of eicosanoids in the regulation of cellular and humoral responses, including Diptericin expression through the Imd pathway (31, 43). Photorhabdus is known to inhibit PLA2 (44), but a variety of parasitic nematodes also secrete proteins that could similarly interfere with eicosanoid synthesis through their ability to bind fatty acids, including arachidonic acid, which could sequester necessary eicosanoid precursors (45). Similar proteins have also been found in the ES products of Steinernema carpocapsae (10) and the transcriptome of activated H. bacteriophora (14). Interference with this pathway would be consistent with the findings presented here and an efficient way for the parasite to simultaneously suppress multiple immune responses.

#### DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

#### REFERENCES

### AUTHOR CONTRIBUTIONS

EK conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared the figures, and reviewed drafts of the paper. JH, DO'H, and IE conceived and designed the experiments, supervised the project, reviewed drafts of the paper, and prepared the manuscript.

### FUNDING

This work was supported by a George Washington University facilitating fund awarded to DO'H, JH, and IE.

#### ACKNOWLEDGMENTS

We thank members of the Department of Biological Sciences at George Washington University (GWU) for critical reading of the manuscript, and Kyle Devine for rearing the flies.

nematode Heterorhabditis bacteriophora. PLoS ONE. (2013) 8:e69618. doi: 10.1371/journal.pone.0069618

nematode Heterorhabditis bacteriophora. ACS Chem Biol. (2012) 7:961–6. doi: 10.1021/cb300056q

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Kenney, Hawdon, O'Halloran and Eleftherianos. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Expression of Human Mutant Huntingtin Protein in Drosophila Hemocytes Impairs Immune Responses

Yu-Hsien Lin1,2 \*, Houda Ouns Maaroufi1,2, Emad Ibrahim1,2, Lucie Kucerova1 and Michal Zurovec1,2 \*

*1 Biology Centre of the Czech Academy of Sciences, Institute of Entomology, Ceske Budejovice, Czechia, 2 Faculty of Science, University of South Bohemia, Ceske Budejovice, Czechia*

The pathogenic effect of mutant HTT (mHTT) which causes Huntington disease (HD) are not restricted to nervous system. Such phenotypes include aberrant immune responses observed in the HD models. However, it is still unclear how this immune dysregulation influences the innate immune response against pathogenic infection. In the present study, we used transgenic *Drosophila melanogaster* expressing mutant HTT protein (mHTT) with hemocyte-specific drivers and examined the immune responses and hemocyte function. We found that mHTT expression in the hemocytes did not affect fly viability, but the numbers of circulating hemocytes were significantly decreased. Consequently, we observed that the expression of mHTT in the hemocytes compromised the immune responses including clot formation and encapsulation which lead to the increased susceptibility to entomopathogenic nematode and parasitoid wasp infections. In addition, mHTT expression in *Drosophila* macrophage-like S2 cells *in vitro* reduced ATP levels, phagocytic activity and the induction of antimicrobial peptides. Further effects observed in mHTT-expressing cells included the altered production of cytokines and activation of JAK/STAT signaling. The present study shows that the expression of mHTT in *Drosophila* hemocytes causes deficient cellular and humoral immune responses against invading pathogens. Our findings provide the insight into the pathogenic effects of mHTT in the immune cells.

Keywords: Huntington's disease, immunity, infection, Drosophila melanogaster, phagocytosis, cytokines, antimicrobial peptide (AMPs)

#### INTRODUCTION

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an abnormal expansion of CAG trinucleotide in the Huntingtin (htt) gene. Mutant HTT protein (mHTT) contains an extended polyglutamine tract encoded by 40 to over 150 CAG repeats, which causes cytotoxicity and leads to neurodegeneration; this results in involuntary movement, cognitive impairment, and psychiatric abnormalities (1). Although many clinical symptoms of HD are related to neuronal dysfunction, emerging evidence indicates that the expression of mHTT in non-neuronal cells of the brain or in the peripheral tissues also contributes to the pathogenesis of HD (2). Abnormal phenotypic effects caused by the dysfunction of non-neuronal cells have been described in cardiac cells, muscles, the endocrine system, adipose tissue, testes and immune cells of HD patients, and also in mouse HD models (2, 3).

#### Edited by:

*Susanna Valanne, University of Tampere, Finland*

#### Reviewed by:

*Jenny Sassone, Vita-Salute San Raffaele University, Italy Ioannis Eleftherianos, George Washington University, United States*

\*Correspondence:

*Yu-Hsien Lin<EMAIL_ADDRESS>Michal Zurovec<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology*

Received: *20 June 2019* Accepted: *25 September 2019* Published: *16 October 2019*

#### Citation:

*Lin Y-H, Maaroufi HO, Ibrahim E, Kucerova L and Zurovec M (2019) Expression of Human Mutant Huntingtin Protein in Drosophila Hemocytes Impairs Immune Responses. Front. Immunol. 10:2405. doi: 10.3389/fimmu.2019.02405*

**36**

Abnormalities related to the immune system were observed in a number of studies of HD patients (4). The expression of mHTT in both brain and peripheral immune cells (microglial and myeloid cells) induces the NF-κB signaling pathway which elevates levels of pro-inflammatory cytokines and chemokines, leading to systemic inflammation (5). In addition, macrophages isolated from HD model mice exhibited migration deficits, and microglia showed a delayed response to laser-induced injury in the brain (6). Although several studies proposed that the immune cell response is impaired in HD, this phenomenon is still poorly characterized in relation to host responses to pathogens. One recent study reported increased proliferation of a parasite, Toxoplasma gondii, in HD model mice, causing premature mortality and thus suggesting that expression of mHTT in immune cells may suppress immune responses (7).

Drosophila melanogaster has been long-term established as a HD model. In vivo experiments have revealed that the ectopic overexpression of mutant human htt (exon 1 with expanded CAG repeats) in the neural tissue of transgenic flies causes neurodegeneration (8, 9). The mechanisms of cellular pathology observed in the HD flies seem similar to those in human patients, including the suppression of mitochondrial function, transcriptional dysregulation, and neuronal apoptosis (10, 11). Genetic screening for disease modifiers in HD model flies led to the identification of the effects of sumoylation and HSP70 chaperone machinery on neurodegeneration. The subsequent confirmation that these pathways are involved in the pathology of human patients validates the Drosophila model for investigating HD (12, 13). Furthermore, since the tissue-specific expression of transgenes in Drosophila can be easily controlled using the UAS-Gal4 system, Drosophila have also been used to study the effects of HD on non-neuronal cells, including glial cells, photoreceptors, cardiac cells, and salivary glands (14–18).

