Mechanisms directing stress-specific outputs from a regulatory hub - Hog1 in Candida albicans

Cells exist in dynamic environments and must constantly tune their physiology to ever changing environmental conditions. Cells perceive many of these challenges as environmental stresses. They first detect these environmental stresses, and then transduce these signals via biological circuitry that culminates in the activation of the appropriate cellular response and ultimately cellular adaptation. Key components of this biological circuitry are evolutionarily conserved from yeasts to humans. They include proteins called stress-activated protein kinases (SAPKs) which become chemically activated (phosphorylated) in response to a stress, and subsequently chemically activate (phosphorylate) their targets, thereby leading to the activation of the cellular response.

These SAPKs are central to stress adaptation, responding to many types of stress and triggering the appropriate cellular response. This is the case in human cells (e.g. for the p38 SAPK) and in the pathogenic yeast Candida albicans (e.g. for the orthologous Hog1 SAPK). The interesting conundrum is how a SAPK is able act as a regulatory hub, distinguishing different input signals and then activating the appropriate stress-specific cellular response. What specifies the different inputs, allowing the programming of the differential outputs? We address this conundrum in this project, by dissecting the Hog1 SAPK in the major fungal pathogen of humans, Candida albicans.

Recently we discovered that the Candida albicans Hog1 SAPK is subjected to additional chemical modifications and that these newly discovered modifications occur in a stress-specific manner. Therefore, we hypothesize that these stress-specific modifications provide the molecular code that programmes the accurate regulation of specific downstream cellular outputs. In this project we will exploit a powerful combination of high throughput genomic technologies and precision molecular tools to test this hypothesis and dissect the molecular activation and function of this Hog1 SAPK.

This is important because it will increase our understanding of stress signalling in eukaryotic cells. Furthermore it is important because this stress signalling promotes the ability of Candida albicans to cause infection. Most individuals carry Candida in their microflora where it causes minimal damage in most healthy individuals. However this yeast is a frequent cause of "superficial infections" (thrush) in individuals when host defences become disturbed, and in intensive care patients it can cause life-threatening system-wide infections of the bloodstream and internal organs, over 40% of which are fatal. A limited repertoire of antifungal drugs is available, and clinicians require more effective therapies to help their patients. Research efforts to develop such therapies are enhanced by a greater understanding of the pathobiology of Candida, revealing potential weaknesses that can be targeted by these therapies. This is why the company Novabiotics is interested in the output of this project. By revealing how the Hog1 SAPK works, there is an excellent chance that we will uncover how Candida resists host defences and antifungal drugs, and how this resistance can be circumvented.

Stress-activated protein kinase (SAPK) pathways are important stress signalling modules found in all eukaryotic cells. These pathways comprise of a protein kinase cascade that activates the SAPK by phosphorylation at a conserved TGY motif. However, TGY phosphorylation alone is not sufficient to explain how SAPKs programme stress-specific outputs in response to diverse stress inputs. The Hog1 SAPK is critical for the adaptation of the pathogenic fungus Candida albicans to a diverse range of stresses, and is essential for its virulence. Notably, in addition to TGY phosphorylation, we have found that C. albicans Hog1 is subjected to three further posttranslational modifications in a stress-specific fashion: (a) phosphorylation at T179, (b) oxidation, and (c) S-nitrosylation. Our working hypothesis is that these stress-specific SAPK modifications modulate the stress-specific outputs of Hog1. In this project we will test this, firstly by establishing the impact of blocking these modifications upon the ability of Hog1 to drive stress-specific adaptation in C. albicans. We will then perform complementary interaction, expression and chemogenetic screens to define the Hog1 interactome under different stress conditions. This will allow us to test our prediction that Hog1 phosphorylation, oxidation and S-nitrosylation programme stress-dependent interactions. In addition, the global characterisation of environmentally contingent outputs of the Hog1 interactome will facilitate the identification of those components vital in mediating stress-specific downstream responses. Finally we will establish the impact of the novel posttranslational modifications of Hog1, and the Hog1-dependent stress-specific responses identified in this study, on C. albicans-host interactions and disease progression using well established tools and infection models.

To achieve the maximum impact for our project we will:
1. Ensure that our scientific observations and datasets are disseminated effectively across the academic community
2. Protect and exploit commercially valuable intellectual property that arises during the course of the project.
3. Contribute to public outreach programmes with a view to enhancing the public understanding of science
4. Further enhance our collaborations in the UK and abroad.
5. Provide an excellent training for our PDRAs in medical mycology, molecular biology, genomics and infection biology.

ACADEMIC DISSEMINATION: We will ensure that our work is disseminated broadly across the academic community through publication in leading journals, presentations at the top international conferences in our field, collaboration with international colleagues, the release of datasets through recognised public repositories and our personal websites, 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 biological question of relevance to human health and we will protect and exploit potentially valuable findings. The relevance to human health lies at two levels.
A. We will establish how the Hog1 SAPK regulates key fitness attributes in a major pathogen of humans. 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 the most prevalent cause of life-threatening systemic fungal infections in immunocompromised patients. Candida is the fourth leading cause of hospital-acquired bloodstream infections with a mortality rate above 40%, exceeding that of all Gram-negative bacterial septicaemias. Furthermore, systemic fungal infections significantly extend the average length of stay of intensive care patients, having a major impact upon medical care budgets (~1000 Euros per day per infected patient). Efforts to extend and improve the efficacy of antifungal therapies depend on a better understanding of fungal pathobiology. This project directly addresses this need because stress adaptation is critical for fungal virulence and disease progression, and our project is likely to provide information that leads to improvements in antifungal therapies. This view is reinforced by the support from NovoBiotics, an SME developing novel antimicrobial peptides. Our work will reveal mechanisms by which Hog1-related mechanisms promote the resistance of Candida to these peptides, thereby allowing Novabiotics to develop approaches that circumvent these resistance mechanisms. This principle can be extended to other antifungal therapies because Hog1 also promotes resistance to other types of antifungal drug.
B. SAPKs are highly conserved and the mechanisms we are examining in C. albicans are conserved in human cells. Hence, our findings will be translatable to human systems where the SAPKs p38 and JNK1 regulate cytokine responses, T cell proliferation, apoptosis and differentiation as well as stress adaptation. All of these processes underpin human health and represent attractive therapeutic targets.

PUBLIC OUTREACH: We will continue our contributions to local and national public outreach programmes as detailed in the Pathways to Impact.

COLLABORATIONS: This project involves the establishment of a new international collaboration (with Morten Grotli, University of Gothenburg) which provides access to novel molecules that facilitate a chemogenomic screen for Hog1 interactors. This new collaboration strengthens our network of eminent international collaborators.

TRAINING: We will provide an excellent research training in fungal genomics, molecular and cell biology and infection biology for our PDRAs. Also, we will enhance their career prospects by providing training in transferable skills and networking.