The function of Cecropins were not assessed in this manuscript. We also assessed the role of AMPs in the melanization response, wound clotting, and hemocyte populations. Hemocyte counting i. Altogether, our study suggests that Drosophila AMPs are primarily immune effectors, and not regulators of innate immunity. A Chromosomal locations of AMP genes that were deleted.
Expression normalized with iso w -UC set to a value of 1. Pellet densities are reported for all systemic infections in OD at nm. This prompted us to explore the role of antimicrobial peptides in defence against Gram-positive bacteria and fungi. Collectively, our data demonstrate that AMPs are major immune effectors in defence against Gram-negative bacteria and have a less essential role in defence against bacteria and fungi.
Having shown that AMPs as a whole significantly contribute to fly defence, we next explored the contribution of individual peptides to this effect. To tackle this question in a systematic manner, we performed survival analyses using fly lines lacking one or several AMPs, focusing on pathogens with a range of virulence that we previously showed to be sensitive to the action of AMPs.
This includes the yeast C. Given seven independent AMP mutations, over combinations of mutants are possible, making a systematic analysis of AMP interactions a logistical nightmare. Therefore, we designed an approach that would allow us to characterize their contributions to defence by deleting groups of AMPs. To this end, we generated three groups of combined mutants: A flies lacking Defensin Group A ; Defensin is regulated by Imd signalling but is primarily active against Gram-positive bacteria in vitro Imler and Bulet, C Flies lacking the two antifungal peptide genes Metchnikowin and Drosomycin Group C, mostly regulated by the Toll pathway.
By screening these seven genotypes as well as individual mutants, we were able to assess potential interactions between AMPs of different groups, as well as decipher the function of individual AMPs. We first applied this AMP-groups approach to infections with the relatively avirulent yeast C. We have no explanation for this interaction, but this could be due to i a better canalization of the immune response by preventing the induction of ineffective AMPs, ii complex biochemical interactions amongst the AMPs involved affecting either the host or pathogen or iii differences in genetic background generated by additional recombination.
We then investigated the individual contributions of Metchnikowin and Drosomycin to survival to C. We observed that Group C deficient flies eventually succumb to uncontrolled C. We next analyzed the contribution of AMPs in resistance to infection with the moderately virulent Gram-negative bacterium P. Thus, we again observed a better survival rate with the co-occurring loss of Group A and C peptides see possible explanation above. In this case, Group A flies were susceptible while AC flies were not. At this time point, most AMP mutants had significantly higher bacterial loads compared to wild-type flies.
Collectively, the use of various compound mutants reveals that several Imd-responsive AMPs, notably Drosocin, Attacins, and Diptericins, jointly contribute to defence against P. Use of individual mutant lines, however, revealed a pattern overtly different from P. This indicates that a full transcriptional output of Diptericin is required over the course of the infection to resist P. Altogether, our results suggest that only the Diptericin gene family, amongst the many AMPs regulated by the Imd pathway, provides the full AMP-based contribution to defence against this bacterium.
Bacterial counts confirm that the susceptibility of these Diptericin mutants arises from an inability of the host to suppress bacterial growth Figure 5C. Collectively, our study shows that Diptericins are critical to resist P. An exclusive role for Diptericins was also found for the more virulent P. In the course of our exploration of AMP-pathogen interactions, we identified another highly specific interaction between E.
Use of compound mutants revealed that alone, Group B flies were already susceptible to E. C By 18hpi, bacterial loads in individual Drosocin mutants or Rel E20 flies are significantly higher than wild-type. E Drosocin mutants in an alternate genetic background yw are susceptible to E. We chose to further explore the AMPs deleted in Group B flies, as alone this genotype already displayed a strong susceptibility.
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We confirmed the high susceptibility of Drosocin mutant flies to E. Thus, we recovered two highly specific AMP-pathogen interactions: Diptericins are essential to combat P. Despite the recent emphasis on innate immunity, little is known on how immune effectors contribute individually or collectively to host defence, exemplified by the lack of in depth in vivo functional characterization of Drosophila AMPs.
