Abstract
Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading microorganisms. These short, cationic peptides have been implicated in many biological processes, primarily involving innate immunity. In vitro studies have shown AMPs kill bacteria and fungi at physiological concentrations, but little validation has been done in vivo. We utilised CRISPR gene editing to delete all known immune inducible AMPs of Drosophila, namely: 4 Attacins, 4 Cecropins, 2 Diptericins, Drosocin, Drosomycin, Metchnikowin and Defensin. Using individual and multiple knockouts, including flies lacking all 14 AMP genes, we characterize the in vivo function of individual and groups of AMPs against diverse bacterial and fungal pathogens. We found that Drosophila AMPs act primarily against Gram-negative bacteria and fungi, acting either additively or synergistically. We also describe remarkable specificity wherein certain AMPs contribute the bulk of microbicidal activity against specific pathogens, providing functional demonstrations of highly specific AMP-pathogen interactions in an in vivo setting.
Introduction
While innate immune mechanisms were neglected during the decades where adaptive immunity captured most of the attention, they have become central to our understanding of immunology. Recent emphasis on innate immunity has, however, mostly focused on the first two phases of the immune response: microbial recognition and associated downstream signaling pathways. In contrast, how innate immune effectors individually or collectively contribute to host resistance has not been investigated to the same extent. The existence of multiple effectors that redundantly contribute to host resistance has hampered their functional characterization by genetic approaches1. The single mutation methodology that still prevails today has obvious limits in the study of immune effectors, which often belong to large gene families. As such, our current understanding of the logic underlying the roles of immune effectors is only poorly defined. As a consequence, the key parameters that influence host survival associated with a successful immune response are not well characterized. In this paper, we harnessed the power of the CRISPR gene editing approach to study the function of Drosophila antimicrobial peptides in host defence both individually and collectively.
Antimicrobial peptides (AMPs) are small, cationic, usually amphipathic peptides that contribute to innate immune defence in plants and animals2–4. They display potent antimicrobial activity in vitro by disrupting negatively-charged microbial membranes, but AMPs can also target specific microbial processes5–7. Their expression is induced to very high levels upon challenge to provide microbicidal concentrations in the μM range. Numerous studies have revealed unique roles that AMPs may play in host physiology, including anti-tumour activity8,9, inflammation in aging10–12, involvement in memory13,14, mammalian immune signaling15,16, wound-healing17,18, regulation of the host microbiota19,20, tolerance to oxidative stress21,22, and of course microbicidal activity1,2,23. The fact that AMP genes are immune inducible and expressed at high levels has led to the common assumption they play a vital role in the innate immune response24. However, little is known in most cases about how AMPs individually or collectively contribute to animal host defence. In vivo functional analysis of AMPs has been hampered by the sheer number and small size of these genes, making them difficult to mutate with traditional genetic tools (but e.g. see25,26).
Since the first animal AMPs were discovered in silk moths27, insects and particularly Drosophila melanogaster have emerged as a powerful model for characterizing their function. There are currently seven known families of inducible AMPs in D. melanogaster. Their activities have been determined either in vitro by using peptides directly purified from flies or produced in heterologous systems, or deduced by comparison with homologous peptides isolated in other insect species: Drosomycin and Metchnikowin show antifungal activity28,29; Cecropins (four inducible genes) and Defensin have both antibacterial and some antifungal activities30–33; and Drosocin, Attacins (four genes) and Diptericins (two genes) primarily exhibit antibacterial activity6,34–37. In Drosophila, these AMPs are produced either locally at various surface epithelia in contact with environmental microbes38–40, or secreted systemically into the hemolymph, the insect blood. During systemic infection, these 14 antimicrobial peptides are strongly induced in the fat body, an organ analogous to the mammalian liver.