The present study aimed to survey the physiological impact of mHTT expression in Drosophila hemocytes. We used the Drosophila UAS-Gal4 system to express mHTT with hemocytespecific drivers and investigated the effect of mHTT on survival, hemocyte development, and susceptibility to pathogens. We also expressed mHTT in a Drosophila macrophage-like cell line, S2 cells, and assessed the effect of mHTT on phagocytic activity, ATP levels, antimicrobial peptides, and production of cytokines. Our results suggest that the expression of mHTT in hemocytes does not directly affect survival but causes immune dysregulation, which leads to an impaired immune response against pathogenic invasion.

#### RESULTS

#### Expression of mHTT in Hemocytes Did Not Affect Larval Viability but Decreased the Number of Circulating Hemocytes

In order to characterize the effects of mHTT in Drosophila hemocytes, we used a tissue-specific UAS-Gal4 system by expressing wild-type human HTT (Q20) or mutant HTT (Q93) under the control of a pan-neuronal driver, elav-gal4, or hemocyte drivers, hml-gal4, and he-gal4. The flies devoid of plasmatocytes (phagoless) generated by expressing pro-apoptosis genes, rpr and hid with hml-gal4 were used as negative control (19, 20). The results showed that the ectopic expression of Q93 with the pan-neuronal driver (elav-gal4) decreased both the eclosion rate and the longevity of the adult flies, but not the rate of pupariation (**Figure 1**). The expression of Q20 and Q93 with both hemocyte drivers (hml-gal4 and he-gal4) had no effect on pupariation and eclosion rates. Furthermore, the differences in longevity between Q20 and Q93 flies were not significant, and their survival rate was higher than phagoless flies (**Figure 1C**). These results indicated that the hemocyte-specific expression of mHTT did not influence fly viability, unlike its expression in the brain.

Although the expression of mHTT did not affect fly survival, we observed a significant decrease in the number of circulating hemocytes. In first-instar larvae, the number of hemocytes differed significantly only in the phagoless flies (**Figure 2A**). However, a reduced amount of circulating and sessile hemocytes was apparent in the Q93 mutants from second-instar larvae. As shown in **Figure 2B**, the circular hemocyte numbers in Q93 larvae were still higher than in phagoless flies, showing about 50% of the numbers observed in the Q20 control (**Figure 2B**). These results showed that the expression of mHTT with two different hemocyte-specific drivers reduced the number of hemocytes.

#### Expression of mHTT in Hemocytes Impaired the Immune Response to Parasites

To examine whether mHTT expression in Drosophila hemocytes affects the innate immune response and whether such larvae are still able to restrain parasite development, we tested the sensitivity of such flies to entomopathogenic nematode and parasitoid wasp infections, which are two Drosophila pathogenic models for examining the cellular immune response (21, 22). Early third-instar larvae expressing mHTT (Q93), wild-type HTT (Q20) or phagoless were infected with nematode species, Heterorhabditis bacteriophora or Steinernema carpocapsae, which contain the bacterial symbionts Photorhabdus luminescens and Xenorhabdus nematophila, respectively. Mortality was calculated at 24 and 48 h post-infection. As shown in **Figure 3**, both phagoless and Q93 larvae displayed significantly higher mortality than Q20 controls. Previous studies revealed that the formation of hemolymph clot is an important innate immune response against entomopathogenic nematode infection in Drosophila (23, 24). To determine whether the expression of mHTT in the hemocytes caused clotting defects, we used an established bead aggregation assay (24, 25). Compared to the larvae expressing normal HTT (Q20), the hemolymph collected from mHTT (Q93)-expressing larvae displayed poor bead aggregation similar to phagoless larvae (**Figure 3E**). This results indicated that the expression of mHTT suppresses the clotting activity and thus increases the susceptibility to nematode infection.

Similarly, we infected Drosophila larvae with a parasitoid wasp, Leptopilina boulardi and calculated the number of emerged

fly and wasp adults. The number of eclosed Drosophila adults was not significantly different between Q20 and Q93 driven by hml-gal4 and he-gal4, while phagoless showed lower eclosion rates than both Q20 and Q93 (**Figures 4A,B**). However, the number of emerged wasps were significantly higher in both Q93 and phagoless flies, thus indicating that a greater number of wasps overwhelmed the immune reaction of Q93 hosts and successfully developed to adult stage. In addition, the higher number of wasp eggs successfully hatched in both Q93 and phagoless larvae (**Figure 4C**); these results indicated that Q93 and phagoless larvae have less efficient immune reaction against wasp infection. Since the encapsulation and melanization are major defense mechanisms against parasitoid wasp infection, we quantified the number of the melanized capsules to assess the immune activity after 72 h post-infection. We found that there were more intact melanized capsules in Q20 larvae (79%) than in those expressing Q93 (51.6%) or in phagoless (17.7%) (**Figures 4D,G** left). We also observed a higher amount of melanization pieces in Q93 or phagoless individuals than in Q20 larvae (**Figures 4E,G** middle). The formation of such defective capsules was described previously in immune-deficient mutant flies (26). Moreover, 37% of the infected phagoless larvae formed no melanization capsules compared to Q20 (0%) or Q93 (4.8%) infected larvae (**Figures 4F,G** right). These results could explain a lower proportion of phagoless adults successfully eclosed after wasp infection (**Figures 4A,B**). Taken together, our results suggest, that mHTT expression impairs the innate immune reactions to nematode and parasitoid wasp infections due to the deficient cellular immune responses such as clot formation and encapsulation.

