Antimicrobial Properties of a Peptide Derived from the Male Fertility Factor kl2 Protein of Drosophila melanogaster

Round 1

Reviewer 1 Report

The article elaborates on the antimicrobial properties of MFF-p1 peptide derived from the male fertility factor kl2. I find it very interesting; however, the authors have to address some minor issues to the manuscript before it is accepted for publication.

The article has a lot of formatting shortcomings, e.g. (i) sometimes the authors use tabs at the beginning of a paragraph and sometimes not, (ii) sometimes they use an underline in the description of a figure and sometimes not, (iii) Table 1 is in general badly formatted, huge font size!, moved to the left and the same is true for Figure 2 and Figure 3. All Figures should be formatted in the same way, the same font type and size etc. In this form, they are unacceptable.

Line 27-28: the species name should be in one line

Line 39: the citation [2] should be in the same line as the sentence it concerns

Line 46: not AMP but AMPs – plural

Line 54: the citation [21-23] should be in the same line as the sentence it concerns

Line 71: Without any introduction chemerin 8(VR-15) is mentioned? As it is important for the study please elaborate on it e.g. one or two sentences in the introduction section.

Line 188: The time points should be explained in the text either for clarity.

Line 209-215: I would elaborate on the description of the paragraph, it is not clear.

Line 219-226: The description of the figure is inadequate, the abbreviations contained in the charts are missing in the description, I would advise that the control and other samples be in the same colour for clarity. There is also a white space before line 222 and 223?

Line 260: There is unnecessary comma before the word ‘then’

Line 262: There should be comma before ‘the word ‘there’

Line 276: the species name should be in one line

Line 336: ‘2h’ should be in one line

Line 428: Correct citation

Author Response

Reviewer 1

The article elaborates on the antimicrobial properties of MFF-p1 peptide derived from the male fertility factor kl2. I find it very interesting; however, the authors have to address some minor issues to the manuscript before it is accepted for publication.

The article has a lot of formatting shortcomings, e.g.

(i) sometimes the authors use tabs at the beginning of a paragraph and sometimes not,

It is corrected now.

(ii) sometimes they use an underline in the description of a figure and sometimes not,

It is corrected now.

(iii) Table 1 is in general badly formatted, huge font size!, moved to the left and the same is true for Figure 2 and Figure 3. All Figures should be formatted in the same way, the same font type and size etc. In this form, they are unacceptable.

Table 1 and Figures 2 and 3 are formatted now.

Line 27-28: the species name should be in one line

It is corrected now.                                    

Line 39: the citation [2] should be in the same line as the sentence it concerns

It is corrected.

Line 46: not AMP but AMPs – plural

It is corrected now.

Line 54: the citation [21-23] should be in the same line as the sentence it concerns

It is corrected now.

Line 71: Without any introduction chemerin 8(VR-15) is mentioned? As it is important for the study please elaborate on it e.g. one or two sentences in the introduction section.

We added one sentence to the Introduction regarding chemerin-8.

Line 188: The time points should be explained in the text either for clarity.

It is Corrected now.

Line 209-215: I would elaborate on the description of the paragraph, it is not clear.

We added one paragraph (line 211-222) to make clear how we tested antibacterial properties of MFF-kl2 in flies.

Line 219-226: The description of the figure is inadequate, the abbreviations contained in the charts are missing in the description, I would advise that the control and other samples be in the same colour for clarity. There is also a white space before line 222 and 223?

We added descriptions of abbreviations to the Figure legends and changed colours.

Line 260: There is unnecessary comma before the word ‘then’

Corrected

Line 262: There should be comma before ‘the word ‘there’

Corrected

Line 276: the species name should be in one line

It is corrected now.

Line 336: ‘2h’ should be in one line

Corrected

Line 428: Correct citation

Corrected

Reviewer 2 Report

Manuscript entitled “Antimicrobial properties of a chemerin-related peptide from the male fertility factor kl2 of Drosophila melanogaster” describes antimicrobial properties of two peptides MFF-p1 and Z-p. The conceived and experimental design, are very good. Investigation concerning antimicrobial peptides (AMPs) are very important especially because of the constant increase of bacterial resistance on antibiotics. Data presented in this study are detail and complete from identifying peptide candidates for analysis through their synthesis, Drosophila melanogaster infections with three selected bacteria strains (E. coli, S. aureus or B. subtilis). All presented experiments are with appropriate controls. Experiments were carefully designed and logically conducted. Obtained results demonstrated role of MFF-kl2 in antimicrobial defense in D. melanogaster. Since this peptide is a functional analog of the human chemerin derived peptide p4 future study are important for understanding of antimicrobial mechanisms of action and activity. I would suggest acceptance of the manuscript in the present form.

