Human serum potently induces hyphal development of the polymorphic fungal pathogen Candida albicans, a phenotype that contributes critically to infections. The fungal adenylyl cyclase Cyr1p is a key component of the cAMP/PKA-signaling pathway that controls diverse infection-related traits, including hyphal morphogenesis. However, identity of the serum hyphal inducer(s) and its fungal sensor remain unknown. Our initial analyses of active serum fractions revealed signs of bacterial peptidoglycan (PGN)-like molecules. Here, we show that several purified and synthetic muramyl dipeptides (MDPs), subunits of PGN, can strongly promote C. albicans hyphal growth. Analogous to PGN recognition by the mammalian sensors Nod1 and Nod2 through their leucine-rich-repeat (LRR) domain, we show that MDPs activate Cyr1p by directly binding to its LRR domain. Given the abundance of PGN in the intestine, a natural habitat and invasion site for C. albicans, our findings have important implications for the mechanisms of infection by this pathogen.

SummaryMany Ras GTPases activate their effectors through binding at a conserved Ras association (RA) domain. An example is the activation of the budding yeast adenylate cyclase Cyr1 by Ras1 and Ras2. Candida albicans Ras1 is speculated to similarly activate Cdc35, the orthologue of Cyr1, for hyphal development. Here, we have investigated whether the RA domain mediates Ras1-Cdc35 interaction and how this interaction regulates cAMP levels and morphogenesis. Yeast two-hybrid assays suggested that Ras1 interacts only with the RA but not any other identifiable domains of Cdc35. The Ras1-RA interaction was further confirmed by in vitro binding assays of purified RA domain and Ras1 and by co-immunoprecipitation of Ras1 and Cdc35 from cell lysates. Substituting Ala for the conserved residue K 338 or L 349 in the RA domain or deleting the RA domain abolished the Ras1-RA or Ras1-Cdc35 interactions. cdc35 mutants with the RA domain deleted or carrying K388A or L349A mutation exhibited rather normal yeast growth but were completely defective in hyphal morphogenesis. Further, the mutants contained nearly wild-type levels of cAMP during yeast growth but were unable to increase it upon hyphal induction. These results suggest an essential role for the RA-mediated Ras1-Cdc35 interaction in raising cellular cAMP levels for hyphal morphogenesis.

SummaryA key virulence trait of Candida albicans is its ability to undergo the yeast-to-hyphal growth transition in response to environmental signals. This transition critically requires a rapid activation of the adenylyl cyclase Cyr1 to generate a cAMP spike. However, the identity of the signal sensors and mechanisms of signal processing and integration remain largely unclear. Recent evidence suggests that some sensors are embedded in Cyr1 itself. To test this hypothesis, we asked whether purified Cyr1 can respond to hyphal induction. Here, we report that Cyr1 co-purifies with Cap1 and G-actin as a tripartite complex which can increase cAMP synthesis in response to hyphal inducing signals in an actindependent manner. Cap1 binds Cyr1 and G-actin through its N-and C-terminus respectively. Deleting the G-actin binding sites or treating the complex with the actin toxin latrunculin A or cytochalasin A inhibits the activation of cAMP synthesis. Strains expressing Cap1 mutants lacking the G-actin binding site are impaired in both cAMP synthesis and hyphal morphogenesis. Thus, our findings reveal an essentially intact sensor/effector apparatus composed of Cyr1, Cap1 and G-actin. Furthermore, G-actin's regulatory role in this apparatus may prove to be the missing link whereby cellular actin status knowingly influences cAMP-mediated cellular processes.