Mechanism of EGFR activation by a novel fungal pore-forming toxin

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. This microbial-epithelial encounter results in either quiescence in the case of harmless 'commensal' microbes, or activation of immunity and damage protection mechanisms in the case of disease-causing 'pathogenic' microbes. When these normal host responses are disrupted, a plethora of complications can arise that have implications beyond infection, as conditions including cancer and autoimmune disease are associated with abnormal interactions between a microbe and its host. 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 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 now the third most common hospital-acquired bloodstream infection and are a serious burden to healthcare provision. Given that the vast majority of C. albicans infections are acquired through mucosal surfaces, it is of paramount importance to understand how epithelial tissues at these surfaces detect and restrict this medically-important pathogen to the commensal state. Identifying these mechanisms is of considerable value not only for fungal infections but also for controlling other microbes at mucosal surfaces.

Our recent work has been instrumental in understanding the mechanisms by which epithelial cells identify disease-causing C. albicans and how this causes damage induction and immune activation in the host. We previously showed that C. albicans infection leads to epithelial damage and the secretion of immune activators to induce local mucosal immunity to protect against C. albicans infection. Recently, we found that to activate this protective immune process, epithelial cells target a specific protein (Ece1p) on the 'invasive' form of C. albicans known as hyphae. We now demonstrate that a specific region of the Ece1p protein (named Ece1-III) acts as a novel pore-forming toxin (PFT) when present at high concentration. This is the first PFT identified in a human pathogenic fungus. The pore forming abilities of Ece1-III enable C. albicans to cause epithelial damage and are recognised by epithelial cells to activate immunity. Furthermore, we show that this newly identified fungal PFT activates a specific epithelial surface receptor (EGFR), which induces epithelial immunity and the protective responses that protect against fungal-induced damage. We have demonstrated that immune activation occurs as a result of direct interaction between Ece1-III and EGFR. However, we found that Ece1-III is also able to activate EGFR indirectly through a complex series of events that take place at the epithelial cell surface as part of a response to cell damage. The nature of these surface events leading indirectly to EGFR activation is unknown, although pore formation seems to be essential for their activation. Given that EGFR is the key receptor mediating both epithelial immunity and damage protection, the aim of this proposal is to determine precisely how pore-formation by this novel fungal PFT indirectly activates EGFR and to demonstrate the importance of these activation events during C. albicans mucosal infection in vivo.

This project will provide new insights into the mechanism of action of fungal PFTs and to our understanding of epithelial tissue responses to 'pathogenic' fungi during mucosal immunity. Further, it will identify novel host-microbe interactions that can be targeted not only for new, more effective antifungal therapies, but also for defining innovative approaches to combat the action of bacterial PFTs and mucosal infections in general.

The ability of mucosal tissues to discriminate between commensal and pathogenic microbes is essential to human health. Recently, we have identified several epithelial signalling mechanisms instrumental in identifying the commensal-pathogen switch of Candida albicans, a major life-threatening fungal pathogen of humans. These activation mechanisms target the hyphal form of the fungus, resulting in the induction of epithelial immunity and protection against damage. We have now identified a C. albicans hyphal protein (Ece1p) and an internal 32 aa sequence (Ece1-III) as a novel pore-forming toxin (PFT), which induces cellular damage and activates epithelial immunity. Furthermore, we have identified EGFR as the target receptor for this newly discovered fungal PFT and provide evidence that this PFT-EGFR interaction event appears critical in the epithelial recognition of the commensal-pathogen switch of C. albicans. We demonstrate that C. albicans Ece1-III can activate EGFR directly and indirectly. Direct activation is independent of pore formation and occurs via direct binding of Ece1-III to EGFR. Indirect EGFR activation is dependent on pore formation but is the result of a complex series of surface-mediated signalling events. Given that EGFR is a key receptor mediating both epithelial immunity and damage protection during C. albicans infection, we aim to identify precisely how this novel fungal PFT indirectly activates EGFR. Murine experiments will also be performed as a basis for future studies to demonstrate that the Ece1-III-mediated EGFR activation mechanism that we have demonstrated in vitro has a bearing on fungal infection and mucosal protection in vivo. This project will provide substantial new insights to our understanding of fungal PFTs, epithelial-microbial interactions, and will also identify new microbial and epithelial targets for the development of new drugs, immune-based treatments, vaccines and adjuvants to prevent mucosal diseases.

Mucosal diseases continue to be major causes of death, suffering and economic loss and have serious impacts on the welfare of global communities. The impact of mucosal-acquired 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 ~30 million infections/year (4x more than tuberculosis and 8x more than HIV: WHO). Candida infections are also the third most common hospital-acquired bloodstream infection 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 200,000 deaths/year. Furthermore, 50% of HIV+ and 90% of AIDS patients suffer from oral candidiasis, equating to ~2 million cases/year. Yearly healthcare costs in the USA for fungal infections are ~$3 billion, of which Candida infections account for $2 billion. EU healthcare costs are estimated to be similar. Therefore, Candida pathogens and C. albicans in particular (which 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 host and 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, the epithelial receptor (EGFR), the signalling circuits it activates and C. albicans Ece1p/Ece1-III can all be targeted for the development of more effective immune-based antifungal treatments and for future therapies to combat mucosal infections in general. For example, Ece1-III possesses unique properties that impart a dual role for exploitation; firstly as a novel vaccine target (given its role in pathogenesis) to induce protective mucosal immunity against C. albicans infections and, secondly, as a novel mucosal adjuvant given its ability to activate epithelial cells and mucosal immunity. Furthermore, EGFR and its signalling circuits can be exploited to develop strategies to stimulate protective immunity against a multitude of mucosal pathogens. The project will also impact on the diagnostics industry, since Ece1p 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 Ece1-III as an identifiable 'risk factor' of pathogenesis and infection. In summary, this project will have an 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, as well as 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 healthcare in the UK as well as human health and quality of life on a global scale.