The rhoptry proteome of Eimeria tenella sporozoites.

Proteins derived from the rhoptry secretory organelles are crucial for the invasion and survival of apicomplexan parasites within host cells. The rhoptries are club-shaped organelles that contain two distinct subpopulations of proteins that localise to separate compartments of the organelle. Proteins from the neck region (rhoptry neck proteins, RON) are secreted early in invasion and a subset of these is critical for the formation and function of the moving junction between parasite and host membranes.

Assessing transmission blockade in Plasmodium spp.

Here we describe a series of methods that can be used to assess the activities of "vaccines," drugs, and genetically modified vectors, for their abilities to inhibit transmission of Plasmodium from its vertebrate to its mosquito hosts. The selection of method to be used is determined by the purpose of the experiment, which can include the determination of the site/time of activity, and/or the potential reduction in transmission achieved.

Laboratory maintenance of rodent malaria parasites.

We provide a series of protocols that have been used for the cyclic transmission of rodent malaria parasites in the laboratory. This is now possible both in vivo and in vitro. We focus on the least "resource intensive" and generic methods that we find applicable to any parasite-host combination.

Proteomic comparison of four Eimeria tenella life-cycle stages: unsporulated oocyst, sporulated oocyst, sporozoite and second-generation merozoite.

We report the proteomes of four life-cycle stages of the Apicomplexan parasite Eimeria tenella. A total of 1868 proteins were identified, with 630, 699, 845 and 1532 found in early oocysts (unsporulated), late oocysts (sporulated), sporozoites and second-generation merozoites, respectively. A multidimensional protein identification technology shotgun approach identified 812 sporozoites, 1528 merozoites and all of the oocyst proteins, whereas 2-D gel proteomics identified 230 sporozoites and 98 merozoite proteins.

Characterisation of Plasmodium invasive organelles; an ookinete microneme proteome.

Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy.

Plasmodium male development gene-1 (mdv-1) is important for female sexual development and identifies a polarised plasma membrane during zygote development.

Successful development of Plasmodium sexual stages is essential for parasite survival, but the genes involved are poorly understood. We 'knocked out' the male development gene-1 (mdv-1) locus in Plasmodium berghei and found it to be important in female gametocyte activation. Indirect immunofluorescence assays show MDV-1 has a punctate cytoplasmic distribution in gametocytes.

The proteome of Toxoplasma gondii: integration with the genome provides novel insights into gene expression and annotation.

Background: Although the genomes of many of the most important human and animal pathogens have now been sequenced, our understanding of the actual proteins expressed by these genomes and how well they predict protein sequence and expression is still deficient. We have used three complementary approaches (two-dimensional electrophoresis, gel-liquid chromatography linked tandem mass spectrometry and MudPIT) to analyze the proteome of Toxoplasma gondii, a parasite of medical and veterinary significance, and have developed a public repository for these data within ToxoDB, making for the first time proteomics data an integral part of this key genome resource.

Results: The draft genome for Toxoplasma predicts around 8,000 genes with varying degrees of confidence.

Determining the protein repertoire of Cryptosporidium parvum sporozoites.

The genome of the intracellular parasite Cryptosporidium parvum has recently been sequenced, but protein expression data for the invasive stages of this important zoonotic gastrointestinal pathogen are limited. In this paper a comprehensive analysis of the expressed protein repertoire of an excysted oocyst/sporozoite preparation of C. parvum is presented.

Identification of a very large Rab GTPase family in the parasitic protozoan Trichomonas vaginalis.

Rab proteins are pivotal components of the membrane trafficking machinery in all eukaryotes. Distinct Rab proteins locate to specific endomembrane compartments and genomic studies suggest that Rab gene diversity correlates with endomembrane system complexity; for example unicellular organisms generally possess 5-20 Rab family members and the size of the repertoire increases to 25-60 in multicellular systems. Here we report 65 open reading frames from the unicellular protozoan Trichomonas vaginalis that encode distinct Rab proteins (TvRabs), indicating a family with complexity that rivals Homo sapiens in number.

Characterisation of blood coagulation factor XI T475I.

PCR-SSCP and DNA sequence analysis of a factor XI (FXI) deficient patient (FXI:C 39 U/dL; FXI:Ag 27 U/dL) identified a C to T transition in exon 12 of the FXI gene (F11 c.1521C>T) that predicts the substitution of Thr475 by Ile (FXI T475I) within the serine protease domain of FXI. This mutation destroys a consensus sequence for N-linked glycosylation, N473-Y-T475, known to be utilized in vivo.

Molecular evolution of the vertebrate blood coagulation network.

In mammalian blood coagulation 5 proteases, factor VII (FVII), factor IX (FIX), factor X (FX), protein C (PC) and prothrombin act with two cofactors factor V and factor VIII to control the generation of fibrin. Biochemical evidence and molecular cloning data have previously indicated that blood coagulation involving tissue factor, prothrombin and fibrinogen is present in all vertebrates. Using degenerate RT-PCR we have isolated and characterized novel cDNAs with sequence identity to the blood coagulation serine proteases and cofactors from chicken and the puffer fish (Fugu rubripes).