Taking advantage of new gene editing approaches, we developed a systematic mutation approach to study the function of Drosophila AMPs. With seven distinct mutations, we were able to generate a fly line lacking 10 AMPs that are known to be strongly induced during the systemic immune response. Using a systemic mode of infection that induces AMP expression in the fat body and hemocytes, we found that most flies lacking a single AMP family exhibited a higher susceptibility to certain pathogens consistent with their in vitro activity. In most cases, the susceptibility of single mutants was slight, and the contribution of individual AMPs could be revealed only when combined to other AMP mutations as illustrated by the susceptibility of Drosocin, Attacin, and Diptericin combined mutants to P.
Thus, the use of compound rather than single mutations provides a better strategy to decipher the contribution of AMPs to host defence. Future studies should investigate the role of AMPs in these local epithelial immune responses. However, a method of deciphering the contributions of the different downstream effectors to the specificity of these pathways remained out of reach, as mutations in these immune effectors were lacking.
Our study shows that AMPs contribute greatly to resistance to Gram-negative bacteria. It would be interesting to know if the specificity of AMPs to primarily combatting Gram-negative bacteria is also true in other species. Based on our study and Clemmons et al. Thus, the susceptibility of these two pathways to different sets of microbes not only reflects specificity at the level of recognition, but can now also be translated to the activities of downstream effectors. Our collection of AMP mutant fly lines placed us in an ideal position to investigate AMP interactions in an in vivo setting.
Synergistic loss of resistance may arise in two general fashions: first, co-operation of AMPs using similar mechanisms of action may breach a threshold microbicidal activity whereupon pathogens are no longer able to resist. This may be the case for the synergistic effect of Diptericins and Attacins against P. It is commonly thought that the innate immune response lacks the specificity of the adaptive immune system, which mounts directed defences against specific pathogens.
Here, we provide a demonstration in an in vivo setting that such a strategy may actually be employed by the innate immune system. Remarkably, we recovered not just one, but two examples of exquisite specificity in our laborious but relatively limited assays. Diptericin has previously been highlighted for its important role in defence against P. We also show that Drosocin is specifically required for defence against E.
This striking finding validates previous biochemical analyses showing Drosocin in vitro activity against several Enterobacteriaceae, including E. However, we find support for an alternative hypothesis that suggests AMP diversity may be due to highly specific interactions between AMPs and subsets of pathogens that they target. Nevertheless, it seems these immune effectors play non-redundant roles in defence. By providing a long-awaited in vivo functional validation for the role of AMPs in host defence, we also pave the way for a better understanding of the functions of immune effectors.
Our approach of using multiple compound mutants, now possible with the development of new genome editing approaches, was especially effective to decipher the logic of immune effectors. Finally, our set of isogenized AMP mutant lines provides long-awaited tools to decipher the role of AMPs not only in systemic immunity, but also in local immune responses, and the various roles that AMPs may play in aging, neurodegeneration, anti-tumor activity, regulation of the microbiota and more, where disparate evidence has pointed to their involvement.
P-element mediated homologous recombination according to Baena-Lopez et al.
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Plasmids were provided by Mickael Poidevin. It was subsequently shown that Cec SK6 is a complex aberration at the Cecropin locus that retains a wild-type copy of the Cecropin cluster. These bacterial pellets were diluted to the desired optical density at nm OD as indicated. Aspergillus fumigatus was grown at room temperature on Malt Agar, and spores were collected in sterile PBS rinses, pelleted by centrifugation, and then resuspended to the desired OD in PBS.
The entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae were grown on Malt Agar at room temperature until sporulation. For infections with B. At least two replicate survival experiments were performed for each infection, with 20—35 flies per vial on standard fly medium without yeast. Survivals were scored twice daily, with additional scoring at sensitive time points. Direct comparisons were performed using Log-Rank tests in Prism seven software. The effect size and direction is included as the CoxPH hazard ratio HR where relevant, with a positive effect indicating increased susceptibility.
