Candidalysin is a fungal peptide toxin secreted by Candida albicans during epithelial invasion, where it contributes to host membrane disruption and tissue damage (Moyes et al., 2016). CaL is generated from the polyprotein precursor Ece1, which contains the CaL precursor peptide alongside seven additional non-candidalysin peptides (NCEPs). Proper proteolytic processing of Ece1 and the extracellular release of CaL are essential for its pathogenic activity (Richardson et al., 2018; Mueller et al., 2024). In this study, we applied fluorescence correlation spectroscopy (FCS) as a sensitive tool to characterize both the behavior of fluorescently labeled candidalysin and its impact on lipid bilayers. FCS allowed us to quantify diffusion dynamics at nanomolar concentrations, providing insight into toxin binding, accumulation, and aggregation at the membrane interface. Using Alexa Fluor 647-labeled CaL, we compared diffusion in aqueous solution versus supported lipid bilayers (SLBs). In parallel, we examined how NCEPs influence CaL self-assembly by pre-incubating the peptides prior to FCS measurements. Importantly, we extended this approach to probe lipid mobility directly. By monitoring the diffusion of fluorescent lipid analogs in SLBs, we determined how CaL incorporation alters bilayer dynamics. These experiments revealed that toxin binding not only changes its own diffusion profile but also measurably changes lipid mobility, highlighting membrane reorganization as a key step in the pathogenic mechanism. Together, our results demonstrate how FCS can dissect the interplay between candidalysin and lipid bilayers at the molecular level, providing new perspectives on how fungal peptide toxins compromise epithelial membrane integrity.
Zhurgenbayeva et al. (Sun,) studied this question.