#### Reduced Phagocytic Activity and ATP Levels in mHTT Cells

To find out whether mHTT expression could cause a detrimental effect on hemocyte functions, we expressed mHTT or wildtype HTT in Drosophila S2 cells. The S2 cell line consists of macrophage-like cells with phagocytic activity and the ability to produce antimicrobial peptides (AMPs) (27). We transfected the cells with four different recombinant constructs encoding green fluorescent protein (GFP) fused to HTT repeats under an inducible metallothionein promoter. We created stable cell lineages and confirmed that the S2 cells expressed HTT-fusion proteins by observing the GFP. As shown in **Supplemental Figure 1**, most of the cells in all cell lineages were positive for the fluorophore. Furthermore, the cells containing the mHTT Q46, Q72, and Q97 constructs (all except wild-type Q25) showed formation of mHTT aggregates.

We further treated the HTT-expressing cells with E. coli particles conjugated by pH-sensitive dye (pHrodo) to examine their phagocytic activity. This causes bright fluorescence to be visible after particle engulfment in the acidic environment of phagolysosome. The results showed that after inducing mHTT expression, the fluorescence signals were significantly lower in Q46, Q72, and Q97 mHTT-expressing cells but not in cells expressing wild-type Q25 HTT (**Figure 5A**). Quantification of the cells containing fluorescent signals showed a significant

reduction (20–30%) of fluorescent-positive cells in mHTTexpressing cells compared to the control cells (Q25) (**Figure 5B**), thus supporting the hypothesis that expression of mHTT in immune cells impairs phagocytic activity.

The phagocytic capacity of immune cells has been associated with mitochondrial activity (28–30); mHTT has been shown to cause impairment of energy metabolism and mitochondrial dysfunction in human peripheral blood cells (31). To test whether mHTT can also impair the energy metabolism of Drosophila immune cells, we measured the ATP levels in S2 lineages after mHTT induction. The results showed that ATP levels significantly decreased in cells expressing Q72 and Q97 mHTT after 72 h of induction (**Figure 6A**). The ATP levels in cells expressing Q46, Q72, and Q97 mHTT were further reduced after 120 h of induction (**Figure 6B**). This indicated that the expression of mHTT reduces ATP levels, which may further limit the cellular immune responses against pathogenic infection.

The studies in human and mouse have demonstrated that the expression of Bcl-2 family proteins associated with mitochondrial dysfunction is activated by mHTT expression (32). To assess whether the level of Drosophila Bcl-2 proteins is also altered by mHTT expression, we compared the transcription levels of two of Bcl-2 genes, buffy and debcl, in Q25- and Q97 expressing S2 cells (**Figure 6C**). We found that buffy expression is five times higher in Q97- than in Q25- expressing cells, but we did not detect any significant difference in debcl mRNA

FIGURE 3 | hemocyte ablation (*hml* > *UAS-rpr, hid*) were used as negative control. Bead aggregation assay was used for assessing the clotting activity (E). Hemolymph was collected from Q93, Q20, and *Phagoless* (*hml-gal4*) larvae, mixed with a bead suspension, and the aggregates were quantified by ImageJ software. All the experiments were performed in five to six independent replicates. Data are presented as averages ± SEM. Significances were analyzed by ANOVA with Fisher LSD *post-ho*c test; significant differences among treatment groups are marked with different letters (*P* < 0.05).

level. Different from pro-apoptotic function of debcl, buffy was suggested to play an anti-apoptotic role under stress conditions which is similar to mammalian Bcl-2 proteins (33, 34). We conclude that the alternation of ATP synthesis and buffy expression indicate the abnormality of mitochondrial function in mHTT expressing cells, and the induction of buffy might be a protective mechanism for preventing the cell death caused by mitochondrial dysfunction.

#### Upregulation of Cytokines Expression and Downstream JAK/STAT Signaling in mHTT Expression Cells

It has been reported that the level of cytokines and chemokines are abnormally increased in the plasma of HD patients (35). Consistently, the production of cytokines from monocytes and macrophages of HD patients have shown hyper-activation after lipopolysaccharide stimulation (36). To test whether mHTT has a similar effect in Drosophila, we used Schneider 2 (S2) cells and measured the effect of mHTT expression on three Drosophila cytokines, upd1, upd2, and upd3, as well as dome, jak (hop) and downstream targets of JAK-STAT signaling (**Figure 7A**). The results showed that the expression of cytokine upd3 is significantly increased in Q97 mHTT-expressing cells compared to Q25 controls (**Figure 7B**). In addition, the expression of dome receptor and four downstream targets, tep1, totA, totB, and totC were also significantly increased in Q97-expressing cells. These results indicated that the expression of mHTT induced the production of cytokines and activates JAK/STAT signaling.

### Decreased Antimicrobial Peptide (AMP) Production in Response to Bacteria

Drosophila Toll and Imd pathways control the humoral immune response against invasive microorganisms by regulating the induction of downstream AMP genes in both hemocytes and the fat body (37). To examine whether AMP induction was affected by mHTT expression in Drosophila S2 cells, we treated mHTT-expressing cells with a mixture of heat-inactivated Gramnegative bacteria, Escherichia coli, and Gram-positive bacteria, Micrococcus luteus. The induction of AMPs was assessed using qPCR. As shown in **Figure 8**, there was no difference in the expression of AMPs between Q25 and Q97 in the absence of bacterial treatment. In contrast, all AMP genes were significantly induced in both Q25- and Q97-expressing cells at 8 h after bacterial treatment. However, AMP induction levels were significantly lower in cells expressing Q97 (**Figure 8A**). We further assessed the AMP expression levels under in vivo condition after infecting larvae with phytopathogenic bacteria, Erwinia carotovora carotovora 15 (Ecc15). We examined the expression levels of dpt, dptB, attA, and cecA which were known as being highly induced after Ecc15 infection (38). Our results of larval infections showed that except for attA, the induction levels of dpt, dptB, and cecA in Q93 or phagoless larvae were significantly lower than in Q20 controls (**Figure 8B**). These results confirm that the induction of AMPs in response to bacteria was significantly suppressed in mHTT-expressing cells or larvae.

