Actin-like proteins 3 and 5a in Plasmodium life cycle progression

• Applicant: Ross Douglas, Ph.D.
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 518746970

The actin superfamily consists of multiple members that have diverse functions ranging from polymerisation, cytoskeleton regulation, protein folding, chromatin remodelling and carbohydrate metabolism. Despite these diverse functions there is a common core, the actin fold, which acts as an ATP binding site. The unique sequence insertions outside of the common core result in the specific roles of each superfamily member. "Actin related proteins" (Arps) are another member of this superfamily that are famously involved in trafficking, actin filament branching and chromatin remodelling. Within this subfamily there are Arps that only exist in apicomplexan parasites and are thus referred to as "actin-like proteins" (Alps). Given their uniqueness and prevalence in apicomplexan parasites, as well as our own preliminary evidence, I hypothesize that Alps play critical roles in parasite progression at different developmental steps. Further, these Alps contain unique sequence insertions that we predict mediate these specialist functions. Our group is interested in the contribution of these Alps in parasite progression across the malaria parasite life cycle. Here, I propose the study of Alp3 and 5a with a view to understand their structure-function relationships with different methods and at different scales. Firstly, we will characterize the overall functions of Alp3 and 5a using complimentary in vitro biochemical and in vivo gene knockout approaches. The Alps will be heterologously expressed with the aim to determine their structures while the corresponding genes will be knocked out in the parasite and the subsequent phenotype fully characterized in a mouse-mosquito model of infection (including transcriptomic methods). Secondly, the sub-cellular localization of the respective proteins will be assessed by tagging with fluorescent proteins. These tagged versions will also be used for pull down experiments to identify potential Alp-interactors. Thirdly, to begin to elucidate the structure-function relationships of these Alps, we intend to mutate Alp insertions and assess the consequences of these changes biochemically and in parasite progression. The proposed work involves a synergistic interplay between the in vitro and in vivo methods that will provide mechanistic insight into Alp function. Such advances will allow for improved understanding of actin superfamily members that, despite sharing a common structural core, function differently and thus our findings will shed important light on actin evolution and diversity.

• DFG Programme: Research Grants