Abstract
Malaria continues to be one of the major causes of morbidity and mortality in the developing countries. The development of the resistance against the available antimalarial drugs and scarcity of effective vaccines have demanded the urgent need of finding new antimalarial targets. The severe form of malaria is caused by Plasmodium falciparum, which manifests a complex life cycle involving various morphologically and functionally distinct forms within two different hosts - human and Anopheles mosquitoes. In order to thrive in two distinctive host’s environment, these parasites employ different mechanisms to regulate their tightly coordinated gene expression. This thesis project is focused on exploring the regulation, which is mediated by guanine-rich DNA secondary structures, predominantly G-quadruplexes. These structures are found in wide range of organisms and are involved in gene regulation such as transcription, DNA replication and telomeric maintenance. Recently, they are also found to be involved in the process of virulence to evade the host’s immune response in numerous pathogens such as bacteria, protozoa and viruses. In Plasmodium, the G-quadruplex forming motifs are found to be enriched in the telomeric and sub-telomeric regions, where the virulence genes are present. The G4 existence in these AT biased genome points towards their role in the mechanism of gene regulation and antigenic variation. However, there is a lack of experimental evidence to support this hypothesis. The aim of this project is to provide the first comprehensive survey of the G4-interactome in order to understand the role of G4-mediated regulatory mechanisms in Plasmodium biology. Using a combination of unbiased approaches (Yeast one-hybrid and DNA pull-down assay), we have identified ~152 putative G4 interacting proteins in Plasmodium falciparum. The orthologs of some of these proteins were shown to interact with G4s, thus strengthening our results. Furthermore, to understand how these candidates contribute to G4 mediated regulatory processes, we have selected and characterized two proteins (GBP2 and DNAJ) to perform functional studies following validation of their binding properties. These proteins are shown to play an important role in Plasmodium biology. In this study, we have found that the GBP2 is a dispensable protein that interacts with the selected G4. Even though the deletion of the gene is not lethal to the parasites, it still affects the expression of var genes. Whereas putative DNAJ is an essential protein and its deletion results into the arrest of the parasites at the late stages of erythrocytic cycle. Collectively, this study sheds light on this understudied DNA structure based regulatory mechanism and provide the first systematic survey of the G4 interactome. Given their essential role in parasite development further characterization of obtained candidates will likely generate new targets for antimalarial drugs that will in the long term contribute to the eradication of the disease.