Identification of karyopherin β as an immunogenic antigen of the malaria parasite using immune mice and human sera

A differential immunoscreening of the λgt11 Plasmodium falciparum genomic expression library was carried out using anti-P. yoelii sera (convalescent-phase mouse sera) and immune sera collected from healthy adults, to identify novel cross-reactive and possibly protective antigens of the parasite. One clone, with an insert size of 1132 bp that reacted strongly with both the sera was selected. The insert was found to be a part of the P. falciparum karyopherin β (PfKβ) homologue. RT-PCR and Northern blot analysis confirmed the expression of PfKβ in the blood stages of the parasite. The ~110 kDa protein was localized in the cytoplasm at the ring and trophozoite, and in the parasitophorous vacuole at the schizont stage. Two large fragments of PfKβ representing the N- and C-terminal halves were expressed in E. coli. The recombinant proteins were highly immunogenic in mice, and also found to be the target for immune response in natural infections of Plasmodium spp. Anti-sera against the protein showed a low level of anti-parasitic activity. Immunization with recombinant PfKβ fragments was only partially protective against a heterologous challenge infection in mice. Our results show that the parasite releases a highly immunogenic, cytoplasmic protein into the host which may not contribute to the development of protective immunity

Transcriptome Analysis Reveals Strain-Specific and Conserved Stemness Genes in Schmidtea mediterranea

The planarian Schmidtea mediterranea is a powerful model organism for studying stem cell biology due to its extraordinary regenerative ability mediated by neoblasts, a population of adult somatic stem cells. Elucidation of the S. mediterranea transcriptome and the dynamics of transcript expression will increase our understanding of the gene regulatory programs that regulate stem cell function and differentiation. Here, we have used RNA-Seq to characterize the S. mediterranea transcriptome in sexual and asexual animals and in purified neoblast and differentiated cell populations. Our analysis identified many uncharacterized genes, transcripts, and alternatively spliced isoforms that are differentially expressed in a strain or cell type-specific manner. Transcriptome profiling of purified neoblasts and differentiated cells identified neoblast-enriched transcripts, many of which likely play important roles in regeneration and stem cell function. Strikingly, many of the neoblast-enriched genes are orthologs of genes whose expression is enriched in human embryonic stem cells, suggesting that a core set of genes that regulate stem cell function are conserved across metazoan species

In vivo gene silencing in Plasmodium berghei - a Mouse Malaria Model

RNA interference (RNAi) has emerged as a specific and efficient tool to silence gene expression in a variety of organisms and cell lines. An important prospect for RNAi technology is its possible application in the treatment of diseases using short interfering RNAs (siRNAs). However, the effect of siRNAs in adult animals and their potential to treat or prevent diseases are yet to be fully investigated. The main goal of the present study is to find out whether it was possible to carry out RNAi on circulating malaria parasite in vivo. To trigger RNAi in mouse malaria parasite, we used siRNAs corresponding to cysteine protease genes of Plasmodium berghei (berghepain-1 & 2). Intravenous injections of berghepains' siRNAs in infected animal resulted in characteristic enlargement of food vacuole in circulating parasites. Protein analysis of these treated parasites showed substantial accumulation of hemoglobin, which is reminiscent of the effect observed upon treating Plasmodium falciparum with different cysteine protease inhibitors. Parasites treated with berghepain 1 & 2 siRNAs showed marked reduction in the levels of their cognate mRNAs, thereby suggesting specific inhibition of berghepains' gene expression in vivo. We also observed the generation of ~25 nt RNA species from berghepains' mRNAs in the treated parasites, which is a characteristic of an RNAi phenomenon. These results thus provide evidence that beyond its value for validation of gene functions, RNAi may provide a new approach for disease therapy

Identification of karyopherin β as an immunogenic antigen of the malaria parasite using immune mice and human sera

SUMMARY A differential immunoscreening of the λ gt11 Plasmodium falciparum genomic expression library was carried out using anti-P. yoelii sera (convalescent

Immunogenicity and protective efficacy of Escherichia coli expressed Plasmodium falciparum merozoite surface protein-1<SUB>42</SUB> using human compatible adjuvants

The C-terminal 42-kDa fragment of the merozoite surface protein-1 of Plasmodium falciparum (PfMSP-142) was expressed as a recombinant protein in Escherichia coli and purified to near homogeneity. We tested the immunogenicity of recombinant PfMSP-142 in three clinically acceptable adjuvants (Montanide ISA 720, alum and MF59) in mice and in rabbits. High antibody responses were obtained with two adjuvant formulations with IgGl being the predominant immunoglobulin isotype. Significant T-cell proliferation responses were also observed. Competitive enzyme linked immunosorbant assay (ELISA) showed the presence of both invasion and processing inhibitory antibodies in sera obtained from the immunized rabbits. Passive immunizations of mice with anti-PfMSP-142 IgG purified from the rabbit-sera were found to be protective against a parasite challenge with P. berghei/P. falciparum chimeric line (Pb-PfM19) that expresses Plasmodium falciparum MSP-119. These findings may be useful for the development of a malaria vaccine based on Plasmodium falciparum MSP-142

A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression

The process of merozoite release in Plasmodium falciparum involves rupture of the parasitophorous vacuole membrane and erythrocyte plasma membrane. Through the use of protease inhibitors that halt the merozoite release, a number of parasite proteases, especially serine, aspartic, and cysteine proteases, have been implicated in the schizont rupture. To understand the precise role of cysteine proteases in the merozoite release, in the present study, we treated P. falciparum cultures with siRNAs corresponding to falcipain-1, falcipain-2, and falcipain-3, the three papain-family proteases of the parasite. Treatment of malaria parasites with either of the falcipain siRNAs considerably reduced parasite growth. Morphological examination of the siRNA treated parasite cultures revealed that most of the parasites in falcipain-2 siRNA treated cultures were arrested at schizont stage. Analysis of a transgenic P. falciparum line expressing chimeric-GFP upon treatment with falcipain-2 siRNA revealed block in the rupture of erythrocyte membrane at the time of merozoite egression. These results suggest that falcipain-2 is an important parasitic protease that participates in hemoglobin degradation and in the merozoite release

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