Mycoplasma hominis Causes DNA Damage and Cell Death in Primary Human Keratinocytes

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

To Microorganisms

We are thankful for all the editor comments.  The peer review pointed out important aspects of our manuscript and contributed to improving its quality. We are in agreement with the observations that have been done for this editorial group and modifications in the submitted manuscript are presented below:

Reviewer 1

Summary:

The manuscript from Amorim, A.T. et al., demonstrated the effect of Mycoplasma hominis infection on primary human keratinocytes. After M. hominis infection, the proliferation and viability of cells were decreased, the infection induced pro-inflammatory response in the keratinocytes.

Main comments:

- The authors estimate the influence of M. hominis to cellular process in PHK. There is no data about the state of mycoplasma in the manuscript. Does M. hominis get inside the cells? How much? I recommend to measure adhesion and invasion behavior of M. hominis. It defines the nature of host-pathogen interactions.

Answer: We appreciate the consideration. However, as the process of adhesion and invasion are processes extensively explored in the literature, the main focus of our manuscript was the elucidation of the processes triggered by inflammatory events. The recognition of the pathogen by the pattern recognition receptors (receptors responsible for triggering the inflammatory response) is independent of the adhesion and invasion processes (these processes can potentiate but are not fundamental). This was the main reason why we did not perform invasion and adhesion assays. However, we are currently conducting trials using time-lapse microscopy to verify these interactions (results to be published in a future manuscript)

– I do not fully understand what was the purpose of using different MOI and different time of incubation in every experiments. For quantification of viable PHKs, MOI 0.005-0.3 and 72 h of infection; for western-blot, MOI 0.01–0.04 and 48 h of infection; for clonogenic assay MOI 1:30 and 15 days of infection; for qRT-PCR 1 and 48 h of infection; for flow cytometry 6 h nd 48 h of infection.

Answer: We describe in a range because this experiment was made in triplicate. Besides that, we describe each MOI for each experiment because is a challenge to set a precise amount of Mycoplasma in the Mycoplasmology field. For each new infection, the same quantity of mycoplasma hominis is grown in 100 mL, centrifuged, resuspended in cell medium in the same way, and for our control, we plated this concentrate solution of mycoplasma in SP4 agar and count colony number every time before cell infection, and colony number is verified to better estimate mycoplasma cell viable in the moment of infection.

Specific comments:

- Why was line of primary human keratinocytes chosen in the study? Please explain your choice in the Introduction

- Line 74, is this sentence correct?

Answer: We appreciate your consideration. Modifications made to the manuscript

- Line 91-98, It is not clear how long M. hominis was cultivated. Were bacteria in mid-log phase when authors used culture for further experiments?

Answer: We appreciate your consideration. Modifications made to the manuscript

- Line 114-115, “the cells were washed with 1× PBX”. Do you mean PBS?

Answer: We appreciate your consideration. Modifications made to the manuscript

- There is no reference to Figure 2A.1 and 2A.2 in the text. Please use only label A, B, C etc for figures.

Answer: We appreciate your consideration. Modifications made to the manuscript

- Figure 2B, How many replicates were used in western-blot experiments? How can you explain the difference in abundance of p53 in Figure 2B and Figure 2D?

Answer: The western blot result shown in Figure 2B exhibit total p53 levels and is representative of three independent experiments. On the other hand, the result presented in Figure 2D is representative of two independent experiments using the Human Apoptosis Array (R&D Systems). Here the levels of p53 phosphorylation at particular amino acids, and not of the total protein, are shown. In particular, phosphorylation of p53 at serine 46 is a critical event leading to apoptosis.

- The authors used the Human DNA Damage Signaling Pathway kit for quantification of mRNA levels. This array contains primers for TP53 (Tumor protein p53). Expression level of this gene (RT-qPCR data) was not changed after M. hominis infection, but the protein level of p53 was increased. Please discuss this phenomenon in the text.

Answer: The sentence “P53 protein stabilization, without an increase in the transcription levels of its coding mRNA, is a common response related to DNA damage. Here, we showed that M. hominis infection altered the expression profile of some genes related to DNA damage pathways in PHKs, suggesting the activation of DNA damage pathways. Therefore, we can assume that in our model, p53 protein accumulation, in the absence of increased mRNA levels, can be explained, at least in part, by protein stabilization. The molecular mechanisms involved are being investigated in our laboratory [45-48].” was added to the Introduction (lines 385-393)

And we added these 4 papers to the discussion:

45. Lavin, M. F.; Gueven, N. The complexity of p53 stabilization and activation. Cell Death Differ 2006, 13 (6), 941-950. DOI: 10.1038/sj.cdd.4401925.
46. Cheng, Q.; Chen, J. Mechanism of p53 stabilization by ATM after DNA damage. Cell Cycle 2010, 9 (3), 472-478. DOI: 10.4161/cc.9.3.10556.
47. Kruse, J. P.; Gu, W. Modes of p53 regulation. Cell 2009, 137 (4), 609-622. DOI: 10.1016/j.cell.2009.04.050.
48. Haronikova, L.; Olivares-Illana, V.; Wang, L.; Karakostis, K.; Chen, S.; Fåhraeus, R. The p53 mRNA: an integral part of the cellular stress response. Nucleic Acids Res 2019, 47 (7), 3257-3271. DOI: 10.1093/nar/gkz124.

