Single-molecule visualization of candidalysin self-assembly reveals kinetics and interactions behind peptide polymerization

Candidalysin (CL) is a fungal virulence factor secreted by Candida albicans which acts as a mammalian cell-damaging peptide toxin. Previously, we have shown the damaging mechanism relies on spontaneous assembly of CL into complexes in the extracellular space prior to membrane binding and pore formation. Of particular interest are the loops formed by CL polymers, which may be a critical component of pore structure. Using a suite of biophysical techniques, we performed studies of CL assembly rates and probed the interactions that guide CL oligomerization and polymerization. Single-molecule visualization of CL complexes was a crucial factor in understanding the assembly mechanism. To this end, we utilized atomic force microscopy (AFM), a technique capable of direct visualization of CL assemblies in physiologically relevant temperatures and buffer solution. Using AFM, we differentiated the assembly rates of CL particle types, such as linear polymers, loops, and larger complexes with secondary polymerization sites. Additionally, CL interactions were probed under varying conditions, including concentration, temperature, and ionic strength. AFM results were supported by mass photometry and other biochemical techniques. Taken together, these data provide insight into the novel mechanism of this fungal peptide toxin and pave the way for therapeutic development.