Arrest of nuclear division in Plasmodium through blockage of erythrocyte surface exposed ribosomal protein P2.

Malaria parasites reside inside erythrocytes and the disease manifestations are linked to the growth inside infected erythrocytes (IE). The growth of the parasite is mostly confined to the trophozoite stage during which nuclear division occurs followed by the formation of cell bodies (schizogony). The mechanism and regulation of schizogony are poorly understood. Here we show a novel role for a Plasmodium falciparum 60S stalk ribosomal acidic protein P2 (PfP2) (PFC0400w), which gets exported to the IE surface for 6-8 hrs during early schizogony, starting around 26-28 hrs post-merozoite invasion. The surface exposure is demonstrated using multiple PfP2-specific monoclonal antibodies, and is confirmed through transfection using PfP2-GFP. The IE surface-exposed PfP2-protein occurs mainly as SDS-resistant P2-homo-tetramers. Treatment with anti-PfP2 monoclonals causes arrest of IEs at the first nuclear division. Upon removal of the antibodies, about 80-85% of synchronized parasites can be released even after 24 hrs of antibody treatment. It has been reported that a tubovesicular network (TVN) is set up in early trophozoites which is used for nutrient import. Anti-P2 monoclonal antibodies cause a complete fragmentation of TVN by 36 hrs, and impairs lipid import in IEs. These may be downstream causes for the cell-cycle arrest. Upon antibody removal, the TVN is reconstituted, and the cell division progresses. Each of the above properties is observed in the rodent malaria parasite species P. yoelii and P. berghei. The translocation of the P2 protein to the IE surface is therefore likely to be of fundamental importance in Plasmodium cell division.

Organization of Plasmodium falciparum spliceosomal core complex and role of arginine methylation in its assembly.

BACKGROUND: Splicing and alternate splicing are the two key biological processes that result in the generation of diverse transcript and protein isoforms in Plasmodium falciparum as well as in other eukaryotic organisms. Not much is known about the organization of splicing machinery and mechanisms in human malaria parasite. Present study reports the organization and assembly of Plasmodium spliceosome Sm core complex. METHODS: Presence of all the seven Plasmodium Sm-like proteins in the intra-erythrocytic stages was assessed based on the protein(s) expression analysis using immuno-localization and western blotting. Localization/co-localization studies were performed by immunofluorescence analysis on thin parasite smear using laser scanning confocal microscope. Interaction studies were carried out using yeast two-hybrid analysis and validated by in vitro pull-down assays. PfPRMT5 (arginine methyl transferase) and PfSmD1 interaction analysis was performed by pull-down assays and the interacting proteins were identified by MALDI-TOF spectrometry. RESULTS: PfSm proteins are expressed at asexual blood stages of the parasite and show nucleo-cytoplasmic localization. Protein-protein interaction studies showed that PfSm proteins form a heptameric complex, typical of spliceosome core complex as shown in humans. Interaction of PfSMN (survival of motor neuron, tudor domain containing protein) or PfTu-TSN (Tudor domain of Tudor Staphylococcal nuclease) with PfSmD1 proteins was found to be methylation dependent. Co-localization by immunofluorescence and co-immunoprecipitation studies suggested an association between PfPRMT5 and PfSmD1, indicating the role of arginine methylation in assembly of Plasmodium spliceosome complex. CONCLUSIONS: Plasmodium Sm-like proteins form a heptameric ring-like structure, although the arrangement of PfSm proteins slightly differs from human splicing machinery. The data shows the interaction of PfSMN with PfSmD1 and this interaction is found to be methylation dependent. PfPRMT5 probably exists as a part of methylosome complex that may function in the cytoplasmic assembly of Sm proteins at asexual blood stages of P. falciparum.

Proteome analysis reveals a large merozoite surface protein-1 associated complex on the Plasmodium falciparum merozoite surface.

Plasmodium merozoite surface protein-1 (MSP-1) is an essential antigen for the merozoite invasion of erythrocytes. A key challenge to the development of an effective malaria vaccine that can block the erythrocyte invasion is to establish the molecular interaction(s) among the parasite surface proteins as well as with the host cell encoded receptors. In the present study, we applied molecular interactions and proteome approaches to identify PfMSP-1 associated complex on the merozoite surface. Proteomic analysis identified a major malaria surface protein, PfRhopH3 interacting with PfMSP-1(42). Pull-down experiments with merozoite lysate using anti-PfMSP-1 or anti-PfRhopH3 antibodies showed 16 bands that when identified by tandem mass spectrometry corresponded to11 parasite proteins: PfMSP-3, PfMSP-6, PfMSP-7, PfMSP-9, PfRhopH3, PfRhopH1, PfRAP-1, PfRAP-2, and two RAP domain containing proteins. This MSP-1 associated complex was specifically seen at schizont/merozoite stages but not the next ring stage. We could also identify many of these proteins in culture supernatant, suggesting the shedding of the complex. Interestingly, the PfRhopH3 protein also showed binding to the human erythrocyte and anti-PfRhopH3 antibodies blocked the erythrocyte invasion of the merozoites. These results have potential implications in the development of PfMSP-1 based blood stage malaria vaccine.

