Identification and Analysis of Plasmodium falciparum Genes Mediating Cytoadherence

Abstract

Malaria is the most important parasitic disease of man: almost two billion people live in areas in which the most pathogenic human Plasmodium species is transmitted. The parasite P. falciparum accounts for more than 90% of world-wide malaria morbidity and mortality: every year it is responsible for about 500 million clinical malaria cases and for up to 2.7 million deaths world-wide. A characteristic feature of infections with P. falciparum is the ability of infected red blood cells (iRBC) to adhere to venular endothelial cells. This adhesive behaviour is manifested by the iRBC some 16-20 hours into the intraerythrocytic cycle, when the parasite matures to the early trophozoite stage. At this stage, it expresses and transports molecular adhesins to the red cell surface, leaving ring-infected erythrocytes to be the predominant form of parasites found in the peripheral blood. This phenomenon, called sequestration, is proposed to be the key event in vital organ failure, and by adhesion to the microvasculature of the brain, leads to the major life-threatening manifestation of falciparum malaria: cerebral malaria. The major parasite-derived adhesin, incorporated in the iRBC membrane, was detected 1984 and termed Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1). PfEMP-1 was originally described as a strain specific, high molecular weight (250-350 kDa), highly polymorphic protein on the surface of P. falciparum-infected RBCs and it was thought to be involved in adhesion to the already identified receptors on the surface of the endothelial cells, such as CD36, thrombospondin (TSP), inter-cellular adhesion molecule-1 (ICAM-1), and others. Later on (1991), it was shown, that PfEMP-1 undergoes antigenic variation in cloned isolates and that changes in antigenic type are accompanied by changes in the binding specificity for host receptors. Further progress was made 1995 when the genes encoding PfEMP-1 were identified and sequenced: It was shown that each variant antigen type was encoded by a single gene. These genes, termed var genes, were shown to be present at 50-100 copies per haploid genome, to be highly polymorphic, both within a single parasite and between isolates, but to have a similar basic structure. Briefly: this structure includes a cysteine-rich interdomain region (CIDR), 2-5 Duffy binding like domains (DBL) and a acidic terminal sequence (ATS). Nevertheless, the causal proof, that var genes are indeed involved in sequestration in vivo has to be shown. It was the aim of the presented thesis, to undertake new and innovative approaches, in order to identify and analyze possible parasite-derived ligands, involved in sequestration: a schizont-specific P. falciparum cDNA expression library was constructed and introduced in simian COScells by transient transfection. In a first approach, these transfected COScells were used in a newly developed in vitro assay. Screening cells, expressing ICAM-1 or CD36 on their surface were used to identify parasite-derived genes, whose gene products confer adherence to these host cell receptors. In a second approach, the transfected COS 7 cells were screened with antibodies against surface molecules of iRBCs to identify the genes involved. A more direct, third approach was applied by transfecting COS 7 cells with fragments of var genes, identified by PCR amplification using primers binding to conserved var gene domains, such as DBL1 or ATS, in order to analyze the role of var genes in cytoadherence. And finally, in the fourth approach, var gene domains, expressed as 6xhistagged proteins in Escherichia coli, were used to analyze binding of these selected domains (CIDR- and DBL1 domain) to different host receptor molecules, expressed on the surface of Chinese hamster ovary (CHO) cells. With the fourth approach, we were able to show, that the CIDR domain of var genes binds to chondroitin sulphate A (CSA) in a dose dependent manner. This binding could be inhibited by 50 μg of soluble CSA (62.4%) and by chondroitinase ABC treatment (up to 94.4%) of the CHO cells. Unfortunately, all approaches using transfected COS 7 cells, did not lead to the identification of new or known adhesive molecules. In the course of this thesis it became evident, that the AT-richness of the P. falciparum genome (69% in coding and 86% in non coding-regions) might be responsible for this lack of expression of P. falciparum genes in COS 7 cells, since the existence of AUUUA-motifs can mediate mRNA decay in mammalian cells, a motif which is abundant in P. falciparum mRNA. Nevertheless, it was possible during this thesis, to establish the techniques of transient and stable transfection of mammalian cells, to improve screening techniques by the use of green fluorescent protein (GFP) as a reporter system and to pick and isolate single cDNA clones from transfected COS 7 cells.