Mycobacterium abscessus Opsonization Allows an Escape from the Defensin Bactericidal Action in Drosophila

ABSTRACT Mycobacterium abscessus, an intracellular nontuberculous mycobacterium, is considered the most pathogenic species among the group of rapidly growing mycobacteria. The resistance of M. abscessus to the host innate response contributes to its pathogenicity in addition to several virulence factors. We have recently shown in Drosophila that antimicrobial peptides (AMPs), whose production is induced by M. abscessus, are unable to control mycobacterial infection. This could be due to their inability to kill mycobacteria and/or the hidden location of the pathogen in phagocytic cells. Here, we demonstrate that the rapid internalization of M. abscessus by Drosophila macrophages allows it to escape the AMP-mediated humoral response. By depleting phagocytes in AMP-deficient flies, we found that several AMPs were required for the control of extracellular M. abscessus. This was confirmed in the Tep4 opsonin-deficient flies, which we show can better control M. abscessus growth and have increased survival through overproduction of some AMPs, including Defensin. Furthermore, Defensin alone was sufficient to kill extracellular M. abscessus both in vitro and in vivo and control its infection. Collectively, our data support that Tep4-mediated opsonization of M. abscessus allows its escape and resistance toward the Defensin bactericidal action in Drosophila. IMPORTANCE Mycobacterium abscessus, an opportunistic pathogen in cystic fibrosis patients, is the most pathogenic species among the fast-growing mycobacteria. How M. abscessus resists the host innate response before establishing an infection remains unclear. Using Drosophila, we have recently demonstrated that M. abscessus resists the host innate response by surviving the cytotoxic lysis of the infected phagocytes and the induced antimicrobial peptides (AMPs), including Defensin. In this work, we demonstrate that M. abscessus resists the latter response by being rapidly internalized by Drosophila phagocytes. Indeed, by combining in vivo and in vitro approaches, we show that Defensin is able to control extracellular M. abscessus infection through a direct bactericidal action. In conclusion, we report that M. abscessus escapes the host AMP-mediated humoral response by taking advantage of its internalization by the phagocytes.

which per the authors' description, demonstrated that Mab is internalized by Drosophila phagocytic plasmacytes, but was able to survive lysis of the phagocytic plasmacytes by NK cell-like thanocytes. Drosophila were resistant to Mab infection when thanocytes were depleted. Furthermore, they report that their previous work showed that AMPs are induced by Mab infection in Drosophila. In this study, they looked to build upon this finding by investigating the role of AMPs in Mab infection, and how Mab may evade the AMP response in Drosophila. The authors find that opsonization with Tep4 allows internalization of Mab which may allow it to evade host AMPs.
Major critiques -Lines 133 -134 -A major aspect of the conclusions of this manuscript seem to rest upon the concept that the Mab are extracellular when the phagocytic plasmacytes are depleted, however, this is not actually shown. Are there other cell types that could be susceptible to intracellular Mab infection in the absence of the phagocytic plasmacytes? This should be addressed as it's unclear if it already was addressed in the previous under revision manuscript or not.
- Figure 2A -the use of CFUs alone is not adequate to definitively state that Tep4 is involved in internalization. The experimental design would be unable to distinguish between internalized Mab versus Mab bound to the outer surface of the cell line. A complementary method such as flow cytometry or confocal microscopy with quenching should be utilized to make this distinction.
- Figure 2-the manuscript lacks evidence for the efficacy of the Tep4 RNAi -this should be shown in a figure or at a minimum, should be included in the text of the results section.
- Figure 2 -it is stated that Tep4 has been shown to act as an opsonin in the literature but it isn't directly demonstrated here. Results would be strengthened with addition of a western blot showing that Tep4 directly binds to Mab in the Drosophila model.
- Figure 3 and discussion -what is the proposed mechanism by which Tep4 depletion upregulates antimicrobial peptide expression? - Figure 3B-D -it appears that these AMPs are minimally upregulated in the case of Mab infection of control (non Tep4 mutant) Drosophila. Particularly, it appears that Defensin is not upregulated by much if any by Mab infection alone (only upregulated in the absence of Tep4). The authors state that their prior (under revision) work showed that most AMPs are upregulated by Mab infection in Drosophila. This seems inconsistent with what is shown here (though difficult to know for sure without being able to reference the prior study). This should be more thoroughly addressed.
- Figure 4 -extent/efficacy of defensin overexpression should be shown or discussed.
-Lines 229-230 -inhibition of growth is not the same as direct bactericidal activity, results should be stated accordingly. -Stating that defensin is "sufficient" for Mab control may be overstating the conclusions. Would rephrase to indicate that it has an important role in host defense against Mab infection.
Minor critiques -Was a rough or smooth morphotype of Mab used? The discussion implies it was smooth but it is not otherwise discussed. Should at least be included in the materials & methods section. If smooth was used, were any studies attempted using rough morphotype? -Lines 108-119 should be incorporated into the introduction rather than the results section -Line 131 -it should be addressed why Relish mutant flies, but not any of the group B AMP mutants, had decreased survival.
- Figure 2B -why is Mab growth increased in the Tep5 RNAi treatment at day 5? This should be addressed.
- Figure 2D-F -should have each graph labeled with what bacteria is used.
- Figure 3I and J -should include a statistical comparison of Group A/+ and GroupA/+ & Tep4>RNAi Tep4 -are these significantly different or not?
Reviewer #2 (Comments for the Author): Touré et al., showed that AMPs were required to control extracellular Mab, and Defensin was found to be sufficient to kill extracellular Mab both in vitro and in vivo. The data suggest that Tep4-mediated opsonization of Mab allows its escape and resistance towards the Defensin bactericidal action in Drosophila.

