A Stress Hub in Scedosporium apiospermum: The High Osmolarity Glycerol (HOG) Pathway

Round 1

Reviewer 1 Report

1.For the results in "2.1 SaHog1 is highly conserved", could you provide direct, specific data to support your results?

2.Line 157-158, for the conclusion "This effect was reproducible even when different experimental runs were used.", have relevant validation experiments been done?

3.Why were these parameters under stress conditions selected for fungal stress in this study?

4.Lines 232-234, what is the relationship between the distinct kinetics of phosphorylation of SaHog1 and the HOG pathway? What specific effects do the distinct kinetics of phosphorylation of SaHog1 have on the studied HOG pathways?

5.Lines 274-271, the cultivation of the fungus is described here, on the medium culture 9 days, or in a germ-free water in the water? The description needs to be accurate.

6.The name of the species must be in italics.

Author Response

Reviewer #1:

 

1. For the results in "2.1 SaHog1 is highly conserved", could you provide direct, specific data to support your results?

 

REPLY: We would like to thank the reviewer for asking for this. Please note that data were provided in a new figure (Figure 2), which included a cladogram analysis of Hog1 from distant fungal species.

 

2. Line 157-158, for the conclusion "This effect was reproducible even when different experimental runs were used.", have relevant validation experiments been done?

 

REPLY: Yes. We ran at least two separate experimental assays of stress (different cultures of fungi stressed with different preparations of stressed medium). The blot profiles where the « discrete effect » was revealed were reproducible in each of the assays.  

 

3. Why were these parameters under stress conditions selected for fungal stress in this study?

 

REPLY: The current knowledge on the occurrence of Scedosporium in nature (like human-impacted areas, including polluted regions) and in the human host (like the airways of cystic fibrosis patients) infers that these fungi can tolerate and adapt to a wide range of stresses that are likely to encounter in such hostile environments. And in order to check out if the HOG pathway, represented by Hog1 MAPK, is involved in such adaptation, we aimed to test as much as possible different types of chemicals or conditions belonging to groups of stress that are prominent in these hostile environments. For example, acid pH, antifungals, oxidative stress, and hypercapnia all mimic the microenvironment of the airways of cystic fibrosis patients, while the other groups of stress mimic the natural niches. Kindly note that we adjusted some sentences in case these ideas were not clear. 

 

4. Lines 232-234, what is the relationship between the distinct kinetics of phosphorylation of SaHog1 and the HOG pathway? What specific effects do the distinct kinetics of phosphorylation of SaHog1 have on the studied HOG pathways?

 

REPLY: In fact, the protein Hog1 MAPK is representative of the HOG pathway, which is the reason why the pathway is named after it. This implies that the status (activated or not) and the role of the HOG pathway are reflected by those of Hog1 MAPK. Giving an example, when one states that Hog1 MAPK is activated under X stress and involved in X roles, it gives quite the same meaning as when to replace « Hog1 MAPK » with « HOG pathway ». Back to the text, we hypothesized that the kinetics of activation of SaHog1 MAPK seen in western blot reflect the regulatory roles played by the SaHog1 MAPK/HOG pathway.

 

5. Lines 274-271, the cultivation of the fungus is described here, on the medium culture 9 days, or in a germ-free water in the water? The description needs to be accurate.

 

REPLY: We reformulated the sentence so that it provides an accurate description.

 

6. The name of the species must be in italics.

 

REPLY: We are sorry for this typo. Corrections were made.

Reviewer 2 Report

1.-Figure captions 1 and 2 contain a lot of information, it is recommended to summarize.

 

2.-It would be worth including a biochemical method to visualize the reaction of its enzymes in a culture medium.

 

3.-As for the discussion, it can still be improved, including information from other more recent studies.

Author Response

Reviewer #2:

 

1. Figure captions 1 and 2 contain a lot of information, it is recommended to summarize.

 

 REPLY: The recommendation was taken into consideration.

 

2. It would be worth including a biochemical method to visualize the reaction of its enzymes in a culture medium.

 

REPLY: We would like to thank the reviewer for this appreciated comment. Unfortunately, there are still no biochemical methods that visualize the phosphorylation reactions done by the kinases of the HOG pathway. Otherwise, it would be complicated to develop such methods while western blot stands as a golden standard mean that helps visualize the kinase reactions from a final point of view. Functional genomic studies support that there is no way that Hog1 MAPK gets phosphorylated on the TGY motif other than by the upstream kinases of the HOG pathway (Ssk2 MAPKKK and Pbs2 MAPKK). This means that once the phosphorylation of Hog1 MAPK on the TGY motif is detected by western blot, it constitutes strong evidence that the upstream kinases have done their enzymatic reaction (phosphorylation), which culminates in the phosphorylation of the Hog1 MAPK.

 

3. As for the discussion, it can still be improved, including information from other more recent studies.

 

REPLY: We tried to upgrade the discussion as fair as possible. Kindly note that, at the end of 2021, we published a review on the HOG pathway in fungi (reference number 13), which compelled current knowledge on the pathway in four fungal species (S. cerevisiae, in addition to the most frequent clinical pathogens, C. albicans, C. neoformans, and A. fumigatus). We also included almost all types of stress ever reported as activators of the phosphorylation of Hog1 in these species. Since the indicated publishing date, only a few papers on the role of the HOG pathway in stress response in fungi have been out. However, the quasi-totality of these papers does not evoke the stress tested in this paper. Since this paper discusses the phosphorylation of the Hog1 in stress, we followed this methodology; each time we wanted to mention that fungal Hog1 responds to a broad range of stress, we cited our review (reference number 13), otherwise, when we discussed a specific type of stress, we tried to mention the original papers.

Round 2

Reviewer 1 Report

ok