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Invasion of human erythrocytes by Plasmodium falciparum merozoites involves multiple interactions between host receptors and their merozoite ligands. Here we report human Cyclophilin B as a receptor for PfRhopH3 during merozoite invasion. Localization and binding studies show that Cyclophilin B is present on the erythrocytes and binds strongly to merozoites. We demonstrate that PfRhopH3 binds to the RBCs and their treatment with Cyclosporin A prevents merozoite invasion. We also show a multi-protein complex involving Cyclophilin B and Basigin, as well as PfRhopH3 and PfRh5 that aids the invasion. Furthermore, we report identification of a de novo peptide CDP3 that binds Cyclophilin B and blocks invasion by up to 80%. Collectively, our data provide evidence of compounded interactions between host receptors and merozoite surface proteins and paves the way for developing peptide and small-molecules that inhibit the protein-protein interactions, individually or in toto, leading to abrogation of the invasion process.
The prokaryotic ATP-dependent ClpP protease, localized in the relict plastid of malaria parasite, represents a potential drug target. In the present study, we utilized in silico structure-based screening and medicinal chemistry approaches to identify a novel pyrimidine series of compounds inhibiting P. falciparum ClpP protease activity and evaluated their antiparasitic activities. Structure-activity relationship indicated that morpholine moiety at C2, an aromatic substitution at N3 and a 4-oxo moiety on the pyrimidine are important for potent inhibition of ClpP enzyme along with antiparasiticidal activity. Compound 33 exhibited potent antiparasitic activity (EC₅₀ 9.0±0.2μM), a 9-fold improvement over the antiparasitic activity of the hit molecule 6. Treatment of blood stage P. falciparum cultures with compound 33 caused morphological and developmental abnormalities in the parasites; further, compound 33 treatment hindered apicoplast development indicating the targeting of apicoplast.
Protein secretion is an essential process in all eukaryotes including organisms belonging to the phylum Apicomplexa, which includes many intracellular parasites. The apicomplexan parasites possess a specialized collection of secretory organelles that release a number of proteins to facilitate the invasion of host cells and some of these proteins also participate in immune evasion. Like in other eukaryotes, these parasites possess a series of membrane-bound compartments, namely the endoplasmic reticulum (ER), the intermediate compartments (IC) or vesicular tubular clusters (VTS) and Golgi complex through which proteins pass in a sequential and vectorial fashion. Two sets of proteins; COPI and COPII are important for directing the sequential transfer of material between the ER and Golgi complex.
Members of the HtrA family of serine proteases are known to play roles in mitochondrial homeostasis as well as in programmed cell death. Mitochondrial homeostasis and metabolism are crucial for the survival and propagation of the malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin-like protease activity that can be inhibited by its specific inhibitor, ucf-101. A transgenic parasite line was generated, using the HA-glmS C-terminal tagging approach, for localization as well as for inducible knock-down of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during the asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, the ucf-101 treatment did not affect the parasite growth, suggesting the non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase-like protease as well as parasite cell death, suggesting the involvement of protease activity of PfHtrA2 in apoptosis-like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show the specific roles of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating stress-induced cell death.
Proteins that coat Plasmodium falciparum merozoite surface and those secreted from its apical secretory organelles are considered promising candidates for the vaccine against malaria. In the present study, we have identified an asparagine rich parasite protein (PfAARP; Gene ID PFD1105w), that harbors a predicted signal sequence, a C-terminal transmembrane region and whose transcription and translation patterns are similar to some well characterized merozoite surface/apical proteins. PfAARP was localized to the apical end of the merozoites by GFP-targeting approach using an inducible, schizont-stage expression system, by immunofluorescence assays using anti-PfAARP antibodies. Immuno-electron microsopic studies showed that PfAARP is localized in the apical ends of the rhoptries in the merozoites. RBC binding assays with PfAARP expressed on COS cells surface showed that it binds to RBCs through its N-terminal region with a receptor on the RBC surface that is sensitive to trypsin and neuraminidase treatments. Sequencing of PfAARP from different P. falciparum strains as well as field isolates showed that the N-terminal region is highly conserved. Recombinant protein corresponding to the N-terminal region of PfAARP (PfAARP-N) was produced in its functional form in E. coli. PfAARP-N showed reactivity with immune sera from individuals residing in P. falciparum endemic area. The anti-PfAARP-N rabbit antibodies significantly inhibited parasite invasion in vitro. Our data on localization, functional assays and invasion inhibition, suggest a role of PfAARP in erythrocyte binding and invasion by the merozoite.