## DISCUSSION

Peripheral immune dysregulation is considered as one of the clinical features of HD pathogenesis (39). Previous studies in mice and HD patients have suggested that mHTT expression in immune cells accelerates the neurodegenerative process. The activation of pro-inflammatory products in mHTT-expressing microglial cells elevate the reactive oxygen species (ROS) and cause neuroinflammation, which contributes to neurodegeneration (5, 40). Genetic ablation or pharmacologically-blocked cannabinoid receptor 2 (interleukin-6 regulator), as well as drug suppression of the cytokine-responsive kynurenine pathway, can both slow neurodegeneration and improve the phenotype of R6/2 HD mice (41, 42). Since the expression of mHTT in HD mice and human patients is ubiquitous, it is still unclear whether mHTT expression in blood cells directly contributes to the lethal effect of HD. The present study examined mHTT expressed specifically in Drosophila blood cells and assessed its impacts on development and longevity (**Figure 1**). We found that the expression of mHTT in hemocytes did not cause mortality or a shortening in life span, which is in contrast to expression in the brain. Our results, therefore, suggest that expression of mHTT in immune cells does not directly contribute to mortality.

A reduced proliferation of immune cells has been observed in T. gondii-infected HD mice, in which the expansion of CD8+ T-cells in the spleen and brain was significantly suppressed during infection (7). Our results showed that the expression of mHTT in flies with hemocyte-specific drivers causes a significant reduction in the number of circulating hemocytes (**Figure 2**), and this decrease might be caused by dysfunction of mitochondria (**Figure 7**). The mitochondrial abnormalities resulting in metabolic dysregulation in peripheral blood cells of HD patients increase oxidative damage and suppress their antioxidant capacity (40). The activation of caspase-3 and caspase-9 in lymphoblasts of HD patients increases apoptosis under stress conditions (43).

The mHTT-expressing larvae revealed a higher susceptibility to wasp and nematode infections and this phenotype was caused by defects of clot formation and encapsulation (**Figures 3**, **4**). It has been shown that wasp egg recognition by circulating plasmatocytes and their differentiation to lamellocytes for further encapsulation are important processes of the immune response against wasp invasion in Drosophila (44). The production of clotting components from hemocytes also contributes to

FIGURE 4 | Immune challenge with parasitoid wasp infection and encapsulation activity assay. Larvae expressing mHTT Q93 and HTT Q20 with *hml-gal4* (A) or *he-gal4* (B) hemocyte driver were infected with parasitoid wasp, *L*. *boulardi*. *Phagoless* flies (which underwent hemocyte ablation) were used as negative control. Thirty infected larvae were collected and the numbers of eclosed flies and wasps were calculated, indicating the number of flies overcoming the wasp infection and the number of wasps successfully escaping the fly's immune reaction, respectively. Data are presented as average ± SEM with more than 10 biological replicates. The significances of results for (A) were analyzed by ANOVA with a Fisher LSD *post-hoc* test; different letters on the treatment group indicate significant differences at *P* < 0.05. The significances of results for (B) were examined using Student's *t*-test, \*\*\**P* < 0.001, N.S., not significant. For assessing the encapsulation activity, the numbers of fly larvae containing larvae of parasitic wasps (C), intact melanized capsules (D), melanized pieces (E) as well the numbers of the infected larvae without melanization (F) were recorded. The significances of the results were analyzed by Mann-Whitney *U*-test (paired); significant differences among treatment groups are marked with different letters (*P* < 0.05). The photos show examples of intact melanized capsule in Q20 larvae, melanized pieces in Q20 and Q93 larvae and infected *phagoless* without melanization reaction (G).

wound healing and melanization, which are important against nematode or wasp infections (24, 45). mHTT-expressing macrophages and monocytes from HD mice and patients also showed migration defects toward an inflammatory stimulus (6). Hemocyte migration and adhesion are important factors for the development of embryonic macrophages, as well as successful

wound healing and encapsulation during wasp infection (46). Furthermore, decreased phagocytic activity toward bacterial particles (**Figure 5**) and a suppressed induction of antimicrobial peptides (**Figure 8**) can also contribute to immune deficiency against the bacterial symbionts of nematodes (23, 47).

A previous study showed that macrophages isolated from HD patients and R6/2 mice displayed increased phagocytosis when incubated with fluorescent polystyrene beads (48). Our results seemingly differ because we observed reduced phagocytic activity of the Drosophila macrophage-like cells expressing different mHTT fragments (**Figure 5**). Unlike their approach, we tested phagocytic activity using E. coli particles with a pH-sensitive fluorescent dye that can accurately confirm phagosome formation and initiation of the phagolysosome acidification. However, similar to their results, we found that S2 cells expressing mHTT were able to initiate phagocytosis. We tested this by treating the S2 cells with heat-inactivated E. coli labeled with DNA-specific fluorescent dye (without pH-sensor); the results showed that mHTT-expressing cells were indeed able to engulf E. coli (**Supplemental Figure 2**). Thus, our results suggest that mHTT-expressing cells were unable to complete the process of phagocytosis to final phagolysosome acidification. In addition, a defective actin function has been reported in HD mouse immune cells leading to failure of membrane ruffling (6),

FIGURE 6 | Decrease of ATP level and alternation of Bcl-2 protein expression in mHTT-expressing S2 cells. ATP levels of HTT- and mHTT-expressing S2 cells (5 × 104 cells) were measured after 72 (A) and 120 h (B) of copper induction. Each treatment was performed in five independent replicates with (+copper) or without (–copper) copper induction. Data are presented as average ± SEM. Significances were analyzed by ANOVA with Tukey HSD *post-hoc* test; different letters on the treatment group indicate significant differences at *P* < 0.05. The mRNA levels of *Drosophila* Bcl-2 proteins, *buffy* and *debcl* were measured in Q25 HTT and Q97 mHTT-expressing S2 cells after 120 h of induction (C). All the expressions were normalized to *rp49* transcript and Q25 control (11CT). Data are presented as average ± SEM from three independent replicates. *P*-values were determined using Student's *t*-test, \*\*\**P* < 0.001.

which supports our results since actin assembly is required to trigger engulfment and phagolysosome maturation for successful phagocytosis (49).