The systemic production of AMPs is regulated at the transcriptional level by two NF-kB pathways, the Toll and Imd pathways, which are activated by different classes of microbes. The Toll pathway is predominantly responsive to Gram-positive bacteria and fungi, and accordingly plays a major role in defence against these microbes. In contrast, the Imd pathway is activated by Gram-negative bacteria and a subset of Gram-positive bacteria with DAP-type peptidoglycan, and mutations affecting this pathway cause profound susceptibility to Gram-negative bacteria41,42. However, the expression pattern of AMP genes is complex as each gene is expressed with different kinetics and can often receive transcriptional input from both pathways42,43. This ranges from Diptericin, which is tightly regulated by the Imd pathway, to Drosomycin, whose expression is mostly regulated by the Toll pathway41, except at surface epithelia where Drosomycin is under the control of Imd signaling44. While a critical role of AMPs in Drosophila host defence is supported by transgenic flies overexpressing a single AMP33, the specific contributions of each of these AMPs has not been tested. Indeed loss-of-function mutants for most AMP genes were not previously available due to their small size, making them difficult to mutate before the advent of CRISPR/Cas9 technology. Despite this, the great susceptibility to infection of mutants with defective Toll and Imd pathways is commonly attributed to the loss of the AMPs they regulate, though these pathways control hundreds of genes awaiting characterization42. Strikingly, Clemmons et al.45 recently reported that flies lacking a set of uncharacterized Toll-responsive peptides (named Bomanins) succumb to infection by Gram-positive bacteria and fungi at rates similar to Toll-deficient mutants45. This provocatively suggests that Bomanins, and not AMPs, might be the predominant effectors downstream of the Toll pathway; yet synthesized Bomanins do not display antimicrobial activity in vitro46. Thus, while today the fly represents one of the best-characterized animal immune systems, the contribution of AMPs as immune effectors is poorly defined as we still do not understand why Toll and Imd pathway mutants succumb to infection.
In this paper, we took advantage of recent gene editing technologies to delete each of the known immune inducible AMP genes of Drosophila. Using single and multiple knockouts, as well as a variety of bacterial and fungal pathogens, we have characterized the in vivo function of individual and groups of antimicrobial peptides. We reveal that AMPs can play highly specific roles in defence, being vital for surviving certain infections yet dispensable against others. We highlight key interactions amongst immune effectors and pathogens and reveal to what extent these defence peptides act in concert or alone.
Results
Generation and characterization of AMP mutants
We generated null mutants for the fourteen Drosophila antimicrobial peptide genes that are induced upon systemic infection. These include five single gene mutations affecting Defensin (DefSK3), Attacin C (AttCMi), Metchnikowin (MtkR1), Attacin D (AttDSK1) and Drosomycin (DrsR1) respectively, and three small deletions removing both Diptericins DptA and DptB (DptSK1), the four Cecropins CecA1, CecA2, CecB, and CecC (CecSK6) and the gene cluster containing Drosocin, and Attacins AttA & AttB (Dro-AttABSK2). All mutations/deletions were made using the CRISPR editing approach with the exception of Attacin C, which was disrupted by insertion of a Minos transposable element47, and the Drosomycin and Metchnikowin deletions generated by homologous recombination (Fig. 1A). To disentangle the role of Drosocin and AttA/AttB in the Dro-AttABSK2 deletion, we also generated an individual Drosocin mutant (DroSK4); for complete information, see Figure S1. We then isogenized these mutations for at least seven generations into the w1118 DrosDel isogenic genetic background48 (iso w1118). Then, we recombined these eight independent mutations into a background lacking all 14 inducible AMPs referred to as “ΔAMPs.” ΔAMPs flies were viable and showed no morphological defects. To confirm the absence of AMPs in our ΔAMPs background, we performed a MALDI-TOF analysis of hemolymph from both unchallenged and immune-challenged flies infected by a mixture of Escherichia coli and Micrococcus luteus. This analysis revealed the presence of peaks induced upon challenge corresponding to AMPs in wild-type but not ΔAMPs flies. Importantly it also confirmed that induction of most other immune-induced molecules (IMs)49, was unaffected in ΔAMPs flies (Fig. 1B). Of note, we failed to observe two IMs, IM7 and IM21, in our ΔAMPs flies, suggesting that these unknown peptides are secondary products of AMP genes. We further confirmed that Toll and Imd NF-ĸB signaling pathways were intact in ΔAMPs flies by measuring the expression of target genes of these pathways (Fig. 1C-D). This demonstrates that Drosophila AMPs are not signaling molecules required for Toll or Imd pathway activity. We also assessed the role of AMPs in the melanization response, wound clotting, and hemocyte populations. After clean injury, ΔAMPs flies survive as wild-type (Fig. 1 supplement A). We found no defect in melanization (χ2, p = .34, Fig. 1 supplement B) as both adults and larvae strongly melanize the cuticle following clean injury, (Fig. 1 supplement C). Furthermore, we visualized the formation of clot fibers ex vivo using the hanging drop assay and PNA staining50 in hemolymph of both wild-type and ΔAMPs larvae (Fig. 1 supplement D). Hemocyte counting (i.e. crystal cells, FACS) did not reveal any deficiency in hemocyte populations of ΔAMPs larvae (Fig. 1 supplement E, F, and not shown). Altogether, our study suggests that Drosophila AMPs are primarily immune effectors, and not regulators of innate immunity.
