PfCCp proteins of Plasmodium falciparum: gametocyte-specific expression and role in complement-mediated inhibition of exflagellation

The sexual phase of the malaria parasite Plasmodium falciparum is essential for transmission of the disease and is accompanied by the co-ordinated expression of sexual stage proteins. Six of these proteins belong to a highly conserved apicomplexan family of multi-domain adhesion proteins, termed PfCCps. PfCCp1, PfCCp2 and PfCCp3 are co-dependently expressed in the parasitophorous vacuole associated with the gametocyte plasma membrane. PfCCp2 and PfCCp3 also play an essential role for parasite development in the mosquito. We show that the six PfCCp proteins are expressed in stages II-V of gametocytogenesis as well as during early gamete formation. The proteins are expressed in association with the surface of both male and female gametocytes and macrogametes, but are not present in exflagellating microgametes. Further, the newly described protein PfCCp4 co-localizes with the transmission blocking candidate Pfs230, with which it forms a protein complex. In contrast to the phenotypes that are observed following targeted gene disruption of PfCCp2, PfCCp3 or Pfs230, the lack of PfCCp4 expression does not inhibit parasite development in the mosquito vector. This indicates a non-essential role for this protein during parasite transmission. Exflagellation assays revealed that antibodies directed against distinct domains of PfCCp1 through PfCCp4 and PfFNPA support a complement-mediated decrease in gametocyte emergence. We conclude that the six PfCCp proteins are specifically expressed during gametocytogenesis and gamete formation, and that select members may represent prospective candidates for transmission blocking vaccines.     

Plasmodium falciparum: mRNA co-expression and protein co-localisation of two gene products upregulated in early gametocytes

Genes encoding Plasmodium falciparum proteins Pfs16 and Pfpeg3/mdv1, specifically appearing in the parasitophorous vacuole of the early gametocytes, are upregulated at the onset of sexual differentiation. Analysis of asexual development in gametocyte producing and non-producing clones of P. falciparum indicated that these genes are also transcribed at a low level in asexual parasites, although their protein products are not detectable in these stages by immunofluorescence. Immunoelectron microscopic analysis of stage II gametocytes indicated that Pfs16 and Pfpeg3/mdv1 proteins co-localise in the parasitophorous vacuole membrane and in all derived membranous structures (such as the multi-laminate membrane whorls of the circular clefts in the infected erythrocyte cytoplasm and the membranes of the gametocyte food vacuoles). In this analysis both proteins were also observed for the first time in the membrane and in the lumen of distinct cleft-like structures in the erythrocyte cytoplasm.     

Pfnek3: an atypical activator of a MAP kinase in Plasmodium falciparum

The canonical mitogen-activated protein kinase (MAPK) signal cascade was previously suggested to be atypical in the malaria parasite. This raises queries on the existence of alternative mediators of plasmodial MAPK pathways. This study describes, Pfnek3, a malarial protein kinase belonging to the NIMA (Never in Mitosis, Aspergillus) family. Endogenous Pfnek3 is expressed during late asexual to gametocyte stages and lacks some classical protein kinase sequence motifs. Moreover, Pfnek3 is phylogenetically distant from mammalian NIMA-kinases. Recombinant Pfnek3 was able to phosphorylate and stimulate a malarial MAPK (Pfmap2). Contrastingly, this was not observed with two other kinases, Pfmap1 and human MAPK1, suggesting that the Pfnek3-Pfmap2 interaction may be specific for Pfmap2 regulation. In summary, our data reveal a malarial NIMA-kinase with the potential to regulate a MAPK. Possessing biochemical properties divergent from classical mammalian NIMA-kinases, Pfnek3 could potentially be an attractive target for parasite-selective anti-malarials.     

