A glycolysis–calcineurin regulatory axis orchestrates titan cell formation in Cryptococcus neoformans

Abstract 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, hxk2Δ, and hxk2Δ supplemented with cAMP under titan cell-inducing conditions. Transcriptomic analyses revealed significant downregulation of a large subset of calcineurin-responsive genes in the hxk2Δ 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 hxk2Δ mutant, whereas chelation of extracellular calcium with EGTA significantly inhibited titanization in wild-type cells. Using calcium-sensitive dyes, we found that intracellular calcium levels were substantially reduced in the hxk2Δ 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.