Microtubule-associated proteins modulate subpellicular microtubule function in the malaria parasite Plasmodium berghei

Malaria, caused by the unicellular eukaryotic parasite Plasmodium, is transmitted through the bite of an infected Anopheles mosquito. Plasmodium has a complex life cycle that involves the development within different tissues and environments, both within the mosquito and the vertebrate host. This lifestyle requires constant adjustment in cell shape to meet the changing demands of the host environments but also fast parasite motility to evade the host immune mechanisms. In eukaryotic organisms, cell shape is often mediated by microtubules – hollow filamentous structures composed of α- and β-tubulin. Microtubule dynamics and stability are modulated by microtubule-associated proteins (MAPs). MAPs are proteins that bind either externally or internally to the microtubule lattice with the latter named microtubule inner proteins (MIPs). In Plasmodium, the dynamic shape changes and the structural stability of the parasite are mediated by subpellicular microtubules (SPMTs) that lie directly underneath the inner membrane complex (IMC) that subtend the plasma membrane of the parasite. SPMTs are characteristic for the invasive stages of the parasite and hence found in merozoites, ookinetes and sporozoites. They also play a pivotal role in the elongated, falciform-shaped gametocytes of Plasmodium falciparum, where they facilitate cell elongation and maturation. Disruption of SPMT formation, stability or number affects parasite shape and motility, underscoring their critical functional roles. Unlike the microtubules of model organisms, SPMTs exhibit remarkable stability, a property thought to be conferred by MAPs and potentially MIPs. However, the identities and specific functions of these stabilizing proteins remain largely unknown. In this study, I investigated the functional roles of several MAPs across the life cycle of the rodent-infecting Plasmodium berghei. Individual and combinatorial deletions of two MIPs – previously identified in the related parasite Toxoplasma gondii – and one potential outer MAP did not impair parasite development or transmission. Moreover, these deletions did not yield a detectable phenotype under normal conditions. However, cold assays revealed their contribution to SPMT stability under suboptimal environments, which is potentially relevant in natural settings. Exposure of MIP knockout parasites to low temperatures caused SPMT depolymerization and cytosolic dispersion, underscoring their role in maintaining intrinsic microtubule stability under suboptimal temperature conditions. In addition, I investigated the function of an external MAP, SPM3, unique to Plasmodium (and Cryptosporidium). SPM3 was previously found to be important for SPMT arrangement in the human-infecting Plasmodium falciparum. In Plasmodium berghei, spm3 deletion progressively impaired mosquito-stage development, leading to reduced motility in ookinetes and sporozoites, diminished salivary gland invasion, and defective transmission. Ultrastructure expansion microscopy and electron microscopy revealed that SPM3 is required for proper tethering of SPMTs to the IMC, which is in particular essential for the mosquito-to-vertebrate transmission stages. Collectively, these findings illuminate the mechanisms underlying both the intrinsic stability and the IMC tethering of SPMTs, highlighting the indispensable roles of SPMTs in parasite development and infectivity. These findings deepen our understanding of the structural and functional adaptations that enable Plasmodium to navigate through its life cycle and ensures a foundation for future investigations into parasite biology.