Environmental sensitivity of amyloidogenic motifs in fungal NOD-like receptor-mediated immunity: Molecular and structural insights into amyloid assembly

This study investigates the aggregation behavior of amyloidogenic motifs associated with fungal NOD-like receptor (NLR) proteins, focusing on their sensitivity to various environmental conditions. We aimed to develop a minimal model that explains amyloid aggregation, aligning with in vivo observations and the expected role of these motifs in amyloid-based signaling. The purpose was to understand how changes in physicochemical conditions influence amyloid formation, which is crucial for fungal immune responses and has potential applications in controlling fungal infections. To achieve this, two amyloidogenic motifs, PUASM_N and PUASM_C, derived from the fungus Colletotrichum gloeosporioides, were synthesized and subjected to different conditions that simulate their natural environment. These conditions included varying pH levels, peptide concentrations, and surface adsorption properties. The aggregation kinetics, morphology, and secondary structures of the peptides were analyzed using Thioflavin T (ThT) fluorescence assay, transmission electron microscopy (TEM), and Fourier transform infrared micro-spectroscopy (micro-FTIR). The results showed that PUASM_N aggregates rapidly without a lag phase, forming long, structured fibers. In contrast, PUASM_C aggregates more slowly, with a significant lag phase, forming shorter, irregular fibers. The aggregation of PUASM_C was highly sensitive to environmental factors, such as alkaline pH and surface hydrophobicity, which accelerated its aggregation. PUASM_N, however, displayed consistent aggregation behavior under different conditions. Our findings suggest that minor environmental changes can modulate the functional roles of PUASM peptides, potentially aiding Colletotrichum gloeosporioides in regulating its antipathogenic activity in response to environmental challenges.