AMPs are essential for combating Gram-negative bacterial infection
We used these ΔAMPs flies to explore the role that AMPs play in defence against pathogens during systemic infection. We first focused our attention on Gram-negative bacterial infections, which are combatted by Imd pathway-mediated defence in Drosophila1. We challenged wild-type and ΔAMPs flies with six different Gram-negative bacterial species, using inoculation doses (given as 0D600) selected such that at least some wild-type flies were killed (Fig. 2). In our survival experiments, we also include Relish mutants (RelE20) that lack a functional Imd response and are known to be very susceptible to this class of bacteria51. Globally, ΔAMPs flies were extremely susceptible to all Gram-negative pathogens tested (Fig. 2, light blue plots). The susceptibility of AMP-deficient flies to Gram-negative bacteria largely mirrored that of RelE20 flies. For all Gram-negative infections tested, ΔAMPs flies show a higher bacterial count at 18 hours post-infection (hpi) indicating that AMPs actively inhibit bacterial growth, as expected of ‘antimicrobial peptides’ (Fig. 2 supplement A). Use of GFP-expressing bacteria show that bacterial growth in ΔAMPs flies radiates from the wound site until it spreads systemically (Fig. 2 supplement B,C). Collectively, the use of AMP-deficient flies reveals that AMPs are major players in resistance to Gram-negative bacteria, and likely constitute an essential component of the Imd pathway’s contribution for survival against these germs.
Bomanins and to a lesser extent AMPs contribute to resistance against Gram-positive bacteria and fungi
Previous studies have shown that resistance to Gram-positive bacteria and fungi in Drosophila is mostly mediated by the Toll pathway, although the Imd pathway also contributes to some extent41,43,52,53. Moreover, a deletion removing eight uncharacterized Bomanins (BomΔ55C) induces a strong susceptibility to both Gram-positive bacteria and fungi45, suggesting that Bomanins are major players downstream of Toll in the defence against these germs. This prompted us to explore the role of antimicrobial peptides in defence against Gram-positive bacteria and fungi. We first challenged wild-type and ΔAMPs flies with two lysine-type (E. faecalis, S. aureus) and two DAP-type peptidoglycan containing Gram-positive bacterial species (B. subtilis, L. innocua). We observed that ΔAMPs flies display only weak or no increased susceptibility to infection with these Gram-positive bacterial species, as ΔAMPs survival rates were closer to the wild-type than to späztle flies (spzrm7) lacking a functional Toll pathway (Fig. 2, orange plots). Meanwhile, BomΔ55C mutants consistently phenocopied spzrm7 flies, confirming the important contribution of these peptides in defence against Gram-positive bacteria45.
Next, we monitored the survival of ΔAMPs to the yeast Candida albicans, the opportunistic fungus Aspergillus fumigatus and two entomopathogenic fungi, Beauveria bassiana, and Metarhizium anisopliae. For the latter two, we used a natural mode of infection by spreading spores on the cuticle41. ΔAMPs flies were more susceptible to fungal infections with B. bassiana, A. fumigatus, and C. albicans, but not M. anisopliae (Fig. 2, yellow plots). In all instances, BomΔ55C mutants were as or more susceptible to fungal infection than ΔAMPs flies, approaching Toll-deficient mutant levels. 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.
A combinatory approach to explore AMP interactions
The impact of the ΔAMPs deletion on survival could be due to the action of certain AMPs having a specific effect, or more likely due to the combinatory action of coexpressed AMPs. Indeed, cooperation of AMPs to potentiate their microbicidal activity has been suggested by numerous in vitro approaches7,54,55, but rarely in an in vivo context56. 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. albicans and the Gram-negative bacterial species P. burhodogranariea, P. rettgeri, Ecc15, and E. cloacae. Given eight independent AMP mutations, over 250 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: flies lacking the primarily antibacterial Defensin and Cecropins (Group A, mostly regulated by the Imd pathway), flies lacking the antibacterial Proline-rich Drosocin, and the antibacterial Glycine-rich Diptericins and Attacins (Group B, regulated by the Imd pathway), and flies lacking the two antifungal peptide genes Metchnikowin and Drosomycin (Group C, mostly regulated by the Toll pathway). We then combined these three groups to generate flies lacking AMPs from groups A and B (AB), A and C (AC), or B and C (BC). Finally, flies lacking all three groups are our ΔAMPs flies, which are highly susceptible to a number of infections. 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.