Author Response

Reviewer 2

Manuscript entitled “Antimicrobial properties of a chemerin-related peptide from the male fertility factor kl2 of Drosophila melanogaster” describes antimicrobial properties of two peptides MFF-p1 and Z-p. The conceived and experimental design, are very good. Investigation concerning antimicrobial peptides (AMPs) are very important especially because of the constant increase of bacterial resistance on antibiotics. Data presented in this study are detail and complete from identifying peptide candidates for analysis through their synthesis, Drosophila melanogaster infections with three selected bacteria strains (E. coli, S. aureus or B. subtilis). All presented experiments are with appropriate controls. Experiments were carefully designed and logically conducted. Obtained results demonstrated role of MFF-kl2 in antimicrobial defense in D. melanogaster. Since this peptide is a functional analog of the human chemerin derived peptide p4 future study are important for understanding of antimicrobial mechanisms of action and activity. I would suggest acceptance of the manuscript in the present form.

Reviewer 3 Report

The article of Bernadetta Bilska and coauthors is devoted to the study of the proteins from Drosophila melanogaster containing in their structure a sequence homologous to a small peptide, chemerin-8. This peptide is present in the human chemerin structure and responsible for its antimicrobial activity. The authors did a great job and got interesting results. But the text of the article requires revision on a number of important points.

 

Main comment. In the structure of a large multidomain protein from Drosophila, MFF-kl2, a short peptide homologous to chemerin-8 and having antimicrobial activity was found. Chemerin-8 has antimicrobial properties. At the same time, unlike the previous article in which it was shown that chemerin-8 provides the antimicrobial activity of human chemerin, the role of the peptide discovered by the authors in the present study in the MFF-kl2 structure remained unclear.

 

Title

- Chemerin is a protein. To what extent the search for proteins containing in their structure sites homologous to a small peptide (14 amino acids) of chemerin (chemerin-8) with antimicrobial activity can be named as search for chemerin-related peptides?

 

Abstract

- “Here we report a new antimicrobial peptide of Drosophila melanogaster, which was identified as a functional analog of the mammalian antibacterial chemerin p4 peptide.” The paper describes not a new antimicrobial peptide, but a peptide with antimicrobial activity, the sequence of which is part of the protein MFF-kl2 structure.

 

- “Using bioinformatic tools we identified functional analogs of the antibacterial chemerin-p4 peptide in the proteome of D. melanogaster.” Two proteins were found, in the structure of which homologous to chemerin-8 sequence is present.

 

 

Introduction

- lines 41-43 Why are the names of antimicrobial peptides capitalized?

 

- It is still not entirely clear from the text of the article what was the premise for looking for human chemerin-like peptides in the proteome of Drosophila (Introduction and Discussion).

 

 

Results

- In introduction: “Human chemerin-derived peptide p4 located in the central region of chemerin sequence is responsible for antibacterial activity of this protein.” In results: “MFF-p1 peptide matches very well the characteristics of the chemerin-p4 peptide in its fragment directly associated with antibacterial activity [i.e., 8 (VR-15)].” Drosophila proteins (Fig. 2) are compared with chemerin-8, although chemerin-p4 is indicated in the abstract, introduction, and discussion. Please clarify.

 

- How are regular expression (RE) patterns obtained?

 

- Nevertheless, the percentage of homology even between chemerin-8 of only 15 amino acids and isolated peptides in the structure of two Drosophila’s proteins is extremely low. In addition, the found homologous peptides are located in the structures of two completely different proteins. Does it have a biological sense?

 

- Fig. 2 Maybe it makes sense to give the numbering of the human peptide in the structure of chemerin itself, and not in chemerin-8? By analogy with peptides in the structures of Drosophila’s proteins.

 

- lines 109-111 “It is worth noticing though that both MFF-p1 and Z-p1 manifest their amphipathicity not as an extended β-strand, as the chemerin-8 (VR-15) peptide does, but as an α-helix.” This speaks in favor of the low homology of structures of human chemerin-8 and MFF-p1 or Z-p1.

 

- line 119 Figure 3c?

 

- Fig. 5. Photos show cells after incubation with peptides for 2 hours at a concentration of 100 μM. From Fig. 4, for example for S. aureus, it follows that the concentration of cells in PBS, in the presence of ch-p4 or MFF-p1 was different. But in Fig. 5, this difference is not visible.

 

- Fig. 5. Z-p1 is not shown here.

 

- 2.4. The expression of male fertility factor kl-2 is clock-controlled

The antimicrobial activity of the MFF-kl2 protein itself has not been shown. Its very short peptide MFF-p1 was used in tests. Whether this activity is biologically significant when the peptide is in the composition of such a large protein as MFF-kl2 is unknown.