Systematic deletion and site-directed mutagenesis of FHVB2 establish the role of C-terminal amino acid residues in RNAi suppression.

Viruses and siRNA/miRNA machinery of the host cell interact in diverse ways with the virus encoded RNAi suppressor proteins. These interactions have implications on the replication and pathogenicity of the virus and also on the immune response of the host. Suppressor protein B2 of insect Flock House Virus (FHVB2), has been shown to mediate RNAi suppression via N-terminal region by directly binding to dsRNA. We have previously shown that FHVB2 protein also interacts with host Dicer protein via its PAZ domain. In the present study, we performed systematic mutagenesis studies to map the FHVB2 regions involved in mediating suppression of RNAi. Progressive deletion of 17 amino acids from N- and C-terminii of FHVB2 resulted in cumulative decrease in RNAi suppression activity of FHVB2. The deletion of 17 amino acids from the C-terminus resulted in more reduction in RNAi suppression in comparison to the N-terminal deletions. Subsequently, we generated 17 successive point mutants of FHVB2 C-terminus and evaluated the RNAi suppression activity for each of the point mutants. Each point mutation resulted in a significant reduction in RNAi suppression activity of FHVB2. These results provide evidence for the role of C-terminal of FHVB2 in RNAi suppression.

A prodomain peptide of Plasmodium falciparum cysteine protease (falcipain-2) inhibits malaria parasite development.

Falcipain-2 (FP-2), a papain family cysteine protease of Plasmodium falciparum, is a promising target for antimalarial chemotherapy. Designing inhibitors that are highly selective for falcipain-2 has been difficult because of broad specificity of different cysteine proteinases. Because propeptide regions of cysteine proteases have been shown to inhibit their cognate enzymes specifically and selectively, in the present study, we evaluated the inhibitory potential of few falcipain-2 proregion peptides. A 15 residue peptide (PP1) inhibited falcipain-2 enzyme activity in vitro. Studies on the uptake of PP1 into the parasitized erythrocytes showed access of peptide into the infected RBCs. PP1 fused with Antennapedia homeoprotein internalization domain blocked hemoglobin hydrolysis, merozoite release and markedly inhibited Plasmodium falciparum growth and maturation. Together, our results identify a peptide derived from the proregion of falcipain-2 that blocks late-stage malaria parasite development in RBCs, suggesting the development of peptide and peptidometric drugs against the human malaria parasite.

Tudor domain proteins in protozoan parasites and characterization of Plasmodium falciparum tudor staphylococcal nuclease.

RNA-binding proteins play key roles in post-transcriptional regulation of gene expression. In eukaryotic cells, a multitude of RNA-binding proteins with several RNA-binding domains/motifs have been described. Here, we show the existence of two Tudor domain containing proteins, a survival of motor neuron (SMN)-like protein and a Staphylococcus aureus nuclease homologue referred to as TSN, in Plasmodium and other protozoan parasites. Activity analysis shows that Plasmodium falciparum TSN (PfTSN) possesses nuclease activity and Tudor domain is the RNA-binding domain. A specific inhibitor of micrococcal nucleases, 3',5'-deoxythymidine bisphosphate (pdTp) inhibits the nuclease as well as RNA-binding activities of the protein. PfTSN shows a predominant nuclear localization. Treatment of P. falciparum with pdTp, inhibited in vitro growth of both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, while a four fold concentration of pdTp did not have any significant effect on the mammalian cell line, Huh-7D12. Altogether, these results suggest that PfTSN is an essential enzyme in the parasite's life cycle.

Food vacuole targeting and trafficking of falcipain-2, an important cysteine protease of human malaria parasite Plasmodium falciparum.