Total sample size N is given for each experiment as indicated. The native Drosophila microbiota does not readily grow overnight on LB, allowing for a simple assay to estimate bacterial load. Flies were infected with bacteria at the indicated OD as described, and allowed to recover. Bacterial plates were incubated overnight, and colony-forming units CFUs were counted manually.
For C. Flies were infected by pricking flies with a needle dipped in a pellet of either E. Reverse transcription was performed using 0. Values represent the mean from three replicate experiments. Error bars represent one standard deviation from the mean. Primers used in this study can be found in Supplementary file 1. Two methods were used to collect hemolymph from adult flies: in the first method, pools of five adult females were pricked twice in the thorax and once in the abdomen.
Peptide expression was visualized as described in Uttenweiler-Joseph et al. Both methods produced similar results, and representative expression profiles are given. In brief, flies or L3 larvae were pricked, and the level of melanization was assessed at the wound site. We used FACS sorting to count circulating hemocytes. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wendy Garrett as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Mathias Hornef Reviewer 2 ; Lora V Hooper Reviewer 3. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
It has been agreed that the work is of a high standard and that further experimentation is not required. Please address the points of interpretation and the necessary corrections that are included in the critiques below. Please also consider the suggestions of the reviewers for improvements in the manuscript. In the first large-scale loss-of-function study of antimicrobial peptide AMP genes in Drosophila melanogaster , the authors demonstrate a marked difference in the role of AMPs in the two innate immune pathways.
In the Imd pathway, AMPs are essential. None of the AMPs, individually or collectively, is necessary for survival against Gram positive bacteria and fungi. The experiments are carried out to high standards, thorough, and worthy of publication in this journal.
Nevertheless, there are several problems in presentation and interpretation:. Certain AMPs, such as PGLa and magainin-2, act synergistically: each alone is modestly effective but the two together have a potent antimicrobial activity. Here, there is no evidence for synergy between AMPs. The assay is for survival, which is not a quantitative measure of activity.
Consider two fly AMPs. Furthermore, in scenario two, the AMPs could have strong biochemical synergy, with no antimicrobial activity and hence no survival unless the both gene products are present. Given the well-established biochemical definition of synergistic action for certain pairs of AMPs and the data here indicating redundancy, not synergy, among the Drosophila AMPs, the authors should not use "synergy" and "synergistic" to describe their findings.
Previously, the strongest genetic information on AMP function in Drosophila immunity was an elegant study from the same lab Tzou et al. They found that Defensin was protective against B. However, here they find that eliminating Defensin, Attacins, Drosomycin, and all other AMPs has only a slight effect on resistance to two of these three pathogens. The authors are encouraged to point out and discuss these differences. The authors report a "curious" fact: the effect on survival of eliminating a relevant set of AMP genes is ameliorated by eliminating a seemingly irrelevant set of AMP genes.
They offer three explanations, but overlook a fourth that seems probable. Pathogens and hosts share many molecular and cellular features. Specificity against "microbes" is therefore likely to be limited. Furthermore, AMPs are required at quite high concentrations. It could easily be, therefore, that AMPs in general, and certain AMPs in particular, can be toxic to their hosts by, for example, weakening host cell membranes.
The microbicidal activity of defensins stems from the permeabilization of anionic lipid bilayers and the subsequent release of cellular contents and the destruction of the membrane's electrode potential Fig. Activities of antimicrobial peptides. As well as their antimicrobial function 1 , antimicrobial peptides have other potential roles in inflammation and infection 2,3. The mechanism of the antimicrobial activity is explained in the insert. After electrostatic interactions between the negatively charged bacterial wall and the positively charged peptides a , the peptide associates with the membranes, either by insertion as pores b or by forming carpet-like structures that lead to a destabilization of the membrane.
The sources 1 of antimicrobial peptides in the airways are epithelial cells and inflammatory cells. Defensins and LL have a feedback mediator function that targets these cell types 2,3 , influencing the release of mediators and other cellular processes such as proliferation and chemoattraction. The first step of the interaction between the cationic peptide and the anionic microbial cell membrane is thought to involve electrostatic attraction, which is inhibited by high concentrations of salt in the solution.
