Final Report Summary - FUNGISORT (Sorting out host recognition of fungal infection)
The fungus Candida albicans is a common cause of human infections with serious morbidity, but is also a commensal that is part of the normal intestinal microbiota. Innate immune cells must tolerate commensal colonization of C. albicans, yet deal effectively with opportunistic mucosal and systemic invasion. The interplay between the immune system and fungi occurs predominantly at the level of the fungal cell wall, where fungal pathogen-associated molecular patterns (PAMPs) are recognized by Pattern Recognition Receptors (PRRs) of innate immune cells. The cell wall composition of virulent C. albicans hyphae differs from C. albicans yeasts, resulting in differential immune recognition and signalling. The goal of the FungiSort project was to study PRR interactions with fungal PAMPs and to develop new tools that can be applied to determine commensal and pathogenic states of C. albicans. Sortagging is a site-specific protein-labeling technique that can be used for the flexible attachment of affinity handles like biotin or fluorophores. The sortagging technique can be used to label purified proteins or to perform immunoprecipitation experiments with proteins expressed on the surface of living cells. For the FungiSort project, the sortagging technique was introduced in the host laboratory and applied to the C-type lectin receptor DC-SIGN that plays an important role in innate immune recognition of C. albicans. The carbohydrate recognition domain (CRD) of DC-SIGN was expressed with a sortase tag and labeled with biotin of TAMRA and used in pull down and confocal microscopy experiments, respectively. A stable THP1 macrophage cell line expressing DC-SIGN with a sortase tag was used for live cell sortagging and phagocytosis experiments. We identified glycoproteins that are secreted by C. albicans and that interact with DC-SIGN. We are currently investigating if these secreted glycoproteins occur in vivo and whether they have DC-SIGN-mediated immunomodulatory functions. The results of the FungiSort project contribute to our understanding of innate immune recognition of fungal infections and provide new avenues for the development of new diagnostic tools to distinguish commensal and pathogenic fungi.