The novel microtubule-associated CAP-glycine protein Cgp1 governs growth, differentiation, and virulence of Cryptococcus neoformans

Microtubules are involved in mechanical support, cytoplasmic organization, and several cellular processes by interacting with diverse microtubule-associated proteins such as plus-end tracking proteins, motor proteins, and tubulin-folding cofactors. A number of the cytoskeleton-associated proteins (CAPs) contain the CAP-glycine-rich (CAP-Gly) domain, which is evolutionarily conserved and generally considered to bind to alpha-tubulin to regulate the function of microtubules. However, there has been a dearth of research on CAP-Gly proteins in fungal pathogens, including Cryptococcus neoformans, which is a global cause of fatal meningoencephalitis in immunocompromised patients. In this study, we identified five CAP-Gly protein-encoding genes in C. neoformans. Among these, Cgp1 encoded by CNAG_06352 has a unique domain structure containing CAP-Gly, SPEC, and Spc7 domains that is not orthologous to CAPs in other eukaryotes. Supporting the role of Cgp1 in microtubule-related function, we demonstrate that deletion or overexpression of CGP1 alters cellular susceptibility to thiabendazole, a microtubule destabilizer and that Cgp1 is co-localized with cytoplasmic microtubules. Related to the cellular function of microtubules, Cgp1 governs the maintenance of membrane stability and genotoxic stress responses. Deletion of CGP1 also reduces production of melanin pigment and attenuates the virulence of C. neoformans. Furthermore, we demonstrate that Cgp1 uniquely regulates the sexual differentiation of C. neoformans with distinct roles in the early and late stage of mating. Domain analysis revealed that the CAP-Gly domain plays a major role in all Cgp1 functions examined. In conclusion, this novel CAP-Gly protein, Cgp1, has pleotropic roles in regulating growth, stress responses, differentiation, and virulence in C. neoformans.