Interplay of genetic, epigenetic and transcription factors in the regulation of transcriptional variation in Plasmodium falciparum

Abstract

[eng] The most severe form of malaria, caused by Plasmodium falciparum parasites, still kills over half a million people every year, most of them children under the age of five. Despite huge research efforts, reduction in the global burden of disease has stalled in recent years. P. falciparum has a very complex life cycle including, among other steps, sexual reproduction in female Anopheles mosquitos and an asexual intra-erythoricitic development cycle (IDC) inside the human host, which causes the disease. During the IDC, the parasite needs to continuously adapt to changes in its environment including fluctuations in blood temperature, concentration of nutrients and other metabolites, presence of drugs, and a constant fight against the host’s immune system. In this thesis, we have studied the adaptation mechanisms of P. falciparum to this plethora of challenges, with a special focus on clonally variant genes (CVGs). In P. falciparum, CVGs are a set of genes, participating in host-parasite interactions, which can be found both in a transcriptionally active state, characterized by euchromatin, or a transcriptionally silenced state, characterized by heterochromatin. The state of CVGs is inherited by the progeny of a parasite, with stochastic switches occurring at a low frequency. Parasites with the most optimal patterns of CVGs expression are continuously selected as the environment changes, leading to adaptation and survival of the infecting population. In the first paper of this thesis, we have analyzed subcloned parasite populations to characterize, with unprecedented detail, the heterochromatin distribution associated with the active and silenced states of CVGs. This has allowed us to define different kinds of heterochromatin transitions between the active and silenced states of CVGs and has given us new insights on the regulation of var genes (one of the main virulence factors for malaria) and into the regulation of sexual conversion, a process crucial for malaria transmission. Continuing with CVG regulation, in the second paper of the thesis, we have analyzed how patterns of CVG expression are established at the onset of human infections, after passage through transmission stages. Our results suggest a loss of the epigenetic memory during transmission stages and a reset of the heterochromatin patterns that drive CVG expression. Similar patterns of CVG expression arose in different infected individuals, suggesting that the activation probability of a given CVG is an intrinsic property of the gene. In the third paper of the thesis, we have further studied the sexual conversion phenomenon. We have generated a conditional over-expression system for pfap2-g, the CVG that acts as master regulator of sexual conversion, achieving sexual conversion rates of ~90% after induction. Our results have provided new insights on how heterochromatin at different positions affects expression of pfap2-g and have allowed us to characterize the transcriptional profile of the initial stages of sexual commitment with unprecedented sensitivity. Finally, in the fourth paper of this thesis, we have studied the adaptation of the parasite to heat-shock, which happens in natural infections due to fever episodes. We expected CVGs to participate in this phenomenon, but instead we have identified pfap2-hs, a non-clonally variant transcription factor (TF), as the main driver of the heat-shock response in P. falciparum. AP2-HS acts as the functional homolog of HSF1 (a TF that drives the heat-shock response from yeast to mammals, but is absent in P. falciparum), driving a very tight transcriptional response to heat-shock, characterized by the up-regulation of hsp70 and hsp90. Although the presence of directed responses had previously been demonstrated for other cues, it is the first time that the transcription factor driving such a response is identified in P. falciparum. Taken together, the results of this thesis have broadened our knowledge of the regulation of adaptive mechanisms in P. falciparum. Learning about this deadly parasite’s defense mechanisms will be instrumental to design better strategies to fight it back in the future.