Major comments:
Important comment: The authors are advised to incorporate a DOI or other relevant reference information for the unpublished manuscript "Touré et al. in revision" throughout the entirety of their manuscript. This reference contains crucial information that is essential for comprehending the current manuscript and provides evidence for the hypothesis discussed in the article. Without the DOI, I cannot presently recommend either acceptance or rejection for the manuscript.
Q1. The author demonstrated the involvement of Tep4-opsonin in the internalization of Mab into plasmatocytes. However, it should be noted that Drosophila has six different genes for Teps, namely Tep1-Tep6, and each Tep is known to have distinct roles in promoting phagocytosis of different microorganisms. For example, TEP1 functions as an opsonin to promote phagocytosis of both Gram-positive and Gram-negative bacteria, while TEP2, TEP3, and TEP6 promote phagocytosis of Escherichia coli, Staphylococcus aureus, and Candida albicans, respectively, in cultured S2 cells (DOI: 10.1159/000321554). Tep5 is a pseudogene. In addition, it has been reported that Tep2 and Tep6 affect phagocytosis and melanization in flies infected with Photorhabdus (DOI: 10.1080/21505594.2017.1330240).
In this context, it would be valuable to investigate the involvement of other Teps in Mab internalization and provide experimental results to support the findings. If there is a relevant rationale for focusing only on Tep4 among the six different Teps, the authors should describe it in the manuscript.
Q2. Figure 2A describe Tep4 involvement for internalization. However, it is not significant difference between control and Tep4 RNAi (~0.5 log10 reduction) albeit Figure 2C showed significant survival difference. Thus, I feel necessity to check another Teps involveness on this Mab internalization again.
Q3. The Y-axis unit in Figure 2B should be clarified as the number of bacteria (CFU) or on a logarithmic scale if applicable.
Q4. The number of flies tested in Figure 3A is too small size. Only 5 flies look not enough for statistical analysis. The authors should use at least 20 flies for this experiment for better statistical analysis.
Q5. The authors should explain the exception of Diptericin showing a significant difference compared to other AMPs in Figure  3E. Furthermore, also need to explain about Drosomycin and Drosocin in Figure 3F and G.
Minor comments: Line 78: Please clarify what "Dm" stands for in the text.
Lines 79-84: The text in this section is difficult to understand and should be rewritten for clarity. Lines 125-131: It would be better provide information about gene names (single gene mutation) in an independent table to make it easier for readers to follow.
Lines 179-183: The authors should move this information to the Discussion section and include it with lines 313-318 to avoid repetition.
The statistical analysis in Figure 3I and J should be rechecked, as there appears to be a >25% difference between Tep4>RNAi Tep4 and GroupA/+ & Tep4>RNAi Tep4, but the p-value is only <0.05.

