Ras1-Independent High Iron-Mediated Hyphal Formation in Candida albicans

C. albicans small GTPase Ras1 belonging to the cAMP-Protein Kinase A (PKA) signaling pathway is a well-established master regulator of hyphal development, taking its environmental cues from N-acetylglucosamine (GlcNAc) as a carbon source. Iron is also known to induce filamentation in C. albicans. However, the influence of iron availability on Ras1-cAMP-PKA signaling in response to GlcNAc-induced filamentation has never been studied. In this study, we investigated the role of Ras1 in hyphal induction under varying iron conditions, using both in vitro systems and an in vivo model of mucosal colonization in Caenorhabditis elegans. Surprisingly, upon GlcNAc exposure, Δ/Δras1 cells formed true hyphae exclusively under high-iron conditions, whereas its parent strain (CAI4-Ura+) showed hyphal formation irrespective of environmental iron levels. Further analysis revealed that this GlcNAc-mediated hyphal formation under high iron in Δ/Δras1 cells was independent of cAMP levels but required the downstream effectors Efg1 and Tpk2. A similar iron-dependent pattern of hyphal formation in Δ/Δras1 cells was also observed in vivo in C. elegans. Transcriptomic analysis indicated that high iron induced robust expression of hypha-associated genes in Δ/Δras1, accompanied by downregulation of BCY1, a negative regulator of PKA. Overexpression of BCY1 in Δ/Δras1 cells completely blocked the iron-dependent hyphal formation, highlighting a previously unrecognized Ras1-independent, iron-responsive mechanism controlling PKA-mediated filamentation. Collectively, our findings reveal that increased environmental iron availability can bypass Ras1 to regulate hyphal development by limiting Bcy1 levels to allow PKA activation. This provides insights into how C. albicans can exploit iron replete host niches for enhanced pathogenicity, eliminating the need for key modulators such as Ras1.