Periodic Reporting for period 1 - VirulenceControl (The role of an expanded family of exported effector kinases in environmental sensing and regulation of virulence in human malaria.)
Período documentado: 2023-09-01 hasta 2026-02-28
Approximately 300 million clinical episodes and ~ 450,000 deaths are caused by malaria annually, making it one of the deadliest tropical parasitic diseases. Humans can be infected by 6 Plasmodium species, however 95% of all fatalities are caused by a single species, Plasmodium falciparum. The severity of P. falciparum malaria is associated with the method by which the parasite avoids filtration by the spleen. It does so by two means: 1) cytoadhesion of the infected red blood cell (iRBC) to the vascular endothelium and 2) rigidification of the host cell leading to retention of iRBCs in the microvasculature. In both cases, the accumulation of infected erythrocytes results in obstruction of blood flow and a strong local inflammatory response which can cause severe cerebral malaria leading to stroke and death. Cytoadhesion is predominantly mediated by a parasite protein called PfEMP1 that is transported onto the surface of the infected red blood cell. PfEMP1 binds to receptors on endothelial cells in various organs, sequestering the infected cell away from the peripheral circulation. Interestingly, a rapid increase of PfEMP1 surface translocation, cytoadhesion and RBC rigidity on relatively short timeframes (1-2 hours) has been observed upon fever. This implies that in addition to the well-studied differential PfEMP1 variant expression, a yet unknown mechanism must exist that allows the parasite to sense fever in the host and rapidly control its pathogenesis traits in response.
We have recently identified members of a parasite kinase family that are exported into the RBC and phosphorylate human and parasite exported proteins important for controlling cytoadhesion and rigidification. This kinase family, the FIKK kinases, contains ~20 members which are exported into the host cell only by P. falciparum and a sub-genus of 6 related Plasmodium species that infect great apes, the Laverania. None of the other human-infecting Plasmodium species contains any predicted exported kinase, implying their presence may contribute to the particular virulence of P. falciparum. The FIKK kinase family evolved from a single, non-exported ancestor ~ 1 million years ago, and 17 of the 19 FIKKs found in P. falciparum appear to have been conserved across the sub-genus since then. A key conserved component within the Laverania, that distinguishes it from other Plasmodium species, is the presence of PfEMP1, suggesting that at least some FIKKs and PfEMP1 may have co-evolved.
We found several FIKK kinases that do not play an apparent role in RBC remodelling under standard culturing conditions, despite being expressed. This led us to consider that certain FIKKs may be activated during certain environmental conditions present in the host. One of these conditions is fever, a hallmark of severe human malaria which leads to increased PfEMP1 surface presentation and cytoadhesion within hours. Another common environmental condition in the host is the sickle cell trait, known as the HbAS genotype or the related HbAC variant. PfEMP1 surface translocation and cytoadhesion is markedly reduced in RBCs from patients with sickle cell trait, likely as a result of elevated reactive oxygen species (ROS) in these cells that lead to direct oxidation of proteins. ROS also accumulates in RBCs under hypoxic conditions. This is found in areas of high parasite sequestration in the microvasculature, which could therefore lead to a local effect of ROS on parasite infected RBCs. Treatment of iRBCs with diamide, an oxidising agent indeed mimics the ROS effect observed in HbAS cells.
We have recently identified members of a parasite kinase family that are exported into the RBC and phosphorylate human and parasite exported proteins important for controlling cytoadhesion and rigidification. This kinase family, the FIKK kinases, contains ~20 members which are exported into the host cell only by P. falciparum and a sub-genus of 6 related Plasmodium species that infect great apes, the Laverania. None of the other human-infecting Plasmodium species contains any predicted exported kinase, implying their presence may contribute to the particular virulence of P. falciparum. The FIKK kinase family evolved from a single, non-exported ancestor ~ 1 million years ago, and 17 of the 19 FIKKs found in P. falciparum appear to have been conserved across the sub-genus since then. A key conserved component within the Laverania, that distinguishes it from other Plasmodium species, is the presence of PfEMP1, suggesting that at least some FIKKs and PfEMP1 may have co-evolved.
We found several FIKK kinases that do not play an apparent role in RBC remodelling under standard culturing conditions, despite being expressed. This led us to consider that certain FIKKs may be activated during certain environmental conditions present in the host. One of these conditions is fever, a hallmark of severe human malaria which leads to increased PfEMP1 surface presentation and cytoadhesion within hours. Another common environmental condition in the host is the sickle cell trait, known as the HbAS genotype or the related HbAC variant. PfEMP1 surface translocation and cytoadhesion is markedly reduced in RBCs from patients with sickle cell trait, likely as a result of elevated reactive oxygen species (ROS) in these cells that lead to direct oxidation of proteins. ROS also accumulates in RBCs under hypoxic conditions. This is found in areas of high parasite sequestration in the microvasculature, which could therefore lead to a local effect of ROS on parasite infected RBCs. Treatment of iRBCs with diamide, an oxidising agent indeed mimics the ROS effect observed in HbAS cells.