Drosomycin and Metchnikowin additively contribute to defence against the yeast C. albicans
We first applied this AMP-groups approach to infections with the relatively avirulent yeast C. albicans. Previous studies have shown that Toll, but not Imd, contributes to defence against this fungus57,58. Thus, we suspected that the two antifungal peptides, Drosomycin and Metchnikowin, could play a significant role in the susceptibility of ΔAMPs flies to this yeast. Consistent with this, Group C flies lacking Metchnikowin and Drosomycin were more susceptible to infection (p < .001 relative to iso w1118) with a survival rate similar to ΔAMPs flies (Fig. 3A). Curiously, AC deficient flies that also lack Cecropins and Defensin survived better than Group C deficient flies (Log-Rank p = .014). 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, or iii) differences in genetic background generated by additional recombination. We then investigated the individual contributions of Metchnikowin and Drosomycin to survival to C. albicans. We found that both MtkR1 and DrsR1 individual mutants were somewhat susceptible to infection, but notably only Mtk; Drs compound mutants reached ΔAMPs levels of susceptibility (Fig. 3B). This cooccurring loss of resistance appears to be primarily additive (Mutant, Cox Hazard Ratio (HR), p-value: MtkR1, HR = +1.17, p = .008; DrsR1, HR = +1.85, p < .001; Mtk*Drs, HR = -0.80, p =.116). We observed that Group C deficient flies eventually succumb to uncontrolled C. albicans growth by monitoring yeast titre, indicating that these AMPs indeed act by suppressing yeast growth (Fig. 3C).
In conclusion, our study provides an in vivo validation of the potent antifungal activities of Metchnikowin and Drosomycin28,29, and highlights a clear example of additive cooperation of AMPs.
AMPs synergistically contribute to defence against P. burhodogranariea
We next analyzed the contribution of AMPs in resistance to infection with the moderately virulent Gram-negative bacterium P. burhodogranariea. We found that Group B mutants lacking Drosocin, the two Diptericins, and the four Attacins, were as susceptible to infection as ΔAMPs flies (Fig. 4A), while flies lacking the antifungal peptides Drosomycin and Metchnikowin (Toll-regulated, Group C) resisted the infection as wild-type. Flies lacking Defensin and the four Cecropins (Group A) showed an intermediate susceptibility, but behave as wild-type in the additional absence of Toll Group C peptides (Group AC). 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. Flies individually lacking Defensin or the four Cecropins were weakly susceptible to P. burhodogranariea (p = .022 and p = 0.040 respectively), however the interaction term between Defensin and the Cecropins was not significant (DefSK3*CecSK6, HR = -0.28, p = .382), indicating the susceptibility of Group A flies arises from additive loss of resistance (Figure 4 supplement A).
Following the observation that Group B flies were as susceptible as ΔAMPs flies, we sought to better decipher the contribution of each Group B AMP to resistance to P. burhodogranariea. We observed that mutants for Drosocin alone (DroSK4), or the DiptericinA/B deficiency were not susceptible to this bacterium (Fig. 4B). We additionally saw no marked susceptibility of Drosocin-Attacin A/B deficient flies, nor Attacin C or Attacin D mutants (not shown). Interestingly, we found that compound mutants lacking Drosocin and Attacins A, B, C, and D (Fig. 4B: ‘ΔDro, ΔAtt’), or Drosocin and Diptericins DptA and DptB (‘ΔDro, ΔDp’) displayed an intermediate susceptibility. Only the Group B mutants lacking Drosocin, all Attacins, and both Diptericins (ΔDro, ΔAtt, ΔDpt) phenocopied ΔAMPs flies (Fig. 4B), with synergistic interactions observed upon co-occurring loss of Attacins and Diptericins (ΔAtt*ΔDpt: HR = +1.45, p < .001). By 6hpi, bacterial titres of individual flies already showed significant differences in the most susceptible genotypes (Fig. 4C), though these differences were reduced by 18hpi likely owing to the high chronic load P. burhodogranariea establishes in surviving flies24; also see Fig. 2 supplement A.
Collectively, the use of various compound mutants reveals that several Imd-responsive AMPs, notably Drosocin, Attacins, and Diptericins, jointly contribute to defence against P. burhodogranariea infection. A strong susceptibility of Group B flies was also observed upon infection with Ecc15, another Gram-negative bacterium commonly used to infect flies59 (Fig. 4 supplement B).
