Plasmodium Proteins Research Articles

Using increment of diversity to predict mitochondrial proteins of malaria parasite: integrating pseudo-amino acid composition and structural alphabet

Due to the complexity of Plasmodium falciparum (PF) genome, predicting mitochondrial proteins of PF is more difficult than other species. In this study, using the n-peptide composition of reduced amino acid alphabet (RAAA) obtained from structural alphabet named Protein Blocks as feature parameter, the increment of diversity (ID) is firstly developed to predict mitochondrial proteins. By choosing the 1-peptide compositions on the N-terminal regions with 20 residues as the only input vector, the prediction performance achieves 86.86% accuracy with 0.69 Mathew's correlation coefficient (MCC) by the jackknife test. Moreover, by combining with the hydropathy distribution along protein sequence and several reduced amino acid alphabets, we achieved maximum MCC 0.82 with accuracy 92% in the jackknife test by using the developed ID model. When evaluating on an independent dataset our method performs better than existing methods. The results indicate that the ID is a simple and efficient prediction method for mitochondrial proteins of malaria parasite.

The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission.

SummaryAlthough eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified.

Immunization with pre-erythrocytic antigen CelTOS from Plasmodium falciparum elicits cross-species protection against heterologous challenge with Plasmodium berghei.

BackgroundThe Plasmodium protein Cell-traversal protein for ookinetes and sporozoites (CelTOS) plays an important role in cell traversal of host cells in both, mosquito and vertebrates, and is required for successful malaria infections. CelTOS is highly conserved among the Plasmodium species, suggesting an important functional role across all species. Therefore, targeting the immune response to this highly conserved protein and thus potentially interfering with its biological function may result in protection against infection even by heterologous species of Plasmodium.Methodology/Principal FindingsTo test this hypothesis, we developed a recombinant codon-harmonized P. falciparum CelTOS protein that can be produced to high yields in the E. coli expression system. Inbred Balb/c and outbred CD-1 mice were immunized with various doses of the recombinant protein adjuvanted with Montanide ISA 720 and characterized using in vitro and in vivo analyses.Conclusions/SignificanceImmunization with PfCelTOS resulted in potent humoral and cellular immune responses and most importantly induced sterile protection against a heterologous challenge with P. berghei sporozoites in a proportion of both inbred and outbred mice. The biological activity of CelTOS-specific antibodies against the malaria parasite is likely linked to the impairment of sporozoite motility and hepatocyte infectivity. The results underscore the potential of this antigen as a pre-erythrocytic vaccine candidate and demonstrate for the first time a malaria vaccine that is cross-protective between species.

Plasmodium falciparum Tudor Staphylococcal Nuclease interacting proteins suggest its role in nuclear as well as splicing processes

Tudor Staphylococcal Nuclease (p100 or SND1), a member of the micronuclease family is a multifunctional protein that plays a key role(s) in transcription and splicing processes in many eukaryotic cells. PfTudor-SN, a Plasmodium homolog of the human p100 protein is a structurally conserved protein; however molecular details of its function are not yet understood. Our previous studies have shown that PfTudor-SN binds RNA and it is possible to selectively inhibit parasite growth by PfTudor-SN specific drugs. In the present study, we identified the molecular interactions between Plasmodium falciparum Tudor-SN and twelve Plasmodium proteins such as Histone h2A, SPT2 (a transcriptional regulator), a Cold-shock DNA binding protein in a bacterial two-hybrid screen. To get further insight into some of these interactions, we mapped the interaction domain in PfTudor-SN protein using the yeast two-hybrid system. Of these proteins, Plasmodium N-methyl- d-aspartate receptor associated protein, PfUbiquitin conjugating enzyme and Cold-shock DNA binding protein showed interaction with the SN domains of PfTudor-SN. Immuno-localization studies of the interacting proteins showed their presence predominantly in the nucleus, which inevitably suggests the molecular interactions between these proteins and PfTudor-SN. Furthermore, we also identified a molecular interaction between the Tudor domain of PfTudor-SN protein and Plasmodium spliceosomal Sm protein, PfSmD1 advocating the role of PfTudor-SN in the spliceosome assembly. Together, these results suggest multiple role(s) for PfTudor-SN protein mainly in nuclear and splicing processes at asexual blood stages of the malaria parasite.

