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  1. Evaluation Summary:

    This manuscript is of interest to several fields, in particular to microbiologists and structural biologists interested in pore-forming proteins and peptides. The data presented reveal insights into the mode of action of a newly identified peptide toxin secreted by Candida albicans (candidalysin). Using different techniques the authors propose and test a model for membrane perforation by candidalysin and identify an intriguing inactive mutant. While the presented data supports the main conclusions of the paper some of the initial assumptions need further assessment while the described mutants could benefit from more extensive characterization.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Candilysin (CL) is a virulent factor in infections by the fungus Candida albicans.
    This manuscript aims to determine the mechanism(s) by which CL interacts with and permeabilizes the membranes of host cells.

    The strengths of this manuscript are that it uses a comprehensive biophysical toolkit to address this question, and it interest also lies in revealing a previously unknown mode of membrane permeabilization by toxins, in that CL is shown to oligomerize in solution and that these oligomers next define membrane permeabilisation.

    Apart from a few minor weaknesses that can be addressed by clarification, the main weakness is that it is unclear - in the authors' interpretation - what defines the size of linear oligomers/polymers that are presumed to next join on the membrane, and if this size is a robust aspect of CL or a more arbitrary result related to the specific experimental conditions covered in this manuscript.

    Overall, the manuscript convincingly demonstrates the formation/presence of CL oligomers in solution and that these oligomers play a role in membrane permeation. Their remain some question marks, however, about the authors' conclusion exactly how this permeation occurs.

    This is an exciting paper in terms of suggesting a new biophysical mechanism of membranes pore formation that is relevant in the context of fungal infections.

  3. Reviewer #2 (Public Review):

    The authors investigated the mechanism of a fungal cytolytic peptide (candidalysin) produced by the opportunistic pathogen Candida albicans. Using different techniques, the authors show that candidalysin is able to form stable oligomers which are then able to spontaneously polymerize and insert in membranes. Imaging the preformed oligomers using electron microscopy and atomic force microscopy the authors propose the peptide can form polymers in two different orientations. In addition, the authors identify a single point mutation that prevents polymer formation and is inactive on epithelial cells.

    The authors convincingly show that synthetic candidalysin assembles in solution into oligomers of different sizes using mass spectrometry, analytical ultracentrifugation and mass photometry. Following this initial oligomerization candidalysin further assembles into long linear polymers. While the oligomerization data is quite convincing the assumption that the oligomer is an 8mer is less convincing and would require further investigation. However, the stoichiometry of this initial building block does not impact the main conclusions of the paper.

    Using electron microscopy and atomic force microscopy the authors image the polymers and characterize their structure. AFM was used to measure volume increase which led to the conclusion that polymerization occurs stepwise by the addition of a basic structural unit. Visualization of the polymers by AFM on supported lipid bilayers identify two different conformations. While the AFM can show pores that perturb the membrane it is unable to visualize bound linear polymers or polymers embedded in the membrane that do not perforate. The author used detergent solubilization to reveal polymers formed however more controls are needed to show that the structures seen are not affected by the detergent solubilization.

    Mutagenesis of residues on one side of the peptide led to the identification of an inactive mutant G4W. The data clearly shows that the mutant is inactive and unable to trigger any response when used on cells however further characterization of the mutants is lacking which again could strengthen the conclusions presented.

    The main conclusions of the work, that candidalysin requires spontaneous oligomerization and further polymerization in order to assemble and disrupt membranes. The use of AFM and EM is clear and convincing. Identification of an inactive mutant can further our understanding of the different steps towards membrane disruption.

    The initial assumption that the main building block is an α-helical octamer. While the methods used by the authors clearly show that a building block oligomer is required for oligomerization the nature of the building block is less clear. Further experiments would be needed to confirm the stoichiometry before a model can be built. The identified mutants are not clearly presented in the text which makes their interpretation difficult.

    This work clearly advances our understanding of the mechanism of membrane disruption by candidalysin. In addition, if the inactive mutant that was identified is blocked from polymerization it could be of great interest to structural biologists in order to elucidate the structure of the oligomeric building block.

  4. Reviewer #3 (Public Review):

    This paper represents an important contribution to the field of pore-forming toxins, in particular peptide toxins. In nature, several families of pore-forming proteins are known, each of them showing the unique structure of monomeric units as well as a unique mechanism of pore formation that usually involves significant conformational changes in molecules upon membrane insertion. The mechanism proposed in this study is completely different from the ones that we know to date. The authors use an array of complementary biophysical approaches as well as simulations to show this. It also nicely shows the capability of methodological approaches such as AFM or CD, beyond the modules used on average in other studies. This adds strength to the topic of the paper as well as gives the reader ideas on how these approaches could be used in their studies as well (educational aspect).

    In terms of weakness, there are some details that should be better addressed. For example, the model of the CL peptide is only poorly presented, the orientation of N and C termini is not proposed on figures describing polymers of 8-mer, which is important. A hypothesis on how flipping from unrimmed to rimmed pore happens would be welcome. The lipid specificity is not addressed, and in fact, experiments with different approaches are in some cases done with different lipids, with no explanation why. The discussion is rather weak. The authors compare this novel mechanism of pore formation to other families of pore-forming proteins, but there seems to be some lack of insight in other mechanisms of action (and structures of soluble proteins and their respective pores). However, I believe that this study importantly paves the path to further more detailed studies of CL pore-forming mechanism.