Responses to cellular stress in malaria parasites

Malaria remains a global health burden with almost half of the worldŐs population being at risk of infection. While the disease can be caused by several Plasmodium species, this thesis focuses on the most virulent species P. falciparum. One of many challenges in the eradication of malaria is the parasiteŐs great adaptability to environmental changes. In this thesis, we aimed to investigate adaptational strategies used by P. falciparum to cope with nutrient deprivation and drug induced cellular stress.

In recent years, extracellular vesicles (EVs) have been implicated as facilitators of intercellular communication. In malaria patients, an increase in their abundance in plasma samples has been associated to disease severity. Additionally, their transcriptomal cargo was proposed to modulate processes such as host immune evasion and gametogenesis commitment. Here, we exposed intraerythrocytic parasite cultures to short-term nutrient deprivation and could detect an increase in EV release from ring stage parasitized red blood cells. Additionally, we observed a change in the transcriptomal profile of EVs upon glucose and amino acid deprivation which specifically affected the human miRNA and parasitic tRNA fragment populations. The functional implications of these observations remain to be elucidated but similar observations were made in other protozoan parasites, suggesting a potentially conserved role for the release of EVs in response to changing environments.

Next, we investigated the potential of EVs derived from uninfected red blood cells to deliver antisense oligonucleotides to parasite cultures in order to target specific transcripts of interest. Our data indicates that this approach is well tolerated by parasite cultures and that the effect on transcript levels is titratable. Surprisingly, transcript abrogation required an invasion event and thus we hypothesized that the exhibited effect might be due to merozoites aiding EV entry into the red blood cell. The therapeutic potential of EVs to deliver different kinds of nucleic acids to modify gene expression has been proposed in various eukaryotic systems. Hence, the herein proposed method might aid reverse genetic investigations in P. falciparum and presents a possible solution to overcome current bioavailability challenges such as the parasiteŐs intracellularity.

There is little knowledge on how the parasite can sense nutrient deprivation such as amino acid starvation and mount its stress response accordingly. Due to the absence of compartments of the in eukaryotes conserved mTOR complex, it was proposed that the parasite employs a non-canonical stress-sensing pathway to adjust its protein synthesis on a systemic level. In this thesis, we present a stress response pathway facilitated by the regulation of tRNA charging upon amino acid starvation. The latter led to a selective ribosomal stalling of isoleucine (Ile)-rich transcripts, which were implicated in processes such as proliferation and lipid synthesis. Notably, isoleucine is the only amino acid, which cannot be acquired through hemoglobin digestion. This led us to hypothesize, that the parasite enters a hibernatory state through stalling of Ile-rich transcripts to ensure survival until amino acids are sufficiently supplied in the host serum. Furthermore, we propose that the intraerythrocytic parasite specifically adapted its proteome to one of its major amino acid sources - the hostŐs hemoglobin. These findings do not only propose a novel nutrient sensing pathway, but also give new insights into potential drivers of protein evolution.

Previously, short-term hibernation as a mechanism to respond to cellular stress was not only reported for parasites upon nutrient deprivation, but also upon drug treatment such as artemisinin. In our last study, we investigated the anti-parasitic effect of a potential new antimalarial called sevuparin. Notably, the heparin-derivative was proposed to be well tolerated in clinical trials and has been previously implicated to block parasite invasion and cytoadhesion. In our study, we could see that sevuparin also impacted intracellular parasite development and reduced multiplication, but also affected virulence features such as PfEMP1 surface expression. Through further investigations of these observations, we showed that heparinoids impair maintenance of cellular homeostasis of the infected red blood cell, possibly through inhibition of glycolysis and consequential perturbations of cation and metabolite levels. Thus, our study results revealed a multimodal effect of sevuparin, further supporting this heparinoid as a promising therapeutic candidate.

Collectively, this thesis provides new insights into the adaptational strategies used by P. falciparum to cope with environmental changes. Additionally, we adopted a variety of new techniques, which might advance genetic analyses of the parasite in the future. Moreover, this thesis furthers the understanding of the parasiteŐs biology and its gene regulatory machinery, which might aid the development of novel therapeutic strategies.