Identification of three CCp genes in Babesia divergens: Novel markers for sexual stages parasites

Babesia divergens, a tick-borne protozoan parasite of red blood cells, is the main agent of bovine and human babesiosis in Europe. Very few data are available concerning its life cycle and sexual reproduction inside the tick vector, Ixodes ricinus. The aim of this study was to define some markers of the B.divergens sexual stage. An in silico post-genomic approach was used to analyze genomic, transcriptomic and proteomic data and to select specific sexual stage proteins of the related apicomplexan genus Plasmodium. Three proteins, based on sequence identity between the available genomes of Plasmodium and Babesia spp., were chosen, as members of a highly conserved and apicomplexan sexual stages specific protein family (CCp) potentially involved in adhesive functions. Degenerate primers were used to amplify and clone three B.divergens orthologs (bdccp1, bdccp2, and bdccp3) corresponding to newly identified genes in this parasite. The opportunities offered by such markers to study parasite development in its vector are discussed.

Predicting malaria interactome classifications from time-course transcriptomic data along the intraerythrocytic developmental cycle

Even though a vaccine for malaria infections has been under intense study for many years, it has resisted several different lines of attack attempted by biologists. More than half of Plasmodium proteins still remain uncharacterized and therefore cannot be used in clinical trials. The task is further complicated by the metamorphic life-cycle of the parasite, which allows for rapid evolutionary changes and diversity among related strains, thus making precise targeting of the appropriate proteins for vaccination a technical challenge. We propose an automated method for predicting functions for the malaria parasite, which capitalizes on the importance of the intraerythrocytic developmental cycle data and expression changes during its five phases, as determined computationally by our segmentation algorithm. Our method combines temporal gene expression profiles with protein-protein interaction data, sequence similarity scores, and metabolic pathway information to produce a set of predicted protein functions that can be used as targets for vaccine development. We use a Bayesian approach, which assigns a probability of having (or not having) a particular function to each protein, given the various sources of evidence. In our method, each data source is represented by either a functional linkage graph or a categorical feature vector. The methods are tested on Plasmodium falciparum, the species responsible for the deadliest malaria infections. The algorithm was able to assign meaningful functions to 628 out of 1439 previously unannotated proteins, which are first-choice candidates for experimental vaccine research. We conclude that analyzing time-course gene expression profiles in separate phases leads to much higher prediction accuracy when compared with Pearson correlation coefficients computed across the time course as a whole. Additionally, we demonstrate that temporal expression profiles alone are able to improve the predictive power of the integrated data.

Proteomic Profiling of Plasmodium falciparum through Improved, Semiquantitative Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtain a snapshot of the proteome at any specific time. However, its application to study the Plasmodial (malaria parasite) proteome is still limited due to inefficient extraction and detection methods and the extraordinarily large size of some proteins. Here, we report an optimized protein extraction method, the most appropriate methods for Plasmodial protein quantification and 2-DE detection, and finally protein identification by mass spectrometry (MS). Linear detection of Plasmodial proteins in a optimized lysis buffer was only possible with the 2-D Quant kit, and of the four stains investigated, Flamingo Pink was superior regarding sensitivity, linearity, and excellent MS-compatibility. 2-DE analyses of the Plasmodial proteome using this methodology resulted in the reliable detection of 349 spots and a 95% success rate in MS/MS identification. Subsequent application to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots, which constituted 57 and 49 proteins from the Plasmodial ring and trophozoite stages, respectively. This study additionally highlights the presence of various isoforms within the Plasmodial proteome, which is of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle.

The malarial CDK Pfmrk and its effector PfMAT1 phosphorylate DNA replication proteins and co-localize in the nucleus

Cyclin-dependent kinases (CDKs) have an established role in metazoans and yeast in DNA replication, transcription and cell cycle regulation. Several CDKs and their effectors have been identified in the malaria parasite Plasmodium falciparum and their biological functions are beginning to be investigated. Here we report results from the functional characterization of Pfmrk and its effector PfMAT1. We validated the interactions between Pfmrk and PfMAT1 and pinpointed their intracellular location. Co-immunoprecipitation studies demonstrated physical interaction between the two proteins and identified the C-terminal domain of PfMAT1 as the Pfmrk activator domain. Immunofluorescence analyses using GFP and RFP-tagged versions of Pfmrk and PfMAT1, respectively, demonstrated the co-localization of these two proteins to the parasite nucleus. Bacterial two-hybrid screen of a P. falciparum cDNA library using Pfmrk as the bait identified two plasmodial DNA replication proteins, PfRFC-5 and PfMCM6, as interactors with Pfmrk. We demonstrate that that these two proteins are substrates of Pfmrk-mediated phosphorylation and that PfMAT1 confers substrate specificity to the Pfmrk kinase complex. Collectively, these data suggest a role for Pfmrk in the nucleus of the parasite presumably in regulation of the DNA replication machinery.