- Line 324-327, This text is not consistent with the results shown in the Figure 3A. “After 1 h of infection, CCL2, CSF2, CXCL10, IFNB1, IL6, CXCL8, IRF1, NFKBIA, TLR4, and TNF were up-regulated”. Only nine genes are indicated as significant up-regulated in the Figure 3A. “After 48 h, CCL2, CD14, CD180, IL6, and TNF were up-regulated, whereas IFNB1 and IL10 were down-regulated (Figure 4B).” There is no Figure 4B in the text. Please check carefully your text and labels of your figures.

Answer: We appreciate your consideration. Modifications made to the manuscript

- Figure 3B, Please use axis with the gap for better understanding of cytokines levels in control samples

Answer: We appreciate your consideration. Modifications made to the manuscript

- Line 406-410, The authors correctly describe the possible effect of mycoplasma metabolism on cell viability and physiology. Why didn't the authors test the hypothesis of an increase pH of the media during infection? This is a very simple experiment (just add Phenol red in the media) that will corroborate the assumption. And in general, the question remains whether M. hominis cells grow and divide during infection.

The manuscript was revised again by a native English speaker. We reinforce our acknowledgments for all comments, and we hope that this revised manuscript could be published by this editorial group.

Yours sincerely,

Lucas Marques

Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript, the authors investigate the pathological processes of M. hominis in vivo using primary human keratinocytes (PHKs) as opposed to immortalized and transformed cells. The results are effectively presented, and the experimental protocol is generally well-designed. The following factors, however, need to be taken into account.

1. 1. To determine the viability of infected cells, a hemocytometer was used. However, since this method does not distinguish between apoptotic and necrotic cells, we cannot conclude that the reduced cell count was caused by apoptosis.  I propose supplementing an LDH leakage assay to rule out necrosis caused by M. hominis-induced cytotoxicity at high concentrations.

2. line 239-240,  there is only one cureve for Fig. 1B, what is the precise MOI? In addition，the description of the doses of M. hominis is vague in line 106

3. line 324-325, Since TLR2 is the most important and universal receptor for sense mycoplasmas, why not detect it?

Minor comments:

1. There are an excessive number of numbered affiliations on the title page; in fact, there were only two institutions listed (1, 2, 4, 8 and 9, and 3, 5 and 6 are the same organization)

2. The length and width of the picture should be proportional (Fig.1C)

Author Response

To Microorganisms

We are thankful for all the editor comments.  The peer review pointed out important aspects of our manuscript and contributed to improving its quality. We are in agreement with the observations that have been done for this editorial group and modifications in the submitted manuscript are presented below:

Reviewer 2

In this manuscript, the authors investigate the pathological processes of M. hominis in vivo using primary human keratinocytes (PHKs) as opposed to immortalized and transformed cells. The results are effectively presented, and the experimental protocol is generally well-designed. The following factors, however, need to be taken into account.

1. To determine the viability of infected cells, a hemocytometer was used. However, since this method does not distinguish between apoptotic and necrotic cells, we cannot conclude that the reduced cell count was caused by apoptosis.  I propose supplementing an LDH leakage assay to rule out necrosis caused by M. hominis-induced cytotoxicity at high concentrations.
2. line 239-240,  there is only one curve for Fig. 1B, what is the precise MOI? In addition，the description of the doses of M. hominis is vague in line 106

Answer: We describe in a range because this experiment was made in triplicate. Besides that, we describe each MOI for each experiment because is a challenge to set a precise amount of Mycoplasma in the Mycoplasmology field. For each new infection, the same quantity of mycoplasma hominis is grown in 100 mL, centrifuged, resuspended in cell medium in the same way, and for our control, we plated this concentrate solution of mycoplasma in SP4 agar and count colony number every time before cell infection, and colony number is verified to better estimate mycoplasma cell viable in the moment of infection.

3. line 324-325, Since TLR2 is the most important and universal receptor for sense mycoplasmas, why not detect it?

Answer: Activation of TLR2 was observed in the study but was not statistically significant. Ureaplasmal or mycoplasmal LAMPs have been shown to interact with TLR2 and TLR4, leading to an inflammatory response. The increased expression of TLR4 after Mollicutes infections may be associated with the induction of autophagy in the cells

Minor comments:

1. There are an excessive number of numbered affiliations on the title page; in fact, there were only two institutions listed (1, 2, 4, 8 and 9, and 3, 5 and 6 are the same organization)

Answer: We appreciate your consideration. Modifications made to the manuscript

2. The length and width of the picture should be proportional (Fig.1C)

Answer: We appreciate your consideration. Modifications made to the manuscript

The manuscript was revised again by a native English speaker. We reinforce our acknowledgments for all comments, and we hope that this revised manuscript could be published by this editorial group.

Yours sincerely,

Lucas Marques

Author Response File: Author Response.pdf