 

- Figure 7. Bactericidal properties of Male fertility factor kl2.

The content of the figure does not show this.

 

- What is about the characteristics of mutant flies (act>kl-2 RNAi) - their physiology, viability, resistance to abiotic stress, and others. Has anything else been affected by silencing this gene?

 

Discussion

- lines 239-241 “Based on these findings, we used NCBI PHI BLAST to search the NR Protein database to identify novel AMPs, from D. melanogaster, matching characteristics of the antimicrobial chemerin-p4 peptide. We found two such peptides: MFF-p1(coded by kl-2 gene) and Z-p1 (coded by Zizimin).” The discovered amino acid sequences in the structure of the proteins are not antimicrobial peptides.

 

- lines 277-284 “We propose a mechanism in which MFF-kl2 controls the microbial burden by direct antibacterial properties of its antimicrobial peptide MFF-p1.”

It is not clear how this is possible, given that the antimicrobial peptide MFF-p1 is only 14 amino acids, and the protein MFF-kl2 consists of 4459 amino acids.

 

 

Materials and Methods

- 4.5. Antimicrobial assays

Why was the incubation of cells with peptides carried out only 2 hours, including for the determination of MICs. And why was very high peptide concentration (100 μM) used?

 

- 4.7. qRT-PCR

Why was one housekeeping gene used?

Author Response

Reviewer 3

The article of Bernadetta Bilska and coauthors is devoted to the study of the proteins from Drosophila melanogaster containing in their structure a sequence homologous to a small peptide, chemerin-8. This peptide is present in the human chemerin structure and responsible for its antimicrobial activity. The authors did a great job and got interesting results. But the text of the article requires revision on a number of important points.

 

Main comment. In the structure of a large multidomain protein from Drosophila, MFF-kl2, a short peptide homologous to chemerin-8 and having antimicrobial activity was found. Chemerin-8 has antimicrobial properties. At the same time, unlike the previous article in which it was shown that chemerin-8 provides the antimicrobial activity of human chemerin, the role of the peptide discovered by the authors in the present study in the MFF-kl2 structure remained unclear.

A functional homology between chemerin and MFF-kl2 is rather clear. Chemerin is a multifunctional protein and its antimicrobial properties were recently described because of its short peptide chemerin-8 containing 15 amino acids. In our study we found antibacterial activity of Male fertility factor kl2 protein in addition to its role in spermatogenesis, spermiogenesis and male fertility. The antibacterial activity  of this protein depends on a short peptide MF-p1 which we identified in this protein. MFF-kl2 was detected in males of Drosophila, in their abdomen when it is important for movement of the sperm flagellum and in addition may protects sperm against bacteria. 

Title

- Chemerin is a protein. To what extent the search for proteins containing in their structure sites homologous to a small peptide (14 amino acids) of chemerin (chemerin-8) with antimicrobial activity can be named as search for chemerin-related peptides?

According to the reviewer’s comment, we changed the title as follows: “Antimicrobial properties of a peptide derived from the Male fertility factor kl2 protein of Drosophila melanogaster “.

Abstract

- “Here we report a new antimicrobial peptide of Drosophila melanogaster, which was identified as a functional analog of the mammalian antibacterial chemerin p4 peptide.” The paper describes not a new antimicrobial peptide, but a peptide with antimicrobial activity, the sequence of which is part of the protein MFF-kl2 structure.

We agree with the reviewer. The sentence has been modified for clarity to: “Here we report antimicrobial properties of a peptide derived from the Male fertility factor kl2 (MFF-kl2) protein of Drosophila melanogaster, which was identified as a functional homolog of the mammalian antibacterial chemerin-p4 peptide. 

- “Using bioinformatic tools we identified functional analogs of the antibacterial chemerin-p4 peptide in the proteome of D. melanogaster.” Two proteins were found, in the structure of which homologous to chemerin-8 sequence is present.

The sentence has been modified: “Using bioinformatic tools we found functional analogs of the antibacterial chemerin-p4 peptide in the proteome of D. melanogaster.”

- lines 41-43 Why are the names of antimicrobial peptides capitalized?

This is accepted spelling rule for names of protein of D. melanogaster.

- It is still not entirely clear from the text of the article what was the premise for looking for human chemerin-like peptides in the proteome of Drosophila (Introduction and Discussion).

AMPs of insects are crucial for immune responses, however, other proteins may also have antimicrobial properties beside other functions what we have learned in case of chemerin. Looking for such proteins in Drosophila we used already known sequences of antibacterial peptides of chemerin (chemerin-p4 and chmerin-8) assuming that similar peptides in Drosophila should have some sequence homology and similar biochemical properties.