Diptericins alone contribute to defence against P. rettgeri
We continued our exploration of AMP interactions using our AMP groups approach with the fairly virulent P. rettgeri (strain Dmel), a strain isolated from wild-caught Drosophila hemolymph60. We were especially interested by this bacterium as previous studies61,62 have shown a correlation between susceptibility to P. rettgeri and a polymorphism in the Diptericin A gene pointing to a specific AMP-pathogen interaction. Use of compound mutants revealed only loss of Group B AMPs was needed to reach the susceptibility of ΔAMPs and RelE20 flies (Fig. 5A). Use of individual mutant lines however revealed a pattern strikingly different from that P. burhodogranariea, as the sole Diptericin A/B deficiency caused susceptibility similar to Group B, ΔAMPs, and RelE20 flies (Fig. 5B,C). We further confirmed this susceptibility using a DptA RNAi construct (Fig. 5 supplement A, B). Moreover, flies carrying the DptSK1 mutation over a deficiency (Df(2R)Exel6067) were also highly susceptible to P. rettgeri (Fig. 5D). Interestingly, flies that were heterozygotes for DptSK1 or the Df(2R)Exel6067 that have only one copy of the two Diptericins were markedly susceptible to infection with P. rettgeri (Fig. 5D). This indicates that a full transcriptional output of Diptericin is required over the course of the infection to resist P. rettgeri infection (Fig. 5E). 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. To test this hypothesis, we generated a fly line lacking all the AMPs except DptA and DptB (ΔAMPs+Dpt). Strikingly, ΔAMPs+Dpt flies have the same survival rate as wild-type flies, further emphasizing the specificity of this interaction (Fig. 5B). Bacterial counts confirm that the susceptibility of these Diptericin mutants arises from an inability of the host to suppress bacterial growth (Fig. 5C).
Collectively, our study shows that Diptericins are critical to resist P. rettgeri, while they play an important but less essential role in defence against P. burhodogranariea infection. We were curious whether Diptericin’s major contribution to defence observed with P. rettgeri could be generalized to other members of the genus Providencia. An exclusive role for Diptericins was also found for the more virulent P. stuartii (Fig. 5 supplement C), but not for other Providencia species tested (P. burhodogranariea, P. alcalifaciens, P. sneebia, P. vermicola) (data not shown).
Drosocin is critical to resist infection with E. cloacae
In the course of our exploration of AMP-pathogen interactions, we identified another highly specific interaction between E. cloacae and Drosocin. Use of compound mutants revealed that alone, Group B flies were already susceptible to E. cloacae. Meanwhile, Group AB flies reached ΔAMPs levels of susceptibility, while Group A and Group C flies resisted as wild-type (Fig. 6A). The high susceptibility of Group AB flies results from a synergistic interaction amongst Group A and Group B peptides in defence against E. cloacae (A*B, HR = +2.55, p = .003).
We chose to further explore the AMPs deleted in Group B flies, as alone this genotype already displayed a strong susceptibility. Use of individual mutant lines revealed that mutants for Drosocin alone (DroSK4) or the Drosocin-Attacin A/B deficiency (Dro-AttABSK2), but not AttC, AttD, nor DptSK1 (not shown), recapitulate the susceptibility observed in Group B flies (Fig. 6B). At 18hpi, both DroSK4 and ΔAMPs flies had significantly higher bacterial loads compared to wild-type flies, while RelE20 mutants were already moribund with much higher bacterial loads (Fig. 6C). Indeed, the deletion of Drosocin alone drastically alters the fly’s ability to control the otherwise avirulent E. cloacae with inoculations at OD=200 (~39,000 bacteria, Fig. 6A-C) or even OD=10 (~7,000 bacteria, Fig. 6 supplement A).
We confirmed the high susceptibility of Drosocin mutant flies to E. cloacae in various contexts: transheterozygote flies carrying DroSK4 over a Drosocin deficiency (Df(2R)BSC858) that also lacks flanking genes including AttA and AttB ((Fig. 6D), the DroSK4 mutations in an alternate genetic background (yw, Fig. 6E), and, Drosocin RNAi (Fig. 6 supplement B,C). Thus, we recovered two highly specific AMP-pathogen interactions: Diptericins are essential to combat P. rettgeri infection, while Drosocin is paramount to surviving E. cloacae infection.
Discussion
A combinatory approach to study AMPs
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 eight distinct mutations, we were able to generate a fly line lacking the 14 AMPs that are inducible during the systemic immune response. A striking first finding is that ΔAMPs flies were perfectly healthy and have an otherwise wild-type immune response. This indicates that in contrast to mammals15, Drosophila AMPs are not likely to function as signaling molecules. Most flies lacking a single AMP family exhibited a higher susceptibility to certain pathogens consistent with their in vitro activity. We found activity of Diptericins against P. rettgeri, Drosocin against E. cloacae, Drosomycin and Metchnikowin against C. albicans, and Defensin and Cecropin against P. burhodogranariea (Fig. 4 supplement A). 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. burhodogranariea. Thus, the use of compound rather than single mutations provides a better strategy to decipher the contribution of AMPs to host defence.
