[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.
Full-text · Article · Mar 2004 · Molecular and Biochemical Parasitology
[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.
Full-text · Article · Aug 2003 · Molecular Microbiology
[Show abstract][Hide abstract]ABSTRACT: A survey of Plasmodium falciparum infection and clone multiplicity in Ghanaian children was carried out to study the effect of the onset of the malaria transmission season on disease incidence. Fortnightly blood samples were collected from 40 children living in the rural town of Dodowa, between February and August 1998. P. falciparum parasite densities were calculated and PCR genotyping was carried out using the polymorphic MSP-1 and MSP-2 genes as target loci for the estimation of the number of parasite clones in each sample. The average clone number was estimated using maximum likelihood techniques and the minimum number of clones per patient was analysed for the effects of age, sex, season, minimum number of clones per child, level of parasitaemia and parasite genotype. The statistical analysis indicated that the more clones a child carried, the more likely they were to have a clinical malaria episode. This was true after adjusting for age and season effects and for the measured circulating parasitaemia. The probability of clinical disease also increased if the MSP-1 MAD 20 and the MSP-2 FC 27 alleles were present. This longitudinal analysis thus indicates that the probability of a Ghanaian child having a symptomatic malaria episode is positively associated with both increasing numbers and novel types of P. falciparum clones.