Candidalysins: Mechanistic function of a novel family of fungal peptide toxins

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. Cytolytic proteins and peptide toxins are critical virulence factors of bacterial pathogens and play a major role in bacterial disease. Until very recently, human pathogenic fungi were not known to possess such toxins. The human fungal pathogen Candida albicans, the causative agent of thrush, causes millions of infections annually in people worldwide. An explanation for precisely how this fungus is able to cause mucosal disease has remained at the forefront of medical mycology and immunology for several decades. We have recently identified a secreted peptide toxin (Candidalysin) as an essential factor required for C. albicans infection at mucosal surfaces. This is the first cytolytic peptide toxin to be discovered in any human pathogenic fungus. Our work has shown that Candidalysin interacts with the membrane of epithelial cells, causing membrane damage, inflammation and destruction of host tissues. Production of this toxin by C. albicans is now understood to be a critically important step that facilitates mucosal infection and disease progression. Importantly, we have now identified similar Candidalysin toxins in the related fungal species C. dubliniensis and C. tropicalis, which are also pathogenic. Together with C. albicans, these fungi represent an immense health burden on the global population. The discovery of three Candidalysins identifies these toxins as a conserved family used by pathogenic Candida species to cause disease. Currently, there are no vaccines for Candida infections and the incidence of fungal pathogens acquiring resistance to commonly prescribed antifungal drugs is rising. Given their essential role in mucosal infection, understanding how this family of fungal toxins damage mucosal surfaces is critically important, and will pave the way towards therapeutic intervention that enables us to control and prevent disease progression. This proposal aims to identify exactly how Candidalysins interact with epithelial cell membranes to cause damage and infection. The findings of this work will be pivotal to our future understanding of fungal pathogenesis and will enable us to develop new strategies to engage with fungal infections to control their impact on the human population. This project will identify the Candidalysin family as 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.

The ability of microbes to cause damage to the human host is closely associated with infection and disease progression. We have recently identified a novel family of secreted peptide toxins instrumental to the pathogenicity of Candida albicans (C. albicans), C. dubliniensis and C. tropicalis, three major life-threatening fungal pathogens of humans. This proposal will (i) define the mechanism of Candidalysin activity on epithelial cells and artificial membranes using a combination of biophysical, electrophysiological and phenotypic analyses, (ii) determine the influence of membrane phospholipids on Candidalysin activity using phospholipid arrays and quantifying the permeabilisation of artificial DOPC membranes containing different combinations of phospholipids, (iii) identify essential regions required for Candidalysin activity by comparing the activity of a panel of mutant Candidalysin toxins with their respective wild type controls, and (iv) define the contribution of Candidalysins and hyphal maintenance to epithelial activation in vitro and fungal pathogenesis in vivo using a series of C. albicans knock-in and Candidalysin-negative mutants. This project will provide essential new understanding to basic fungal biology and the mechanisms that underpin Candidalysin-induced epithelial damage. It will also identify Candidalysin as a new target for the development of new drugs, adjuvants, vaccines, 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 ~75 million infections/year. Furthermore, 50% of HIV+ patients suffer from oral candidiasis, equating to ~2 million cases/year. 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. 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 (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. As such, our landmark discovery of the Candidalysins represents a seminal advance in our understanding of fungal infection. A major long-term impact goal of this project is commercial exploitability of our data. The Candidalysins and the epithelial components this novel family of toxins interacts with can be targeted for the development of more effective preventative and antifungal treatments. For example, Candidalysin 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, Candidalysin lacking the C-terminal KR motif 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 Candidalysin 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 Candidalysin 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 Candidalysin as an identifiable 'risk factor' of pathogenesis and infection, and thus when antifungal treatment is warranted. Finally, the identification of the first cytolytic toxin family in Candida has opened the door for a new field of research in toxin discovery in medically important fungi, with broad implications for elucidating multiple pathogen infection mechanisms across the fungal kingdom. Also, given that Candidalysin may act similar to peptide toxins such as melittin (honey bee) and magainin 2 (African frog), this work may provide broader insights into the basic biology underpinning toxin activity in a range of different organisms. 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.