Dear Professor Ljungdahl,

Thank you very much for submitting your manuscript "Proline catabolism is a key factor facilitating Candida albicans pathogenicity" for consideration at PLOS Pathogens. I reviewed the manuscript and read the three reviews that you had submitted. The reviewers appreciated the attention to an important topic and I feel that you have addressed their comments satisfactorily. However, after reading the manuscript closely, I have identified some additional issues that must be addressed. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the following recommendations.

1.  P5C should be defined in the abstract
2. C. parapsilosis seemed to grow almost as poorly as C. dubliniensis (Fig. 1A). This should be commented on. I would also soften the statements that virulence correlates with ability to utilize proline because C. dubliniensis and C. parapsilosis are virulent in humans.
3. Line 184. Define SGL
4. Fig 2E. Need statistical comparison between Am and each of the proline isomers/analogs to show which are significantly different from baseline, especially for the put3 mutant.  This is important because it is not clear whether there are any significant changes in Put2GFP expression in the put3 mutant (unlike what is stated in the text).
5. Fig. 3 and 4. The panels should be arranged so that they can be read from left to right and then top to bottom.  For example, panel B should be placed to the right of panel A in the same row.
6. Fig. 3E shows that the presence of proline causes the same drop in O2 consumption in the put1 cells as in the put2 cells. However, proline has a much greater inhibitory effect on the growth of the put2 mutant than the put1. These findings do not appear to support the conclusion that the growth inhibitory effect of proline in the put2 mutant is due to a block in respiration (lines 274-6). This conclusion also needs to be modified in the Discussion (586-590). Also, statistical comparisons between the different strains of C. albicans needs to performed and indicated for Fig. 3E.
7. Fig. 3G. If phloxine B is a measure of viability, one would expect that the colonies with phloxine B staining would be smaller that those that don’t stain because nonviable cells obviously don't proliferate. Why are the colonies of the put1 and put1/2 mutants larger than those of the WT cells, especially in YPD, even though they are phloxine B positive? This observation raises the concern that phloxine B staining may not be a good measure of viability.
8. Fig. 5 A and B. Need statistical analysis of the survival curves (using Kaplan Meier statistics).  Is the survival of flies/mice infected with the put1 mutant significantly different from the put2 mutant?  This is stated in the manuscript, but likely not supported by statistical analysis.
9. Fig. 5C. The organ fungal burden of mice infected with put1 and put2 mutants should be compared statistically to evaluate the possibility that the put1 mutant achieves lower organ fungal burden than the put2 mutant.
10. Lines 395-6. Should compare the median survival, not mean survival because median survival is less affected by outliers.
11. Fig. 5D. The magnification in the lower panels is not sufficient to see any organisms in the kidneys infected with the put1 and put2 mutants, and the possible filamentation defect of the put3 mutant is also not evident. The sections show mainly the renal pelvis. Were there visible lesions in the renal cortex? If so, how did they look?
12. The conclusion that “especially Put2” is require for virulence is not supported by the data because there were no statistically significant differences between put1 and put2 mutants in the in vivo infection models.  The in vitro models clearly did not mimic the in vivo models because deletion of PUT3 had a much greater effect in vitro than in vivo.
13. The extended methods section should be moved from the supplemental data section into the main text.
14. Fig. 6. While the in vivo imaging data are nice, a major weakness is that the labeled strains were auxotrophic (arg- his-). It is known that even though strains with amino acid auxotrophies can appear to filament normally in vivo, these auxotrophies can interact with other mutations (e.g., the put2 deletion), leading to filamentation defects that are not apparent in fully prototrophic strains. For this reason, these studies should be repeated with fully prototrophic strains. Also, it would aid in the interpretation of the results to show data with both the WT and put2 mutant, and optimally the put1 mutant, at the same time points. In panel E, only the WT stain is shown at 4 h. Results with the put mutants should also be shown.
15. The statement the glutamate and glutamine are not readily converted to proline in fungal cells (lines 537-540) should be softened because the authors did not show this (by measuring intracellular proline levels) and there are alternative explanations for why glutamate and glutamine did not induced PUT enzymes.
16. The discussion in lines 604-614 are based on the presumption that proline, arginine, and ornithine are the only stimuli that induce PUT3 expression. An alternative possibility is that other factors that are present in the microniche of the phagosome are able to induce PUT3 expression. Because the investigators did not measure the amino acid levels in the phagosome, the conclusions in this section should be be softened.
17. Line 636 is incorrect.  Hematogenously disseminated candidiasis is establishing a pyelonephritis (kidney infection), not candiduria, which represents simple colonization of the bladder.

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Sincerely,

Scott G. Filler, M.D.

Guest Editor

PLOS Pathogens

Alex Andrianopoulos

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Kasturi Haldar

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​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

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Dear Professor Ljungdahl,

We are pleased to inform you that your manuscript 'Proline catabolism is a key factor facilitating Candida albicans pathogenicity' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Scott G. Filler, M.D.

Guest Editor

PLOS Pathogens

Alex Andrianopoulos

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):