Deciphering subcellular localization-dependent functions of Hog1 MAPK in <i>Cryptococcus neoformans</i>

Abstract The Hog1 mitogen-activated protein kinase regulates stress adaptation, sexual differentiation, and virulence traits by dynamically shuttling between the cytoplasm and nucleus in the fungal pathogen Cryptococcus neoformans, a leading cause of fatal fungal meningoencephalitis worldwide. However, how spatial partitioning governs Hog1 pathway specificity remains poorly understood. Here, we generated genetically engineered strains expressing localization-restricted Hog1 variants including membrane-tethered and constitutively nuclear, and we compared their phenotypic traits with those of a fully functional Hog1–mCherry strain. The membrane-tethered Hog1 restored thermotolerance and antifungal resistance in the hog1Δ mutant, whereas nuclear Hog1 was necessary for osmotic and endoplasmic reticulum (ER) stress response and for capsule and melanin biosynthesis. These localization-specific effects were corroborated by the expression profiles of genes involved in glycerol biosynthesis, under osmotic stress or fludioxonil treatment. Measurement of intracellular glycerol revealed that the plasma membrane-tethering of Hog1 caused excessive accumulation, underscoring the importance of spatiotemporal regulation of Hog1 activity. Furthermore, nuclear Hog1 uniquely reinstated osmoadaptation and conferred tunicamycin tolerance independent of canonical Ire1-Hxl1 splicing, suggesting a nucleus-centric module for ER stress protection. Conversely, membrane-tethered Hog1 enhanced glycerol accumulation and restored amphotericin B resistance while increasing azole susceptibility despite normal ergosterol levels, implicating localization-biased regulation upstream of sterol abundance. In the developmental program, membrane-tethered Hog1 dominantly suppressed the Cpk1-mediated mating. Collectively, these findings demonstrate that Hog1 compartmentalization is a key determinant of stress adaptation, antifungal resistance, differentiation, and virulence regulation in C. neoformans.