of Pfdelta-COP partly overlaps that of the endoplasmic reticulum (ER)-located protein, PfERC, and partly that of the trans-Golgi-associated protein, PfRab6. Treatment of ring-stage plasmodium-infected erythrocytes with brefeldin A (BFA) inhibited development of the ER structure within the parasite cytosol and prevented the trafficking of the P. falciparum erythrocyte membrane protein-1, PfEMP1, to the erythrocyte cytosol. The Pfdelta-COP and PfSec31p populations each appear to be associated with the restricted ER structure in brefeldin-treated rings. When more mature stage parasites were treated with BFA, erythrocyte cytosol-located populations of parasite proteins were not reorganised, however, the overlap between Pfdelta-COP and PfERC in parasite cytosol was more complete suggesting a possible redistribution of the Golgi compartment into the ER. These data support the suggestion that both COPI and COPII proteins are involved in the trafficking of proteins within the parasite cytoplasm. However, only COPII proteins are exported to the erythrocyte cytosol to establish a vesicle-mediated protein trafficking pathway to the erythrocyte membrane.     

Properties of the Plasmodium falciparum homologue of a protective vaccine candidate of Plasmodium yoelii.

We describe an unusual tryptophan-rich protein of Plasmodium falciparum that contains threonine-rich repeats. The protein is encoded by a 2.5 kb gene with a two-exon structure including a short AT-rich intron that is spliced out of the mature message. The 5' end of the gene encodes a hydrophobic region, which is assumed to be a signal peptide. The peptide sequence is characterised by a tryptophan-rich region and a block of degenerate threonine repeats. The protein is synthesised throughout the asexual life cycle and has an apparent molecular weight of approximately 94 kDa. It has a variable molecular weight in different strains of P. falciparum. Length polymorphisms can be found in the intron region and the second exon. Four single nucleotide mutations are localised in the tryptophan-rich region and two were found in the threonine-repeat block. Homology searches based on gene structure and amino acid sequence revealed a relationship with a P. yoelii antigen that has been used successfully in vaccine studies. Thus, this P. falciparum antigen should be considered an additional candidate for assessment in vaccination against the asexual blood-stages of P. falciparum.     

Identification of the Plasmodium chabaudi homologue of merozoite surface proteins 4 and 5 of Plasmodium falciparum

Previous studies of Plasmodium falciparum have identified a region of chromosome 2 in which are clustered three genes for glycosylphosphatidylinositol (GPI)-anchored merozoite surface proteins, MSP2, MSP5, and MSP4, arranged in tandem. MSP4 and MSP5 both encode proteins 272 residues long that contain hydrophobic signal sequences, GPI attachment signals, and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. Nevertheless, the remainder of their protein coding regions are quite dissimilar. The locations and similar structural features of these genes suggest that they have arisen from a gene duplication event. Here we describe the identification of the syntenic region of the genome in the murine malaria parasite, Plasmodium chabaudi adami DS. Only one open reading frame is present in this region, and it encodes a protein with structural features reminiscent of both MSP4 and MSP5, including a single EGF-like domain. Accordingly, the gene has been designated PcMSP4/5. The homologue of the P. falciparum MSP2 gene could not be found in P. chabaudi; however, the amino terminus of the PcMSP4/5 protein shows similarity to that of MSP2. The PcMSP4/5 gene encodes a protein with an apparent molecular mass of 36 kDa, and this protein is detected in mature stages of the parasite. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites and developing and free merozoites. The PcMSP4/5 gene is transcribed in both ring and trophozoite stages but appears to be spliced in a stage-specific manner such that the central intron is spliced from the mRNA in the parasitic stage in which the protein is expressed.     

The putative gene for the first enzyme of glutathione biosynthesis in Plasmodium berghei and Plasmodium falciparum

The putative gene for gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, has been characterized both in Plasmodium berghei and Plasmodium falciparum. Protein sequence comparison between these two species reveals large conserved regions sharing more than 80% similarity, separated by less conserved portions. When the comparison is extended to known gamma-glutamylcysteine synthetases from other eukaryotes, a number of high similarity blocks are observed which may help in identifying sequence essential for protein function.     

Blood group A antigen is a coreceptor in Plasmodium falciparum rosetting

The malaria parasite Plasmodium falciparum utilizes molecules present on the surface of uninfected red blood cells (RBC) for rosette formation, and a dependency on ABO antigens has been previously shown. In this study, the antirosetting effect of immune sera was related to the blood group of the infected human host. Sera from malaria-immune blood group A (or B) individuals were less prone to disrupt rosettes from clinical isolates of blood group A (or B) patients than to disrupt rosettes from isolates of blood group O patients. All fresh clinical isolates and laboratory strains exhibited distinct ABO blood group preferences, indicating that utilization of blood group antigens is a general feature of P. falciparum rosetting. Soluble A antigen strongly inhibited rosette formation when the parasite was cultivated in A RBC, while inhibition by glycosaminoglycans decreased. Furthermore, a soluble A antigen conjugate bound to the cell surface of parasitized RBC. Selective enzymatic digestion of blood group A antigen from the uninfected RBC surfaces totally abolished the preference of the parasite to form rosettes with these RBC, but rosettes could still form. Altogether, present data suggest an important role for A and B antigens as coreceptors in P. falciparum rosetting.     

