title: A Combined Molecular, Cell and Structural Biology Approach Towards Characterising Malaria Alveolins
Creators: name: Coghlan, MP
Publisher: name: London School of Hygiene & Tropical Medicine
Year: 2020

description: Intermediate filament (IF)-­‐based cytoskeletal networks in metazoans have key roles in cell architecture and plasticity, and as mechanical stress absorbers. Much less is known about IFs in protozoans. Alveolins are a family of putative IF proteins found exclusively in apicomplexan parasites (causative agents of diseases such as malaria, toxoplasmosis, cryptosporidiosis), dinoflagellate algae and ciliates. All alveolins share functional domains that are characterized by possessing 12 amino-­‐acid tandem repeats. These ‘alveolin’ modules resemble conserved domains found in other protozoan cytoskeletal proteins like articulins. The demonstrated essential nature of alveolins in malaria parasite development, their expression throughout the life cycle, and their absence in vertebrates makes them potentially attractive drug targets for malaria treatment, prophylaxis and transmission control. Moreover, such drugs could be active against a broad range of other apicomplexan parasites, as well as against related pathogenic protozoans. In this context, a better understanding of the core architecture of the Plasmodium alveolins and their assembly mechanisms is important. This project set out to study the structural requirements of the alveolins and their conserved domains for assembly of the protein into the IF network, and for their functional contribution to cell shape, tensile strength and motility in live malaria parasites, using the Plasmodium berghei mouse malaria model. The results reveal, based on the ookinete and sporozoite-­‐ expressed alveolin IMC1h, that the ‘alveolin’ module is required for recruitment into the cortical cytoskeleton, consistent with the notion that it holds the properties for IF formation. In addition, the carboxy-­‐terminal conserved domain of IMC1h, structurally unrelated to the ‘alveolin’ module, is implicated in facilitating parasite motility through direct or indirect interactions with the motility apparatus. In addition, a structural biology approach was undertaken, aimed at determining the core atomic structure of the alveolins, with various techniques at hand to try and determine both tertiary and secondary structures formed by these proteins. Bioinformatic-­‐based analyses indicated that the ‘alveolin’ module is structurally ordered, and adopts a predominantly β-­‐ strand architecture. High level expression, in soluble form, of various P. berghei alveolin domains in bacteria was achieved as amino-­‐terminal fusions with the protein tag NusA. However, further purification of these recombinant alveolins was severely hampered by problems with solubility after cleavage of the NusA tag, or after concentration, resulting in protein precipitation. Whilst these problems have thus far precluded structural analyses by biophysical means, the observations could reflect actual physical properties of the alveolins and the way by which these molecules assemble in the cell into the insoluble IF network structure, possibly via the intermittent formation of shorter oligomers (protofilaments). Work is ongoing to optimise purification protocols.