Alfica Sehgal

Peculiarities of Host Cholesterol Transport to the Unique Intracellular Vacuole Containing Toxoplasma

The intracellular protozoan Toxoplasma gondii is auxotrophic for low‐density lipoprotein (LDL)‐derived cholesterol (C). We previously showed that T. gondii scavenges this essential lipid from host endolysosomal compartments and that C delivery to the parasitophorous vacuole (PV) does not require transit through host Golgi or endoplasmic reticulum. In this study, we explore the itinerary of C from the host endolysosomes to the PV. Labeled C incorporated into LDL is rapidly detected in intravacuolar parasites and partially esterified by the parasites. In contrast to diverse mammalian organelles, the post‐endolysosomal transfer of C to the PV does not involve the host plasma membrane as an intermediate. Nevertheless, the PV membrane is accessible to extracellular sterol acceptors, suggesting C trafficking from intracellular parasites to host plasma membrane. C movement to the PV requires temperatures permissive for vesicular transport, metabolic energy and functional microtubules. Host caveolae vesicles and the sterol carrier protein‐2 do not participate in this process. Proteolytic treatment of purified PV or free parasites abolishes C acquisition by the parasites. Altogether, these results support a vesicular transport system from host endolysosomes to the PV, and a requirement for PV membrane and parasite plasma membrane proteins in C delivery to T. gondii.

The P Domain of the P0 Protein of Plasmodium falciparum Protects against Challenge with Malaria Parasites

ABSTRACT Monoclonal antibodies (MAbs) specific for the P domain of the Plasmodium falciparum P0 phosphoriboprotein (PfP0) blocked the invasion of RBCs by P. falciparum. Vaccination with this P-domain peptide protected mice upon malaria parasite challenge. The absolute specificity of the MAbs and the PfP0 P peptide makes them potential protective malaria reagents.

Surface expression of the conserved ribosomal protein P0 on parasite and other cells

Invasion-blocking antibodies against the ribosomal phosphoprotein P0 of the human malarial parasite Plasmodium falciparum (PfP0) have been identified through a differential immunoscreen using immune and patient sera from malaria endemic regions of Eastern India [1]. Purified IgG from rabbit sera, raised against different domains of the PfP0 protein, show no effect on the intraerythrocytic stages of Plasmodium falciparum in culture, but inhibit the invasion of erythrocytes by merozoites in a concentration dependent manner [2,3]. The same antibodies against the PfP0 protein protect mice against challenge with a virulent strain of Plasmodium yoelii [4]. We have recently demonstrated this protein to be present on the surface of merozoites and gametocytes of Plasmodium [3,4]. The phosphoprotein P0 is highly conserved across eukaryotic organisms [5], and is related to the family of phosphoproteins P1 and P2, due to the highly homologous carboxy-terminal 22 amino acid domain [6]. The ribosomal function of P0 is mediated through a pentameric P12·P0·P22 complex, which forms the stalk of the large ribosomal subunit at the GTPase center [7]. P0 interacts with the elongation factor eEF2 [8], and is essential for the ribosomal activity and cell viability in yeast [9]. Through deletion analysis of the P0 protein, the ribosomal function has been mapped to amino acid position 185–230 in yeast P0 [10]. Since P0 is a very conserved protein [5], we wondered whether the surface expression of P0 is an exclusive property of Plasmodium cells, or if it is a general property of the P0 protein in other organisms as well. The presence of this protein on the host cell surface will have serious implications with the usage of this protein in a malaria vaccine. This question also has a direct bearing on the mechanism of translocation of this protein to the surface, as to whether it is a unique phenomenon for Plasmodium. Therefore, we decided to investigate the presence of this protein on different cell types. In this paper we report the localization of P0 protein on the surface of another Apicomplexan parasite Toxoplasma, yeast and mammalian cell lines using mono-specific cross-reactive anti-PfP0 antibodies. In order to test for cross-reactivity of anti-PfP0 sera with different cell types, Western Blot analysis was carried out with total protein extract of these cell types. Anti-PfP0 antisera were raised in rabbits against recombinant GST-fusion proteins, PfP0N (amino acid 17–61) and PfP0C (amino acid 61–316), and purified as described earlier [3]. Specific single band reactivities of 38, 33, 34 and 38 kD proteins were observed with Toxoplasma gondii, yeast (Saccharomyces cere isiae, strain EG103), Chinese Hamster Ovary (CHO) cells and human leukocyte protein preparations respectively, using Abbre iations: GST, glutathione-S-transferase; IPTG, isopropylthio-galactosidase; PfP0, Plasmodium falciparum phosphoriboprotein P0; PfP0N, amino-terminal domain of PfP0; PfP0C, carboxy-terminal domain of PfP0. * Corresponding author. Tel: +91-22-215-2971/2979 x 2570; fax: +91-22-215-2110/2181. E-mail address: sharma@tifr.res.in (S. Sharma). 1 Present address: Lecturer, Agricultural Science Unit, Indian Statistical Institute, 203, B.T. Road, Calcutta 700 035, India.

Translocation of ribosomal protein P0 onto the Toxoplasma gondii tachyzoite surface.

