Remodeling of sporozoite microtubules during Plasmodium liver stage development

Abstract

Microtubules, composed of - and {beta}-tubulin polymers, are essential components of the eukaryotic cytoskeleton. They maintain cellular shape and structural integrity and play critical roles in cell division and in intracellular vesicular transport. In Plasmodium, the parasite that causes malaria, nuclear replication during the liver stage is among the fastest known in eukaryotic cells and relies heavily on microtubules for DNA segregation and cytoskeletal organization. Despite their importance, the role of microtubules in liver stage development remains largely unexplored. Here, we investigated microtubule dynamics during liver stage development using a combination of cell and molecular biology techniques, expansion microscopy, and live-cell imaging. By employing antibodies specific for {beta}-tubulin post-translational modifications (PTMs), we found that the Plasmodium sporozoites subpellicular microtubules (SSPM) persist during liver infection, giving rise to liver stage parasite microtubule bundles (LSPMB). These LSPMB form multimeric tubulin structures within hepatocytes and are redistributed to the hemi-spindle poles of parasite nuclei during schizogony. Deletion of the C-terminal region encompassing all known Plasmodium -tubulin PTM sites prevented sporozoite migration from the mosquito midgut to the salivary glands, effectively blocking parasite transmission. Using Plasmodium microtubule-specific depolymerisation drugs, we found that while LSPMB are stable in sporozoites, they exhibit dynamic behavior during hepatocyte infection. Given the regulatory role of PTMs in microtubule dynamics, we generated parasite mutants by substituting and deleting key {beta}-tubulin C-terminal residues involved in PTMs. Substitution of the polyglutamylation site with alanine and deletion of the C-terminal tyrosination/detyrosination motifs impaired parasite growth during liver infection. Together, our findings reveal extensive microtubule remodeling during liver stage development and establish -tubulin C-terminal modifications as critical regulators of both intracellular development and parasite transmission of Plasmodium parasites.