We have published 2 major articles on the findings so far. In the first study (Belda et al., 2025, Nature Microbiology) we described the evolution of the FIKK kinase family, and that each of the ~20 members very likely fullfils independent functions. By solving the structure of one FIKK kinase, we could identify critical regions in the FIKK kinase domain that is rapidly evolving that explains how the kinase family could diversify across the Laverania sub-genus of Plasmodium parasites in evolutionary short time. Finally, we used the conserved ATP binding site across the FIKKs in collaboration with GSK to screen for compounds that would inhibit the kinase family as a whole to obtain pan-FIKK inhibitors. We identified several compounds, but further work will be required (outside of this ERC grant) to achieve higher selectivity.
The second study currently available as a published preprint (Jones et al., 2025, eLife), describes the specific increase of phosphorylated exported proteins after a heat-stress that mimicks physiological fever temperatures.
Using these carefully selected conditions, we then tested the hypothesis that FIKK kinases drive a fever-specific response in the infected RBC cell that drives in the proposed PfEMP1 increase on the surface of infected RBCs. We found that non-destructive heat shock leads to increased PfEMP1 on the surface of the RBC, accompanied by an increase in cytoadhesion. We then measured the phosphorylation state of exported and RBC proteins after a non-destructive heatshock (39C) and found a highly significant enrichment of increased phosphorylation on parasite exported proteins.
The two FIKK kinases, that together drive ~40% of the changes in phosphorylation after a mimicked fever shock, are however not important for increased PfEMP1 surface levels. But these 40% of phosphorylation sites have previously been attributed to the function of these FIKK kinase. Therefore, that these do not play a role in response to fever temperatures is maybe not surprising.
We also tested an alternative hypothesis, that increased PfEMP1 on the surface of the RBCs may be caused by increased protein trafficking. We found that some proteins are exported into the red blood cell faster and earlier upon infection by the parasite when it experienced a heat stress. We hypothesize that other FIKKs play a role in this process, which is subject to ongoing work.
A second major aim of the project was to test how reactive oxygen species affect FIKK function. Through a series of biochemical assays in vitro and in parasite cell culture, we identified reactive cysteines in the FIKK kinases, which render them sensitive to reactive oxygen species. Through structural modeling we identified several key residues important for the sensitivity and we have started to design chemical probes that specificially inhibit FIKK kinases through this mechanism (cys-reactive probes). Such compounds will allow us to monitor the oxydation states of FIKK kinases in various infection settings (HbAS cells, low oxygen levels) found frequently in human hosts.
The second study currently available as a published preprint (Jones et al., 2025, eLife), describes the specific increase of phosphorylated exported proteins after a heat-stress that mimicks physiological fever temperatures.
Using these carefully selected conditions, we then tested the hypothesis that FIKK kinases drive a fever-specific response in the infected RBC cell that drives in the proposed PfEMP1 increase on the surface of infected RBCs. We found that non-destructive heat shock leads to increased PfEMP1 on the surface of the RBC, accompanied by an increase in cytoadhesion. We then measured the phosphorylation state of exported and RBC proteins after a non-destructive heatshock (39C) and found a highly significant enrichment of increased phosphorylation on parasite exported proteins.
The two FIKK kinases, that together drive ~40% of the changes in phosphorylation after a mimicked fever shock, are however not important for increased PfEMP1 surface levels. But these 40% of phosphorylation sites have previously been attributed to the function of these FIKK kinase. Therefore, that these do not play a role in response to fever temperatures is maybe not surprising.
We also tested an alternative hypothesis, that increased PfEMP1 on the surface of the RBCs may be caused by increased protein trafficking. We found that some proteins are exported into the red blood cell faster and earlier upon infection by the parasite when it experienced a heat stress. We hypothesize that other FIKKs play a role in this process, which is subject to ongoing work.
A second major aim of the project was to test how reactive oxygen species affect FIKK function. Through a series of biochemical assays in vitro and in parasite cell culture, we identified reactive cysteines in the FIKK kinases, which render them sensitive to reactive oxygen species. Through structural modeling we identified several key residues important for the sensitivity and we have started to design chemical probes that specificially inhibit FIKK kinases through this mechanism (cys-reactive probes). Such compounds will allow us to monitor the oxydation states of FIKK kinases in various infection settings (HbAS cells, low oxygen levels) found frequently in human hosts.
We are in the midths of the project and found exciting data that show sensibility of FIKK kinases to elevated ROS levels. We also find increased phosphorylation upon heat stress, which may be linked to increased FIKK kinase activity, or changes in substrate specificity. Increased cytoadhesion upon temperatures commonly found in patients suggests that this could be a major contribution to disease pathogenesis. Antipyretic treatments have shown some promising results for survival of severe malaria cases. At least part of the symptom alleviation could be due to decreased bound parasite mass.