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In this study, the authors investigate the role of antimicrobial peptides and the opsonin Tep4 in control of M. abscessus (Mab) infection in Drosophila. This manuscript builds upon the findings of a prior manuscript that is evidently in revision at present, which per the authors' description, demonstrated that Mab is internalized by Drosophila phagocytic plasmacytes, but was able to survive lysis of the phagocytic plasmacytes by NK cell-like thanocytes. Drosophila were resistant to Mab infection when thanocytes were depleted. Furthermore, they report that their previous work showed that AMPs are induced by Mab infection in Drosophila. In this study, they looked to build upon this finding by investigating the role of AMPs in Mab infection, and how Mab may evade the AMP response in Drosophila. The authors find that opsonization with Tep4 allows internalization of Mab which may allow it to evade host AMPs. with what is shown here (though difficult to know for sure without being able to reference the prior study). This should be more thoroughly addressed. - Figure 4 -extent/efficacy of defensin overexpression should be shown or discussed.
-Lines 229-230 -inhibition of growth is not the same as direct bactericidal activity, results should be stated accordingly. -Stating that defensin is "sufficient" for Mab control may be overstating the conclusions.
Would rephrase to indicate that it has an important role in host defense against Mab infection.
Minor critiques -Was a rough or smooth morphotype of Mab used? The discussion implies it was smooth but it is not otherwise discussed. Should at least be included in the materials & methods section. If smooth was used, were any studies attempted using rough morphotype? -Lines 108-119 should be incorporated into the introduction rather than the results section -Line 131 -it should be addressed why Relish mutant flies, but not any of the group B AMP mutants, had decreased survival. - Figure 2B -why is Mab growth increased in the Tep5 RNAi treatment at day 5? This should be addressed. - Figure 2D-F -should have each graph labeled with what bacteria is used.
- Figure 3I and J -should include a statistical comparison of Group A/+ and GroupA/+ & Tep4>RNAi Tep4 -are these significantly different or not?

RESPONSE TO REVIEWERS SPECTRUM00777-23
Dear Dr. Fabienne Girard-misguich: Thank you for submitting your manuscript to Microbiology Spectrum.
Your manuscript has been evaluated by two reviewers. Although the reviewers were generally enthusiastic about the manuscript, they raised several issues that need to be resolved before we can proceed further in the editorial process.
In addition, they both noted that the manuscript is largely based on another study (Touré et al. In revision) which they would like to have access to and which they think should be published before the present study. I urge you to be scrupulous in meeting this expectation.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.
Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
The ASM Journals program strives for constant improvement in our submission and publication process. We would like to thank Reviewer #1 for his/her insightful comments and constructive suggestions, which we address below.

RESPONSE TO REVIEWERS SPECTRUM00777-23
- Figure 2-the manuscript lacks evidence for the efficacy of the Tep4 RNAi -this should be shown in a figure or at a minimum, should be included in the text of the results section.
We apologize for not including the efficacy of the two Tep4-RNAi (whole flies and S2 cells) used in the Figure 2 of the original manuscript. We have included these in the revised manuscript (pages 8 and 9). They correspond to the new Figure S1B and D, which you will find below.
- Figure 2 -it is stated that Tep4 has been shown to act as an opsonin in the literature but it isn't directly demonstrated here. Results would be strengthened with addition of a western blot showing that Tep4 directly binds to Mab in the Drosophila model.
We agree with Reviewer #1 that an in vivo demonstration of Tep4 involvement in Mab internalization would bring a plus but, unfortunately, it is technically impossible in adult Drosophila. Indeed, one limitation of this model is the difficulty in collecting sufficient numbers of phagocytic plasmatocytes by dissection. Tep proteins have been described as opsonins by counting percentage of internalized microbes in S2 cells using RNAi (DOI: 10.1007/978-1-59745-204-5_24). In

RESPONSE TO REVIEWERS SPECTRUM00777-23
addition, even in larvae in which cell collection is less challenging, Tep4 involvement in P. aeruginosa opsonization was demonstrated by counting the fraction of bacteria internalized by plasmatocytes (DOI 10.15252/embr.201744880).
- Figure 3 and discussion -what is the proposed mechanism by which Tep4 depletion upregulates antimicrobial peptide expression?
The proposed mechanism is that Mab might be more extracellular in Tep4-deficient flies and thus would stimulate more Toll and Imd pathways. To test this hypothesis, we quantified the transcript levels of Peptidoglycan recognition protein (PGRP)-SB1 and -SD, which encode two of the main equivalents of bacterial specific Pattern Recognition Receptors (PRR) in Drosophila. We found that both transcripts were upregulated. We have included these results in the revised manuscript (page 10). These correspond to the new Figure S2A and S2B which you will find below.
- Figure 3B-D -it appears that these AMPs are minimally upregulated in the case of Mab infection of control ( Defensin is a major effector of the Drosophila humoral response when Mab is extracellular.
One hypothesis is that even without induction, the concentration of Defensin in the hemolymph is sufficient to kill Mab.
Indeed, AMPs can have concentrations ranging from 10 to 500 µM in the Drosophila hemolymph (DOI:

RESPONSE TO REVIEWERS SPECTRUM00777-23
10.1159/000086648). Knowing that Defensin can have bactericidal action in a beginning concentration of 1µM (our work, DOI: 10.1159/000086648), it is possible that even without induction, its basal amount can kill Mab, which could explain the increased sensitivity of Defensin mutants to extracellular Mab.
- Figure 4 -extent/efficacy of defensin overexpression should be shown or discussed.
We apologize for not including the graph in the original manuscript. We have added this in the revised version which corresponds to Figure S3 that you will find below.
-Lines 229-230 -inhibition of growth is not the same as direct bactericidal activity, results should be stated accordingly.
The corresponding section has been rewritten in the revised manuscript (page 9) based on these suggestions.

RESPONSE TO REVIEWERS SPECTRUM00777-23
-Stating that defensin is "sufficient" for Mab control may be overstating the conclusions. Would rephrase to indicate that it has an important role in host defense against Mab infection.
The corresponding section has been rewritten in the revised manuscript (page 9) based on these suggestions.
Minor critiques -Was a rough or smooth morphotype of Mab used? The discussion implies it was smooth but it is not otherwise discussed. Should at least be included in the materials & methods section. If smooth was used, were any studies attempted using rough morphotype?
We apologize for not including this information in our original manuscript. In this study, we only used the smooth morphotype. We have clarified this point in the revised manuscript by naming it S-M. abscessus. In the future, it would be interesting to test whether the rough morphotype behaves differently; however, in this study, we would like to focus only on the smooth morphotype .
-Lines 108-119 should be incorporated into the introduction rather than the results section We have included this in the Introduction section of the revised manuscript (page 11).
-Line 131 -it should be addressed why Relish mutant flies, but not any of the group B AMP mutants, had decreased survival.
As the production of Defensin is also dependent on the Imd pathway, Relish loss of function might impact Defensin production. The decreased survival of Imd mutant flies could be related to the latter consequence, as Defensin is important for controlling extracellular Mab. This part has been addressed in the Discussion section of the revised manuscript (page 13).
- Figure 2B -why is Mab growth increased in the Tep5 RNAi treatment at day 5? This should be addressed.
In the new Figure 2D (former Figure 2B), Mab growth is increased at day 5 in Tep5-RNAi compared to Tep4-RNAi and not compared to the untreated control. We apologize for having present the comparison Tep4-RNAi versus Tep5-RNAi at day 5 on the original Figure. We have added the non-significant comparison between Tep5-RNAi and untreated cells, as for the other time points to avoid confusion for the readers.

RESPONSE TO REVIEWERS SPECTRUM00777-23
- Figure 2D-F -should have each graph labeled with what bacteria is used.
The Figure has been modified accordingly.
- Figure 3I and J -should include a statistical comparison of Group A/+ and GroupA/+ & Tep4>RNAi Tep4 -are these significantly different or not?
These statistical comparisons have been added to the corresponding graphs. There is a significant difference for the Group A but not the Group C.

RESPONSE TO REVIEWERS SPECTRUM00777-23
Reviewer #2 (Comments for the Author): Touré et al., showed that AMPs were required to control extracellular Mab, and Defensin was found to be sufficient to kill extracellular Mab both in vitro and in vivo. The data suggest that Tep4-mediated opsonization of Mab allows its escape and resistance towards the Defensin bactericidal action in Drosophila.
We would like to thank Reviewer #2 for his/her insightful comments and constructive suggestions, which we address below.
Major comments: Important comment: The authors are advised to incorporate a DOI or other relevant reference information for the unpublished manuscript "Touré et al. in revision" throughout the entirety of their manuscript. This reference contains crucial information that is essential for comprehending the current manuscript and provides evidence for the hypothesis discussed in the article. Without the DOI, I cannot presently recommend either acceptance or rejection for the manuscript.
We agree with Reviewer #2 that the paper that was in revision contains crucial information for a full understanding of the current manuscript. The paper has now been published in PLOS Pathogens and is accessible under In combination with other data of the paper, this also suggests that Tep4 is most likely a major opsonin for Mab. These data are included in the revised manuscript (page 14) and in the new Figure 2A which you will find below.

RESPONSE TO REVIEWERS SPECTRUM00777-23
Q2. Figure 2A describe Tep4 involvement for internalization. However, it is not significant difference between control and Tep4 RNAi (~0.5 log10 reduction) albeit Figure 2C showed significant survival difference. Thus, I feel necessity to check another Teps involveness on this Mab internalization again.
The observed reduction in internalization is statistically significant, although we cannot exclude the involvement of other actors. However, the results presented in Figure 2A suggest The reduction we observed is also in the same orders of magnitude as this observed by the authors of the seminal paper describing the involvement of Teps in the internalization of different bacteria in S2 cells (DOI: 10.1371/journal.pbio.0040004). This is illustrated by the figure in this paper which you will find below.

RESPONSE TO REVIEWERS SPECTRUM00777-23
Q3. The Y-axis unit in Figure 2B should be clarified as the number of bacteria (CFU) or on a logarithmic scale if applicable.
The Y-axis in the Figure 2B corresponds to a logarithmic representation of the number of bacteria per milliliter. We have renamed it "Log (CFU/mL)" to avoid confusion.
Q4. The number of flies tested in Figure 3A is too small size. Only 5 flies look not enough for statistical analysis. The authors should use at least 20 flies for this experiment for better statistical analysis.
We have counted the bacterial loads of 20 flies per condition. This result is presented in the graph below and corresponds to Figure 3A in the revised manuscript.

RESPONSE TO REVIEWERS SPECTRUM00777-23
Q5. The authors should explain the exception of Diptericin showing a significant difference compared to other AMPs in Figure 3E. Furthermore, also need to explain about Drosomycin and Drosocin in Figure 3F and G.
We agree with Reviewer #2 that it is surprising that, in contrast to the other tested Imd-related AMPs, Diptericin was less induced in infected Tep4 mutants than in wild-type flies. This observation could be related to the isoform (Diptericin-A) that we amplified during qRT-PCR. We mentioned that this observation was surprising in the revised manuscript (page 10). However, since group B AMPs, to which Diptericin belongs, do not seem to affect M. abscessus even when the bacterium is extracellular, we do not feel it is necessary to investigate this difference in Diptericin regulation compared to other Imd-dependent AMPs.
The fact that Drosomycin is not upregulated on day 3 after infection with 10 CFU of M. abscessus is consistent with our previous work (DOI:10.1371/journal.ppat.1011257). Indeed, we only observed the induction of Drosomycin expression after infection with 1000 CFU.
Finally, although the difference is not huge, we observed a significant increase in the amount of Drosocin transcripts in the infected Tep4 mutant compared to that in the infected control. This is consistent with what we observed with other Imd-related AMPs except for Diptericin.

Minor comments:
Line 78: Please clarify what "Dm" stands for in the text.
We apologize for leaving this typeface in the manuscript. This has been removed from the revised manuscript.
Lines 79-84: The text in this section is difficult to understand and should be rewritten for clarity.
The section has been rewritten for better understanding (pages 4-5).

RESPONSE TO REVIEWERS SPECTRUM00777-23
The mutations have been correctly named in the revised manuscript (page 7).
Lines 125-131: It would be better provide information about gene names (single gene mutation) in an independent table to make it easier for readers to follow.
As requested, we have included a Table that recapitulates the different mutations in Figure 1.
Lines 179-183: The authors should move this information to the Discussion section and include it with lines 313-318 to avoid repetition.
This paragraph has been moved to the Discussion section accordingly.
The statistical analysis in Figure 3I and J should be rechecked, as there appears to be a >25% difference between Tep4>RNAi Tep4 and GroupA/+ & Tep4>RNAi Tep4, but the p-value is only <0.05.
We have checked this statistical analysis. The p-value is 0.0307. We believe that despite the difference of more than 25% on day 10, this "low" p-value may be related to the fact that the analysis takes into account the entire duration of the experiment. As observed in the figure, the difference only starts from day 6.

RESPONSE TO REVIEWERS SPECTRUM00777-23
Staff Comments: To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgibin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary.
Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. <b>Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. </b>" Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
May 5, 2023 1st Revision -Editorial Decision May 5, 2023 Dr. Fabienne Girard-misguich Universite de Versailles Saint-Quentin-en-Yvelines Versailles France Re: Spectrum00777-23R1 (Mycobacterium abscessus opsonization allows an escape from the Defensin bactericidal action in Drosophila) Dear Dr. Fabienne Girard-misguich: Congratulations! Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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