 

Results

- In introduction: “Human chemerin-derived peptide p4 located in the central region of chemerin sequence is responsible for antibacterial activity of this protein.” In results: “MFF-p1 peptide matches very well the characteristics of the chemerin-p4 peptide in its fragment directly associated with antibacterial activity [i.e., 8 (VR-15)].” Drosophila proteins (Fig. 2) are compared with chemerin-8, although chemerin-p4 is indicated in the abstract, introduction, and discussion. Please clarify.

We explained in the Introduction that chemerin-p4 and its fragment chemerin-8 have both antibacterial properties: “In the previous study it was shown, that the shorter peptide within p4, chemerin-8 (VR-15), that correspond to the sequence Val 66-Arg 80 of chemerin is responsible for antimicrobial activity of p4 [24]. To better understand the relevance of chemerin-derived antimicrobial peptides in controlling microbial burden, we used D. melanogaster as a model looking for peptides that share similar sequence homology and biochemical features with the antimicrobial peptides chemerin-p4 and chemerin-8 (VR-15).”

 

- How are regular expression (RE) patterns obtained?

           The details concerning RE patterns are given in lines 320-325. The RE pattern described in PL236566 corresponded to the distribution of residues with specific physico-chemical properties maintaining the antibacterial properties of synthetic analogues of the chemerin-p4 peptide (Godlewska U, Bilska B, Zegar A, Brzoza P, Borek A, Murzyn K, et al. The antimicrobial activity of chemerin-derived peptide p4 requires oxidative conditions. J Biol Chem. 2019;294. doi:10.1074/jbc.RA118.005495; Banas M, Zabieglo K, Kasetty G, Kapinska-Mrowiecka M, Borowczyk J, Drukala J, et al. Chemerin Is an Antimicrobial Agent in Human Epidermis. PLoS One. 2013;8: 2–9. doi:10.1371/journal.pone.0058709)

 

- Nevertheless, the percentage of homology even between chemerin-8 of only 15 amino acids and isolated peptides in the structure of two Drosophila’s proteins is extremely low. In addition, the found homologous peptides are located in the structures of two completely different proteins. Does it have a biological sense?

We agree that homology is low and it is written in the manuscript: “ While there are no statistically significant signs of sequence similarity due to homology (…) ” , however, we also found homologous biochemical properties of MFF-p1 and Z-p1 to chemerin-p4. That is why we tested their antimicrobial properties and detected them in case of MFF-p1 of the MFF-kl2 protein. It is possible that Z-p1 needs additional mechanisms to show antibacterial defense. In our opinion it is interesting that different proteins beside their regular functions may have antibacterial properties by carrying specific antibacterial peptides.

- Fig. 2 Maybe it makes sense to give the numbering of the human peptide in the structure of chemerin itself, and not in chemerin-8? By analogy with peptides in the structures of Drosophila’s proteins.

The original Figure 2 presents a direct result of the PHI BLAST program. The output of this program contains the start and end positions of the matching fragment of the query and database sequences. If we change this, i.e. instead of chemerin-8 (VR-15) peptide, there will be chemerin protein and its corresponding sequence coordinates, it will not illustrate the course of the actual analysis. Instead, we referred to the correspondence between the chemerin- 8(VR-15) and full chemerin sequence in the Figure 2 caption.

 

- lines 109-111 “It is worth noticing though that both MFF-p1 and Z-p1 manifest their amphipathicity not as an extended β-strand, as the chemerin-8 (VR-15) peptide does, but as an α-helix.” This speaks in favor of the low homology of structures of human chemerin-8 and MFF-p1 or Z-p1.

Homology is, by definition, a binary property and therefore should not be a subject for evaluation. In the work, we identified functional analogues of human chemerin peptides and not their homologues. The quoted sentence relates to the properties of the newly discovered peptides of D. melanogaster and it is not related to the consideration of the evolutionary origin of these peptides.

- line 119 Figure 3c?

This mistake is corrected (Figure 3 bottom).

- Fig. 5. Photos show cells after incubation with peptides for 2 hours at a concentration of 100 μM. From Fig. 4, for example for S. aureus, it follows that the concentration of cells in PBS, in the presence of ch-p4 or MFF-p1 was different. But in Fig. 5, this difference is not visible.

For TEM imaging, bacteria, after the treatment, were centrifuged and a pellet was fixed and embedded in Epon. Next ultrathin sections were cut and observed in TEM. So, the concentration of cells on an image depends on the obtained pellet and sections prepared for TEM. TEM images can only show differences in cell morphology but not the real concentration of cells. However, pellets were smaller in groups treated with peptides comparing with controls.

 

- Fig. 5. Z-p1 is not shown here.