Characterization and Structural Studies of the Plasmodium falciparum Ubiquitin and Nedd8 Hydrolase UCHL3

Like their human hosts, Plasmodium falciparum parasites rely on the ubiquitin-proteasome system for survival. We previously identified PfUCHL3, a deubiquitinating enzyme, and here we characterize its activity and changes in active site architecture upon binding to ubiquitin. We find strong evidence that PfUCHL3 is essential to parasite survival. The crystal structures of both PfUCHL3 alone and in complex with the ubiquitin-based suicide substrate UbVME suggest a rather rigid active site crossover loop that likely plays a role in restricting the size of ubiquitin adduct substrates. Molecular dynamics simulations of the structures and a model of the PfUCHL3-PfNedd8 complex allowed the identification of shared key interactions of ubiquitin and PfNedd8 with PfUCHL3, explaining the dual specificity of this enzyme. Distinct differences observed in ubiquitin binding between PfUCHL3 and its human counterpart make it likely that the parasitic DUB can be selectively targeted while leaving the human enzyme unaffected.

The host targeting motif in exported Plasmodium proteins is cleaved in the parasite endoplasmic reticulum

During the blood stage of its lifecycle, the malaria parasite resides and replicates inside a membrane vacuole within its host cell, the human erythrocyte. The parasite exports many proteins across the vacuole membrane and into the host cell cytoplasm. Most exported proteins are characterized by the presence of a host targeting (HT) motif, also referred to as a Plasmodium export element (PEXEL), which corresponds to the consensus sequence RxLxE/D/Q. During export the HT motif is cleaved by an unknown protease. Here, we generate parasite lines expressing HT motif containing proteins that are localized to different compartments within the parasite or host cell. We find that the HT motif in a protein that is retained in the parasite endoplasmic reticulum is cleaved and N-acetylated as efficiently as a protein that is exported. This shows that cleavage of the HT motif occurs early in the secretory pathway, in the parasite endoplasmic reticulum.

Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte

During their intraerythrocytic development, malaria parasites export hundreds of proteins to remodel their host cell. Nutrient acquisition, cytoadherence and antigenic variation are among the key virulence functions effected by this erythrocyte takeover. Proteins destined for export are synthesized in the endoplasmic reticulum (ER) and cleaved at a conserved (PEXEL) motif, which allows translocation into the host cell via an ATP-driven translocon called the PTEX complex. We report that plasmepsin V, an ER aspartic protease with distant homology to the mammalian processing enzyme BACE, recognizes the PEXEL motif and cleaves it at the correct site. This enzyme is essential for parasite viability and ER residence is essential for its function. We propose that plasmepsin V is the PEXEL protease and is an attractive enzyme for antimalarial drug development.

In silico and biological survey of transcription-associated proteins implicated in the transcriptional machinery during the erythrocytic development of Plasmodium falciparum

BackgroundMalaria is the most important parasitic disease in the world with approximately two million people dying every year, mostly due to Plasmodium falciparum infection. During its complex life cycle in the Anopheles vector and human host, the parasite requires the coordinated and modulated expression of diverse sets of genes involved in epigenetic, transcriptional and post-transcriptional regulation. However, despite the availability of the complete sequence of the Plasmodium falciparum genome, we are still quite ignorant about Plasmodium mechanisms of transcriptional gene regulation. This is due to the poor prediction of nuclear proteins, cognate DNA motifs and structures involved in transcription.ResultsA comprehensive directory of proteins reported to be potentially involved in Plasmodium transcriptional machinery was built from all in silico reports and databanks. The transcription-associated proteins were clustered in three main sets of factors: general transcription factors, chromatin-related proteins (structuring, remodelling and histone modifying enzymes), and specific transcription factors. Only a few of these factors have been molecularly analysed. Furthermore, from transcriptome and proteome data we modelled expression patterns of transcripts and corresponding proteins during the intra-erythrocytic cycle. Finally, an interactome of these proteins based either on in silico or on 2-yeast-hybrid experimental approaches is discussed.ConclusionThis is the first attempt to build a comprehensive directory of potential transcription-associated proteins in Plasmodium. In addition, all complete transcriptome, proteome and interactome raw data were re-analysed, compared and discussed for a better comprehension of the complex biological processes of Plasmodium falciparum transcriptional regulation during the erythrocytic development.

