Regulation of Als3 and Nrg1 during morphogenesis in Candida albicans
Candida albicans is an opportunistic human pathogen. Several factors contribute to the virulence of this fungus. These include phenotypic switching, biofilm formation, the secretion of hydrolytic enzymes, the production of adhesion molecules, and the ability to undergo reversible morphological changes from yeast to (pseudo)hyphae in response to the host physiological conditions. Multiple signalling pathways link the relevant environmental signals to hyphal growth, and several downstream transcription factors promote or repress genes involved in the yeast-to-hypha transition. A number of genes are induced specifically during hyphal development. ALS3 encodes a hypha-specific cell wall protein of the Agglutinin-Like Sequence family of adhesins. The first aim of this project was to study the transcriptional regulation of ALS3. Previous work in our laboratory identified two sequences that bind the transcriptional repressor Nrgl (NREs), and that are required for repression under yeast inducing conditions. Moreover, a region of 150 bp between -471 and -321 was defined as being important for ALS3 activation (A1 region). However, no short sequence or known response element within this activation region, in isolation, was sufficient for hypha- specific activation of ALS3. In this study we identified an additional activation region (A2) located between -1438 and -1050. Furthermore, we found that the transcription factors Efgl, Bcrl and Teel are required for the activation of ALS3 under serum- inducing conditions. Also, Cphl contributes to the hypha-specific activation of ALS3, but it is not essential for expression. Teel does not act directly through the five putative Teel binding sites present in the ALS3 promoter, which are not functional, but mediates activation of ALS3 indirectly via Bcrl. Under conditions that promote yeast growth, ALS3 expression is repressed by Nrgl, Tupl and Rfgl, but not by Ssn6 or the mating factor al. The morphogenetic repressor Nrgl targets the global repressor Tupl to the promoters of hypha-specific genes and other genes to inhibit the yeast-to-hypha transition. The second aim of our project was to study the regulation of Nrgl. Previous work in our laboratory showed that, as expected, NRG1 mRNA levels decrease during hyphal development. However, the down-regulation of NRG1 was too slow to account for the rapid induction of hyphal growth. In this study we found that Nrgl is a phosphoprotein that is found phosphorylated in yeast cells and becomes rapidly and transiently dephosphorylated in serum-induced cells. In addition, Efgl is required for Nrgl- mediated repression via the NRE under non-inducing conditions, whereas the release of Nrgl-mediated repression under inducing conditions is dependent on components of the cAMP-PKA pathway. Furthermore, we found that the Nrgl-mediated repression of an NRE-containing reporter construct in yeast cells requires both Tupl and Ssn6, although this might not always be the case for native promoters. Altogether, the results of this project show that several signalling pathways converge at the promoter of the hypha-specific gene ALS3 to regulate its expression. The activity of one of the negative regulators of ALS3, the morphogenetic repressor Nrgl, appears to be modulated both at the transcriptional and post-translational level. Furthermore, the cAMP-PKA pathway seems to link positive and negative regulation of morphogenesis in C. albicans.