 The legend has been modified as suggested.

- 2.4. The expression of male fertility factor kl-2 is clock-controlled

The antimicrobial activity of the MFF-kl2 protein itself has not been shown. Its very short peptide MFF-p1 was used in tests. Whether this activity is biologically significant when the peptide is in the composition of such a large protein as MFF-kl2 is unknown.

MFF-kl2 protein, which could be tested in vitro is not commercially available, therefore we did in vivo experiments, and used transgenic flies with the silenced kl-2 gene encoding MFF-kl2. Our results showed that experimental flies with this gene silencing have a reduced ability to defense bacterial infection.

 

- Figure 7. Bactericidal properties of Male fertility factor kl2. The content of the figure does not show this.

 This figure is changed and more information is added to the legend.

- What is about the characteristics of mutant flies (act>kl-2 RNAi) - their physiology, viability, resistance to abiotic stress, and others. Has anything else been affected by silencing this gene?

We have not tested physiology and viability of these flies. However, we observed no changes in viability of mutant flies and controls. It has also been reported that male fertility factors affect male fertility but not viability.

Discussion

- lines 239-241 “Based on these findings, we used NCBI PHI BLAST to search the NR Protein database to identify novel AMPs, from D. melanogaster, matching characteristics of the antimicrobial chemerin-p4 peptide. We found two such peptides: MFF-p1(coded by kl-2 gene) and Z-p1 (coded by Zizimin).” The discovered amino acid sequences in the structure of the proteins are not antimicrobial peptides.

In this section of the manuscript we used "novel" to underline previously undescribed, new function of known proteins, at least in the case of MFF-kl2 and MFF-p1 of the MFF-kl2. MFF-p1 can be synthesized and used for further testing as AMP.

- lines 277-284 “We propose a mechanism in which MFF-kl2 controls the microbial burden by direct antibacterial properties of its antimicrobial peptide MFF-p1.” It is not clear how this is possible, given that the antimicrobial peptide MFF-p1 is only 14 amino acids, and the protein MFF-kl2 consists of 4459 amino acids.

We change this sentence as follows : “We propose a mechanism in which MFF-kl2 may control the microbial burden by direct antibacterial properties of its antimicrobial peptide MFF-p1.” Since MFF-p1 has antibacterial properties they may depend of its small antimicrobial peptide MFF-p1. Unfortunately, this mechanism is unknown but our in vivo experiment showed that flies with the silenced kl2 gene had a reduced ability to defense bacterial infection. Moreover, for comparison, chemerin is also a large protein but its antibacterial potential depends only on 15 aa (Banas, Magdalena, et al. "Chemerin is an antimicrobial agent in human epidermis." PloS one 8.3 (2013): e58709).

Materials and Methods

- 4.5. Antimicrobial assays

Why was the incubation of cells with peptides carried out only 2 hours, including for the determination of MICs. And why was very high peptide concentration (100 μM) used?

All experiments were carried out after 2 hours of incubation to compare activity of MFF-p1 and Z-p1 with chemerin-derived peptides, that were fully active in that condition (based on the previously published data). The high peptide concentration (100 μM) was used to determine if peptides display any antimicrobial properties.

- 4.7. qRT-PCR

Why was one housekeeping gene used?

In studies on circadian rhythms in gene expression of D. melanogaster, this gene is usually used as a reference gene. We know that Rpl32 expression is clock-independent and constant over the day. This reference gene was used in the following studies:

1. Rodriguez, J., Tang, C. H. A., Khodor, Y. L., Vodala, S., Menet, J. S., & Rosbash, M. (2013). Nascent-Seq analysis of Drosophila cycling gene expression. Proceedings of the National Academy of Sciences, 110(4), E275-E284.
   
   
2. Shukla, A. K., Johnson, K., & Giniger, E. (2021). Common features of aging fail to occur in Drosophila raised without a bacterial microbiome. iScience, 102703.
   
   
3. Abaquita, A. L., Damulewicz, M., Bhattacharya, D., & Pyza, E. (2021). Regulation of Heme Oxygenase and Its Cross-Talks with Apoptosis and Autophagy under Different Conditions in Drosophila. Antioxidants, 10(11), 1716.
   
   
4. Damulewicz, M., Loboda, A., Bukowska-Strakova, K., Jozkowicz, A., Dulak, J., & Pyza, E. (2015). Clock and clock-controlled genes are differently expressed in the retina, lamina and in selected cells of the visual system of Drosophila melanogaster. Frontiers in cellular neuroscience, 9, 353.

