Candida albicans Zcf37, a zinc finger protein, is required for stabilization of the white state

Candida albicans, the most prevalent human fungal pathogen, can switch stochastically between white and opaque phases. In this study, we identified Zcf37, a zinc finger protein, as a new regulator of white-opaque switching. Deletion of ZCF37 increased white-to-opaque switching frequency and stabilized the opaque state. Overexpression of ZCF37 promoted conversion of opaque cells to white phase, but needed existence of Efg1, a key regulator required for maintenance of the white state. Deletion of EFG1 abolished the effect of ectopically expressed Zcf37 on opaque-to-white switching, whereas ectopic expression of EFG1 promoted white cell formation without presence of Zcf37. Our results suggest that Zcf37 acts as an activator of white cell formation and a repressor of opaque state and functions upstream of Efg1.     

Invasive filamentous growth of Candida albicans is promoted by Czf1p-dependent relief of Efg1p-mediated repression

Filamentation of Candida albicans occurs in response to many environmental cues. During growth within matrix, Efg1p represses filamentation and Czf1p relieves this repression. We propose that Czf1p interacts with Efg1p, altering its function. The complex regulation of filamentation may reflect the versatility of C. albicans as a pathogen.     

A potential phosphorylation site for an A-type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans

Efg1p in the human fungal pathogen Candida albicans is a member of the conserved APSES class of proteins regulating morphogenetic processes in fungi. We have analyzed the importance for hyphal morphogenesis of a putative phosphorylation site for protein kinase A (PKA), threonine-206, within an Efg1p domain highly conserved among APSES proteins. Alanine substitution of T206, but not of the adjacent T207 and T208 residues, led to a block of hypha formation on solid and in liquid media, while a T206E exchange caused hyperfilamentation. The extent of the morphogenetic defect caused by the T206A mutation depended on hypha-induction conditions. Extragenous suppression of mutations in signaling components, including tpk2 and cek1 mutations, was achieved by wild-type- and T206E-, but not by the T206A-variant-encoding allele of EFG1. All muteins tested were produced at equal levels and at high production levels supported pseudohyphal formation. The results are consistent with a role of Efg1p as a central downstream component of a PKA-signaling pathway including Tpk2p or other PKA isoforms. Threonine-206 of Efg1p is essential as a putative phosphorylation target to promote hyphal induction by a subset of environmental cues.     

Inhibition of yeast-to-hypha transition in Candida albicans by phorbasin H isolated from Phorbas sp.

Phorbasin H is a diterpene acid of a bisabolane-related skeletal class isolated from the marine sponge Phorbas sp. In this study, we examined whether phorbasin H acted as a yeast-to-hypha transition inhibitor of Candida albicans. Growth experiments suggest that this compound does not inhibit yeast cell growth but inhibits filamentous growth in C. albicans. Northern blot analysis of signaling pathway components indicated that phorbasin H inhibited the expression of mRNAs related to cAMP–Efg1 pathway. The exogenous addition of db-cAMP to C. albicans cells had no influence on the frequency of hyphal formation. The expression of hypha-specific HWP1 and ALS3 mRNAs, both of which are positively regulated by the important regulator of cell wall dynamics Efg1, was significantly inhibited by the addition of phorbasin H. This compound also reduced the ability of C. albicans cells to adhere in a dose-dependent manner. Our findings suggest that phorbasin H impacts the activity of the cAMP–Efg1 pathway, thus leading to an alteration of C. albicans morphology.     

Crk1, a novel Cdc2-related protein kinase, is required for hyphal development and virulence in Candida albicans

Both mitogen-activated protein kinases and cyclin-dependent kinases play a role in hyphal development in Candida albicans. Using an oligonucleotide probe-based screen, we have isolated a new member of the Cdc2 kinase subfamily, designated Crk1 (Cdc2-related kinase). The protein sequence of Crk1 is most similar to those of Saccharomyces cerevisiae Sgv1 and human Pkl1/Cdk9. In S. cerevisiae, CRK1 suppresses some, but not all, of the defects associated with an sgv1 mutant. Deleting both copies of CRK1 in C. albicans slows growth slightly but leads to a profound defect in hyphal development under all conditions examined. crk1/crk1 mutants are impaired in the induction of hypha-specific genes and are avirulent in mice. Consistent with this, ectopic expression of the Crk1 kinase domain (CRK1N) promotes filamentous or invasive growth in S. cerevisiae and hyphal development in C. albicans. The activity of Crk1 in S. cerevisiae requires Flo8 but is independent of Ste12 and Phd1. Similarly, Crk1 promotes filamentation through a route independent of Cph1 and Efg1 in C. albicans. RAS1(V13) can also activate filamentation in a cph1/cph1 efg1/efg1 double mutant. Interestingly, CRK1N produces florid hyphae in ras1/ras1 strains, while RAS1(V13) generates feeble hyphae in crk1/crk1 strains.