[Show abstract][Hide abstract] ABSTRACT: Improved Plasmodium falciparum cDNA expression libraries were constructed by combining mRNA oligo-capping with in vitro recombination and directional cloning of cDNA inserts into a plasmid vector that expresses sequences as thioredoxin fusion proteins. A novel procedure has also been developed for the rapid identification of seropositive clones on high-density filters, using direct labelling of P. falciparum immune immunoglobulin with fluorescein isothiocynate (FITC). This approach combines the advantages of recombination-assisted cDNA cloning with high throughput, non-radioactive serological screening of expression libraries. Production of replicate colony matrices allows the identification of antigens recognised by different pools with different specificities from residents of a malaria endemic region. Analyses of DNA sequences derived from sero-reactive colonies indicate that this is an effective method for producing recombinant proteins that react with antibodies from malaria-exposed individuals. This approach permits the systematic construction of a database of antigenic proteins recognised by sera from malaria-exposed individuals.
Molecular and Biochemical Parasitology 03/2004; 133(2):267-74. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytoadhesion of infected red blood cells (iRBC) is mediated through parasite-encoded, clonally variant surface antigens (VSA) and is a central process in the pathogenesis of Plasmodium falciparum malaria. Pregnancy-associated malaria (PAM) has been linked to VSA-mediated adhesion of iRBC to the glycosaminoglycan chondroitin sulphate A (CSA) in the placental intervillous space. Several studies have pointed to members of the PfEMP1 VSA family as mediators of CSA-specific iRBC sequestration in the placenta. Here, we report marked upregulation of a single var gene in several P. falciparum parasite isolates after selection for adhesion to CSA in vitro. The gene belongs to a highly conserved and common var gene subfamily (var2csa). The var2csa genes are structurally distinct from all other var genes in the parasite genome in lacking both CIDR and DBL-gamma domains. These domains have previously been implicated in PfEMP1-mediated adhesion to CD36 and CSA. We also show that var2csa was transcribed at higher levels in three placental parasite isolates compared with transcription in parasites from peripheral blood of two children with P. falciparum malaria. This var gene thus has the properties expected of a gene encoding the parasite adhesion molecule that initiates the pathology associated with PAM.