Functional characterisation of the FIKK kinase family of Plasmodium falciparum
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Author(s)
Belda, Hugo Marie Jean-Raymond
Type
Thesis
Abstract
Key to P. falciparum virulence is its capacity to remodel the host erythrocyte. Infected erythrocytes become rigid and cytoadhere to the vascular endothelium leading to the disease symptoms and preventing their filtration by the spleen. Unlike other human- infecting Plasmodium species, P. falciparum exports a family of 18 FIKK kinases into the host cell.
Here, a conditional knockout strategy based on the DiCre/LoxPint technology was used to study 4 FIKK kinases (FIKK4.1, FIKK7.1, FIKK10.1 and FIKK11) and identify their potential targets by quantitative phosphoproteome analysis. The deletion of FIKK4.1 led to a significant reduction in the phosphorylation of host cytoskeletal proteins and parasite proteins involved in remodelling. The characterisation of FIKK4.1 KO parasites confirmed its role both in the rigidification of the infected erythrocytes and in the trafficking of the adherence-mediating virulence factor PfEMP1 to the host cell surface.
Additionally, recombinant versions of several FIKK kinase domains were used to identify potential pan-FIKK inhibitors. When tested in vitro, these compounds showed activity on both P. falciparum and P. knowlesi, raising concerns regarding their specificity. A whole genome sequencing on drug-resistant parasites did not allow to identify additional targets. Moreover, it was shown that the compounds were not active on the FIKK kinases in culture due to the high intra-erythrocytic ATP concentration.
Using the recombinant FIKK kinase domains it was also shown that FIKK kinases possess distinct substrate specificity. Whereas most of them conserved the ancestral basophilicity, some evolved to phosphorylate preferentially acidic motifs. Strikingly, FIKK13 was found to be a tyrosine kinase, a feature supposed to be absent in Plasmodium. Finally, by studying the FIKK kinases from another Plasmodium species closely related to P. falciparum, it was shown that FIKK kinases substrate specificity is conserved across species of the Laverania clade.
Here, a conditional knockout strategy based on the DiCre/LoxPint technology was used to study 4 FIKK kinases (FIKK4.1, FIKK7.1, FIKK10.1 and FIKK11) and identify their potential targets by quantitative phosphoproteome analysis. The deletion of FIKK4.1 led to a significant reduction in the phosphorylation of host cytoskeletal proteins and parasite proteins involved in remodelling. The characterisation of FIKK4.1 KO parasites confirmed its role both in the rigidification of the infected erythrocytes and in the trafficking of the adherence-mediating virulence factor PfEMP1 to the host cell surface.
Additionally, recombinant versions of several FIKK kinase domains were used to identify potential pan-FIKK inhibitors. When tested in vitro, these compounds showed activity on both P. falciparum and P. knowlesi, raising concerns regarding their specificity. A whole genome sequencing on drug-resistant parasites did not allow to identify additional targets. Moreover, it was shown that the compounds were not active on the FIKK kinases in culture due to the high intra-erythrocytic ATP concentration.
Using the recombinant FIKK kinase domains it was also shown that FIKK kinases possess distinct substrate specificity. Whereas most of them conserved the ancestral basophilicity, some evolved to phosphorylate preferentially acidic motifs. Strikingly, FIKK13 was found to be a tyrosine kinase, a feature supposed to be absent in Plasmodium. Finally, by studying the FIKK kinases from another Plasmodium species closely related to P. falciparum, it was shown that FIKK kinases substrate specificity is conserved across species of the Laverania clade.
Version
Open Access
Date Issued
2020-10
Date Awarded
2021-04
Copyright Statement
Creative Commons Attribution Licence
License URL
Advisor
Treeck, Moritz
Tate, Edward
Publisher Department
Francis Crick Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)