The second step is the permeabilization of the membrane. One mechanism of permeabilization is thought to involve the formation of ion pores. The existence of pores, their dimensions and electrical properties have been described for model bilayers and various cell types [ 41 , 42 ]. Additionally, defensin-related cell death has been related to interference with protein synthesis or DNA damage. Functional studies on antimicrobial activity have primarily been restricted to experiments in vitro with purified components. In fact, the evidence that defensins actually contribute to innate immunity in vivo is largely indirect.
In addition to their antimicrobial activity, defensins and cathelicidins can bind to lipopolysaccharide and inactivate the biological functions of this endotoxin. Bacterial resistance to antimicrobial peptides is a rare phenomenon. Mechanisms that result in the development of resistance involve modifications of outer cell wall components, such as lipopolysaccharide [ 45 ], teichoic acids [ 46 ], or phosphoicholine [ 47 ], and the modulation of efflux pumps [ 48 ].
Beside their role as endogenous antibiotics, antimicrobial peptides have functions in inflammation, wound repair, and regulation of the adaptive immune system Fig. Human neutrophil defensins have been described as being cytotoxic to various cell types [ 49 ], as inducing cytokine synthesis in airway epithelial cells [ 50 ], monocytes [ 51 ], and T lymphocytes [ 52 ], as increasing the release of SLPI from respiratory epithelial cells [ 53 ], and as decreasing intracellular glutathione concentration [ 54 ].
Further, they increase the binding of bacteria to epithelial cells [ 55 ] and induce the liberation of histamine from mast cells. On the basis of their activity in vitro , their patterns of expression and gene regulation, and their involvement in pathways of innate immunity, there is strong suggestive evidence that antimicrobial peptides serve as host defense substances not only by direct antimicrobial activity but also as mediators. Animal experiments with the use of a human bronchial xenograft model with the genetic depletion of hBD-1 by antisense oligonucleotides [ 17 ], overexpression of antimicrobial peptides in animal models of infection [ 30 ], or the above-mentioned experiments with matrilysin knockout mice [ 12 ] support this view.
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An assessment of the relative contribution of individual proteins or peptides to the host defense is difficult to accomplish. The concentrations of antimicrobial peptides and proteins at the site of action e. A functional analysis of purified peptides and proteins in vitro does not reflect the complexity of component interactions, such as synergism and antagonism between multiple substances. The story of cystic fibrosis CF research over the past decade has provided important lessons about the relation between a defect in an ion channel and a breach of the host defense system of the airways.
An obvious defect of the host defense system of the respiratory tract is evident from clinical studies and was evaluated in several models in vitro or ex vivo [ 17 , 59 ]. The link between the defect in ion transport and decreased host defense is less obvious and remains speculative at present reviewed in [ 60 ]. Antimicrobial peptides might have a role in this pathogenesis, either by being inactivated by increased salt concentration in secretions of CF airways or by being absent from the ASF owing to alteration of the secretory apparatus caused by the dysfunctional CF transmembrane conductance regulator CFTR.
Beside their host defense function during infections, the proinflammatory activity of antimicrobial peptides is likely to have negative consequences too. On the basis of the activities of antimicrobial peptides, it is obvious that these substances affect the inflammatory process Fig.
Owing to the lack of detailed knowledge of their functional spectrum in vivo , it is hard to decide which peptide antibiotic in which concentration results in a proinflammatory or anti-inflammatory activity. On the one hand, defensins attract inflammatory cells such as neutrophils, B-cells, and macrophages, and activate these and other cell types, including epithelial cells.
Defensins might lead to an imbalance of the redox system by reducing glutathione levels in epithelial airway cells and might disturb the protease-antiprotease system by binding to proteinase inhibitor serpin family members. On the other hand, defensins might also exhibit anti-inflammatory activities by induction of the secretion of IL [ 61 ] or SLPI [ 53 ], or by facilitating the binding of microorganisms to epithelia with subsequent clearance of the microorganisms by a bactericidal activity of the cell.
