SURFACE ANTIGENS AND VIRULENCE IN PLASMODIUM FALCIPARUM MALARIA

Plasmodium falciparum is an intracellular protozoan that may cause severe forms of malaria. It is a major world health hazard and reaps the highest toll among the children and pregnant mothers of the developing world. An Anopheles mosquito vector injects the pathogen when taking a blood meal. After multiplication in cells of the liver, the parasite escapes and infects red blood cells in a cyclic manner and this is when the clinical manifestations of malaria as a disease become apparent. The parasite causes the infected red blood cells to adhere to each other (rosetting) and to the blood vessel walls (cytoadherence) by exporting highly variable and adhesive PfEMP1 proteins to the erythrocyte surface. Different immunological and genetic properties of the host as well as parasite specific clonal phenotypes determine the outcome of the encounter. We have investigated the parasite side of these events by performing a clinical case-control study where we sampled a score of isolates from patients with severe or mild malaria. These investigations took part in two endemic areas in Uganda; Apac a small rural community in the northern part of the country and Kampala, the capital. The work was partitioned into four different headings where we aimed to explore different aspects of what separates the parasites we found in severe disease patients from the uncomplicated group. Primarily we sought to characterize the sequences of the var genes encoding PfEMP1 expressed in the different patient groups. Through implementing a semiquantitative PCR amplifying cDNA, massive scale sequencing and a bioinformatics pipeline we could identify degenerated amino-acid motifs that were either statistically overrepresented in PfEMP1 sequence tags sampled from patients with severeor mild malaria. These were put in a structural-functional context through 3D modeling and potential sites for receptor interaction were identified. The expression of the var genes in the fresh isolates was further explored in a temporal context to understand the timing of var gene transcription in the patient. We compared semi-and absolutely quantified var genes in a subset of the Ugandan isolates. We chose an approach where we constructed a quantitative-PCR assay that enumerated the amounts of individual var genes in a heterogeneous solution. The transcription patterns were individual to each isolate and we found that dominance of genes could flux between developmental stages. In a separate study, two of the isolates were chosen to be included in a larger genomics survey of the entire genome by use of a 70-mer oligonucleotide micro-array platform. Size fractionated gDNA from a panel of parasites were hybridized under stringent conditions and cross referenced against the 3D7AH1 genome parasite. The assay could identify a number of gene copy number polymorphisms that were associated to proliferative properties of the parasites, drug resistance or putative invasion related genes. The microarray results were confirmed by PCR and fluorescent in situ DNA hybridization. Finally we studied the growth of fresh in vitro adapted field isolates of children from severeor mild malaria and found a correlation between the level of rosetting and their multiplication rates. By disrupting rosetting with either antiimmunoglobulin antibodies, heparan sulfate or antibodies to PfEMP1 we could also block the growth of the parasite facilitated by PfEMP1 rosetting. Taken together, these findings argue that P. falciparum specific phenoand genotypes exist that may predispose for the development of severe malaria. In conclusion, we have found specific molecular evidence for inter-parasite differences in P. falciparum that in the future may be exploited in intervention strategies. LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to by their roman numerals. I. Normark J, Nilsson D, Ribacke U, Winter G, Moll K, Wheelock CE, Bayarugaba J, Kironde F, Egwang TG, Chen Q, Andersson B, Wahlgren M. PfEMP1-DBL1alpha amino acid motifs in severe disease states of Plasmodium falciparum malaria. Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15835-40. II. Ribacke U, Mok BW, Wirta V, Normark J, Lundeberg J, Kironde F, Egwang TG, Nilsson P, Wahlgren M.Genome wide gene amplifications and deletions in Plasmodium falciparum. Mol Biochem Parasitol 2007 Sep;155(1):33-44. III. Blomqvist K, *Normark J*, Nilsson D, Ribacke U, Orikiriza J, Trillkott P, Byarugaba J, Egwang TG, Kironde K, Andersson B, Wahlgren M Developmental Shift in var gene Transcription in Fresh Plasmodium falciparum isolates. Manuscript IV. Ribacke U, Moll K, Normark J, Vogt AM, Chen Q, Flaberg E, Szekely L, Hultenby K, Egwang TG, Wahlgren M Merozoite invasion in Plasmodium falciparum malaria is facilitated by PfEMP1 mediated rosetting. Manuscript * These authors contributed equally to this work