Functional characterization of the alveolins, a family of cytoskeleton proteins of malaria parasites.

Malaria remains one of the most serious infectious parasitic diseases in humans. A shared cellular feature of the motile and invasive stages (zoites) of malaria parasites is the presence of a unique cortical cytoskeletal structure named the subpellicular network (SPN), which forms an internal cytoskeletal basket providing mechanical strength to the cell. Malaria parasites have three distinct zoite stages: the merozoite (blood stage); the ookinete (early mosquito stage); and the sporozoite (late mosquito and liver stage). A family of intermediate filament proteins, named alveolins, comprise main building blocks of the SPN. Several Plasmodium alveolins have been shown to be differentially expressed across zoite stages, and to have functionally equivalent and essential roles involved in parasite morphogenesis, tensile strength, motility and infectivity. In this thesis, genetically altered parasite lines expressing disrupted, fluorescent protein-tagged or mutated alveolins were generated in the mouse malaria model P. berghei, to further characterize select alveolin family members with respect to their life stage expression, subcellular localization and trafficking, and their contribution to parasite development, infectivity and mosquito transmission. Through structure-function analysis, the contributions of conserved cysteine motifs to alveolin function and post-translational lipid modification (palmitoylation) were also investigated. Re-examination of the alveolin repertoire identified 13 alveolin family members whose conserved 'alveolin' domains possess tandem repeats of typically 12 amino acids. The results show two alveolins, IMC1c and IMC1e, to be expressed in all three zoite stages and to be essential for blood stage parasite development. Moreover, IMC1c and IMC1e display different temporal recruitment to the SPN. By contrast, the alveolin IMC1d is expressed only in ookinetes and is functionally dispensable. We show that the cysteine motif in the alveolin IMC1c is the site of S-palmitoylation, but is functionally redundant. By contast, the cysteine motifs of IMC1a are important for sporozoite development and infectivity. The combined results suggest that while alveolins have a shared core architecture and overall cytoskeletal function, differences in life stage expression, protein expression level, recruitment to the SPN, and palmitoylation status ensure that each family member makes a unique contribution to parasite development.