Density-dependent impact of the human malaria parasite Plasmodium falciparum gametocyte sex ratio on mosquito infection rates

Malaria parasites produce male and female life cycle stages (gametocytes) that must fertilize to achieve successful colonization of the mosquito. Gametocyte sex ratios have been shown to be under strong selection pressure both as an adaptive response to a worsening blood environment for transmission and according to the number of co-infecting clones in the vertebrate. Evidence for an impact of sex ratio on the transmission success of Plasmodium falciparum has, however, been more controversial. Theoretical models of fertilization predict that increasingly male sex ratios will be favoured at low gametocyte densities to ensure fertilization. Here, we analyse in vitro transmission studies of P. falciparum to Anopheles gambiae mosquitoes and test this prediction. We find that there is a discernible effect of sex ratio on transmission but which is dependent upon the gametocyte density. While increasingly male sex ratios do give higher transmission success at low gametocyte densities, they reduce success at higher densities. This therefore provides empirical confirmation that sex ratio has an immediate impact on transmission success and that it is density-dependent. Identifying the signals used by the parasite to alter its sex ratio is essential to determine the success of transmission-blocking vaccines that aim to impede the fertilization process.     

Plasmodium falciparum RuvB1 is an active DNA helicase and translocates in the 5′–3′ direction

RuvB family of protein contains two similar kinds of proteins i.e. RuvB1 and RuvB2 from yeast to human. These proteins belong to the AAA + class of proteins and are critical components of several multiprotein complexes involved in diverse cellular activities. There are two RuvB proteins annotated in the Plasmodium database but the identification of the third protein recently by our lab has raised the question why Plasmodium falciparum contains three RuvB proteins instead of two. Hence the biochemical characterizations of these proteins have become essential to understand the role of these proteins in the malaria parasite. Recently we have reported the characterization of the recombinant PfRuvB3, which contains ATPase activity but lacks DNA helicase activity. In the present study we report the phylogenetic analysis and detailed biochemical characterization of one of the other RuvB homologue RuvB1 from P. falciparum. PfRuvB1 shows considerable homology with human as well as yeast RuvB1 and contains Walker motif A and Walker motif B. The activity analysis of this protein revealed that PfRuvB1 is an ATPase and this activity increased significantly in the presence of ss-DNA. PfRuvB1 also contains DNA helicase activity and translocates preferentially in 5′ to 3′ direction. In vivo investigation of PfRuvB1 revealed that it is constitutively expressed during all the stages of intraerythrocytic cycle of P. falciparum and localizes mainly to the nucleus. These studies will make important contribution in understanding the role of RuvB protein in P. falciparum.     

Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine-pyrimethamine in Mali

Sulfadoxine-pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.     

(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate

The effects of drugs on Plasmodium falciparum transmission stages may reduce the spread of parasites in the population and contribute to malaria control. Detailed quantitative studies on (sub)microscopic gametocytaemia have become feasible with the availability of real-time Pfs25 quantitative Nucleic Acid Sequence-based Amplification (QT-NASBA), which can be used to detect gametocyte densities above 20 gametocytes per millilitre from in vitro cultures. Gametocyte dynamics were investigated in children with uncomplicated P. falciparum malaria after treatment with sulphadoxine-pyrimethamine (SP) or a combination of SP and artesunate (SP+AS), in a 28-days drug efficacy study. This study demonstrated that gametocyte prevalence in 873 samples from symptomatic Kenyan children was 2.8 times higher by QT-NASBA compared with microscopy. Microscopy-positive cases showed a significant correlation with QT-NASBA for gametocyte density. At enrolment, gametocyte prevalence was 86% by QT-NASBA compared with 22% by microscopy. Gametocytes were detected in 97% of children in at least one blood sample and in 38% of children in all samples obtained during the 28-days follow-up. Both the risk of gametocyte carriage and gametocyte density were considerably higher after treatment with SP compared with SP+AS. Gametocyte prevalence and density decreased with time in the SP+AS group, but not in the SP-treated children. Our data suggest that the potential of malaria transmission remains high even after treatment with artemisinin combination therapy, although prevalence and density of gametocytes is lower after SP+AS.     

Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi

During its life cycle the malarial parasite Plasmodium forms three invasive stages which have to invade different and specific cells for replication to ensue. Invasion is vital to parasite survival and consequently proteins responsible for invasion are considered to be candidate vaccine/drug targets. Plasmodium perforin-like proteins (PPLPs) have been implicated in invasion because they contain a predicted pore-forming domain. Ookinetes express three PPLPs, and one of them (PPLP3) has previously been shown to be essential for mosquito midgut invasion. In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection. Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel. The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.     

Adhesion protein complexes of malaria gametocytes assemble following parasite transmission to the mosquito

During differentiation in the human, the gametocytes of the malaria parasite Plasmodium falciparum display a remarkable number of adhesive proteins on their plasma membrane. These include the PfCCp protein family of six secreted proteins that assemble to multimeric protein complexes (MPCs) within the gametocyte parasitophorous vacuole. We now show that the PfCCp-based MPCs are linked to the gametocyte plasma membrane via interactions with Pfs230, a binding-partner of the GPI-anchored Pfs48/45. Upon onset of gametogenesis, which takes place after gametocyte uptake by blood-feeding mosquitoes, GPI-anchored Pfs25 joins the MPC, providing an additional link of its components to the plasma membrane. Gametogenesis also initiates cleavage of Pfs230 at its N-terminal site, resulting in its increased interaction with the MPC. Either lack of Pfs230 or impaired Pfs230 processing causes proteolysis of the PfCCp proteins and release from the MPC. Our data point to MPC assembly as a crucial step for sexual reproduction.     

High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in the Gambia

The purpose of this work was to determine the infectivity to mosquitoes of genetically diverse Plasmodium falciparum clones seen in natural infections in the Gambia. Two principal questions were addressed: (i) how infectious are gametocytes of sub-patent infections, particularly at the end of the dry season; and (ii) are all clones in multiclonal infections equally capable of infecting mosquitoes? The work was carried out with two cohorts of infected individuals. Firstly, a group of 31 P. falciparum-infected people were recruited in the middle of the dry season (May, 2003), then examined for P. falciparum at the beginning (August 2003) and middle (October, 2003) of the transmission season. On each occasion, we examined the genotypes of asexual forms and gametocytes by PCR and RT-PCR, as well as their infectivity to Anopheles gambiae using membrane feeds. One individual gave rise to infected mosquitoes in May, and two in August. Different gametocyte genotypes co-existed in the same infection and fluctuated over time. The mean multiplicity of infection was 1.4, 1.7 and 1.5 clones in May, August and October, respectively. Second, a group of patients undergoing drug-treatment during August 2003 was tested for asexual and gametocyte genotypes and their infectivity to mosquitoes. Forty-three out of 100 feeds produced infections. The genetic complexity of the parasites in mosquitoes was sometimes greater than that detectable in the blood on which the mosquitoes had fed. This suggested that gametocytes of clones existing in the blood below PCR detection limits at the time of the feed were at least as infectious to the mosquitoes as the more abundant clones. These findings emphasise the crucial role of gametocyte complexity and infectivity in generating the remarkable diversity of P. falciparum genotypes seen in infected people, even in an area of seasonal transmission.     

Regulated oligomerisation and molecular interactions of the early gametocyte protein Pfg27 in Plasmodium falciparum sexual differentiation

Gametocytes of the protozoan Plasmodium falciparum ensure malaria parasite transmission from humans to the insect vectors. In their development, they produce the abundant specific protein Pfg27, the function and in vivo molecular interactions of which are unknown. Here we reveal a previously unreported localisation of Pfg27 in the gametocyte nucleus by immunoelectron microscopy and studies with HaloTag and Green Fluorescent Protein fusions, and identify a network of interactions established by the protein during gametocyte development. We report the ability of endogenous Pfg27 to form oligomeric complexes that are affected by phosphorylation of the protein, possibly through the identified phosphorylation sites, Ser32 and Thr208. We show that Pfg27 binds RNA molecules through specific residues and that the protein interacts with parasite RNA-binding proteins such as EF1α and PfH45. We propose a structural model for Pfg27 oligomerisation, based on the sequence and structural conservation here recognised between Pfg27 and sterile alpha motif. This study provides a molecular basis for Pfg27 to establish an interaction network with RNA and RNA-binding proteins and to govern its dynamic oligomerisation in developing gametocytes.     