AMPs and Bomanins are essential contributors to Toll and Imd pathway mediated host defence
The Toll and Imd pathways provide a paradigm of innate immunity, illustrating how two distinct pathways link pathogen recognition to distinct but overlapping sets of downstream immune effectors1,63. 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. Consistent with this, ΔAMPs flies are almost as susceptible as Imd-deficient mutants to most Gram-negative bacteria. In contrast, flies lacking AMPs were only slightly more susceptible to Gram-positive bacteria and fungal infections compared to wild-type flies, and this susceptibility rarely approached the susceptibility of Bomanin mutants. This may be due to the cell walls of Gram-negative bacteria being thinner and more fluid than the rigid cell walls of Gram-positive bacteria64, consequently making Gram-negative bacteria more prone to the action of pore-forming cationic peptides. 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.45, we can now explain the susceptibility of Toll and Imd mutants at the level of the effectors, as we show that mutations affecting Imd-pathway responsive antibacterial peptide genes are highly susceptible to Gram-negative bacteria while the Toll-responsive targets Drosomycin, Metchnikowin, and especially the Bomanins, confer resistance to fungi and Gram-positive bacteria. 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. It remains to be seen how Bomanins contribute to the microbicidal activity of immune-induced hemolymph, as attempts to synthesize Bomanins have not revealed direct antimicrobial activity46. It should also be noted that many putative effectors downstream of Toll and Imd remain uncharacterized, and so could also contribute to host defence beyond AMPs and Bomanins.
AMPs act additively and synergistically to suppress bacterial growth in vivo
In the last few years, numerous in vitro studies have focused on the potential for synergistic interactions of AMPs in microbial killing7,54,56,65–70. Our collection of AMP mutant fly lines placed us in an ideal position to investigate AMP interactions in an in vivo setting. While Toll-responsive AMPs (Group C: Metchnikowin, Drosomycin) additively contributed to defence against the yeast C. albicans, we found that certain combinations of AMPs have synergistic contributions to defence against P. burhodogranariea. 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 our observations of synergy amongst Diptericins and Attacins against P. burhodogranariea, as only co-occurring loss of both these related glycine-rich peptide families36 led to complete loss of resistance. Alternatively, synergy may arise due to complementary mechanisms of action, whereupon one AMP potentiates the other AMP’s ability to act. For instance, the action of the bumblebee AMP Abaecin, which binds to the molecular chaperone DnaK to inhibit bacterial DNA replication, is potentiated by the presence of the pore-forming peptide Hymenoptaecin71. Drosophila Drosocin is highly similar to Abaecin, including O-glycosylation of a critical threonine residue2,72, and thus likely acts in a similar fashion. Furthermore, Drosophila Attacin C is maturated into both a glycine-rich peptide and a Drosocin-like peptide called MPAC73. As such, co-occuring loss of Drosocin, MPAC, and other possible MPAC-like peptides encoded by the Attacin/Diptericin superfamily may be responsible for the synergistic loss of resistance in Drosocin, Attacin, Diptericin combined mutants.
AMPs can act with great specificity against certain pathogens
It is commonly thought that the innate immune response lacks the specificity of the adaptive immune system, which mounts directed defences against specific pathogens. Accordingly for innate immunity, the diversity of immune-inducible AMPs can be justified by the need for generalist and/or co-operative mechanisms of microbial killing. However, an alternate explanation may be that innate immunity expresses diverse AMPs in an attempt to hit the pathogen with a “silver bullet:” an AMP specifically attuned to defend against that pathogen. 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. rettgeri62, but it was previously unknown whether other AMPs may confer defence in this infection model. Astoundingly, flies mutant for all other inducible AMPs resisted P. rettgeri infection as wild-type, while only Diptericin mutants succumbed to infection. This means that Diptericins do not co-operate with other AMPs in defence against P. rettgeri, and are solely responsible for defence in this specific host-pathogen interaction. Moreover, +/DptSK1 heterozygote flies were nonetheless extremely susceptible to infection, demonstrating that a full transcriptional output over the course of infection is required to effectively prevent pathogen growth. A previous study has shown that ~7hpi appears to be the critical time point at which P. rettgeri either grows unimpeded or the infection is controlled24. This time point correlates with the time at which the Diptericin transcriptional output is in full-force41. Thus, a lag in the transcriptional response in DptSK1/+ flies likely prevents the host from reaching a competent Diptericin concentration, indicating that Diptericin expression level is a key factor in successful host defence.