Consistent with previous observations in HD mice and patients, we also found that Drosophila hemocyte cytokine udp3 was upregulated in mHTT-expressing cells (**Figure 8**). Udp3 binds to the JAK/STAT signaling receptor, Dome, and initiates phosphorylation cascades which translocate the transcription factor, Stat92E, into the nucleus and activates downstream target genes (**Figure 7A**) (50). Two selected downstream target genes, tep1 and totA, were highly expressed in mHTT-expressing cells (**Figure 7B**). Notably, we found that the induction of antimicrobial peptides was significantly suppressed in mHTTexpressing cells after bacterial treatments, which has not yet been observed in other HD models. It is known that several human antimicrobial peptides are expressed in blood cells including neutrophils and macrophages (51). Since the transcriptomic analysis in HD blood cells has shown dysregulation of transcription in large genomic regions (52), further studies will be needed to understand whether the production of antimicrobial peptides is impaired in the blood cells of patients or HD mice during infection.

In summary, the present study demonstrates immune dysregulation in flies expressing mHTT in hemocytes (**Figure 9**). This expression does not directly cause a lethal effect, although it does reduce the number of circulating hemocytes and decrease ATP levels. Cytokine expression and downstream JAK/STAT signaling are activated upon mHTT expression, which has also been observed in HD patients and mice. In addition, the induction of antimicrobial peptides as well as the immune response against different pathogenic infections are impaired in mHTT-expressing Drosophila cells. The present study introduces a system for studying the tissue-specific effects of mHTT in Drosophila immune cells. Further studies can be applied to clarify the molecular interaction between mHTT and antimicrobial peptide pathways (Toll and IMD signaling) as well as the mechanisms of phagocytosis suppression.

### MATERIALS AND METHODS

#### Fly Stocks

Flies were reared at 25◦C on standard cornmeal medium. The fly strains used were UAS-Q20Httexon1111F1L and UAS-Q93Httexon14F132 obtained from Prof. Lawrence Marsh (UC Irvine, USA) (8), which contain 20 (wild-type) and 93 (mutant HTT) polyglutamine repeats, respectively. The pan-neuronal driver, elav-gal4[C155], and hemocyte drivers, he-gal4 and hml-gal4, were obtained from Bloomington Drosophila Stock Center and Dr. Tomas Dolezal (University of South Bohemia), respectively (53–55). Hemocyte-ablated flies (phagoless) were used as negative controls and were generated by overexpressing pro-apoptotic proteins (UAS-rpr, -hid) with hml-gal4 (19, 20, 56).

#### Developmental and Longevity Assay

Thirty first-instar larvae collected from a juice plate were transferred into vials to measure the number of pupae and adults for each replicate. For the longevity assay, about 20–30 newly emerged male adults were collected for each replicate and maintained at 29◦C. Q93 expression driven by pan-neuronal driver, elav-gal4 and phgoless flies were used as positive controls for longevity assay. Since expression of Q93 driven by elavgal4 (X chromosome insertion) results in high mortality of male progeny (dosage compensation) (57), female progeny were used forrecording the longevity. The number of dead flies was counted

every day. All the experiments were performed in at least six independent replicates.

### Circulating Hemocyte Counting

Circulating hemocytes were obtained from larvae by cuticle tearing in Ringer's buffer with thiourea to prevent melanization (25 µl of buffer per 6 larvae). The number of hemocytes expressing GFP (hml-gal4 or he-gal4 > UAS-gfp) were counted using a hemocytometer. At least five independent replicates were analyzed for each genotype.

## Parasitoid Wasp Infection, Eclosion, and Encapsulation Assay

Leptopilina boulardi parasitoid wasps were obtained from Dr. Jan Hrcek (Biology Center CAS) and maintained by infecting ˇ wild-type Drosophila larvae. For the wasp infection assay, forty larvae (second instar) were transferred onto a dish containing cornmeal food, and three female wasps were then placed onto the dish and allowed to attack for 72 h. After infection, 30 infected larvae were collected from the dish and transferred into a vial containing cornmeal for each replicate. Each genotype was tested in at least 10 independent replicates. The total number of eclosed flies and wasps were calculated (26). For the encapsulation assay, the infected larvae were dissected 72 h post-infection and the number of larvae containing intact melanized capsules, broken melanized pieces as well as wasp larvae was recorded.

### Nematode Infection

Two nematode species, Steinernema carpocapsae and Heterorhabditis bacteriophora, were used in this study, under previously described maintenance conditions (58). For the infection assay, nematodes were combined with autoclaved

FIGURE 8 | Suppression of antimicrobial peptides (AMPs) induction after bacterial treatment in the mHTT-expressing S2 cells and larvae. (A) Q25 and Q97-expressing S2 cells were incubated with *E. coli* and *M. luteus* (Q25\_B and Q97\_B) or without bacteria (Q25\_C and Q97\_C) for 8 h, and the expression levels of AMPs was measured. The AMPs expressions were normalized to *rp49* expression and Q25 control. (B) Larvae expressing mHTT Q93 or HTT Q20 under *hml-gal4* hemocyte drivers as well as *Phagoless* mutants were infected with ECC15-GFP and their expression of AMPs was determined after 8 h. The expressions were normalized to *rp49* transcripts and non-infected controls. The AMPs expression levels of non-infected controls for each genotype were set to one. All the data are presented as average ± SEM from three independent replicates. The significances were analyzed by ANOVA with Fisher LSD *post-hoc* test; different letters on the treatment group indicate significant differences at *P* < 0.05.

immune cells of HD patients and mice.

water to achieve a concentration of 25 infective juveniles per 10 µl. Then, 10 µl of nematode suspension was applied to paper and placed in each wells of a 96-well plate. Individual larvae were transferred to each well where they stayed in contact with the nematodes, and the plate was covered with Parafilm. The infection was conducted at 25◦C in the dark. Each experimental replicate consisted of 32 early third-instar larvae (72 h after egg hatching), and all experiments were done at least in five replicates. The number of dead larvae were counted after 24 and 48 h of infection (23, 24).

#### Bead Aggregation Assay

The bead aggregation assay was described in our previous study (24). Briefly, 2.5 µl of hemolymph was collected from six late third-instar larvae, mixed with BSA-blocked bead suspension (tosylactivated Dynabeads M-280, Invitrogen), diluted in Drosophila Ringer (pH 6.0) in a well of cavity diagnostic slide (Thermo Scientific) and covered with round cover glass. Pictures were taken with a Nikon SMZ-745T stereomicroscope associated with a CANON EOS 550D. The images were analyzed and quantified with the ImageJ graphics software with the "Analyze Particles" module.