Malaria proteases are attractive anti-malarial targets because of their roles in parasite development and infection. Falcipain-2 (FP-2), a food vacuole cysteine protease in Plasmodium falciparum, is involved in hemoglobin degradation and cleavage of cytoskeletal elements. To understand the route of trafficking and identify the signals involved in trafficking to food vacuole, we have generated transgenic parasites expressing green fluorescent protein (GFP) fusion proteins comprising of N-terminal regions of falcipain-2 fused to GFP. Using falcipain2-GFP chimeras and anti-falcipain-2 antibody, we show that falcipain-2 is trafficked through a classical vesicle mediated secretory pathway involving endoplasmic reticulum and Golgi-like apparatus. Photobleaching and confocal microscopy techniques reveal that falcipain-2 is carried to the food vacuole in the form of cytostomal vesicles. We identify an N-terminal sequence (1-120aa) of falcipain-2, sufficient for its transport to the food vacuole. Analysis of sequences of few other food vacuole targeted proteins suggests a common mechanism for protein trafficking to food vacuole of malaria parasite.

Design and synthesis of protein-protein interaction mimics as Plasmodium falciparum cysteine protease, falcipain-2 inhibitors.

Small peptides that mimic the protein-protein interactions between falcipain-2 and egg white cystatin, an endogenous inhibitor of cysteine proteases, were designed and synthesized and their effects on falcipain-2 activity were analyzed. The mimics are characterized by the presence of different linkers: γ-aminobutyric acid, cis-4-aminocyclohexane carboxylic acid and a macrocycle formed by GABA and two cysteines joined by a disulfide linkage. Some of these compounds showed falcipain-2 inhibition in the micromolar range and produced morphological abnormalities in the Plasmodium food vacuole. Although these peptides are less potent than cystatin, considering the reduction of amino acid residues and the capacity to cross membranes, this approach could be an interesting starting point for the development of a new class of anti-malarial drugs.

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.

Voltage gated calcium channels negatively regulate protective immunity to Mycobacterium tuberculosis.

Mycobacterium tuberculosis modulates levels and activity of key intracellular second messengers to evade protective immune responses. Calcium release from voltage gated calcium channels (VGCC) regulates immune responses to pathogens. In this study, we investigated the roles of VGCC in regulating protective immunity to mycobacteria in vitro and in vivo. Inhibiting L-type or R-type VGCC in dendritic cells (DCs) either using antibodies or by siRNA increased calcium influx in an inositol 1,4,5-phosphate and calcium release calcium activated channel dependent mechanism that resulted in increased expression of genes favoring pro-inflammatory responses. Further, VGCC-blocked DCs activated T cells that in turn mediated killing of M. tuberculosis inside macrophages. Likewise, inhibiting VGCC in infected macrophages and PBMCs induced calcium influx, upregulated the expression of pro-inflammatory genes and resulted in enhanced killing of intracellular M. tuberculosis. Importantly, compared to healthy controls, PBMCs of tuberculosis patients expressed higher levels of both VGCC, which were significantly reduced following chemotherapy. Finally, blocking VGCC in vivo in M. tuberculosis infected mice using specific antibodies increased intracellular calcium and significantly reduced bacterial loads. These results indicate that L-type and R-type VGCC play a negative role in M. tuberculosis infection by regulating calcium mobilization in cells that determine protective immunity.

Falcipain-1, a Plasmodium falciparum cysteine protease with vaccine potential.

Cysteine proteases (falcipains) of Plasmodium falciparum are potential targets for antimalarial chemotherapy, since they have been shown to be involved in important cellular functions such as hemoglobin degradation and invasion/rupture of red blood cells during parasite life cycle. The role of falcipain-1 at the asexual blood stages of the parasite still remains uncertain. This is mainly due to a lack of methods to prepare this protein in an active form. In order to obtain biologically active falcipain-1, a number of falcipain-1 constructs were designed and a systematic assessment of the refolding conditions was done. We describe here the expression, purification, and characterization of a falcipain-1 construct encoding mature falcipain-1 and 35 amino acids from the C-terminal of the pro domain. Recombinant falcipain-1 was overexpressed in the form of inclusion bodies, solubilized, and purified by Ni(2+)-nitrilotriacetic acid affinity chromatography under denaturing conditions. A systemic approach was then followed to optimize refolding parameters. An optimum refolding condition was obtained, and the yield of the purified refolded falcipain-1 was approximately 1 mg/liter. Activity of the protein was analyzed by fluorometric and gelatin degradation assays. Immunolocalization studies using anti-falcipain-1 sera revealed a distinct staining at the apical end of the P. falciparum merozoites. Previous studies using falcipain-1-specific inhibitors have suggested a role of falcipain-1 in merozoite invasion. Based on its localization and its role in invasion, we analyzed the immunogenicity of falcipain-1 in mice, followed by heterologous challenge with Plasmodium yoelii sporozoites. Our results suggest a possible role of falcipain-1 in merozoite invasion.