We also show that Drosocin is specifically required for defence against E. cloacae. This striking finding validates previous biochemical analyses showing Drosocin in vitro activity against several Enterobacteriaceae, including E. cloacae37. As ΔAMPs flies are more susceptible than Drosocin single mutants, other AMPs also contribute to Drosocin-mediated control of E. cloacae. As highlighted above, Drosocin is similar to other Proline-rich AMPs (e.g. Abaecin, Pyrrhocoricin) that have been shown to target bacterial DnaK6,7. Alone, these peptides still penetrate bacteria cell walls through their uptake by bacterial permeases71,74. Thus, while Drosocin would benefit from the presence of pore-forming toxins to enter bacterial cells71, the veritable “stake to the heart” is likely the plunging of Drosocin itself into vital bacterial machinery.
On the role of AMPs in host defence
It has often been questioned why flies should need so many AMPs1,4,75. A common idea, supported by in vitro experiments7,65,70, is that AMPs work as cocktails, wherein multiple effectors are needed to kill invading pathogens. 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. Burgeoning support for this idea also comes from recent evolutionary studies that show Drosophila and vertebrate AMPs experience positive selection62,72,75–81, a hallmark of host-pathogen evolutionary conflict. Our functional demonstrations of AMP-pathogen specificity, using naturally relevant pathogens60,82, suggest that such specificity is fairly common, and that certain AMPs can act as the arbiters of life or death upon infection by certain pathogens. This stands in contrast to the classical view that the AMP response contains such redundancy that single peptides should have little effect on organism-level immunity4,61,75,83. 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. Understanding the role of AMPs in innate immunity holds great promise for the development of novel antibiotics18,84,85, insight into autoimmune diseases86–89, and given their potential for remarkably specific interactions, perhaps in predicting key parameters that predispose individuals or populations to certain kinds of infections61,75,76. Finally, our set of isogenized AMP mutant lines provides long-awaited tools to decipher the role of AMPs not only in immunity, but also in the various roles that AMPs may play in aging, neurodegeneration, anti-tumour activity, regulation of the microbiota and more, where disparate evidence has pointed to their involvement.
Materials and Methods
Drosophila genetics and mutant generation
The DrosDel48 isogenic w1118 (iso w1118) wild type was used as a genetic background for mutant isogenization. Alternate wild-types used throughout include Oregon R (OR-R), w1118 from the Vienna Drosophila Resource Centre, and the Canton-S isogenic line Exelexis w1118, which was kindly provided by Brian McCabe. BomΔ55C mutants were generously provided by Steven Wasserman, and BomΔ55C was isogenized into the iso w1118background. RelE20 and spzrm7 iso w1118 flies were provided by Luis Teixeira51,91. Prophenoloxidase mutants (ΔPPO) are described in Dudzic et al.92. P-element mediated homologous recombination according to Baena-Lopez et al.93 was used to generate mutants for Mtk (MtkR1) and Drs (DrsR1).
Plasmids were provided by Mickael Poidevin. Attacin Cmutants (AttCMi, #25598), the Diptericin deficiency (Df(2R)Exel6067, #7549), the Drosocin deficiency (Df(2R)BSC858, #27928), UAS-Diptericin RNAi (DptRNAi, #53923), UAS-Drosocin RNAi (DroRNAi, #67223), and Actin5C-Gal4 (ActGal4, #4414) were ordered from the Bloomington stock centre (stock #s included). CRISPR mutations were performed by Shu Kondo according to Kondo and Ueda94, and full descriptions are given in Figure S1. In brief, flies deficient for Drosocin, Attacin A, and Attacin B (Dro-AttABSK2), Diptericin A and Diptericin B (DptSK1), and Cecropins CecA1, CecA2, CecB, CecC (CecSK6) were all produced by gene region deletion specific to those AMPs without affecting other genes. Single mutants for Defensin (DefSK3), Drosocin (DroSK4), and Attacin D (AttDSK1) are small indels resulting in the production of short (80-107 residues) nonsense peptides. Mutations were isogenized for a minimum of seven generations into the iso w1118 background prior to subsequent recombination.
Microbial culture conditions
Bacteria were grown overnight on a shaking plate at 200rpm in their respective growth media and temperature conditions, and then pelleted by centrifugation at 4°C. These bacterial pellets were diluted to the desired optical density at 600nm (OD) as indicated. The following bacteria were grown at 37°C in LB media: Escherichia coli strain 1106, Salmonella typhimurium, Enterobacter cloacae ß12, Providencia rettgeri strain Dmel, Providencia burhodogranariea strain B, Providencia stuartii strain DSM 4539, Providencia sneebia strain Dmel, Providencia alcalifaciens strain Dmel, Providencia vermicola strain DSM 17385, Bacillus subtilis, and Staphylococcus aureus. Erwinia carotovora carotovora (Ecc15) and Micrococcus luteus were grown overnight in LB at 29°C. Enterococcus faecalis and Listeria innocua were cultured in BHI medium at 37°C. Candida albicans strain ATCC 2001 was cultured in YPG medium at 37°C. 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.