 

 

 

 

Round 2

Reviewer 3 Report

In my opinion, the article requires conceptual revision (in the Discussion section). And unfortunately, I do not agree with the author’s opinion that “A functional homology between chemerin and MFF-kl2 is rather clear.” and that “In our study we found antibacterial activity of Male fertility factor kl2 protein in addition to its role in spermatogenesis, spermiogenesis and male fertility. The antibacterial activity of this protein depends on a short peptide MF-p1 which we identified in this protein.” The manuscript does not contain sufficient data to support this. If similar information is published in other articles, please provide links.

The situation with chemerin and the protein MFF kl2, in the structure of which the authors found ch-8 (VR-15)-like peptide exhibiting antibacterial activity, is different. The antibacterial activity of chemerin was determined [16]. And it was shown that the middle part of the сhemerin Val66-Pro85 [16] or Val66-Pro80 [24] provides the antimicrobial activity of this multifunctional human protein. In this article, the authors showed only the antibacterial activity of the peptide fragment of drosophila protein MFF kl2, but not MFF kl2 itself. An increase in kl-2 gene expression and an increase of the concentration of bacteria in the tissues of infected flies with the silenced expression of this gene undoubtedly indicate the important role MFF kl2 in protecting flies from infection but does not indicate bactericidal activity of this protein. It should also be noted that the percentage of identity between peptide ch-8 (VR-15) and peptide MFF-p1 is only 21%. And it is well known that short peptides containing a large number of cationic residues exhibit affinity for negatively charged cell membranes and exhibit antimicrobial activity. Some additional remarks and comments are given below.

Discussion

- lines 239-241 “Based on these findings, we used NCBI PHI BLAST to search the NR Protein database to identify novel AMPs, from D. melanogaster, matching characteristics of the antimicrobial chemerin-p4 peptide. We found two such peptides: MFF-p1(coded by kl-2 gene) and Z-p1 (coded by Zizimin).” Z-p1 did not possess antibacterial activity. Please correct.

- lines 253-258 “…..MFF-p1 possesses bactericidal activity against E. coli and B. subtilis, probably due to the features that include a positive net charge (+4) and ability to adopt an amphipathic structure. The antimicrobial effect of MFF-p1 against S. aureus was limited likely by differences in cell wall structure between Gram-positive and Gram-negative bacteria, where the thickness of peptidoglycan layer contributes to susceptibility to antimicrobial challenge mediated by AMPs [32].” But B. subtilis is Gram-positive bacterium as well as S. aureus. Please correct.

- lines 282-284 “We propose a mechanism in which MFF-kl2 may control the microbial burden by direct antibacterial properties of its antimicrobial peptide MFF-p1.” I cannot agree with the opinion of the authors. “Since MFF-p1 has antibacterial properties they may depend on its small antimicrobial peptide MFF-p1. Unfortunately, this mechanism is unknown but our in vivo experiment showed that flies with the silenced kl2 gene had a reduced ability to defense bacterial infection. Moreover, for comparison, chemerin is also a large protein but its antibacterial potential depends only on 15 aa (PloS one 8.3 (2013): e58709).” In this article 2013, using a panel of overlapping chemerin-derived synthetic peptides, authors determined the sequence which mediated chemerin antibacterial activity. Chemerin consists of 143 aa, but MFF-kl2 consists of 4459 aa. Chemerin is a multifunctional protein implicated in the chemotaxis of immune cells, sharing a similar tertiary structure with antibacterial cathelicidins. MFF-kl2 involves in microtubule-based movement as the authors described. As an additional argument. Many recombinant AMPs are produced by expression in E. coli cells and their toxicity is leveled by the expression of AMP with a fusion partner, for example, thioredoxin.

Results

- In the search for homologous sequences the authors used the peptide ch-8 (VR-15) (Fig. 2), but in the tests comparing the antimicrobial activity of newly discovered peptides, the peptide ch-p4 was used. Please explain, why not ch-8 (VR-15) was used in the tests.

- Figure 7. “Gene expression (A) and bactericidal properties of MFF kl2 (B) in response to microbial challenge.” Wrong figure legend, because antibacterial activity of MFF kl2 was not shown in this figure and in this article in general. Silencing the expression of the kl-2 gene can lead to various events and the data obtained are not direct evidence of the bactericidal action of MFF kl2. Please correct.

 

 

 

 

 

Author Response

In my opinion, the article requires conceptual revision (in the Discussion section). And unfortunately, I do not agree with the author’s opinion that “A functional homology between chemerin and MFF-kl2 is rather clear.” and that “In our study we found antibacterial activity of Male fertility factor kl2 protein in addition to its role in spermatogenesis, spermiogenesis and male fertility. The antibacterial activity of this protein depends on a short peptide MF-p1 which we identified in this protein.” The manuscript does not contain sufficient data to support this. If similar information is published in other articles, please provide links.

