The C. albicans virulence factor Candidalysin polymerizes in solution to form membrane pores and damage epithelial cells.
The pathogenic fungus Candida albicans causes severe invasive candidiasis. C. albicans infection requires the action of the virulence factor Candidalysin (CL), which damages the plasma membrane of the target human cells. However, the molecular mechanism that CL uses to permeabilize membranes is poorly understood. We employed complementary biophysical, modeling, microscopy, and cell biology methods to reveal that CL forms membrane pores using a unique molecular mechanism. Unexpectedly, it was observed that CL readily assembles into linear polymers in solution. The basic structural unit in polymer formation is a CL 8-mer, which is sequentially added into a string configuration. Finally, the linear polymers can close into a loop. Our data indicate that CL loops spontaneously insert into the membrane to become membrane pores. We identified a CL mutation (G4W) that inhibited the formation of polymers in solution and prevented formation of pores in different synthetic lipid membranes systems. Studies in epithelial cells showed that G4W CL failed to activate the danger response signaling pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of thousands of CL pores by atomic force microscopy revealed the co-existence of simple depressions and complex pores decorated with protrusions. Imaging and modeling indicate that the two types of pores are formed by CL molecules assembled into alternate orientations. We propose that this structural rearrangement represents a maturation mechanism that might stabilize pore formation to achieve more robust cellular damage. Taken together, the data show that CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution directly leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.