Systemic infections and survival
Systemic infections were performed by pricking 3-5 day old adult males in the thorax with a 100 μm thick insect pin dipped into a concentrated pellet of bacteria or fungal spores. Infected flies were subsequently maintained at 25°C for experiments. For infections with B. bassiana and M. anisopliae, flies were anaesthetized and then shaken on a sporulating plate of fungi for 30s. 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. Comparisons of iso w1118 wild-type to ΔAMPs mutants were made using a Cox-proportional hazard (CoxPH) model, where independent experiments were included as covariates, and covariates were removed if not significant (p > .05). Direct comparisons were performed using Log-Rank tests in Prism 7 software. The effect size and direction is included as the CoxPH hazard ratio (HR) where relevant, with a positive effect indicating increased susceptibility. CoxPH models were used to test for synergistic contributions of AMPs to survival in R 3.4.4. Total sample size (N) is given for each experiment as indicated.
Quantification of microbial load
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. At the indicated time postinfection, flies were anaesthetized using CO2 and surface sterilized by washing them in 70% ethanol. Ethanol was removed, and then flies were homogenized using a Precellys™ bead beater at 6500rpm for 30 seconds in LB broth, with 300ul for individual samples, or 500uL for pools of 5-7 flies. These homogenates were serially diluted and 150uL was plated on LB agar. Bacterial plates were incubated overnight, and colony-forming units (CFUs) were counted manually. Statistical analyses were performed using One-way ANOVA with Sidak’s correction. P-values are reported as < 0.05 = *, < 0.01 = **, and < 0.001 = ***. For C. albicans, BiGGY agar was used instead to select for Candida colonies from fly homogenates.
Gene expression by qPCR
Flies were infected by pricking flies with a needle dipped in a pellet of either E. coli or M. luteus (OD600 = 200), and frozen at -20°C 6h and 24h post-infection respectively. Total RNA was then extracted from pooled samples of five flies each using TRIzol reagent, and re-suspended in MilliQ dH2O. Reverse transcription was performed using 0.5 micrograms total RNA in 10 μl reactions using PrimeScript RT (TAKARA) with random hexamer and oligo dT primers. Quantitative PCR was performed on a LightCycler 480 (Roche) in 96-well plates using Applied Biosystems™ SYBR™ Select Master Mix. 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 Table S1. Statistical analyses were performed using one-way ANOVA with Tukey post-hoc comparisons. P-values are reported as not significant = ns, < 0.05 = *, < 0.01 = **, and < 0.001 = ***. qPCR primers and sources11,72,95 are included in Table S1.
MALDI-TOFpeptide analysis
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. Wounded flies were then spun down with 15μL of 0.1% trifluoroacetic acid (TFA) at 21000 RCF at 4°C in a mini-column fitted with a 10μm pore to prevent contamination by circulating hemocytes. These samples were frozen at -20°C until analysis, and three biological replicates were performed with 4 technical replicates. In the second method, approximately 20nL of fresh hemolymph was extracted from individual adult males using a Nanoject, and immediately added to 1μL of 1% TFA, and the matrix was added after drying. Peptide expression was visualized as described in Üttenweiller-Joseph et al.49. Both methods produced similar results, and representative expression profiles are given.
Melanization and hemocyte characterization, image acquisition
Melanization assays90 and peanut agglutinin (PNA) clot staining50 was performed as previously described. 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. For sessile crystal cell visualization, L3 larvae were cooked in dH2O at 70°C for 20 minutes, and crystal cells were visualized on a Leica DFC300FX camera using Leica Application Suite and counted manually.
Author contributions
MAH, AD and BL designed the study. MAH and AD performed DrosDel isogenization and recombination. MP and SK supplied critical reagents. MAH performed the experiments, and CC provided experimental support. MAH and BL analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We would like to thank Marc Moniatte for assistance with MALDI-TOF analysis, Claudia Melcarne for assistance with hemocyte characterization, and Igor Iatsenko for help in preparation of critical reagents. Brian Lazzaro generously provided Providencia species used in this study. We would like to thank Hannah Westlake for useful comments on the manuscript. MAH would like to extend special thanks to Jan Dudzic for many illuminating discussions had over coffee.