According to the Reviewer’s comments we made changes in the Discussion regarding “functional homology” and now we write about antibacterial activity of MF-p1 and that MFF-kl2 protein, in addition to its other functions, protects flies against bacteria. Regarding the importance of MFF-kl2 in the antibacterial protection we used transgenic flies with the silenced kl2 gene and those flies were less protected against bacteria than control. We used GAL4-UAS system that is commonly used in Drosophila to study function of a particular gene and its protein, so using RNAi to silence the kl2 gene in all cells under the actin promotor we did not affect other proteins.

The situation with chemerin and the protein MFF kl2, in the structure of which the authors found ch-8 (VR-15)-like peptide exhibiting antibacterial activity, is different. The antibacterial activity of chemerin was determined [16]. And it was shown that the middle part of the сhemerin Val66-Pro85 [16] or Val66-Pro80 [24] provides the antimicrobial activity of this multifunctional human protein. In this article, the authors showed only the antibacterial activity of the peptide fragment of drosophila protein MFF kl2, but not MFF kl2 itself. An increase in kl-2 gene expression and an increase of the concentration of bacteria in the tissues of infected flies with the silenced expression of this gene undoubtedly indicate the important role MFF kl2 in protecting flies from infection but does not indicate bactericidal activity of this protein. It should also be noted that the percentage of identity between peptide ch-8 (VR-15) and peptide MFF-p1 is only 21%. And it is well known that short peptides containing a large number of cationic residues exhibit affinity for negatively charged cell membranes and exhibit antimicrobial activity. Some additional remarks and comments are given below.

We showed in the present study, as the Reviewer noticed,  that the peptide MFF-p1, derived from MFF kl2, shows antibacterial activity. We have not tested the antibacterial activity of the MFF-kl2 protein, however, working on Drosophila we could use GAL4-UAS system to study the function of MFF-kl2. This is the advantage of studying various protein functions in Drosophila, because their levels can be precisely decreased or increased in all cells or only in specific ones. We showed in living flies that they are not protected against bacteria when the level of the whole protein MFF-kl2 is decreased.

The Reviewer admits that “an increase of the concentration of bacteria in the tissues of infected flies with the silenced expression of this gene undoubtedly indicate the important role MFF kl2 in protecting flies from infection”. We observed this results after decreasing the level of MFF-kl2 only, but not other proteins, so it indicates the role of MFF-kl2 in protecting flies against bacteria.

Discussion

- lines 239-241 “Based on these findings, we used NCBI PHI BLAST to search the NR Protein database to identify novel AMPs, from D. melanogaster, matching characteristics of the antimicrobial chemerin-p4 peptide. We found two such peptides: MFF-p1(coded by kl-2 gene) and Z-p1 (coded by Zizimin).” Z-p1 did not possess antibacterial activity. Please correct.

This sentence is corrected as follows: “Based on these findings, we used NCBI PHI BLAST to search the NR Protein database to identify peptides from D. melanogaster, matching characteristics of the antimicrobial chemerin-p4 peptide. We found two such peptides: MFF-p1(coded by kl-2 gene) and Z-p1 (coded by Zizimin).”

- lines 253-258 “…..MFF-p1 possesses bactericidal activity against E. coli and B. subtilis, probably due to the features that include a positive net charge (+4) and ability to adopt an amphipathic structure. The antimicrobial effect of MFF-p1 against S. aureus was limited likely by differences in cell wall structure between Gram-positive and Gram-negative bacteria, where the thickness of peptidoglycan layer contributes to susceptibility to antimicrobial challenge mediated by AMPs [32].” But B. subtilis is Gram-positive bacterium as well as S. aureus. Please correct.

Although both B. subtilis and S. aureus are Gram-positive bacteria, it has been found that cell walls of these bacteria species show differences in their synthesis and properties

[Yokoyama et al. (1986). Structure and functions of linkage unit intermediates in the biosynthesis of ribitol teichoic acids in Staphylococcus aureus H and Bacillus subtilis W23. European journal of biochemistry, 161(2), 479-489;

Brown  et al. (2010) Staphylococcus aureus and Bacillus subtilis W23 make polyribitol wall teichoic acids using different enzymatic pathways." Chemistry & biology 17.10: 1101-1110.]

