Functional characterisation of Plasmodium actin-like proteins Alp1 and Alp2b

Abstract

Malaria, the disease caused by Plasmodium parasites transmitted via Anopheles mosquitoes, remains a significant global health threat, responsible for over 600,000 deaths annually. The current treatments are constrained by inefficacy and the spread of drug resistance, which highlights the necessity for alternative strategies to prevent parasite transmission. The actin-like proteins (Alps), Alp1 and Alp2b, are unique actin-related proteins and specific to Apicomplexa. They play critical roles in Plasmodium transmission, yet their exact functions are still unknown. The aim of this thesis was therefore to characterise these Alps through elucidation of their specific biological functions, especially during the mammal-to-insect transmission stages. <br> The production of recombinant P. berghei Alp1 and Alp2b was attempted using the E. coli system to allow in vitro biochemical assays. Despite sufficient expression levels, purification and solubilisation of Alp1 and Alp2b, respectively, required further technical optimisation. <br> The partial and full knock-out of Alp1 in the rodent model species P. berghei demonstrated the essential role of this protein in facilitating ookinete gliding motility, with its C-terminus having no effect on either parasite growth or motility. Notably, alp1 from human-pathogenic species P. falciparum could not rescue P. berghei motility when complemented as cDNA, underlining the potential significance of the introns. Cellular localisation of Alp1 was investigated through generation of the Alp1-3xHA line. Despite the critical role of Alp1 in gliding motility, no anticipated spatial association with the cytoskeletal motor machinery was observed. The actin chromobody was successfully utilised to visualise diverse structures of actin filaments in ookinetes. It further revealed that the absence of Alp1 leads to a moderate stabilisation of actin filaments. This suggests that Alp1 may contribute to the dynamic turnover of actin filaments, which ultimately affects the operation of the gliding motor machinery. <br> Alp2b is crucial for microgametogenesis and exflagellation. Here, a quantitative assay was established to precisely compare the exflagellation activity of different Alp2b mutants. While structurally similar P. falciparum Alp2b was partially functional in P. berghei, the exchange of the Alp2b D-loop or H-plug with the equivalent structures of actin completely arrested exflagellation. This result indicates that the Alp2b-specific larger D-loop and H-plug are functionally critical. More selective mutations in each region identified critical amino acid residues within the D-loop, while the conserved residues at the tip of the H-plug are interchangeable with the corresponding actin sequence. Collectively, this shows that Alp2b function relies on specific conserved amino acid residues within its unique regions, while some residues may also contribute to structural integrity. The deletion of Alp2b in P. berghei resulted in a delay in axonemal development and the absence of nuclear segregation. Consequently, the immotile gametes were unable to exflagellate. <br> Altogether, this thesis demonstrates how Alp1 influences actin filament stability in ookinetes and its absence inhibits gliding motility, as well as how Alp2b facilitates crucial steps of male gametogenesis while its function depends on specific residues of unique regions. In both cases, these Alps are essential for Plasmodium transmission and thus provide a firm basis for potential transmission-blocking drug development. Production of recombinant Alps, as pursued in this thesis, would be of great benefit in this context. Finally, this study offers the first insights into the roles of these unique members of the actin superfamily, which would also contribute to the advancement of current cytoskeleton research.