A Conserved Metabolic Network Regulates Titan Cell Formation in Cryptococcus neoformans
Cryptococcus neoformans is an opportunistic fungal pathogen that causes pulmonary infections and life-threatening meningoencephalitis in immunocompromised individuals. In addition to the polysaccharide capsule and melanin, titan cell formation is a key virulence trait that promotes immune evasion and disease progression. Despite the established role of titan cells in C. neoformans pathogenesis, the molecular mechanisms governing their formation remain poorly understood. Here, we demonstrate that glycolysis is critical for titan cell formation in C. neoformans. Pharmacological inhibition or genetic disruption of glycolysis significantly impaired titanization, whereas exogenous cAMP add-back restored the defect. To elucidate the underlying mechanism, we performed comparative RNA-seq analyses of wild- type, hxk2{Delta}, and hxk2{Delta} supplemented with cAMP under titan cell inducing conditions. Transcriptomic analyses revealed significant downregulation of a large subset of calcineurin- responsive genes in the hxk2{Delta} mutant, many of which were restored upon cAMP supplementation. Consistent with these findings, pharmacological inhibition of calcineurin using FK506 or cyclosporin A markedly reduced titan cell formation, establishing an essential role for calcineurin signaling in this morphological transition. Furthermore, supplementation with CaCl2 rescued the titanization defect of the hxk2{Delta} mutant, whereas chelation of extracellular calcium with EGTA significantly inhibited titanization in wild-type cells. Using the calcium-sensitive dye, we found that intracellular calcium levels were substantially reduced in the hxk2{Delta} mutant and were restored by CaCl2 or cAMP supplementation. Collectively, our findings uncover a previously unrecognized glycolysis-calcineurin regulatory axis that governs titan cell formation and establishes a direct mechanistic link between central carbon metabolism, calcium homeostasis and fungal morphogenesis. SummaryTitan cell formation is a critical virulence trait that promotes immune evasion and disease progression of Cryptococcus neoformans. However, the mechanisms governing this morphological transition remain poorly understood. Here, we demonstrate that glycolysis is essential for titan cell formation and uncover a previously unrecognized link between glycolysis, calcium homeostasis, and calcineurin signaling. Comparative transcriptomics, pharmacological perturbation, calcium rescue experiments, and intracellular calcium measurements reveal that disruption of glycolysis attenuates calcineurin signaling by reducing intracellular calcium levels, thereby impairing titanization. These findings establish a novel glycolysis-calcineurin regulatory axis controlling fungal morphogenesis in C. neoformans.