Mechanisms by which 3'-phosphorylated phosphoinositides (3'-PIPs) regulate the development of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are poorly understood. The catabolic process of autophagy, which is dependent on autophagy-related proteins (ATGs), is one of the major targets of 3'-PIPs in yeast and mammals. In the present study, we identified autophagy-related protein ATG18 as an effector of 3'-PIPs in these parasites. Pfalciparum ATG18 (PfATG18) and Tgondii ATG18 (TgATG18) interact with 3'-PIPs but exhibited differences in their specificity of interaction with the ligand PIP. The conditional knockdown of Tgondii or Pfalciparum ATG18 (Tg/PfATG18) impaired replication of parasites and resulted in their delayed death. Intriguingly, ATG18 depletion resulted in the loss of the apicomplexan parasite-specific nonphotosynthetic plastid-like organelle apicoplast, which harbors the machinery for biosynthesis of key metabolites, and the interaction of ATG18 to phosphatidylinositol 3-phosphate (PI3P) was critical for apicoplast inheritance. Furthermore, ATG18 regulates membrane association and apicoplast localization of ATG8. These findings provide insights into a novel noncanonical role of ATG18 in apicoplast inheritance. This function of ATG18 in organelle biogenesis is unprecedented in any organism and may be conserved across most apicomplexan parasites.IMPORTANCE Typically, autophagy is a catabolic process utilized by cells for their survival upon encountering nutrient-limiting conditions. The autophagy machinery is very tightly regulated, and autophagy-related genes (ATGs) play a pivotal role in this process. In the present study, we report a novel noncanonical function of autophagy-related protein ATG18 in inheritance of the nonphotosynthetic plastid-like organelle apicoplast in apicomplexan parasites Plasmodium and Toxoplasma ATG18 depletion in these parasites resulted in "delayed death," which was the result of loss of apicoplast and impaired parasite division. Pf/TgATG18 interact with 3'-phosphorylated PIPs, which guide their cellular localization in the parasite, which is essential for their function.
The P-loop NTPases constitute one of the largest groups of globular protein domains that play highly diverse functional roles in most of the organisms. Even with the availability of nearly 300 different Hidden Markov Models representing the P-loop NTPase superfamily, not many P-loop NTPases are known in Plasmodium falciparum. A number of characteristic attributes of the genome have resulted into the lack of knowledge about this functionally diverse, but important class of proteins.
Eukaryotic cellular machineries are intricately regulated by several molecular mechanisms involving transcriptional control, post-translational control and post-translational modifications of proteins (PTMs). Reversible protein phosphorylation/dephosphorylation process, which involves kinases as well as phosphatases, represents an important regulatory mechanism for diverse pathways and systems in all organisms including human malaria parasite, Plasmodium falciparum. Earlier analysis on P. falciparum protein-phosphatome revealed presence of 34 phosphatases in Plasmodium genome. Recently, we re-analysed P. falciparum phosphatome aimed at identifying parasite specific phosphatases.
Glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) is an antioxidant enzyme trafficked to the apicoplast, a secondary endosymbiotic organelle, in Plasmodium falciparum. Apicoplast trafficking signals usually consist of N-terminal signal and transit peptides, but the trafficking signal of PfTPxGl appears to exhibit important differences. As transfection is a protracted process in P. falciparum, we expressed the N terminus of PfTPxGl as a GFP fusion protein in a related apicomplexan, Toxoplasma gondii, in order to dissect its trafficking signals. We show that PfTPxGl possesses an N-terminal signal anchor that takes the protein to the endoplasmic reticulum in Toxoplasma-this is the first step in the apicoplast targeting pathway. We dissected the residues important for endomembrane system uptake, membrane anchorage, orientation, spacing, and cleavage. Protease protection assays and fluorescence complementation revealed that the C terminus of the protein lies in the ER lumen, a topology that is proposed to be retained in the apicoplast. Additionally, we examined one mutant, responsible for altered PfTPxGl targeting in Toxoplasma, in Plasmodium. This study has demonstrated that PfTPxGl belongs to an emergent class of proteins that possess signal anchors, unlike the canonical bipartite targeting signals employed for the trafficking of luminal apicoplast proteins. This work adds to the mounting evidence that the signals involved in the targeting of apicoplast membrane proteins may not be as straightforward as those of luminal proteins, and also highlights the usefulness of T. gondii as a heterologous system in certain aspects of this study, such as reducing screening time and facilitating the verification of membrane topology.
The circumsporozoite protein (CSP) is the major surface protein of the sporozoite stage of malaria parasites and has multiple functions as the parasite develops and then migrates from the mosquito midgut to the mammalian liver. The overall structure of CSP is conserved among Plasmodium species, consisting of a species-specific central tandem repeat region flanked by two conserved domains: the NH2-terminus and the thrombospondin repeat (TSR) at the COOH-terminus. Although the central repeat region is an immunodominant B-cell epitope and the basis of the only candidate malaria vaccine in Phase III clinical trials, little is known about its functional role(s). We used the rodent malaria model Plasmodium berghei to investigate the role of the CSP tandem repeat region during sporozoite development. Here we describe two mutant parasite lines, one lacking the tandem repeat region (ΔRep) and the other lacking the NH2-terminus as well as the repeat region (ΔNΔRep). We show that in both mutant lines oocyst formation is unaffected but sporozoite development is defective.
Plasmodium parasites, the causative agents of malaria, possess a distinctive membranous structure of flattened alveolar vesicles supported by a proteinaceous network, and referred to as the inner membrane complex (IMC). The IMC has a role in actomyosin-mediated motility and host cell invasion. Here, we examine the location, protein interactome and function of PhIL1, an IMC-associated protein on the motile and invasive stages of both human and rodent parasites. We show that PhIL1 is located in the IMC in all three invasive (merozoite, ookinete-, and sporozoite) stages of development, as well as in the male gametocyte and locates both at the apical and basal ends of ookinete and sporozoite stages. Proteins interacting with PhIL1 were identified, showing that PhIL1 was bound to only some proteins present in the glideosome motor complex (GAP50, GAPM1-3) of both P. falciparum and P. berghei. Analysis of PhIL1 function using gene targeting approaches indicated that the protein is required for both asexual and sexual stages of development. In conclusion, we show that PhIL1 is required for development of all zoite stages of Plasmodium and it is part of a novel protein complex with an overall composition overlapping with but different to that of the glideosome.
The human malaria parasite, Plasmodium falciparum, takes up numerous host cytosolic components and exogenous nutrients through endocytosis during the intra-erythrocytic stages. Eps15 homology domain-containing proteins (EHDs) are conserved NTPases, which are implicated in membrane remodeling and regulation of specific endocytic transport steps in eukaryotic cells. In the present study, we have characterized the dynamin-like C-terminal Eps15 homology domain containing protein of P. falciparum (PfEHD). Using a GFP-targeting approach, we studied localization and trafficking of PfEHD in the parasite. The PfEHD-GFP fusion protein was found to be a membrane bound protein that associates with vesicular network in the parasite. Time-lapse microscopy studies showed that these vesicles originate at parasite plasma membrane, migrate through the parasite cytosol and culminate into a large multi-vesicular like structure near the food-vacuole. Co-staining of food vacuole membrane showed that the multi-vesicular structure is juxtaposed but outside the food vacuole. Labeling of parasites with neutral lipid specific dye, Nile Red, showed that this large structure is neutral lipid storage site in the parasites. Proteomic analysis identified endocytosis modulators as PfEHD associated proteins in the parasites. Treatment of parasites with endocytosis inhibitors obstructed the development of PfEHD-labeled vesicles and blocked their targeting to the lipid storage site. Overall, our data suggests that the PfEHD is involved in endocytosis and plays a role in the generation of endocytic vesicles at the parasite plasma membrane, that are subsequently targeted to the neutral lipid generation/storage site localized near the food vacuole.
