β-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal β-1,3-glucans. Ac specificallyanchors curdlan or laminarin, indicating the presence of surface β-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a β-1,3-glucan outer layer, showed significantadhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble β-1,3-glucan substantially inhibited this adhesion, indicating the involvement of β-1,3-glucan recognition. Biotinylated β-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the β-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identifiedseveral Ac proteins capable of binding β-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identifiedproteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinityto β-1,3-glucans. These findingsunderscore the complexity of binding via β-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.