Regulation of hyphal development by protein kinase A, stress-responsive MAP kinases, and calcineurin via transcription factors Sfl1 and Sfl2 in Candida albicans.

UNLABELLED: Candida albicans is a major human fungal pathogen whose ability to undergo reversible morphological transitions between yeast and hyphal growth forms represents a key virulence trait. While the cAMP-protein kinase A (PKA) pathway is essential for initiating hyphal growth in vitro, it is dispensable for filamentation in vivo, yet the molecular mechanisms underlying PKA-dependent and -independent hyphal development remain incompletely understood. Sfl1 and Sfl2 are homologous heat shock transcription factors that antagonistically regulate hyphal development, with Sfl1 repressing and Sfl2 promoting filamentation. Here, we use site-specific mutagenesis to dissect how PKA, stress-responsive MAP kinases, and the phosphatase calcineurin regulate Sfl1 and Sfl2 function. Serine-to-alanine (S-to-A) substitutions at predicted PKA phosphorylation sites activated both factors, while serine-to-aspartate (S-to-D) mutations inhibited their activity. SFL1PKA A cells suppressed hyphal initiation and failed to downregulate NRG1, a key repressor of hyphal development. Genetic inactivation of Sfl1 bypassed Tpk2 requirements; however, S-to-A substitutions at the predicted PKA sites in the hyphal regulator Efg1 blocked hyphal initiation regardless of Sfl1 status. SFL2PKA DD reduced hyphal formation while SFL2PKA AA enhanced filamentation compared to wild-type SFL2. Environmental stresses regulate these factors through distinct post-translational mechanisms: phosphomimetic mutations at MAPK sites destabilized Sfl1 and promoted hyphal initiation even in SFL1PKA A cells, whereas Sfl2 lacks equivalent MAPK sites but contains calcineurin-binding motifs critical for filamentation under salt stress. This study reveals how Sfl1 and Sfl2 integrate nutritional and stress signals to control hyphal morphogenesis through both PKA-dependent and -independent regulatory mechanisms.

IMPORTANCE: Candida albicans exists as a commensal yeast in healthy individuals but becomes an invasive pathogen when host immunity is compromised. Its ability to switch between yeast and hyphal forms is crucial for pathogenesis. While the cAMP-protein kinase A (PKA) pathway is essential for hyphal induction in vitro, filamentation occurs independently of PKA during host infection. This study elucidates how the transcriptional regulators Sfl1 and Sfl2 integrate nutritional and stress signals to control morphological transitions. Through site-specific mutagenesis of conserved target sites for protein kinase A, stress-responsive MAP kinases, and the phosphatase calcineurin in Sfl1 and Sfl2, we demonstrate their roles in orchestrating hyphal development. These findings advance our understanding of how C. albicans modulates its morphology in response to host conditions, providing mechanistic insights into the regulatory networks important for both commensal colonization and invasion.