Transcriptional regulation of Iron and Copper homeostasis in Candida albicans

The ability of the human pathogen, Candida albicans, to acquire copper and iron from the uncongenial host environment is essential for virulence. Studies in our laboratory have led to identification of the copper-sensing transactivator CaMac1p that regulates genes involved in both copper and iron homeostasis. Described here are our findings of the molecular mechanism involved in the action of Mac1p. Experiments using yeast two-hybrid assays, suggests that the C-terminus domain is involved in protein - protein interactions and that CaMac1p can form homo-dimers in the presence of copper, however it does not undergo intramolecular interactions. The results also indicated that it is likely that the N-terminus domain increases the protein binding activity of the C-terminus domain. For the first time CaMac1 protein was successfully purified and using gel-shift assays CaMac1p was also demonstrated to bind to its own promoter which confirmed the hypothesis that CaMAC1 is self-regulated. This copper-responsive regulation is a key difference when compared to the ScMAC1 (Saccharomyces cerevisiae MAC1) gene, which is constitutively transcribed. The presence of one CuRE (copper response element) site allowed for optimal activation demonstrating the sites are not synergistically associated unlike in S. cerevisiae. Although CaMAC1 also plays a role in iron homeostasis, the exact mechanism of iron responsive gene regulation in C. albicans was as yet unknown; therefore we also studied the role of the putative transcription factor Sef1p as a potential regulator of iron acquisition genes. A novel role of SEF1 in copper homeostasis was detected where Sef1p was found to be involved directly or indirectly in regulation of cupric (as well as ferric) reductase CaFRE7 and copper uptake gene CaCTR1. Hence RT-PCR was also used to analyse the interactions of Sef1p with the other copper and iron responsive regulators - Mac1p and Sfu1p. The transcriptional activator Mac1p was observed to function in a manner different from its traditional role as a positive regulator. CaMac1p acted as a repressor and regulated SFU1 in a copper responsive manner by directly binding to its promoter region. This is the first time that functional evidence (using EMSA) has been provided for this. Sef1p was also found to function together with Mac1p to co-regulate the expression of CaMAC1 itself. This affirms that C. albicans portrays an interdependent regulatory circuit, referred to as the ‘Feed forward loop’. This study elucidates the co-dependent role of these regulators linking iron and copper acquisition which play a vital role in the virulence of C. albicans.