Appropriate Time for Primaquine Treatment to Reduce Plasmodium falciparum Transmission in Hypoendemic Areas

Artemesinin-combination therapies (ACT) for falciparum malaria reduce gametocyte carriage, and therefore reduce transmission. Artemisinin derivatives will act against only young gametocytes whereas primaquine acts on mature gametocytes which are present usually in the circulation at the time when the patient presents for treatment. Both artemisinin derivatives and primaquine have short half-lives, less than 1 hr and 7 hr, respectively. Therefore, asexual parasites or young gametocytes remain after completed ACT. A single dose of primaquine (0.50-0.75 mg base/kg) at the end of ACT can kill only mature gametocytes but cannot kill young gametocytes (if present). Remaining asexual forms after completion of ACT course, e.g., artesunate-mefloquine for 3 days, may develop to mature gametocytes 7-15 days later. Thus, an additional dose of primaquine (0.50-0.75 mg base/kg) given 2 weeks after ACT completion may be beneficial for killing remaining mature gametocytes and contribute to more interruption of Plasmodium falciparum transmission than giving only 1 single dose of primaquine just after completing ACT.     

Computational prediction of protein interactions related to the invasion of erythrocytes by malarial parasites

Background

The invasion of red blood cells (RBCs) by malarial parasites is an essential step in the life cycle of Plasmodium falciparum. Human-parasite surface protein interactions play a critical role in this process. Although several interactions between human and parasite proteins have been discovered, the mechanism related to invasion remains poorly understood because numerous human-parasite protein interactions have not yet been identified. High-throughput screening experiments are not feasible for malarial parasites due to difficulty in expressing the parasite proteins. Here, we performed computational prediction of the PPIs involved in malaria parasite invasion to elucidate the mechanism by which invasion occurs.

Results

In this study, an expectation maximization algorithm was used to estimate the probabilities of domain-domain interactions (DDIs). Estimates of DDI probabilities were then used to infer PPI probabilities. We found that our prediction performance was better than that based on the information of D. melanogaster alone when information related to the six species was used. Prediction performance was assessed using protein interaction data from S. cerevisiae, indicating that the predicted results were reliable. We then used the estimates of DDI probabilities to infer interactions between 490 parasite and 3,787 human membrane proteins. A small-scale dataset was used to illustrate the usability of our method in predicting interactions between human and parasite proteins. The positive predictive value (PPV) was lower than that observed in S. cerevisiae. We integrated gene expression data to improve prediction accuracy and to reduce false positives. We identified 80 membrane proteins highly expressed in the schizont stage by fast Fourier transform method. Approximately 221 erythrocyte membrane proteins were identified using published mass spectral datasets. A network consisting of 205 interactions was predicted. Results of network analysis suggest that SNARE proteins of parasites and APP of humans may function in the invasion of RBCs by parasites.

Conclusions

We predicted a small-scale PPI network that may be involved in parasite invasion of RBCs by integrating DDI information and expression profiles. Experimental studies should be conducted to validate the predicted interactions. The predicted PPIs help elucidate the mechanism of parasite invasion and provide directions for future experimental investigations.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0393-z) contains supplementary material, which is available to authorized users.     