Plasmodial heat shock proteins: targets for chemotherapy

Heat shock proteins act as molecular chaperones, facilitating protein folding in cells of living organisms. Their role is particularly important in parasites because environmental changes associated with their life cycles place a strain on protein homoeostasis. Not surprisingly, some heat shock proteins are essential for the survival of the most virulent malaria parasite, Plasmodium falciparum. This justifies the need for a greater understanding of the specific roles and regulation of malarial heat shock proteins. Furthermore, heat shock proteins play a major role during invasion of the host by the parasite and mediate in malaria pathogenesis. The identification and development of inhibitor compounds of heat shock proteins has recently attracted attention. This is important, given the fact that traditional antimalarial drugs are increasingly failing, as a consequence of parasite increasing drug resistance. Heat shock protein 90 (Hsp90), Hsp70/Hsp40 partnerships and small heat shock proteins are major malaria drug targets. This review examines the structural and functional features of these proteins that render them ideal drug targets and the challenges of targeting these proteins towards malaria drug design. The major antimalarial compounds that have been used to inhibit heat shock proteins include the antibiotic, geldanamycin, deoxyspergualin and pyrimidinones. The proposed mechanisms of action of these molecules and the pathways they inhibit are discussed.

Plasmod-PPI: A web-server predicting complex biopolymer targets in plasmodium with entropy measures of protein–protein interactions

We can define structural indices of polymer or biopolymer complex structures and use them in the prediction of new drug targets in parasites. For instance, Plasmodium falciparum causes the most severe form of Malaria and kills up to 2.7 million people annually whereas Plasmodium vivax is geographically the most widely distributed cause with more than 80 million clinical cases. Due to drug resistance and toxicity, discovering novel drug targets is mandatory; such as Protein–Protein Complexes unique in this pathogen and not present in human host (pPPCs). Additionally, the 3D structure of an increasing number of Plasmodium proteins is being reported in public databases making easier the development of bioinformatics models to predict pPPCs. In addition, some PPCs expressed both in parasite and human, such as DHFR synthase, play a significant role in drug resistance in both Malaria and Human Cancer. However, there are no general models to predict pPPCs using indices of PPC biopolymer structure. Therefore, we introduced herein new Markov Chain numerical descriptors of protein–protein Interactions (PPIs) based on electrostatic entropy measures and calculated these parameters for 5257 pairs of proteins (774 pPPCs and 4483 non-pPPCs) from more than 20 organisms, including parasite and human hosts. We found a simple Classification Tree with high Accuracy, Sensitivity, and Specificity (90.2–98.5%) both in training and independent test sub-sets and implemented this predictor in the user-friendly web server PlasmodPPI freely available at http://miaja.tic.udc.es/Bio-AIMS/PlasmodPPI.php.

Prediction of mitochondrial proteins of malaria parasite using split amino acid composition and PSSM profile

The rate of human death due to malaria is increasing day-by-day. Thus the malaria causing parasite Plasmodium falciparum (PF) remains the cause of concern. With the wealth of data now available, it is imperative to understand protein localization in order to gain deeper insight into their functional roles. In this manuscript, an attempt has been made to develop prediction method for the localization of mitochondrial proteins. In this study, we describe a method for predicting mitochondrial proteins of malaria parasite using machine-learning technique. All models were trained and tested on 175 proteins (40 mitochondrial and 135 non-mitochondrial proteins) and evaluated using five-fold cross validation. We developed a Support Vector Machine (SVM) model for predicting mitochondrial proteins of P. falciparum, using amino acids and dipeptides composition and achieved maximum MCC 0.38 and 0.51, respectively. In this study, split amino acid composition (SAAC) is used where composition of N-termini, C-termini, and rest of protein is computed separately. The performance of SVM model improved significantly from MCC 0.38 to 0.73 when SAAC instead of simple amino acid composition was used as input. In addition, SVM model has been developed using composition of PSSM profile with MCC 0.75 and accuracy 91.38%. We achieved maximum MCC 0.81 with accuracy 92% using a hybrid model, which combines PSSM profile and SAAC. When evaluated on an independent dataset our method performs better than existing methods. A web server PFMpred has been developed for predicting mitochondrial proteins of malaria parasites ( http://www.imtech.res.in/raghava/pfmpred/).

Paludisme d’importation : pièges diagnostiques et tests de diagnostic rapide

Imported malaria is a disease prevalent in France: 6,000 to 8,000 cases a year, of which 15% are pediatric cases. Despite this high incidence, the diagnosis is often delayed. The patient may then evolve to a severe form. This diagnostic delay is due to non-specific clinical symptoms in children. The most common symptoms are fever and digestive disorders (diarrhea, vomiting). In the absence of thrombocytopenia, the laboratory tests are not very useful for diagnosis. Parasitological examinations are dependent on the experience of the biologist, particularly in cases of low parasitemia as those observed in children who have received partial chemoprophylaxis. The recent introduction of rapid tests based on the detection of Plasmodium proteins, allows emergency remedy to this problem. The blood smears remains the gold standard and has to be used to confirm the results of rapid tests. If rapid tests improve the detection of Plasmodium in 2009, it remains mandatory to evoke the diagnosis of malaria in any febrile child coming from an endemic area.