Post-transcriptional and post-translational modifications play a major role in Plasmodium life cycle regulation. Lysine methylation of histone proteins is well documented in several organisms, however in recent years lysine methylation of proteins outside histone code is emerging out as an important post-translational modification (PTM). In the present study we have performed global analysis of lysine methylation of proteins in asexual blood stages of Plasmodium falciparum development. We immunoprecipitated stage specific Plasmodium lysates using anti-methyl lysine specific antibodies that immunostained the asexual blood stage parasites. Using liquid chromatography and tandem mass spectrometry analysis, 570 lysine methylated proteins at three different blood stages were identified. Analysis of the peptide sequences identified 605 methylated sites within 422 proteins. Functional classification of the methylated proteins revealed that the proteins are mainly involved in nucleotide metabolic processes, chromatin organization, transport, homeostatic processes and protein folding. The motif analysis of the methylated lysine peptides reveals novel motifs. Many of the identified lysine methylated proteins are also interacting partners/substrates of PfSET domain proteins as revealed by STRING database analysis. Our findings suggest that the protein methylation at lysine residues is widespread in Plasmodium and plays an important regulatory role in diverse set of the parasite pathways.
Reduced sensitivity of the human malaria parasite, Plasmodium falciparum, to Artemisinin and its derivatives (ARTs) threatens the global efforts towards eliminating malaria. ARTs have been shown to cause ubiquitous cellular and genetic insults, which results in the activation of the unfolded protein response (UPR) pathways. The UPR restores protein homeostasis, which otherwise would be toxic to cellular survival. Here, we interrogated the role of DNA-damage inducible protein 1 (PfDdi1), a unique proteasome-interacting retropepsin in mediating the actions of the ARTs. We demonstrate that PfDdi1 is an active A2 family protease that hydrolyzes ubiquitinated proteasome substrates. Treatment of P. falciparum parasites with ARTs leads to the accumulation of ubiquitinated proteins in the parasites and blocks the destruction of ubiquitinated proteins by inhibiting the PfDdi1 protease activity. Besides, whereas the PfDdi1 is predominantly localized in the cytoplasm, exposure of the parasites to ARTs leads to DNA fragmentation and increased recruitment of the PfDdi1 into the nucleus. Furthermore, we show that Ddi1 knock-out Saccharomycescerevisiae cells are more susceptible to ARTs and the PfDdI1 protein robustly restores the corresponding functions in the knock-out cells. Together, these results show that ARTs act in multiple ways; by inducing DNA and protein damage and might be impairing the damage recovery by inhibiting the activity of PfDdi1, an essential ubiquitin-proteasome retropepsin.
The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface.
Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to 'Pv-fam-a' family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle.
The beta subunits (beta1, beta2, and beta5) of 20S proteasome and HslV/ClpQ are ATP-dependent threonine proteases present in eukaryotes and prokaryotes, respectively that control levels of key regulatory proteins in the cell. The orthologue of prokaryotic HslV protease in Plasmodium falciparum (PfHslV) is a novel drug target candidate that has no homolog in the human host. In the present study, the PfHslV was expressed, localized and biochemically characterized. The recombinant PfHslV harbored threonine protease specific activity as well as chymotrypsin like and peptidyl glutamyl peptide hydrolase activities. All the three activities could be inhibited by respective specific inhibitors. The protein was localized in the cytosol of the parasite as a soluble protein by Western immunoblotting of parasite fractions and by immuno-fluorescence microscopy. Activity of the protease in the parasite was ascertained by following the degradation of GFP in a transgenic parasite line expressing fusion protein of GFP and Arc-repressor gene, a known target of HslV protease in the prokaryotes. A model structure of PfHslV was constructed based on the crystal structure of Escherichia coli HslV to assess the structural homology. Availability of the structure model of PfHslV may facilitate identification or designing of novel and specific drugs against PfHslV. The in vitro protease assays with recombinant PfHslV and the transgenic parasite line generated in the present study may be exploited in the screening of novel inhibitors to evaluate their anti-malarial activity.