Identification and biochemical characterisation of a protein phosphatase 5 homologue from Plasmodium falciparum

We report the identification of a new serine/threonine phosphatase from Plasmodium falciparum at the DNA and protein levels. A 1.8 kb cDNA fragment encoding the protein phosphatase was identified via PCR amplification. The sequence has a coding capacity of 594 amino acids. Immunoblot analysis of P. falciparum extracts showed that antibodies generated against the His₆-fusion protein recognise a protein of approximately 80 kDa. The deduced amino acid sequence shares 55% identity with a mouse protein, identified as Protein Phosphatase 5 (PP5). We show that the P. falciparum PP5 homologue (PfPP5) has all structural and functional characteristics of this class of enzymes. It contains three tetratricopeptide repeats (TPR) and a nuclear targeting sequence at its N-terminus and a highly conserved C-terminal catalytic domain. Southern blot results are compatible with the existence of PfPP5 as a single copy gene. Purified recombinant protein, like the native protein enriched from P. falciparum extracts exhibited phosphatase activity that can be enhanced by both arachidonic and oleic acids, but not by myristic or stearic acid. In addition, the activity is inhibited by okadaic acid (OA) with an IC₅₀ of 4 nM. Immunofluorescence microscopy has localised PfPP5 preferentially to the nucleus. The function of PfPP5 is presently unclear, but like other PP5s of many eukaryotic organisms, it may have important regulatory functions in the parasite cell cycle.     

Plasmodium falciparum MLH is schizont stage specific endonuclease

Malaria is one of the most important infectious diseases in many regions around the world including India. Plasmodium falciparum is the cause of most lethal form of malaria while Plasmodium vivax is the major cause outside Africa. Regardless of considerable efforts over the last many years there is still no commercial vaccine against malaria and the disease is mainly treated using a range of established drugs. With time, the malaria parasite is developing drug resistance to most of the commonly used drugs. This drug resistance might be due to defective mismatch repair in the parasite. Previously we have reported that the P. falciparum genome contains homologues to most of the components of mismatch repair (MMR) complex. In the present study we report the detailed biochemical characterization of one of the main component of MMR complex, MLH, from P. falciparum. Our results show that MLH is an ATPase and it can incise covalently closed circular DNA in the presence of Mn(2+) or Mg(2+) ions. Using the truncated derivatives we show that full length protein MLH is required for all the enzymatic activities. Using immunodepletion assays we further show that the ATPase and endomuclease activities are attributable to PfMLH protein. Using immunofluorescence assay we report that the peak expression of MLH in both 3D7 and Dd2 strains of P. falciparum is mainly in the schizont stages of the intraerythrocytic development, where DNA replication is active. MMR also contributes to the overall fidelity of DNA replication and the peak expression of MLH in the schizont stages suggests that MLH is most likely involved in correcting the mismatches occurring during replication. This study should make a significant contribution in our better understanding of DNA metabolic processes in the parasite.     

A novel dual Dbp5/DDX19 homologue from Plasmodium falciparum requires Q motif for activity

Helicases are ubiquitous essential enzymes which have significant role in the nucleic acid metabolism. Using in silico approaches in the recent past we have identified a number of helicases in the Plasmodium falciparum genome. In the present study we report purification and detailed characterization of a novel helicase from P. falciparum. Our results indicate that this helicase is a homologue of Dbp5 and DDX19 from yeast and human, respectively. The biochemical characterization shows that it contains DNA and RNA unwinding, nucleic acid dependent ATPase and RNA binding activities. It is interesting to note that this enzyme can unwind DNA duplexes in both 5' to 3' and 3' to 5' directions. Using truncated derivatives we further show that Q motif is essentially required for all of its activities. These studies should make an important contribution in understanding the enzymes involved in nucleic acid metabolism in the parasite.     

Human serum haptoglobin is toxic to Plasmodium falciparum in vitro

Innate immune responses are important in the control of malaria, particularly in those who have not yet mounted an effective adaptive response. Here we report that the human serum acute phase protein, haptoglobin, is toxic to Plasmodium falciparum cultured in vitro. This effect is phenotype dependent and occurs during the trophozoite phase of the asexual life cycle. We propose that the increased levels of haptoglobin seen in the acute phase response may be protective against malaria in humans.     

Susceptibility of Plasmodium falciparum cyclic AMP-dependent protein kinase and its mammalian homologue to the inhibitors

Cyclic AMP-dependent protein kinase (protein kinase A, PKA) is a key element in many cell signaling pathways. An essential role of Plasmodium falciparum PKA (PfPKA) activity was reported in the intraerythrocytic growth of the malaria parasite. However, molecular characterization of PfPKA using purified recombinant proteins has not yet been performed. Here, we report the first successful purification of the enzymatically active PKA catalytic subunit of P. falciparum (PfPKA-C) using a wheat germ cell-free expression system. Interestingly, parasite enzymatic activity was weakly inhibited as compared with the inhibition of mammalian PKA catalytic subunit (PKA-C) by the specific PKA inhibitor, H89. Furthermore, PfPKA-C was only slightly inhibited by protein kinase inhibitor (PKI). These results suggest that substrate sites of PfPKA-C may be different from those of mammalian PKA-Cs. In addition, potential PKI corresponding to malarial PKA-C would also be different from those of mammalian cells.