It is also likely that antimicrobial peptides in the airway secretions modulate the cytokine profile of lymphocytes towards T helper type 1 or 2 cells. This could have a direct effect on the development of bronchial hyper-responsiveness. Additionally, defensins have been shown to release histamine from mast cells and might induce hyper-responsiveness by their cationic charge [ 62 ]. These experimental results do not draw a complete picture of the functions that antimicrobial peptides have in inflammatory or infectious disease; however, they indicate that they fulfil not only an epiphenomenal bystander role but are linked to the underlying pathogenetic processes.
The intriguing idea of developing antimicrobial peptides as innovative antibiotics has been followed up by several biotechnological companies. With the use of protein-biochemical methods and recombinant DNA technology, the structures of naturally occurring peptides serve as starting points for the development of new drugs. Several derivatives of antimicrobial peptides have been through the pharmaceutical process, including human phase I-III studies. The use of human antimicrobial peptides as drugs is restricted so far by the still unknown biological function of these molecules and the high costs of the generation of sufficient amounts.
On the basis of their functions that have been elucidated so far, antimicrobial peptides might not serve only as antibiotics, but also as modulators of inflammation or anti-LPS medication. Antimicrobial peptides have emerged as effector substances of the innate immune system involving activities not only as endogenous antibiotics but also as mediators of inflammation.
Several important topics will have to be addressed in the future:. The identification of novel antimicrobial peptides. It is likely that human families of antimicrobial peptides consist of multiple molecules. Progress in the Human Genome Project will also reveal ways of shortcutting conventional bioscreening procedures for the identification of host defense substances.
Analysis of the biologically relevant functions of antimicrobial peptides. Beside experiments in vitro that give the first molecular insight into the function of peptide antibiotics, a broader approach involving genetic animal models is necessary to interpret results in vitro in the context of a whole organism. Evaluation of the function of antimicrobial peptides in airway and other diseases will provide insights into the corresponding pathogenesis. Development of antimicrobial peptides as drugs.
Studying the biology of antimicrobial peptides might permit the development of novel therapeutics for infectious or inflammatory diseases. Fearon D, Locksley R: The instructive role of innate immunity in the aquired immune response. Science , 50— N Engl JMed , — In Disorders of the Respiratory Tract in Children. Philadelphia: Saunders; , — Curr Opin Immunol , 13— New Engl JMed , — Infect Immun , — Cell , — Natural peptide antibiotics of human neuitrophils.
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FEBS Lett , 1—5. Eur J Biochem , — J Biol Chem , — Isolation, characterization, primary structure, and fungistatic effects on Candida albicans. Am J Hum Genet , — Arch Oral Biol , — J Immunol , — Biochem Biophys Res Commun , — Immunol Rev , 27— Lemaitre B, Reichhart JM, Hoffmann JA: Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Mechanism of bactericidal activity. Biochemistry , — J Leukoc Biol , — Eur Respir J , s.
J Infect Dis , — J Leukoc Biol , 9— J Allergy Clin Immunol , — Hyperresponsiveness due to airway inflammation. Boman HG: Peptide antibiotics and their role in innate immunity. Annu Rev Immunol , 61— Download references. Correspondence to Robert Bals. Reprints and Permissions. Search all BMC articles Search. Abstract One component of host defense at mucosal surfaces seems to be epithelium-derived antimicrobial peptides. Introduction The survival of a multicellular organism in a world laden with microorganisms depends on a network of host defense mechanisms, involving several levels of interacting systems.
Several possible consequences result from the contact of microorganisms with host tissue: 1. Antimicrobial peptides as effector substances of the innate immune system A first line of defense against pathogenic insult is called the innate immune system, which is followed by acquired immune responses associated with the activation of T and B cells aimed against specific antigens [ 1 , 2 ]. Figure 1.
Full size image. Basic biology of antimicrobial peptides Families of human antimicrobial peptides Most antimicrobial peptides are cationic polar molecules with spatially separated hydrophobic and charged regions.