#### Cell Culture

Drosophila Schneider 2 (S2) cells were grown at 25◦C in Shields and Sang medium (Sigma) with 0.1% yeast extract, 0.25% peptone, and 10% heat inactivated fetal bovine serum. To generate stable lines expressing polyglutamine repeats, the S2 cells were transfected with four different Httex1-eGFP pMK33 plasmids (Q25, Q46, Q72, and Q97) containing copperinducible metallothionein promoter (obtained from Dr. Sheng Zhang) (59).

#### Phagocytosis Assay

After induction for 5 days (120 h) with 1 mM copper (CuSO4; Sigma), 100 µl of cell suspension (1 × 106 cells/ml) was transferred to each well of a 96-well plate. Then, 100 µl of pHrodo Red E. coli (1 mg/ml; Thermo Fisher Scientific) was applied to each well for phagocytosis testing. After 8 h of treatment, the supernatant was removed, the cells were washed two times with 1× PBS, and 100 µl of fresh medium was applied. Cells were observed and photographed with a confocal microscope. From the images, the total number of cells and the number of cells displaying red fluorescence were counted. Three experimental repeats for each treatment were done for statistical analysis.

#### ATP Measurement

Cells were treated with 1 mM copper for 3 days (72 h) and 5 days (120 h) to induce mHTT expression. Fifty microliter of a 1 × 106 cells/ml solution (5 × 104 cells) was transferred to each well of a 96-well plate. After removing the supernatant, 60 µl of CellTiter-Glo solution (Promega) was applied to each well for 10 min. Then, 50 µl of the mixture was transferred to each well of 96-well white plates and the intensity of luminescence was then measured. Five independent replicates for each treatment were performed for analysis.

#### Bacterial Infection in vitro and in vivo

Five milliliter of S2 cells (1 × 106 cells/ml) carrying copperinducible Q25 HTT or Q97 mHTT transgenes were incubated in media containing 1 mM CuSO4 for 120 h in 60 mm tissue culture plates. After the induction, the cells were treated for 8 h with 1 ml of bacterial mixture containing Escherichia coli and Micrococcus luteus at an optical density (600 nm) of 1 (OD600 = 1) (37). The cells were then harvested for RNA extraction.

For the in vivo infection, late third instar larvae (96 h after egg hatching) were collected and transferred into a vial with 0.5 g instant Drosophila medium (Formula 4– 24, Carolina Biological Supply) supplemented with 200 µl of bacterial suspension (OD600 = 50) Erwinia carotovora carotovora 15-GFP (ECC15-GFP) and 1,300 µl of distilled water. The larvae were collected for RNA extraction 8 h after the infection (19, 38).

#### RNA Extraction

For in vitro experiments, S2 cells were washed with 1× PBS three times and harvested with 800 µl of RiboZol (VWR). Samples were preserved at −80◦C until RNA purification. For in vivo experiments, 10 larvae were washed by distilled water and homogenized by the pestle motor (Kimble) in 200 µl of RiboZol (VWR Life Science). The sample were then preserved at −80◦C for further RNA purification. RNA was isolated using NucleoSpin RNA columns (Macherey-Nagel) following the manufacturer's instructions and cDNA was synthesized from 2 µg of total RNA using a RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).

### qPCR and Primers

5× HOT FIREPol R EvaGreen R qPCR Mix Plus with ROX (Solis Biodyne) and an Eco Real-Time PCR System (Illumina R ) were used for qPCR. The cDNA was diluted 50 times before use. Each reaction contained 4 µl of EvaGreen qPCR mix, 0.5 µl of forward and reverse primer (10µM), 5 µl of diluted cDNA and ddH2O to adjust the total volume to 20 µl. The list of primers is shown in **Supplemental Table 1**. The expression level was calculated by using the (2−11CT) method. The CT value of target genes were normalized to reference gene, ribosomal protein 49 (rp49).

#### Statistical Analysis

Error bars show standard error of the mean throughout this paper. Significance was established using Student's ttest (N.S., not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001) or one-way ANOVA analysis with Fisher LSD or Tukey HSD post-hoc test. The Mann–Whitney U-test was used for examing the significance of the data on wasp larval hatching and the host encapsulation activities (**Figures 4C–F**). For the statistical analysis of longevity curve, we used online tool OASIS 2 to perform the weighted log-rank test for determining significance (60).

#### DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

### AUTHOR CONTRIBUTIONS

Y-HL conceived the project, performed the experiments and prepared the manuscript. HM performed the hemocyte counting and imaging. EI performed the nematode infections. LK performed the clotting assay. MZ supervised the project and manuscript preparation.

### FUNDING

This work was supported by the grant agency of the University of South Bohemia (065/2017/P to Y-HL), junior grant project GACR (19-13784Y to LK). MZ was a member of the COST action Maximizing impact of research in neurodevelopmental disorders (CA16210).

### ACKNOWLEDGMENTS

We thank Dr. Sheng Zhang (UThealth) for the Httex1-eGFP pMK33 plasmids, Prof. L. Marsh (UC Irvine, USA) for Q20 and Q93 flies, Dr. Tomas Dolezal (University of South Bohemia) for the hemocyte driver line, Dr. Pavel Hyrsl, Pavel Dobes (Masaryk University) for the nematodes, and Dr. Hrcek Jan, Dr. Chia-Hua Lue (Biology Centre CAS), and Dr. Adam Bajgar (University of South Bohemia) for the parasitoid wasps, Dr. Julien Royet (IBDM, France) for bacteria ECC-15.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02405/full#supplementary-material

#### REFERENCES

Supplemental Figure 1 | Expression of four different lengths of HTT-GFP fusion proteins under a fluorescence microscope. mHTT-expressing cells (Q46, Q72, and Q97) showed significant mHTT aggregates after copper induction, while there was no aggregate formation in normal HTT-expressing cells (Q25).

Supplemental Figure 2 | Phagocytosis assay in Q25- and Q97-expressing S2 cells with *E. coli* labeled by DNA-specific dye (Hoechst 33342). Cells expressing mHTT were able to initiate phagocytosis.

