Genomic and transcripto variation in blood stage Plasmodium falciparum

Abstract: Malaria research has entered a postgenomic era since October 2002, when the complete genomic sequence of Plasmodium falciparum strain 3D7 was published. A massive amount of information generated by the P. falciparum genome project has facilitated the development of many novel platforms for profiling different levels of biological aspects. In this thesis, by employing various high throughput approaches, 1 aimed at understanding the genetic basis of phenotype variation in P. falciparum. Genomic diversity of P. falciparum strains originating from different geographical areas was studied using microarray-based comparative genomic hybridization (CGH). Here I show presence of multiple genome widecopy number polymorphisms (CNPs) covering 82 genes. The genes, amplified in up to six copies, encode molecules involved in cell-cycle regulation, cell division, drug resistance, erythrocyte invasion, sexual differentiation and unknown functions. Our results suggest that P. falciparum employs gene duplications and deletions as general strategies to enhance its survival and spread. Even though the P. falciparum subtelomeres are considerably variable, segmental duplication were observed within these regions. High-resolution mapping and CGH data of the subtelomeric regions revealed a block of genes that had been amplified nine times on multiple subtelomeres of P. falciparum. These duplicated segments (SD) are of more than 10 kilobases in size and span six genes, including three known variant gene families: var, rif and pfmc-2tm and three hypothetical genes (n, o-, q-gene). Sequencing data revealed that both inter- and intragenic regions are highly conserved across the species, despite their variation in copy numbers. One of the hypothetical genes within the SD, the n-gene, encoding a PEXEL/VTScontaining two transmembrane proteins was found to be transcribed at an early stage of the asexual erythrocytic cycle and its transcriptional levels were correlated to the gene dosage. The ubiquity and uniqueness of these SDs in the P. falciparum subtelomeres, and the conserved nature of the gene content within, suggests an important role in plasmodia speciation. A major part of the parasite virulence attributes to the extensive host cell modification of blood stages P. falciparum. Two 3D7 isogenic clones with distinct adhesive and antigenic phenotypes (rosettes formation for 3D7S8.4 and CD36 binding for 3D7AH 1 S2) were isolated by micropanipulation. To have a global view of gene expression governing their phenotypes, we have employed a P. falciparum genome array, supplemented with a panel of in-house oligonucleotides, for comparative transcriptomal analysis. Fifteen genes were found highly differentially expressed (greater than a 5-fold change) encoding proteins for apical organellar (Gbph2, GBPrelated antigen), cell-rescue, defense/virulence (RESA-2, RIFIN, PfEMP1), DNA/RNA processing (RNA methylase), erythrocyte invasion (SERA-5) and a number of hypothetical proteins. A number of short and fulllength var transcripts were differentially expressed between the clones; yet, only one full-length transcript was dominant in both rings and trophozoites. In fact, var genes were found at the top of the list of the highly differentially expressed gene in between the two clones and its protein product, PfEMP1, is believed to be the most important determinant of antigenic phenotypes in P. falciparum. To understand better the mechanisms and dynamics of var gene switching, we propagated 3D7S8.4 and 3D7AHIS2 under the same conditions for more than one year without enrichment or panning. We found that, upon long-term culturing, both parasites switch to express a common var gene (var2csa) matched by the loss of PfEMP1 surface expression and host cellbinding. The var2csa gene repositioned in the perinuclear area upon activation away from the telomeric clusters and heterochromatin to express spliced, full-length RNA. We suggest switching to var2csa to be an inherited trait that allows for small populations of P. falciparum to express new var genes. The process may coordinate the variant-antigen repertoire and thus protect against its rapid exhaustion.