Review 2: "A Synthetic Peptide Mimic Kills Candida Albicans and Synergistically Prevents Infection"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review:

The manuscript entitled: “A synthetic peptide mimic kills Candida albicans and synergistically prevents infection” focuses on the combinatorial effect of new peptide polymers and existing anti-fungals.

The authors synthesized four ternary polyacrylamides and examined their antifungal nature against the fungus Candida albicans. Nuclear magnetic resonance and refractive index-based size exclusion chromatography are applied to examine the synthesis and purification of these polymers. They could show that polymer LH (linear, heptyl) is the most effective, and C. albicans antifungal drug-resistant strains are susceptible to this particular polymer. By performing transcriptome profiling with four polymers, they suggest that LH polymer targets protein glycosylation and the fungal membrane, which differs from the other three polymers and the existing drug’s mode of action. The membranolytic potential of polymer LH is determined with C. albicans strain expressing GFP in the cytosol, and the authors could reveal the membranolytic activity of polymer LH by GFP signal loss. Further, using transmission electron microscopy (TEM) they could show a disrupted arrangement of the N-mannan fibrils in the presence of a sub-inhibitory concentration of polymer LH. Their transcriptome analyses also indicated this. Based on these results, they suggest disruption of the mannan layer and cell wall modifications by LH, and these alterations permit macrophage phagocytosis. Further, the treatment of polymer LH reduces the hyphal length and diameter of microcolonies under hypha-inducing conditions. In vitro experiments with the human epithelial cell model revealed that polymer LH alone could not prevent damage to the vaginal epithelial cell; however, the polymer LH is more effective in combination with caspofungin or fluconazole. Using the Galleria in vivo model, they could show a synergistic effect of LH and caspofungin. Finally, the authors conducted in vitro evolution experiment to determine the contribution of LH alone or in combination with existing drugs to resistance development.

Overall, the manuscript presents a nice piece of work and is supported by desired experiments; however, I have a few comments listed below:

1. Transcriptome data is not validated by quantitative methods. At least validation of a few genes related to protein glycosylation or membrane would support transcriptome data.

2. The authors performed a transcriptome study with four polymers. However, the most striking result is obtained with the combination of LH and the existing antifungals. A transcriptome study on the synergistic effect of LH and existing antifungals would give some link to understanding their mode of action. At this stage, it is unclear how the activity of combined drugs (LH + existing drugs) is achieved.

3. Based on the transcriptomic data and TEM, the authors suggest a reduction in the mannan layer; however, mannan quantification is not shown.

In conclusion, this manuscript opens a new direction to use combinations of anti-fungals to treat Candida infection, and thus, it provides a new therapeutic approach.