Supplemental Table 1 | List of qPCR primers used in this study.

and their mutualistic bacteria. J Insect Physiol. (2013) 59:179–85. doi: 10.1016/j.jinsphys.2012.08.003

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Lin, Maaroufi, Ibrahim, Kucerova and Zurovec. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Emerging Mechanisms of Insulin-Mediated Antiviral Immunity in Drosophila melanogaster

Chasity E. Trammell 1,2 and Alan G. Goodman1,3 \*

*1 School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, United States, 2 NIH Biotechnology Graduate Training Program, Washington State University, Pullman, WA, United States, 3 Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, WA, United States*

Arboviruses (arthropod-borne viruses), such as Zika (ZIKV), West Nile (WNV), and dengue (DENV) virus, include some of the most significant global health risks to human populations. The steady increase in the number of cases is of great concern due to the debilitating diseases associated with each viral infection. Because these viruses all depend on the mosquito as a vector for disease transmission, current research has focused on identifying immune mechanisms used by insects to effectively harbor these viruses and cause disease in humans and other animals. *Drosophila melanogaster* are a vital model to study arboviral infections and host responses as they are a genetically malleable model organism for experimentation that can complement analysis in the virus' natural vectors. *D. melanogaster* encode a number of distinct mechanisms of antiviral defense that are found in both mosquito and vertebrate animal systems, providing a viable model for study. These pathways include canonical antiviral modules such as RNA interference (RNAi), JAK/STAT signaling, and the induction of STING-mediated immune responses like autophagy. Insulin signaling plays a significant role in host-pathogen interactions. The exact mechanisms of insulin-mediated immune responses vary with each virus type, but nevertheless ultimately demonstrates that metabolic and immune signaling are coupled for antiviral immunity in an arthropod model. This mini review provides our current understanding of antiviral mechanisms in *D. melanogaster*, with a focus on insulin-mediated antiviral signaling, and how such immune responses pertain to disease models in vertebrate and mosquito species.

#### Edited by:

*Susanna Valanne, Tampere University, Finland*

#### Reviewed by:

*Sara Cherry, University of Pennsylvania, United States Penghua Wang, University of Connecticut Health Center, United States*

> \*Correspondence: *Alan G. Goodman<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology*

Received: *15 October 2019* Accepted: *04 December 2019* Published: *20 December 2019*

#### Citation:

*Trammell CE and Goodman AG (2019) Emerging Mechanisms of Insulin-Mediated Antiviral Immunity in Drosophila melanogaster. Front. Immunol. 10:2973. doi: 10.3389/fimmu.2019.02973* Keywords: innate immunity, RNA interference, JAK/STAT, insulin, STING, West Nile virus, Zika virus, dengue virus

### INTRODUCTION

Mosquitoes are a prominent vector for various arboviruses including West Nile virus (WNV), Zika virus (ZIKV), and dengue virus (DENV). These viruses pose a significant concern to human populations as the mosquitoes' continual encroachment into previously unexposed regions expands (1, 2). This habitat expansion renders more individuals at risk of exposure with limited, if any, treatments available. Climate change has also resulted in alterations in mosquito seasonal activity (3) and feeding behavior (4) resulting in increasing frequency and severity of arboviral cases. There is a direct correlation between the expansion of vector-competent mosquitoes and disease incidence within afflicted regions [reviewed in (5, 6)] indicating that vector activity is a significant risk factor for arboviral disease.

**51**

**Figure 1** outlines the transmission cycle of various arboviruses as they move within host populations and how Drosophila melanogaster can be used to study arboviral immunity for each system. Transmission from mosquito to vertebrates requires a bloodmeal exchange where infected saliva is ejected into the new host. Viral replication then permits the spread of virus from infected host to mosquito to continue the transmission cycle (**Figure 1**). Research is required to identify the signaling responses used in regulating these viruses at the vector and human level. Studies regarding immune responses initiated during the initial bloodmeal exchange (7) are important as this event is a key determinant whether transmission occurs (8). An emphasis as to how immune and nutritional signaling interact with one another is of particular interest as both would be active during ingestion of an infected bloodmeal. Insulin-mediated signaling regulates numerous viruses by inducing activation of canonical immune pathways (9). Because insulin is ingested during the bloodmeal, a recent study has shown that vertebrate insulin is able to regulate the type of innate immune response that occurs during viral infection in insect vector hosts (10).

Previous work has identified the signaling pathways that respond to arboviral infection and their significance with respect to disease outcome and severity (11–13). Drosophila have proven to be a significant model organism for studying arboviruses as many of the signaling pathways identified are conserved amongst insect species [reviewed in (14)]. These studies have utilized the genetic power provided by the Drosophila system to demonstrate the effect that nutritional status poses on host immunity.

Immune responses during various arboviral infections are evolutionarily conserved among insects and include the canonical RNA interference (RNAi) (13, 15, 16), JAK/STAT (10, 12), and STING-mediated signaling (17). These pathways are associated with the insulin/insulin-like growth factor signaling (IIS) pathway and have been established as key determinants in vector competency and disease outcome (9, 18). It has been demonstrated that ingestion of vertebrate insulin regulates whether an RNAi- or JAK/STAT-mediated response is active during infection against WNV (10). STING-mediated immunity has been previously linked to induce JAK/STAT signaling (19) and affects nutritional homeostasis during infection (20, 21) implying that it may be regulated by insulin as well. Since insulin-mediated signaling appears to have a broad impact on insect immunity, recent studies have sought to establish how insulin connects each antiviral pathway to respond to different arboviruses. Because vector competency and transmission is so closely dependent on gut-associated immune signaling, the connection that nutrition and immunity has is indicative of their importance in regulating infection (9, 22). This mini review presents a condensed understanding regarding the major responses that occur during arboviral infection using Drosophila,

studying transmission dynamics and host-pathogen interactions at both vector and human level.

the role that insulin signaling plays, and a summation of current and future efforts taken within this field.

