Central role of Nrg1 in the niche-specific coordination of morphogenesis stress responses and metabolism in the fungal pathogen Candida albicans

The fungus, Candida albicans is medically important because it causes a range of infections in humans, some of which can be lethal. It causes frequent infections of the mouth, especially in young babies, the aged and in AIDS patients. Also, most women suffer at least one 'thrush' infection during their lifetime. Candida also causes life-threatening 'systemic' infections in intensive care patients whose immunological defences are significantly weakened (e.g. in transplant and cancer patients). Considerable research efforts have been devoted to the question 'What makes Candida such a good pathogen?'. Many research groups have focussed their attention on virulence factors. These have been defined as fungal factors that interact directly with the human host to promote infection (i.e. enhance fungal pathogenicity). These virulence factors include the ability of this fungus to switch reversibly between a yeast-like growth form and a hyphal (mycelial) growth form, which is believed to help the fungus invade tissue. Candida switches to hyphal growth in response to specific environmental triggers, which include serum or glucose. This switch is controlled by a complex biological circuitry: some regulatory proteins activate hyphal development in response to these environmental triggers, whilst others repress hyphal development in the absence of these triggers. We have studied a key repressor in some detail - Nrg1. We have shown that Nrg1 is a regulatory protein that prevents hyphal growth in the absence of hypha-inducing environmental triggers. In the course of these studies we have found that, in addition to regulating hyphal development, Nrg1 controls the expression of genes involved in stress protection and carbon metabolism. This is highly significant because these processes are essential for the pathogenicity of Candida. (Human immune cells kill microbial invaders by exposing them to oxidizing chemicals. Candida must be able to protect itself from these oxidative stresses if it is to counteract these host defences successfully and establish an infection. Also, Candida must assimilate nutrients from the host to be able to grow and infect its host. This nutrient assimilation depends upon carbon metabolism.) Hence the regulator Nrg1 seems to be playing a vital role by coordinating a range of important cellular functions that are required for survival in the host (hyphal development, stress protection and metabolism). How does Nrg1 do this? The main objective of this project is to address this question. We have shown that the Nrg1 protein is chemically modified (phosphorylated) and that the level of phosphorylation changes during the yeast-to-hyphal switch. Our hypothesis is that accurate chemical modification is vital for the control of Nrg1 activity, and hence central to the ability of Candida to coordinate its hyphal development, stress protection and metabolism. In this project we will test this hypothesis using a powerful combination of the latest experimental tools. We will define which sites on Nrg1 are modified, identify which enzymes catalyse these modifications, and establish the effects of disrupting these modifications upon the pathogenicity of Candida. These studies will dramatically advance our understanding of how Candida pathogenicity is regulated at the molecular level.

Candida albicans is a major systemic fungal pathogen of humans. Its success as a pathogen depends upon the accurate regulation of virulence factors, such as yeast-hypha morphogenesis, during disease establishment. In addition, the niche-specific regulation of metabolism and stress responses is essential for C. albicans pathogenicity. We identified Nrg1 as a key repressor of hyphal development in C. albicans. Since then, Nrg1 homologues have been implicated in regulating morphogenetic programmes in other fungi. Using a combination of approaches we have dissected the mode of action of Nrg1 in C. albicans. We know that Nrg1 represses hypha-specific genes in a Tup1-dependent fashion via defined promoter elements (NRE's). We also know that Nrg1 activity is regulated in the short and longer terms by post-translational and transcriptional mechanisms, respectively. Nrg1 is a phosphoprotein that is transiently dephosphorylated during hyphal development, and down-regulated by Ras-cAMP signalling. In this project we will define which Nrg1 residues are phosphorylated, which protein kinases catalyse these phosphorylation events, and the impact of these modifications upon Nrg1 activity. This is critical for a complete understanding of how fungal morphogenesis is controlled. More recently our genomic analyses of the cellular roles of this key regulator have revealed that, in addition to regulating yeast-hypha morphogenesis, Nrg1 modulates the expression of stress and metabolic genes in C. albicans. This is highly significant because it suggests that Nrg1 plays a key role in modulating the physiological fitness of this pathogen (central carbon metabolism and stress adaptation) as well as regulating virulence attributes (yeast-hypha morphogenesis). We will test this idea by defining the impact of specific Nrg1 de/phosphorylation events upon these different Nrg1 outputs, and the effects of disrupting these events upon C. albicans pathogenicity.