Mechanisms linking phosphate acquisition, stress resistance, and virulence in the fungal pathogen Candida albicans

Pathogenic microbes must be able to mount robust stress responses to allow them to survive the barrage of chemical weapons released by the host's immune system to kill invading pathogens. The importance of such immune-based defences is highlighted by the fact that patients with weakened immune systems are particularly prone to both bacterial and fungal infections. We recently made the unexpected finding that intracellular phosphate levels, which are controlled by the regulatory factor Pho4, are essential for the human fungal pathogen Candida albicans to survive many stresses encountered during infection such as superoxide stress. Because of this, engineered C. albicans cells lacking Pho4 are very sensitive to killing by macrophages, and are much less virulent than wild-type Candida cells in various infection models. Our pilot studies have shown that the superoxide stress-sensitivity, exhibited by cells lacking Pho4, is due to a previously uncharacterised role for phosphate in regulating copper homeostasis. Copper is an essential metal co-factor for the C. albicans Sod1 superoxide dismutase enzyme, and we find that Sod1 activity is impaired in cells lacking Pho4 resulting in reduced resistance to superoxide stress.

In this proposal we aim to build on these novel connections linking phosphate acquisition and metal homeostasis to the stress resistance and virulence of a major human pathogen, to ask two key questions: (a) how does reduced intracellular phosphate levels de-regulate copper homeostasis resulting in acute sensitivity to superoxide stress, and (b) can the prevention of phosphate acquisition in C. albicans be exploited as a novel therapeutic strategy to help prevent life-threatening infections? This project benefits from our complementary areas of expertise in C. albicans stress responses, infection modelling and the study of metal homeostasis mechanisms. Specifically, we will utilise a powerful combination of precision molecular tools to characterise the pathways that regulate Pho4 in C. albicans, and state-of-the-art technologies to study metal-protein interactions to determine how phosphate impairs copper homeostasis. In addition, we will translate our knowledge of Pho4 regulation in C. albicans and perform a high-throughput screen to identify drugs that inhibit phosphate acquisition. This, in combination with the subsequent testing of such compounds in infection models, has the exciting potential to identify new drugs that inhibit C. albicans pathogenicity.

This project is important for several reasons. First of all it will increase our fundamental understanding of how phosphate acquisition is vital for stress resistance and virulence of a major fungal pathogen of humans. Secondly, as intracellular phosphate homeostasis is emerging as a key virulence determinant in a number of other human and plant fungal pathogens, our findings in C. albicans may have wide reaching consequences for other host-fungus interactions. Finally, it has the potential to identify new drugs that block phosphate acquisition and attenuate fungal virulence. This is important because there is a limited repertoire of antifungal drugs available to treat systemic infections and resistance to such drugs is now a major problem. Indeed, despite the availability of such drugs, C. albicans causes over 400,000 life-threatening systemic infections each year, thus highlighting the urgent need for new effective therapies

Stress responses are essential for the pathogenicity of Candida albicans, a major fungal pathogen of humans. We recently discovered that phosphate homeostasis, regulated by the Pho4 transcription factor, is vital for the resistance of this pathogen to a range of physiologically relevant stresses encountered during infection, including superoxide stress. Consequently, we find that C. albicans cells lacking the Pho4 transcription factor display significantly attenuated virulence in a number of infection models. Our pilot data indicate that the superoxide stress-sensitive phenotype exhibited by pho4 cells is due to a previously uncharacterised role of phosphate in mediating intracellular copper bioavailability, resulting in the impaired activity of the copper-dependent Sod1 superoxide dismutase enzyme. In this proposal we address two important questions emerging from this work; (a) how do reduced intracellular phosphate levels impact on copper homeostasis resulting in acute sensitivity to superoxide stress, and (b) can perturbation of phosphate acquisition in C. albicans be exploited as a novel therapeutic strategy to help prevent life-threatening systemic infections? Firstly we will characterise in-depth the PHO network that regulates Pho4 activation in C. albicans, and define the importance of key regulators in mediating the virulence of this major pathogen. Secondly, we will delineate the mechanisms linking phosphate to copper homeostasis and superoxide stress resistance, by employing state-of-the-art technologies to define the role of phosphate on the intracellular distribution of copper within C. albicans. Finally, we will we will translate our knowledge of PHO pathway regulation in C. albicans and perform a novel screen which, together with infection modelling, has the exciting potential to identify drugs that inhibit phosphate acquisition and attenuate C. albicans virulence.

To achieve the maximum impact for our project we will:
1. Ensure that our scientific findings describing novel connections linking phosphate metabolism and stress resistance, and the potential for targeting phosphate acquisition in preventing fungal virulence, are disseminated effectively across the academic community
2. Protect and exploit any commercially valuable intellectual property relating to new antifungal compounds that arises from this project.
3. Contribute actively to public outreach programmes with a view to enhancing the public understanding of Candida and the impact of fungal infections on public health.
4. Further enhance our collaborations in the UK and abroad.
5. Provide an excellent training for our PDRA in new skills including the biology of metals, antifungal drug discovery and infection modelling.

ACADEMIC DISSEMINATION: We will ensure that our work is disseminated broadly across the academic community through publication in leading journals, presentations at the top national and international conferences in our field, collaboration with international colleagues, and through the activities of the Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology.

RELEVANCE TO HUMAN HEALTH AND COMMERCIAL EXPLOITATION: This project addresses an important area of relevance to human health. Specifically we will determine how phosphate acquisition is vital for stress resistance and virulence in a major pathogen of humans, and we will translate this knowledge to identify drugs that block phosphate acquisition and inhibit fungal virulence.
Fungal infections represent a significant and understudied medical problem (Science vol 336, 647). C. albicans is widespread in humans, a frequent cause of oral thrush and vaginitis, and a leading cause of life-threatening systemic fungal infections in immunocompromised patients. Such systemic infections are associated with an alarming mortality rate above 40%, and over 400,000 cases are reported per annum. Furthermore, systemic fungal infections significantly increase the average length of hospitalisation of intensive care patients, having a major impact upon medical care budgets (~1000 Euros per day per infected patient). Added to this there are only three classes of drug licensed to treat systemic Candida infections and emerging resistance to such drugs is becoming a major problem. Efforts to extend the number of antifungal therapies depend on a better understanding of fungal pathobiology and the translation of this knowledge to identify new drugs. This project directly addresses both these areas. The identification of drugs that disrupt phosphate acquisition and attenuate C. albicans virulence, will be subsequently developed in collaboration with MRC Technology (see Letter of Support).

PUBLIC OUTREACH: We strongly believe that increasing public understanding of scientific research is essential, and will continue our many ongoing contributions to local and national public outreach programmes, as well as initiate a new public outreach activity focussing on anti-fungal drug development (detailed in the Pathways to Impact statement).

COLLABORATIONS: This project involves the establishment of new exciting collaborations with Prof Valeria Culotta (John Hopkins University), an expert in the study of superoxide dismutase enzymes, and with MRC Technology. The collaboration with MRC Technology is facilitating the applicants to translate their research knowledge to actively seek new antifungal agents.

TRAINING: We will provide an excellent research training for our named PDRA, Alison Day, in a range of technologies new to Dr Day including ICP-MS analysis, drug screening and infection modelling. Also, we will enhance her career prospects by providing training in transferable skills and networking.