Proteome mapping of Plasmodium: identification of the P. yoelii remodellome.

Plasmodium associated virulence in the host is linked to extensive remodelling of the host erythrocyte by parasite proteins that form the "remodellome". However, without a common motif or structure available to identify these proteins, little is known about the proteins that are destined to reside in the parasite periphery, the host-cell cytoplasm and/or the erythrocyte membrane. Here, the subcellular fractionation of erythrocytic P. yoelii at trophozoite and schizont stage along with label-free quantitative LC-MS/MS analysis of the whole proteome, revealed a proteome of 1335 proteins. Differential analysis of the relative abundance of these proteins across the subcellular compartments allowed us to map their locations, independently of their predicted features. These results, along with literature data and in vivo validation of 61 proteins enabled the identification of a remodellome of 184 proteins. This approach identified a significant number of conserved remodelling proteins across plasmodium that likely represent key conserved functions in the parasite and provides new insights into parasite evolution and biology.

Cellular immune response from Chagasic patients to CRA or FRA recombinant antigens of Trypanosoma cruzi.

We propose to analyze the relation between the cellular immune response of Chagas' disease patients after in vitro stimulation of peripheral blood mononuclear cells (PBMC) with recombinant antigens cytoplasmatic repetitive antigen (CRA) or flagellar repetitive antigen (FRA) of T. cruzi and the chronic clinical forms of disease. Cells were stimulated using phytohemagglutinin, CRA, FRA, or a soluble antigen of Epimastigota (Ag-Epi) for 24 hr, 72 hr, or 6 days. The proliferation of cells was evaluated after 6 days of culture by quantification of incorporated 3H-thymidine. Cytokines were measured in the supernatants obtained after 24 hr (tumor necrosis factor [TNF]-alpha and interleukin [IL]-4), 72 hr (IL-10), and 6 days (interferon [IFN]-gamma) using enzyme-linked immunosorbent assay (ELISA). Cells of the Chagas patients stimulated with the recombinant antigens exhibited higher proliferation responses compared with that of non-Chagas (NC) individuals. However, when proliferation was compared between patients with the cardiac form (CF) or indeterminate form (IF), it was not possible to establish a difference in the response. So far as the cytokines secreted in the culture supernatants after stimulation in vitro with T. cruzi antigens were concerned, the results showed that CRA, as well as Epi-Ag, were able to stimulate the production of TNF-alpha and IFN-gamma in Chagas patients as compared with NC individuals. However, the cytokine levels after stimulation with the T. cruzi antigens were not different between the patients with CF and IF. CRA was capable of inducing a T helper type 1 (Th1) immune response, with elevated production of TNF-alpha and IFN-gamma in Chagas patients that are carriers of CF and IF clinical forms.

Exploiting calnexin expression on phagosomes to isolate Leishmania parasitophorous vacuoles.

We have developed a simple scheme for the isolation of parasitophorous vacuoles (PVs) that harbor Leishmania parasites. This scheme exploits the observation that PVs display endoplasmic reticulum molecules, including the transmembrane protein calnexin. The presence of calnexin at the surface of the PVs distinguishes them from late endosomal vesicles of comparable density. As a result, PVs can be isolated by calnexin affinity selection from an enriched PV fraction obtained by sucrose density fractionation.

TbRAB1 and TbRAB2 mediate trafficking through the early secretory pathway of Trypanosoma brucei.

The African trypanosome possesses a total of 16 small GTPases of the Rab family, which are involved in control of various membrane transport events. Recently the roles of these proteins in the endocytosis and recycling of the major surface antigen of the bloodstream form, the variant surface glycoprotein (VSG), have been described but little has been reported on the roles of Rab proteins in exocytic pathways in trypanosomatids. Whilst phylogenetic analysis based on sequence similarity indicates a comparatively well conserved core set of Rab proteins, the evolutionary distance of the trypanosome lineage from crown eukaryote model systems requires direct experimental evidence to support these sequence data. By database searching we identified two further Rab genes, TbRAB1 and TbRAB2, which are the trypanosome sequence orthologues of mammalian Rab1 and Rab2, important mediators of ER to Golgi and intra-Golgi transport processes. A remarkably high level of sequence conservation is retained between the trypanosome and higher eukaryote orthologues. By immunolocalisation we find that both TbRAB1 and TbRAB2 reside on membranes in intimate association with the Golgi complex. By heterologous expression in mammalian cells we also demonstrate conservation of targeting information in the TbRAB1 and TbRAB2 proteins, whilst TbRAB1, but not TbRAB2, can complement a Ypt1(ts) conditional mutant in Saccharomyces cerevisiae. The roles of TbRAB1 and TbRAB2 in exocytosis were examined using RNAi. Suppression of TbRAB1 or TbRAB2 was strongly inhibitory to growth and most importantly both TbRAB1 and TbRAB2 were required for normal progression of VSG through the early secretory pathway. These data indicate conservation of function for these proteins between trypanosomes and crown eukaryotes.

PSLAP, a protein with multiple adhesive motifs, is expressed in Plasmodium falciparum gametocytes.

A gene coding for a protein containing two Scavenger Receptor Cysteine-Rich (SRCR) motifs, four Limulus factor C, Coch-5b2 and Lgl1 (LCCL) motifs; and one Polycystin-1, Lipoxygenase and Alpha Toxin (PLAT) motif was cloned from Plasmodium chabaudi and homologues identified in the P. falciparum and P. yoelii genome data bases. At least one of these sequence motifs (SRCR) has adhesive properties in other proteins, therefore, we propose to name this protein PSLAP for Plasmodium SRCR, LCCL Adhesive-like Protein. Southern blotting and chromosome analysis showed that pslap is a single copy gene on chromosome 14 in P. falciparum 3D7. pslap mRNA is strongly expressed in P. falciparum gametocytes, but was undetectable on Northern blots of RNA from the asexual blood stages. Polyclonal antibodies raised to different parts of PSLAP detected a protein expressed in late gametocytes, but not in the early stages of gametocytogenesis or asexual blood stages of P. falciparum. We suggest that PSLAP functions in the mosquito, for example, in modulation of the invertebrate host immune response or in protection against complement factors in the blood meal.

Isolation and characterization of rhoptries of Plasmodium falciparum.

Rhoptries have been isolated from Plasmodium falciparum schizont-infected erythrocytes by isopycnic density centrifugation. Gradient fractions were analyzed by immunoblotting with antibodies against two polypeptides of 140 and 110 kDa, known to be components of the rhoptry. The proteins were present primarily in fractions with a density of 1.16 g ml-1. Electron microscopy of these fractions indicated they were enriched in rhoptries. For the most part, the isolated organelle retained in situ morphology, although some rhoptries were distorted, indicating the structure of some of the organelles is not rigid. Electrophoretic analysis of the rhoptry fractions indicated the presence of a number of proteins, many of which have not been identified to date. Properties of proteins in the isolated rhoptry were examined using the 140 and 110 kDa proteins as representative markers. Both proteins are present in a complex with a 130-kDa protein, as all three co-immunoprecipitate. At the late schizont stage, the rhoptry proteins are present in two distinct forms; a soluble form with an Mr of 480 000 which would correspond to a single copy of the 140/130/110 kDa complex and a form that can be sedimented at 130 000 x g. Properties of the sedimentable form suggest that the proteins are included in structures that resemble membranes. Ionic detergents were required to solubilize the proteins while high concentrations of NaCl and Na2CO3 resulted in only partial solubilization. Furthermore, treatment of disrupted rhoptries with phospholipase A and C resulted in the release of proteins into the soluble form.