A Conserved Metabolic Network Orchestrates Melanin-Dependent Titan Cell Formation in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that primarily causes pulmonary infections, with the potential to cause life-threatening infections including meningoencephalitis in immunocompromised individuals. Key virulence factors including polysaccharide capsule and melanin, facilitate immune evasion and tissue invasion in the host. Recently, titan cell formation has been defined as another important virulence factor of C. neoformans and plays a pivotal role in disease progression. The cyclic AMP-protein kinase A (cAMP-PKA) pathway in C. neoformans has been shown to be an important regulator of virulence-associated processes, including capsule formation, melanin biosynthesis and titan cell formation. However, the upstream signals, critical for the activation of cAMP-PKA pathway in the context of titan cell formation remain poorly understood. In this study, we demonstrate that the central carbon metabolic pathway, glycolysis, is critical for cAMP-dependent titan cell formation. Pharmacological and genetic perturbation of glycolysis significantly attenuated titan cell formation. Remarkably, exogenous addition of cAMP completely reversed the titan cell defects, observed during glycolysis perturbation. Interestingly, melanin deficient strains exhibited a significant attenuation in titan cell formation establishing a novel link between dimorphic switching and melanin biosynthesis in C. neoformans. These findings establish a novel regulatory axis wherein central carbon metabolism orchestrates morphogenetic switching in C. neoformans by regulating the activity of the well-conserved cAMP-PKA pathway. We also demonstrate, for the first time, that melanin biosynthesis which is under the regulatory control of cAMP-PKA pathway, is critical for titan cell formation, providing new insights into the metabolic control of C. neoformans dimorphism.