BPS2026 – Elucidating the role of terminal charges in polymerization of the C. albicans virulence factor candidalysin via atomic force microscopy

Candida albicans is a common fungus that causes a range of maladies from minor epithelial infections to life-threatening invasive candidiasis. The fungus secretes candidalysin (CL), a peptide that causes cell damage and immune activation by permeation of epithelial membranes. The mechanism of CL host cell attack involves peptide assembly into polymers in solution. We examined the assembly mechanism of CL polymers in the absence and presence of model membranes using atomic force microscopy (AFM) in conjunction with other methods. AFM single-molecule imaging showed that CL polymerization involves a convolution of four processes. Self-assembly begins with the formation of a basic subunit, thought to be a barrel-like CL 8-mer that acts as the CL polymer seed. Polymerization proceeds via addition of subunits, and as polymers grow, they can curve and form loops which can further go on to insert into membrane and become pores. Alternatively, secondary polymerization can occur and cause branching or other higher order complex particles, which are also likely to cause membrane leakage upon binding to the bilayer. Interplay between the different rates determines the distribution of CL particle types, indicating a kinetic control mechanism. To elucidate the nature of the self-assembly mechanism, we have selectively altered the free charges residing at each terminal of the CL monomer via N-terminal acetylation (ac-CL) and/or C-terminal amidation (CL-am). Results from AFM analyses are compared with other techniques including mass photometry. Consistent with prior studies altering the Debye length in the solution for the free-terminal construct, our results indicate that electrostatics play a major role in CL looping and subsequent pore formation.