Title: The unexpected consequences of antifungals on innate immune interactions
Funder: The Academy of Medical Sciences

Abstract: Drug-resistant fungi are an emerging clinical problem whose interactions with the immune system are poorly understood. Candida glabrata is a facultative intracellular emerging fungal pathogen that is responsible for ~24% of life-threatening candidiasis cases in the UK. C. glabrata is intrinsically resistant to azole antifungals, such as fluconazole. However, little is known regarding how antifungals influence C. glabrata host-pathogen interactions and innate immune evasion. Furthermore, we have very limited high-throughput tools to study intracellular fungal-host interactions. Therefore, I will leverage my years of expertise in fungal-host interactions to address this problem. Specifically, I will determine the mechanisms driving immune evasion in a drug-resistant fungus and establish a crucial high-throughput platform to evaluate intracellular pathogenesis that facilitates future drug development applications. The fungal cell wall is the first point of contact with the immune system, with several epitopes that influence immune recognition and fungal killing. I have shown that fluconazole treatment promotes epitope remodelling and enhances C. glabrata survival in macrophages. Thus, this proposal will establish how C. glabrata antifungal resistance impacts immune evasion and pathogenesis. I will use my expertise in epitope exposure profiling and macrophage challenge assays to establish how antifungals influence innate immune recognition and fungal killing. I will also establish an imaging cytometry method to address the need for a robust tool to investigate intracellular virulence and identify novel therapeutics. The data and tools developed by this proposal will support MRC and Wellcome Trust Investigator applications and generate impact through academic and drug discovery collaborations.

Lay abstract: Fungal infections are responsible for >1 million deaths worldwide annually. Drug resistant and multi-drug resistant infections are progressively emerging, which is concerning given that we only have four antifungal drug classes for treatment. Candida glabrata is a major fungal pathogen in the USA, Europe, and Asia that is resistant to azole antifungals, a commonly used frontline therapeutic. The immune system plays a critical role in controlling fungal infections. Immune cells recognise specific patterns, or epitopes, in the fungal cell wall, which activate fungal killing mechanisms. I have found that treatment of C. glabrata with fluconazole, a first-generation azole, changes cell wall epitope exposure and unexpectedly leads to enhanced survival of the fungus in immune cells. However, we do not know how other antifungals affect C. glabrata recognition and killing by immune cells. Thus, my research will establish how antifungals, especially azoles, affect C. glabrata interactions with immune cells. These data will provide an understanding of how azole resistance has unexpected consequences for fundamental aspects of fungal infection. Finally, we have limited tools to conduct the necessary studies to identify new drugs for fungal intracellular pathogens. Therefore, I will establish a high-throughput imaging flow cytometry method to address this critical, unmet need. This method will support future research on fungal diseases and facilitate new drug discovery applications for emerging and drug-resistant fungal pathogens.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION