The Drosophila Baramicin polypeptide gene protects against fungal infection

  1. Mark Austin Hanson
  2. Lianne B. Cohen
  3. Alice Marra
  4. Igor Iatsenko
  5. Steven A. Wasserman
  6. Bruno Lemaitre

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Abstract

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A . We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana . Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response.

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  1. Version published to 10.1371/journal.ppat.1009846
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    Reply to the reviewers

    To whom it may concern:

    We are thank the reviewers for their kind assessment of our work and its potential impact. Here we have outlined key points that we plan to address during revisions.

    1. The erect wing story could be investigated a bit further. We agree the erect wing phenotype is intriguing, and will try to improve our understanding. We plan to use fat-body specific c564-Gal4 or BaraA-Gal4 to express UAS-BaraA and attempt to rescue the phenotype. In this way, we will also give insight into whether erect wing can be rescued by immune-tissue or BaraA-endogenous tissue effects. We will note that the cause of erect wing may be due to a lack of BaraA during development and/or during the immune response, which will require careful investigations in the future.
    2. The in vitro antifungal data are modest. We agree. We will perform additional experiments to further corroborate these data to increase confidence in the trends observed.
    3. The nature of the genetic backgro____unds is not clear. We will do our best to explain the genetic background complications in the main text. We use *w; *∆BaraA flies as an independent means of confirming isogenic data (and vice versa). We had to backcross the ∆BaraA mutation with an arbitrary genetic background prior to experiments to remove an off-site mutation that we detected in the antifungal gene *Daisho2 *(formerly IM14). As such, there is no appropriate wild-type control for these flies as the background is mixed. We include OR-R as a generic wild-type representative. OR-R flies survive bacterial infection like *w; ∆BaraA in multiple assays, and so we feel that different immune competences of the genetic backgrounds is unlikely to explain major susceptibilities to fungal infection. We have additional data for * bassiana R444 infection (Fig. 4C-D) with a second wild-type that we can include if desired, which shows similar trends when compared to *w; *∆BaraA. We will also perform additional experiments with newly-generated isogenic flies to increase confidence in the trends, and to better inform on interactions between BaraA and other immune effectors. For other minor points, we will be happy to make suggested changes to improve clarity of the figures or methodology.

    Best regards,

    Mark Hanson and Bruno Lemaitre

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Hanson et al. have set out to investigate the BaraA gene, and show that the gene encodes for several immune induced molecule (IM) peptides, namely IM10, IM12 (and its sub-peptide IM6), IM13 (and its sub-peptides IM5 and IM8), IM22, and IM24. Flies lacking BaraA are viable but susceptible to specific infections, notably by the entomopathogenic fungus Beauveria bassiana. Furthermore, they show that BaraA is antimicrobial and, when combined with the antifungal Pimaricin, it inhibits fungal growth. In principle, this is a nicely written paper with interesting findings. The authors show induction of BaraA with different micro-organisms and where BaraA is expressed, using a BaraA reporter. The exploration of the genomic area, showing the duplication of the BaraA locus is really nice work. Also, the survival experiments show quite clear phenotypes and therefore effects for BaraA.

    Major comments:

    Line 153, related results: Fold induction of BaraA is greater with E. coli (~50) than with C. albicans (~20) or M. luteus (~6) - any comments on this? Also, infection times with these microbes are different - some comments about BaraA kinetics? Based on Fig 1B, BaraA looks to be highly induced by E. coli, although in Fig 1C, after 60h, reporter induction by E. coli is much less than with M. luteus. Some clarification about the kinetics of BaraA in these different infection models is needed.

    Erect wing phenotype in males: This is a bit surprising finding/interpretation. I have also seen erect wings in E. faecalis-infected flies, but I am not sure now in which flies I saw this; I have never tried to quantify this nor made any notes about females/males in this context. I normally use Myd88 RNAi (VDRC #25399) as a control in my experiments, and if they were the ones showing the erect wing phenotype in a prevalent manner, they would also lack BaraA (which is dependent on the Toll pathway function). At the time of doing my experiments, I just interpreted this in the way that the flies looked "sick", they were lifting their wings up and walking around rather than flying. When monitoring my survival experiments, I assumed that the ones with wings up were the ones dying next (the sickest). What is your interpretation; are the flies still ok or very sick, when this erect wing starts to appear?

    Minor comments:

    Wording: In the intro, line 78: "Many of the genes that encode these components of the immune peptidic secretome have remained largely unexplored." - I would say "had remained" until recently - especially the quite recent Bomanin work and work with Daisho1 & 2 have brought about a lot of new information about this "immune peptidic secretome".

    Fig 1A: What is BaraA called in DeGregorio et al? Can't find it (easily) from their lists. Please write BaraA into the Fig 1A graph, to make it clearer. Also, write somewhere in the text or Figure legend what the gene is called in DeGregorio et al (CG33470? CG18278? something else?)

    Line 238 Reference to Supplementary data file 1: In the supplementary data files I downloaded, I can't see the files numbered as data files 1 and so on. Instead, there are folders (Fly stocks, NF-kappaB sites, Primers used) and the files have names. Please clarify that the supplement names match the text.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. State what audience might be interested in and influenced by the reported findings.

    I think the significance of this work is great for Drosophila immunity researchers. The nature and mode of action of many of the Toll pathway -induced peptides is not known, so more information on them is much appreciated by the field. Also, studying molecules with potential antimicrobial activities is also potentially interesting for wider audience.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    The main Drosophila immunity pathways are the Toll and the Imd pathway, and when activated, several immune effector genes are induced. In 2015, a group of Toll pathway target genes was identified by mass spectrometry, that the authors here call "the immune peptidic secretome". (Clemmons AW et al., PLoS Pathogens 2015). Many of these peptide genes have been uncharacterized, although emerging studies have shed light to these findings in the past three years (Lindsay SA et al J. Inn. Imm. 2018; Cohen LB et al. Front.Imm. 2020). This research brings about new information on yet uncharacterized peptides in this group.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Drosophila melanogaster, innate immunity, humoral immunity, cellular immunity Toll pathway, Imd pathway, immune-induced molecules
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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Hanson et al. investigated an antifungal gene they named BaraA, which codes for a protein that is proteolytically processed into 8 smaller peptides. BaraA expression is induced by Toll pathway signaling with minor input from the Imd pathway. It is expressed in the fat body upon immune challenge and expressed in other tissues such as head and eyes. Overexpression of BaraA increased the survival of animals defective in both IMD and Toll pathways. In vitro, the combination of the three major BaraA peptides displayed modest inhibitory effect on fungal pathogens when combined with the antifungal drug Pimaricin, however BaraA peptides alone showed little or no antifungal activity. BaraA deficient mutants showed little to no significant difference in bacterial resistance but appeared to show susceptibility to fungal infection; this fungal susceptibility was independent of the Bomanins. Male BaraA mutants also displayed an erected-wings phenotype when subjected to infection.

    There are 3 key findings:

    •    BaraA overexpression conferred protection against fungal infection.

    •    BaraA-derived peptides displayed antifungal activity in vitro in conjunction with Pimaricin, in vitro

    •    Loss of BaraA decreased fungal resistance.

    Major Concerns:

    The results from the overexpression experiments were clear. However, the second and third findings were less convincing.

    •    The cocktail of IM10-like BaraA peptides showed significant synergy with Pimaricin in killing C. albicans at only one dose out of the five tested, and this combination has modest (19-29%) inhibition on hyphae growth of B. bassiana. The in vitro antifungal experiments might be more compelling if other fungi were examined and/or combinations with other antifungals were investigated, where synergy might be more robust.

    •    The most problematic issue with this data is the control of genetic background in the study of the BaraA mutant strains.  Much of the survival data compares mutant strains (BaraA and/or Bom∆) with Oregon-R as a wildtype.  As best we can tell, the BaraA and Bom strains are not in the genetic background and neither is particularly similar to OR-R.  If the authors can justify the use of OR-R as the wildtype control for these experiments, they should do so explicitly.  Otherwise, these experiments are very difficult to interpret.  This issue is highlighted by other data, where genetic background is carefully controlled, in the iso-w background, and the survival phenotypes are much more mild, and do reach significance is some infections, by log-rank analysis.   All experiments should be performed in this controlled background to enable firm conclusions and interpretations.

    Minor comments:

    •    Figure 1A mined data from a previous published study, which is acceptable, but this data presentation lacks proper description of the methodology, reproducibility, and statistics.

    •    The authors need to clarify the condition of the flies in Figures 1D to G (as well as S1C and D).  Infected?  Baseline?  It is not clear.

    •    There is no visualization of the genomic location of the BaraA deletion, which should be added to figure 2C.

    •    The authors should include the full genotype information for the Bloomington stocks, since the BL numbers may change over time.

    •    In Figure 2C, the authors should include some information about which lines possess the single BaraA locus and which lines have the duplication event.

    •    The author should elaborate on what is known about Dso2 and how the aberrant Dso2 locus might affect their assays. The info here is incomplete and confusing.

    •    Does the Ecc15 strain used in the paper innately resist Ampicillin? If yes, then the result of Ecc15 resisting the combination of IM cocktail and Ampicillin does not reveal much.

    •    It is unclear what the concentration of pimaricin was used for Figure 3E.

    •    The authors should include a clear genetic explanation for their conclusion that BaraA and Bomanins function independently.  The text describing this double mutant analysis could be more informative.

    •    BaraA overexpression significantly improved female survival against M. luteus (Figure S4C, p=0.006), this is interesting but not mentioned in the text.

    •    The author should be clear and consistent about the pathogen source (lab grown vs. commercial) and method of infection (natural infection vs. septic injury).  The authors should explain the difference in virulence between different infection models and methods.

    •    The sex-specific erected wings phenotype is interesting, but does not contribute to the overall significance of the manuscript. The authors should consider moving Figure 6 to the supplement.

    Significance

    This work is a potential step in characterizing the immune effectors downstream of the Toll pathway that contribute to the Drosophila defense against fungal pathogens. These effectors so far have not been characterized and understood. We are familiar with the Toll pathway and its effectors, but in no way are experts.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors use the fruitfly Drosophila melanogaster as a model to study innate immunity. In this manuscript, they study the effects of a set of antimicrobial peptides (AMPs) that are produced by furin cleavage of a larger precursor (Baramicin A, BaraA). Bara A is immune-induced in a Toll-dependent manner and has antifungal activity. Somewhat in line with expression in non-immune tissues, BaraA mutants show ab erect-wing phenotype in males.

    Major comments:

    The experiments are well-presented in a reproducible and statistically sound way. In particular care is taken to control effects of the genetic background. The immune phenotype of BaraA mutants is somewhat subtle but convincing and in line with recent findings by the same authors that some of the recently created CRISPR/Cas mutants in antimicrobial peptides have broader effects while others target intruders in a more specific manner or in combination with other AMPs. These are very relevant studies, which provide a balanced view of innate immunity in particular AMP action. I have one comment about the (BarA dependent, male-specific) erect wing phenotype: this is an interesting observation, which could stimulate work by others, I guess this is one reason why it was included in the manuscript. On its own it stands out a bit in the manuscript since in contrast to other parts, where insight into the underlying mechanisms is provided, this is not the case for the erect wing phenotype. The authors speculate about the non-immune expression, which may be responsible. One might use tissue-specific knockdown or rescue to check up on this (wing muscle or nervous system). This would be cost effective but delay publication for a few months. It depends a bit on the respective journal policy and the plans for further investment of the groups involved whether the phenotype is considered part of BaraA pleiotropism (which I could buy) or is considered too descriptive and should be used later for a later publication. Along similar lines, while sex-specific immune phenotypes are highly interesting, they open up many discussions about the underlying causes, both proximal and ultimate.

    Minor comments:

    The experiments look sound and previous work is mentioned sufficiently. The experimental design and results are easy to follow. I have mentioned some concerns about the erect wing phenotype (see above). Is there any evidence for metabolic regulation of BaraA (TF binding sites for example) in particular in the promoter fragment used for the reporter line? Did any of the fat body drivers show the same effect as the ubiquitous actin driver (this would increase specificity).
    Why was pimaricin used, it seems presently as a representative of membrane-active antifungal drugs, which BaraA peptides are likely not. Still, using combinations with other insect (Drosophila) antifungal AMPs would be more physiological, maybe this was tried and did not work, but should still be discussed. Or do the authors want to imply that physiologically the Daisho peptides or Bomanins have this effect? Perhaps elaborate on this.

    In Fig. 1: part H is missing although mentioned in the legend.

    In the abstract:

    it should be more clearly mentioned that the erect wing phenotype was observed in the mutants. line 27 and 28, replace one "characterized" line 28: contribute line 33: entomopathogenic

    other places:

    line 68: AMPs

    Significance

    Significance:

    The evolutionary relevance and therapeutic potential of AMP synergism is an emerging topic both within insect immunity, innate immunity in general and its use in patient treatment [1, 2]. The latter aspect may be interesting to justify the use of pimaricin. Thus, the work presented here in combination with previous work from the authors leads to a more balanced view of the action of insect AMPs (the authors call that the logic of the Drosophila effector response) with implications for human innate immunity and perhaps even therapy of diseases. Therefore, the data will be interesting for a broad audience. The use of models such as Drosophila, which can be manipulated in a targeted manner has provided insights that are beyond the study of single AMPs in vitro. Still, using overexpression as done in some cases here should be interpreted cautiously and should - if available - compared to data on in vivo concentration of AMPs (the authors try to derive estimates from the MS data), which may be difficult in case there are large local differences.

    My own field:

    primarily insect immunity with a background in mammalian immunity, although I am not able to keep up with all recent development in mammalian immunity.

  6. Version published to 10.1101/2020.11.23.394148v3 on bioRxiv
  7. Version published to 10.1101/2020.11.23.394148v2 on bioRxiv
  8. Version published to 10.1101/2020.11.23.394148v1 on bioRxiv