A major concern in malaria vaccine development is the polymorphism observed among different Plasmodium isolates in different geographical areas across the globe. The merozoite surface protein 1 (MSP-1) is a leading vaccine candidate antigen against asexual blood stages of malaria parasite. To date, little is known about the extent of sequence variation in the Plasmodium vivax MSP-1 gene (Pvmsp-1) among Indian isolates. Since P. vivax accounts for >50% of malaria cases in India and in Colombia, it is essential to know the Pvmsp-1 gene variability in these two countries to sustain it as a vaccine candidate. The extent of polymorphism in Pvmsp-1 gene among Indian and Colombian isolates is described.
Despite significant progress in apicomplexan genome sequencing and genomics, the current list of experimentally validated transcription factors (TFs) in these genomes is incomplete and mainly consists of AP2 family of proteins, with only a limited number of non-AP2 family TFs and transcription-associated co-factors (TcoFs). We have performed a systematic bioinformatics-aided prediction of TFs and TcoFs in apicomplexan genomes and developed the ApicoTFdb database which consists of experimentally validated as well as computationally predicted TFs and TcoFs in 14 apicomplexan species. The predicted TFs are manually curated to complement the existing annotations. The current version of the database includes 1292 TFs which includes experimentally validated and computationally predicted TFs, representing 20 distinct families across 14 apicomplexan species. The predictions include TFs of TUB, NAC, BSD, HTH, Cupin/Jumonji, winged helix and FHA family proteins, not reported earlier as TFs in the genomes. Apart from TFs, ApicoTFdb also classifies TcoFs into three main subclasses: TRs, CRRs and RNARs, representing 2491 TcoFs in 14 apicomplexan species, are analyzed in this study. The database is designed to integrate different tools for comparative analysis. All entries in the database are dynamically linked with other databases, literature reference, protein-protein interactions, pathways and annotations associated with each protein. ApicoTFdb will be useful to the researchers interested in less-studied gene regulatory mechanisms mediating the complex life cycle of the apicomplexan parasites. The database will aid in the discovery of novel drug targets to much needed combat the growing drug resistance in the parasites.
The human malaria parasite, Plasmodium falciparum possesses unique gliding machinery referred to as the glideosome that powers its entry into the insect and vertebrate hosts. Several parasite proteins including Photosensitized INA-labelled protein 1 (PhIL1) have been shown to associate with glideosome machinery. Here we describe a novel PhIL1 associated protein complex that co-exists with the glideosome motor complex in the inner membrane complex of the merozoite. Using an experimental genetics approach, we characterized the role(s) of three proteins associated with PhIL1: a glideosome associated protein- PfGAPM2, an IMC structural protein- PfALV5, and an uncharacterized protein-referred here as PfPhIP (PhIL1 Interacting Protein). Parasites lacking PfPhIP or PfGAPM2 were unable to invade host RBCs. Additionally, the downregulation of PfPhIP resulted in significant defects in merozoite segmentation. Furthermore, the PfPhIP and PfGAPM2 depleted parasites showed abrogation of reorientation/gliding. However, initial attachment with host RBCs was not affected in these parasites. Together, the data presented here show that proteins of the PhIL1-associated complex play an important role in the orientation of P. falciparum merozoites following initial attachment, which is crucial for the formation of a tight junction and hence invasion of host erythrocytes.
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