Parasite virulence and disease severity in Plasmodium falciparum malaria.

Malaria stands out as one of the most important infectious diseases and one of the world’s leading causes of death. Plasmodium falciparum is the parasite responsible for the great majority of severe disease syndromes and mortality, and affects mainly children and pregnant women. Despite intensive research efforts, the understanding of P. falciparum virulence is limited. Infections with the parasite cause everything from asymptomatic parasitemia to severe disease and death, and the reasons for the different disease outcomes are not fully understood. Here we approached this issue by comparing several molecular aspects of parasites with different phenotypic traits as well as clinical isolates from children with severe and uncomplicated disease. Doing so, we first identified a substantial number of gene duplications and deletions in parasite genomes from all over the world. The genes found variable in copy numbers encode molecules with a wide variety of functions, and some of these were shown to have a direct effect on the parasites chances of survival. Apart from suggesting that the parasite regularly duplicate and delete genes to adapt to environmental changes, this also indicates that gene duplications and deletions could render parasites more or less virulent. To increase the understanding of how the virulence-associated adhesion of P. falciparum infected erythrocytes to endothelial cells and uninfected erythrocytes is achieved and regulated, we investigated the entire transcriptomes of parasites with distinct adhesive phenotypes. In a maze of transcriptional differences, receptor preferences for certain PfEMP1 proteins could be elucidated, as well as candidate genes for explorations of new molecules possibly involved in adhesive events or regulation of adhesion mediating proteins. The var genes encoding the original PfEMP1 in these parasites were subsequently shown to switch off when the parasites were cultivated continuously in vitro. Instead, another var gene was turned on, accompanied by low levels of expressed protein and with loss of the adhesive phenotypes. Apart from signifying that a constant selection or immunological stimuli is needed to maintain adhesive traits in P. falciparum, the results also suggested that exposure and adhesion mediated by the maternal malaria associated VAR2CSA protein is regulated on a posttranscriptional level. The gene encoding this protein had previously been reported duplicated in one particular parasite. Using a sensitive allelic discriminative approach we showed that these two gene copies were simultaneously transcribed in single parasites. This contradicts the principle of mutually exclusive expression of var genes in P. falciparum, and adds another layer of complexity upon the understanding of antigenic variation. To identify potentially underlying differences in parasites causing different disease outcomes we also analyzed var gene transcription in clinical isolates from children with severe and uncomplicated malaria. Using a novel analysis approach, we identified small degenerate amino-acid motifs that were over-represented in parasites causing severe disease and in parasites with high rosetting rates. Multiplication rates were analyzed for the same isolates and revealed a higher multiplication potential among severe disease causing parasites. The ability to multiply was also shown to correlate to the rosetting rates of the parasites, and was decreased when rosettes were disrupted with various reagents, suggesting rosetting to facilitate merozoite invasion of erythrocytes. In conclusion, we have identified specific parasite differences that besides increasing the understanding of virulence mechanisms in P. falciparum also present potential candidates for future intervention strategies. LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to in the text by their roman numerals: I. Ribacke U, Mok BW, Wirta V, Normark J, Lundeberg J, Kironde F, Egwang TG, Nilsson P and Wahlgren M. Genome wide gene amplifications and deletions in Plasmodium falciparum. Mol Biochem Parasitol 2007, 155;33-44 II. Mok BW, Ribacke U, Winter G, Yip BH, Tan C-H, Fernandez V, Chen Q, Nilsson P and Wahlgren M. Comparative transcriptomal analysis of isogenic Plasmodium falciparum clones of distinct antigenic and adhesive phenotypes. Mol Biochem Parasitol 2007, 151;184-192 III. Mok BW, Ribacke U, Rasti N, Kironde F, Chen Q, Nilsson P and Wahlgren M. Default pathway of var2csa switching and translational repression in Plasmodium falciparum. PLoS ONE 2008, 3(4);e1982 IV. Brolin K, Ribacke U, Nilsson S, Ankarklev J, Moll K, Wahlgren M and Chen Q. Allelic discrimination of sequence variable gene copies in the haploid genome of Plasmodium falciparum. Manuscript V. Normark J, Nilsson D, Ribacke U, Winter G, Moll K, Wheelock CE, Bayarugaba J, Kironde F, Egwang TG, Chen Q, Andersson B and Wahlgren M. PfEMP1-DBL1α amino acid motifs in severe disease states of Plasmodium falciparum malaria. Proc Natl Acad Sci U S A 2007, 104;15835-15840 VI. Ribacke U, Moll K, Normark J, Vogt AM, Chen Q, Flaberg E, Szekely L, Hultenby K, Egwang TG and Wahlgren M. Merozoite invasion in Plasmodium falciparum malaria is facilitated by PfEMP1 mediated rosetting. Manuscript * These authors contributed equally to this work