Biological function of Candida albicans Ece1 and role in host-microbe interactions

The mucosal (oral, vaginal, gastrointestinal) epithelium is of immense importance in protecting humans against a multitude of infections as it is the initial tissue encountered by the majority of microbes. In health, this microbial-epithelial encounter results in either quiescence in the case of harmless 'commensal' microbes or activation of immunity in the case of disease-causing 'pathogenic' microbes. When these normal responses are disrupted a plethora of complications arise, which have implications beyond infection as conditions including cancer and autoimmune disease are associated with abnormal interactions between host and microbe. Given the importance of these interactions, epithelial cells possess distinct mechanisms enabling mucosal tissues to distinguish between commensal and pathogenic microbes.

The fungus Candida albicans (C.alb) is an example of a microbe that can exist as a commensal in healthy people but becomes a dangerous pathogen causing severe and potentially fatal disease in unhealthy people. Indeed, Candida infections are regularly found to be the third or fourth most common hospital-acquired bloodstream infection, causing death in ~40% of individuals, and are thus a serious burden to healthcare provision. Given that the vast majority of bloodstream C.alb infections are acquired through mucosal surfaces, it is of paramount importance to understand the mechanisms by which C.alb interacts with epithelial tissues in health and to identify the fungal factors that promote infection and disease when normal responses are disrupted. Presently, we have little knowledge of the fungal factors that promote mucosal infections.

Our recent work has been instrumental in understanding the mechanisms by which epithelial cells identify disease-causing C.alb and how this results in immune protection. We previously showed that C.alb infection leads to the secretion of immune activators (cytokines and chemokines) that recruit specific immune cells that protect against C.alb infection. Recently, we found that to activate this protective immune process epithelial cells recognise a specific protein (CaEce1p) on the filamentous, 'invasive' form of C.alb known as hyphae. CaEce1p is a unique hyphal protein consisting of eight repeated fragments of the same size that are individually processed by the enzyme CaKex2p. We provide strong evidence demonstrating that CaEce1p (or its individual fragments) is the critical fungal protein that informs the healthy host of the presence of a microbe that has become dangerous. This proposal aims to demonstrate the function of CaEce1p for C.alb, how CaEce1p is localised to the hyphal surface and, crucially, which fragments of CaEce1p activate epithelial cells. This will provide essential new understanding to basic fungal biology and the mechanisms that underpin C.alb-host interactions in health.

This project will identify CaEce1p as a new target not only for the development of new antifungal drugs but also for the development of new vaccines, adjuvants, diagnostic tests and biomarkers for fungal infections. Of additional interest is the structural similarity between CaEce1p and the hypha-specific Rep1 protein of Ustilago maydis, which is a pathogenic plant fungus that causes smut disease in corn. Thus, the functional data obtained with CaEce1p may also provide valuable groundwork for future studies investigating plant fungal pathogenesis.

The ability of a host organism to discriminate between commensal and pathogenic microbes is essential to health. Recently, we identified an epithelial signalling mechanism instrumental in identifying the commensal-pathogen switch of Candida albicans (C.alb) a major life-threatening fungal pathogen of humans. This activation mechanism targets the hyphal form of C.alb, resulting in induction of proinflammatory cytokines and subsequent protection against mucosal fungal infection. We have now identified the C.alb hyphal protein (CaEce1p) targeted for epithelial recognition of the commensal-pathogen switch. This proposal will (i) characterise the biological function of CaEce1p in C.alb using a combination of C.alb microarrays, bioinformatics and phenotypic analyses, (ii) determine the importance of CaKex2p for CaEce1p processing, localisation and function in C.alb using ECE1-GFP labelled C.alb strains and CaEce1p specific antibodies by fluorescence and electron microscopy, and (iii) ascertain the importance of CaEce1p processing and localisation for epithelial cell activation. This project will provide essential new understanding to basic fungal biology and the mechanisms that underpin C.alb-host interactions in health. It will also identify CaEce1p as a new target for the development of new drugs, vaccines, adjuvants, diagnostic tests and biomarkers for fungal infections.

Fungal infections continue to be major causes of death, suffering and economic loss and have serious impacts on the welfare of global communities. The impact of fungal infections on healthcare and economic expenditures is large and of growing concern. Candida species are the most common fungal pathogens of humans giving rise to severe morbidity in millions of individuals worldwide. Vaginal candidiasis affects ~75% of women during fertile age, equating to ~20 million infections/year (2x more than tuberculosis and 5x more than HIV: WHO 2007). Furthermore, 50% of HIV+ and 90% AIDS patients suffer from oral candidiasis, equating to ~2 million cases/year. Candida infections are also the third or fourth most common hospital-acquired bloodstream infection (depending on the study) and can thus be considered more medically-important than most bacterial infections including Enterococci and Pseudomonas spp. Systemic candidiasis usually results from translocation of Candida across the mucosae and is fatal (~40% mortality) equating to an estimated 100,000 deaths/year. Yearly healthcare costs in the USA for fungal infections are ~$2.6 billion, of which Candida infections account for $1.8 billion. EU healthcare costs are estimated to be similar. Therefore, Candida pathogens and C. albicans in particular (accounts for ~75% of Candida infections) carry an immense health burden and represent a serious socio-economic challenge for worldwide communities. Given this, identification of the fungal mechanisms that can be targeted to prevent or control disease is of high priority for global healthcare provision. Therefore, a major long term impact goal of this project is commercial exploitability of our data. As such, CaEce1p and the epithelial signalling circuits it activates can both be targeted for the development of more effective preventative and antifungal treatments. For example, CaEce1p possesses unique properties that impart a dual role for exploitation, both as a target for intervention (due to its role as a virulence factor) and in activating innate antifungal defences. Furthermore, CaEce1p may represent a novel class of mucosal adjuvant given its ability to activate epithelial cells and mucosal immunity without causing damage. In addition, the signalling circuits activated by CaEce1p interaction with epithelial cells can be exploited for immune-based strategies to combat fungal infections. The project will also impact on the diagnostics industry, since CaEce1p is the first fungal marker identified to date that enables the host to discriminate between commensal and pathogenic C. albicans. This could be exploited in new clinical diagnostic tests that would target CaEce1p as an identifiable 'risk factor' of pathogenesis and infection, and thus when antifungal treatment is warranted. Of additional importance, given the potential similarities between CaEce1p and the hypha-specific Rep1 protein of Ustilago maydis (a fungal pathogen of plants that causes smut disease in corn), any data obtained for CaEce1p interactions with host epithelial cells may also identify potential areas for investigation of U. maydis interactions with plant host cells. In summary, this project will have immediate impact on multiple academic fields but has significant potential in the longer term to impact on pharmaceutical and commercial industries with specific interests in developing new drugs, vaccines and adjuvants to prevent mucosal diseases and in designing new diagnostic tests and biomarkers for mucosal infections. This will have a substantial impact on clinical medicine and disease management, which will ultimately improve the economic competitiveness of the UK and improve human health and quality of life on a global scale.