Detection of new protein domains using co-occurrence: application to Plasmodium falciparum

Hidden Markov models (HMMs) have proved to be a powerful tool for protein domain identification in newly sequenced organisms. However, numerous domains may be missed in highly divergent proteins. This is the case for Plasmodium falciparum proteins, the main causal agent of human malaria. We propose a method to improve the sensitivity of HMM domain detection by exploiting the tendency of the domains to appear preferentially with a few other favorite domains in a protein. When sequence information alone is not sufficient to warrant the presence of a particular domain, our method enables its detection on the basis of the presence of other Pfam or InterPro domains. Moreover, a shuffling procedure allows us to estimate the false discovery rate associated with the results. Applied to P. falciparum, our method identifies 585 new Pfam domains (versus the 3683 already known domains in the Pfam database) with an estimated error rate <20%. These new domains provide 387 new Gene Ontology (GO) annotations to the P. falciparum proteome. Analogous and congruent results are obtained when applying the method to related Plasmodium species (P. vivax and P. yoelii). Supplementary Material and a database of the new domains and GO predictions achieved on Plasmodium proteins are available at http://www.lirmm.fr/~terrapon/codd/. Supplementary data are available at Bioinformatics online.

Disease‐specific biomarker discovery by aptamers

RNA and DNA aptamers developed by an in vitro selection process, Systematic Evolution of Ligands by EXponential enrichment (SELEX), comprise a novel class of high-affinity and specific capture agents, which can be easily modified for cytometry and in vivo applications. A novel application of this technique (Cell SELEX) explores the expression of cell surface epitopes that differ between two given cell types or between healthy and diseased cells. Using whole cells as targets, aptamer libraries can be identified that bind to biomarkers expressed by target cells and not by any other cells. Aptamers have been developed that specifically interact with cell surface epitopes of trypanosomes or distinguish between the differences in molecular signature of somatic and cancer cells. Aside from its use for target cell identification by image and flow cytometry and laser-scanning microscopy, aptamers can be used for ligand-mediated purification and identification of their binding proteins in target cell membranes. In this review, we discuss an approach for the development of aptamers targeting parasite-derived surface proteins of Trypanosoma and Plasmodium.

Age-dependent systemic antibody responses and immunisation-associated changes in mice orally and nasally immunised with Lactococcus lactis expressing a malaria parasite protein

Gram positive food-grade bacteria such as lactococci have significant advantages over attenuated pathogens as vaccine delivery vehicles because of their inherently greater safety. Plasmodium falciparum merozoite surface antigen 2 (MSA2) was expressed in recombinant Lactococcus lactis both intracellularly and covalently anchored to the peptidoglycan of the cell wall (MSA2cP). Balb/c mice of different ages were immunised with the MSA2cP expressing L. lactis in a combined oral and nasal immunisation procedure. Serum IgG antibody responses to MSA2 were higher in young adult Balb/c mice compared to old mice and neonates. The elicited serum IgG antibodies reacted with native MSA2 on the surface of P. falciparum merozoites in an immunofluorescence assay. The serum IgG antibody isotypes in young adult mice were mainly IgG1, IgG2a and IgG2b, while IgG3 tended to be higher in old mice. IgA antibodies to MSA2 were also produced in young mice. Enlarged mesenteric lymph nodes, and more prominent lymphoid tissue in the lamina propria of the ileum and lymphoid follicles in the spleen, were observed in mice fed L. lactis. These findings are relevant for developing L. lactis as a vector to deliver vaccines in human populations.

A newly discovered protein export machine in malaria parasites

Several hundred malaria parasite proteins are exported beyond an encasing vacuole and into the cytosol of the host erythrocyte, a process that is key to the virulence and viability of the causative Plasmodium species. The trafficking machinery responsible for this export is unknown. Here, we identify a Plasmodium Translocon of EXported proteins (PTEX), which is located in the vacuole membrane. The PTEX complex is ATP-powered and comprises HSP101, which is a ClpA/B-like AAA+ ATPase of a type commonly associated with protein translocons, a novel protein termed PTEX150 and a known parasite protein EXP2. EXP2 is the potential channel as it is the membrane-associated component of the core PTEX complex. Two other proteins, a novel protein PTEX88 and a thioredoxin known as TRX2, were also identified as PTEX components. As a common portal for numerous crucial processes, this novel translocon offers an exciting new avenue for therapeutic intervention.