A ribosomal phosphoprotein P0 detected on the surface of the human malarial parasite Plasmodium falciparum (PfP0) has been shown to be recognised by invasion blocking antibodies. Using cross-reactive polyclonal antibodies against PfP0, the surface localisation has also been demonstrated on certain mammalian cells, yeast and Toxoplasma gondii. We sought to characterise the phenomenon of surface localisation in Toxoplasma using T. gondii P0 protein. Sequence analysis of a cDNA clone isolated from a T. gondii library showed marked similarity to PfP0, suggesting that T. gondii expresses an orthologous gene, TgP0. The expression of TgP0 was corroborated by Northern blot analysis revealing a transcript of 1.8 kb in size. Immunofluorescence analysis using anti-PfP0 indicated surface localisation of TgP0. To confirm surface translocation of the TgP0, tachyzoites were transfected with the HA-tagged TgP0 gene followed by immunofluorescence detection of the HA-tag. Surface translocation of transiently expressed TgP0 and blocking of tachyzoite invasion of human foreskin fibroblasts by anti-PfP0 antibodies suggest that P0 protein plays an important role in T. gondii invasion of human cells.

Identification of a hypothetical membrane protein interactor of ribosomal phosphoprotein P0.

The ribosomal phosphoprotein P0 of the human malarial parasitePlasmodium falciparum (PfP0) has been identified as a protective surface protein. InDrosophila, P0 protein functions in the nucleus. The ribosomal function of P0 is mediated at the stalk of the large ribosomal subunit at the GTPase centre, where the elongation factor eEF2 binds. The multiple roles of the P0 protein presumably occur through interactions with other proteins. To identify such interacting protein domains, a yeast two-hybrid screen was carried out. Out of a set of sixty clones isolated, twelve clones that interacted strongly with both PfP0 and theSaccharomyces cerevisiae P0 (ScP0) protein were analysed. These belonged to three broad classes: namely (i) ribosomal proteins; (ii) proteins involved in nucleotide binding; and (iii) hypothetical integral membrane proteins. One of the strongest interactors (clone 67B) mapped to the gene YFL034W which codes for a hypothetical integral membrane protein, and is conserved amongst several eukaryotic organisms. The insert of clone 67B was expressed as a recombinant protein, and immunoprecipitaion (IP) reaction with anti-P0 antibodies pulled down this protein along with PfP0 as well as ScP0 protein. Using deletion constructions, the domain of ScP0, which interacted with clone 67B, was mapped to 60–148 amino acids. It is envisaged that the surface localization of P0 protein may be mediated through interactions with putative YFL034W-like proteins inP. falciparum

Characterization of a differential immunoscreen epitope of Plasmodium falciparum using combinatorial agents

A differential serological screening of a λgt11 cDNA expression library has identified several clones, which react exclusively to sera samples from persons clinically immune to malaria but not to acute malaria patient sera. One such clone, IPf9, has a 315‐bp cDNA insert, which was found to be conserved in different strains of the human and rodent malarial parasite Plasmodium falciparum and Plasmodium berghei, respectively. The induced expression product of IPf9 was used to generate polyclonal sera in rabbits. The IPf9 expression product was also screened with phage surface display combinatorial libraries to isolate reagents that specifically bound to the IPf9 product. The polyclonal antisera and the combinatorial reagents recognized a 50‐kDa protein from P. falciparum, and a 53‐kDa product from P. berghei. Immunofluorescence studies using asexual and sexual stages of P. falciparum showed the protein to be present within the parasite in each of the asexual and sexual stages. The combinatorial reagents showed a partial inhibition in the growth of P. falciparum in vitro. Mice infected with the P. berghei showed the presence of T‐cells that exhibited lymphoproliferation when stimulated with the IPf9 protein. It is suggested that IPf9 protein is a conserved protein epitope, and may be relevant for a protective immune response to malaria.

A protective merozoite protein of Plasmodium falciparum shares an epitope with surface antigens of Paramecium.

A Plasmodium falciparum cDNA expression clone, λPf9, had been identified earlier as a protective epitope, using anti‐λPf9 antibodies and combinatorial phagotopes. A segment of the Pf9 gene showed homology with Paramecium immobilization surface antigens such as 51B, 51A and 156G. A synthetic Pf9‐peptide was designed from this region, and specific antibodies were raised. Each of these anti‐Pf9 antibodies and combinatorial reagents, as well as anti‐Paramecium 51B antibodies, recognized the Pf9‐peptide on ELISA, and the same protein band in parasite immunoblots. The P. falciparum protein was released from the merozoite membrane fraction on treatment with PI‐PLC, indicating the presence of a GPI anchor. Anti‐Pf9‐peptide antibodies specifically inhibited the growth of P. falciparum in culture. Immunofluorescence assays showed the reactivity of anti‐Pf9‐peptide sera with P. falciparum merozoites and gametocytes, as well as on the surface of Paramecium tetraurelia. The Pf9‐peptide was able to induce proliferation of splenic lymphocytes obtained from mice infected with the rodent malarial parasites Plasmodium berghei and Plasmodium yoelii. These results point towards Plasmodium Pf9 as a conserved novel protective protein, sharing an epitope with Paramecium surface antigens.