A relevant in vitro eukaryotic live-cell system for the evaluation of plasmodial protein localization

Understanding the functional genomics and proteomics of plasmodia underpins the development of new approaches to antimalarial chemotherapy. Although genome databanks (e.g. PlasmoDB) and biocomputing tools (e.g. PlasMit, PlasmoAP, PATS) are useful in providing a global albeit predictive view of the myriad of about 5000 genes, only 40% are annotated, with few cases of endorsed subcellular localizations of the corresponding proteins in animal models. Progress in plasmodial protein trafficking has been hampered by the lack of a simple yet reliable method for studying subcellular localization of plasmodial proteins. In this study, we have used a combination of fluorescent markers, organelle-specific probes, phase contrast microscopy, and confocal microscopy to locate a selection of signal peptides from 10 plasmodial proteins in CHO-K1 cells. These eukaryotic cells serve as an in vitro living system for studying the cellular destinations of four mitochondrial-targeted TCA cycle proteins (citrate synthase, CS; isocitrate dehydrogenase, ICDH; branched chain alpha-keto-acid dehydrogenase E1alpha subunit, BCKDH; succinate dehydrogenase flavoprotein-subunit, SDH), two nuclear-targeted proteins (histone deacetylase, HDAC; RNA polymerase, RPOL), two apicoplast-targeted proteins (pyruvate kinase 2, PK2; glutamate dehydrogenase, GDH), and two cytoplasmic resident proteins (malate dehydrogenase, MDH; glycerol kinase, GK). The respective localizations of these malarial proteins have complied with the selected molecular targets, viz. mitochondrial, nuclear and cytoplasmic. Interestingly, MDH that is widely known to be resident in eukaryotic mitochondria was found to be cytoplasmic, probably due to the absence of molecular target sequences. Since the localization of plasmodial proteins is central to the authentication of their pathophysiological roles, this experimental system will serve as a useful a priori approach.     

Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.     

Protein export in Plasmodium parasites: From the endoplasmic reticulum to the vacuolar export machine

It is somewhat paradoxical that the malaria parasite’s survival strategy involves spending almost all of its blood-stage existence residing behind a two-membrane barrier in a host red blood cell, yet giving considerable attention to exporting parasite-encoded proteins back across these membranes. These exported proteins are thought to play diverse roles and are crucial in pathogenic processes, such as re-modelling of the erythrocyte cytoskeleton and mediating the export of a major virulence protein known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), and in metabolic processes such as nutrient uptake and solute exchange. Despite these varied roles most exported proteins have at least one common link; they share a trafficking pathway that begins with entry into the endoplasmic reticulum and concludes with passage across the vacuole membrane via a proteinaceous translocon known as the Plasmodium translocon of exported proteins (PTEX). In this commentary we review recent advances in our understanding of this export pathway and suggest several models by which different aspects of the process may be interconnected.     

Characterisation of the <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> Hsp70–Hsp90 organising protein (PfHop)

Malaria is caused by Plasmodium species, whose transmission to vertebrate hosts is facilitated by mosquito vectors. The transition from the cold blooded mosquito vector to the host represents physiological stress to the parasite, and additionally malaria blood stage infection is characterised by intense fever periods. In recent years, it has become clear that heat shock proteins play an essential role during the parasite's life cycle. Plasmodium falciparum expresses two prominent heat shock proteins: heat shock protein 70 (PfHsp70) and heat shock protein 90 (PfHsp90). Both of these proteins have been implicated in the development and pathogenesis of malaria. In eukaryotes, Hsp70 and Hsp90 proteins are functionally linked by an essential adaptor protein known as the Hsp70–Hsp90 organising protein (Hop). In this study, recombinant P. falciparum Hop (PfHop) was heterologously produced in E. coli and purified by nickel affinity chromatography. Using specific anti-PfHop antisera, the expression and localisation of PfHop in P. falciparum was investigated. PfHop was shown to co-localise with PfHsp70 and PfHsp90 in parasites at the trophozoite stage. Gel filtration and co-immunoprecipitation experiments suggested that PfHop was present in a complex together with PfHsp70 and PfHsp90. The association of PfHop with both PfHsp70 and PfHsp90 suggests that this protein may mediate the functional interaction between the two chaperones.