 

- lines 282-284 “We propose a mechanism in which MFF-kl2 may control the microbial burden by direct antibacterial properties of its antimicrobial peptide MFF-p1.” I cannot agree with the opinion of the authors. “Since MFF-p1 has antibacterial properties they may depend on its small antimicrobial peptide MFF-p1. Unfortunately, this mechanism is unknown but our in vivo experiment showed that flies with the silenced kl2 gene had a reduced ability to defense bacterial infection. Moreover, for comparison, chemerin is also a large protein but its antibacterial potential depends only on 15 aa (PloS one 8.3 (2013): e58709).” In this article 2013, using a panel of overlapping chemerin-derived synthetic peptides, authors determined the sequence which mediated chemerin antibacterial activity. Chemerin consists of 143 aa, but MFF-kl2 consists of 4459 aa. Chemerin is a multifunctional protein implicated in the chemotaxis of immune cells, sharing a similar tertiary structure with antibacterial cathelicidins. MFF-kl2 involves in microtubule-based movement as the authors described. As an additional argument. Many recombinant AMPs are produced by expression in E. coli cells and their toxicity is leveled by the expression of AMP with a fusion partner, for example, thioredoxin.

We proposed a mechanism but if the Reviewer does not agree with our proposed mechanism, we changed this sentence and concluded that MFF-kl2 protects flies against bacterial infection.

Results

- In the search for homologous sequences the authors used the peptide ch-8 (VR-15) (Fig. 2), but in the tests comparing the antimicrobial activity of newly discovered peptides, the peptide ch-p4 was used. Please explain, why not ch-8 (VR-15) was used in the tests.

In the first paragraph of the Results section we write:

“Initially, we performed PHI-BLAST scans of a non-redundant set of Drosophila melanogaster sequences using either chemerin-p4 or chemerin-8 (VR-15) [24] sequences as a query and the corresponding regular expression (RE) pattern (Table 1). These searches resulted in no hits. When the shortened RE patterns (Table 1) were used several dozen hits were observed. Only one of them (MFF-p1, Table 1) had several features which were found previously to be prerequisites for antibacterial activity of chemerin-p4 related peptides, i.e. at least 14 aa long, a net charge at least of +4, and the ability to adopt strongly amphipathic structure [relative hydrophobic moment (rHM) > 0.375].”

In the PHI-BLAST we used either chemerin-p4 or chemerin-8 but because identified MFF-p1 had several features which were found previously to be prerequisites for antibacterial activity of chemerin-p4 related peptides in the tests comparing the antimicrobial activity of newly discovered peptides, the peptide chemerin-p4 was used.

- Figure 7. “Gene expression (A) and bactericidal properties of MFF kl2 (B) in response to microbial challenge.” Wrong figure legend, because antibacterial activity of MFF kl2 was not shown in this figure and in this article in general. Silencing the expression of the kl-2 gene can lead to various events and the data obtained are not direct evidence of the bactericidal action of MFF kl2. Please correct

 

The Figure 7 legend is corrected according to the Reviewer’s comment.

We agree that the kl2 silencing can lead to various events, for example to decrease male fertility, but we have not tested them except the response of transgenic flies to infection.

 

The Figure 7 legend is as follows now:

Figure 7. Gene expression (A) and antibacterial protection of MFF kl2 (B) in response to microbial challenge….

Round 3

Reviewer 3 Report

- lines 252-260 “…..MFF-p1 possesses bactericidal activity against E. coli and B. subtilis, probably due to the features that include a positive net charge (+4) and ability to adopt an amphipathic structure. The antimicrobial effect of MFF-p1 against S. aureus was limited likely by differences in cell wall structure between Gram-positive and Gram-negative bacteria, where the thickness of peptidoglycan layer contributes to susceptibility to antimicrobial challenge mediated by AMPs [32]. Moreover, even between Gram-positive bacteria, as S. aureus and B. subtilis there are differences in the structure and synthesis of cell walls Gram-positive bacteria such as S. aureus and B. subtilis, which may affect responses of these species to their susceptibility to AMPs [33,34]. Please correct.

Author Response

Reviewer's Comment:

• lines 252-260 “…..MFF-p1 possesses bactericidal activity against E. coli and B. subtilis, probably due to the features that include a positive net charge (+4) and ability to adopt an amphipathic structure. The antimicrobial effect of MFF-p1 against S. aureus was limited likely by differences in cell wall structure between Gram-positive and Gram-negative bacteria, where the thickness of peptidoglycan layer contributes to susceptibility to antimicrobial challenge mediated by AMPs [32]. Moreover, even between Gram-positive bacteria, as S. aureus and B. subtilis there are differences in the structure and synthesis of cell walls Gram-positive bacteria such as S. aureus and B. subtilis, which may affect responses of these species to their susceptibility to AMPs [33,34].

According to the Reviewer's suggestion we corrected this sentence as follows:

The antimicrobial effect of MFF-p1 against S. aureus was limited likely by differences in the structure and synthesis of cell walls Gram-positive bacteria such as S. aureus and B. subtilis which may affect their susceptibility to AMPs [33,34].