### RNA INTERFERENCE PATHWAY

One of the most broadly restricting antiviral responses used by insects is the RNAi pathway (13, 16). RNAi signaling occurs in response to detection of viral nucleic acids within the cytosol of infected cells. In Drosophila, recognition of viral nucleic acids by the endonuclease Dicer-2 results in the recruitment of proteins Argonaute-2 (AGO2) and r2d2 to form an RNA-induced silencing complex (RISC) (13, 23). This results in the cleavage and degradation of bound viral nucleic acids (13, 23, 24). While this antiviral response is a significant component of insect immunity against RNA viruses like Sindbis virus (SINV) (24) and ZIKV (25), an arthropod-borne alphavirus and flavivirus, respectively, RNAi has also been shown to respond to DNA viruses like Invertebrate iridescent virus 6 (IIV-6) (26) (**Figure 2A**). While Harsh et al. showed that the loss of the RNAi component, Dicer-2, resulted in increased ZIKV replication and mortality, they linked the increased susceptibility of these flies to dysregulated homeostasis of the gut and fat body. Moreover, studies have shown that another RNAi component, namely AGO2, is dispensable for an antiviral response against ZIKV (17, 25). Additionally, there are flaviviruses that encode viral suppressors of RNAi (VSR) (27). Not only do VSRs function in Drosophila model systems (28), but they also function during WNV, DENV, and Yellow fever virus infections in Culex mosquitoes (29, 30). Thus, the antiviral role of RNAi depends not only on the host but also virus type. Further studies are needed to clarify the antiviral role of RNAi in model and vector organisms, especially with respect to flaviviruses and the VSRs they may encode. Mosquitoes become infected and spread disease via bloodmeal exchanges. Because of the direct role that nutritional acquisition has during infection, its role in antiviral immunity is mediated in part by regulating RNAi. In both insect and mammalian systems, IIS regulates the transcription factor forkhead box O (FOXO), and FOXO is predominately associated with longevity and nutritional signaling as it induces dInR (insulin-like receptor) in Drosophila (31, 32). FOXO possesses a secondary role in host immunity through its induction of RNAi-specific genes. Specifically, FOXO regulates the transcription of Dicer-2 and

FIGURE 2 | Innate immune antimicrobial pathways are conserved in arthropods. Insects utilize RNAi (A), STING-mediated immunity (B), and JAK/STAT signaling (D) in order to effectively respond to various arboviruses at different stages of infection. The IIS pathway (C) is an important mediator in host immunity as it regulates which immune responses are active or suppressed. During times of starvation, RNAi is more active while the bloodmeal provides the needed insulin to suppress RNAi and activate JAK/STAT. STING-mediated immunity has not yet been directly linked to IIS but may be affected by broader nutritional signaling. Each of these pathways, to varying degrees, are conserved in fly, mosquito, and human systems with different efficiencies in responding to viral infection.

AGO2 in Drosophila and is demonstrated to enhance RNAi signaling during Cricket paralysis viral infection (18). Since IIS regulates FOXO transcriptional activity, there is a direct connection between RNAi immunity and insulin signaling. In Drosophila, the IIS pathway is induced by insulin-like peptides (ILPs) binding to dInR (33). Upon binding, a phosphorylation cascade commences that includes phosphorylation of PI3K and Akt. This results in the phosphorylation of nuclear FoxO at three residues, its association with the 14-3-3 chaperone protein, and export into the cytosol (32, 34) (**Figure 2C**). Insulin treatment is demonstrated to result in transcriptional suppression of Dicer-2 and AGO2 to reduce RNAi signaling (10). This insulin-mediated suppression of RNAi proposes that mosquitoes have evolved multiple immune responses to pathogens that is dependent on its nutritional status.

RNAi is evolutionarily conserved across organisms; however, its role in antiviral immunity varies between insects and mammals. While the signaling cascade and proteins involved are conserved, mammals have evolved other sensing mechanisms to detect and respond to viruses. RIG-I and MDA5 RNA sensing are canonical pathways for vertebrate innate immunity and have developed from RNA sensors like Dicer (35). In particular, RIG-I and MDA5 are shown to be critical immune regulators in response to flaviviruses like ZIKV (36) and WNV (37) and alphaviruses like SINV (38). This would imply that while RNAi is still fully functional in mammals, its role in responding to viruses is less stringent than in insect systems [reviewed in (39, 40)]. Other invertebrate and plant species utilize RNAi signaling as a means of antiviral immunity [reviewed in (41, 42)]. Researchers interested in studying RNAi immunity against arboviruses utilize Drosophila as the signaling cascades described are well-conserved in the more relevant mosquito vector. While the functional antiviral role of RNAi varies in mammalian systems, the proteins and signaling events involved are conserved. Because of this, studies utilizing Drosophila have proven it to be a viable model for study in host immunity and other regulatory functions.

#### JAK/STAT PATHWAY

Innate immunity uses various responses to different viruses. These responses include phagocytosis of viral particles or by inducing production of downstream cytokines and antiviral effectors (43). Whereas, the RNAi pathway provides a broad means of protection through the degradation of viral nucleic acids (16), the JAK/STAT pathway is a signaling cascade where detection of infection or other stimuli results in the induction of antiviral effectors like vir-1, Vago, and TotM (12, 24, 44). In Drosophila immunity, this pathway is activated upon detection of infection (45), resulting in induction of the unpaired ligands (46, 47). Unpaired ligands bind to the receptor domeless (48), resulting in the Janus kinase (JAK) ortholog hopscotch to be phosphorylated and form docking sites for phosphorylation and dimerization of the STAT92E transcription factor (12, 49). The activated STAT92E protein complex is imported into the nucleus to induce transcription of downstream antiviral effectors. This includes TotM during early stages of infection (24) and vir-1 during later stages (12) (**Figure 2D**). JAK/STAT has been shown to be involved in the immune response against the insect virus Drosophila C virus but not SINV (24) in Drosophila, and JAK/STAT is antiviral against WNV in both Drosophila and Culex mosquitoes (10, 50).

The connection between JAK/STAT and IIS is not as direct as insulin's effect on the RNAi pathway, but recent research has demonstrated that insulin signaling in insects controls a switch between RNAi- and JAK/STAT-dependent responses. Specifically, when insulin treatment causes transcriptional suppression of RNAi, insects induce enrichment of JAK/STAT (10). This is mediated by insulin's phosphorylation and activation of downstream Akt and ERK proteins during SINV and DENV infection (9, 22) that was further evaluated to induce immunity through JAK/STAT against WNV (10). Insulinmediated induction of JAK/STAT then induces the transcription of downstream antiviral effector proteins vir-1 and TotM (10, 12, 24). JAK/STAT immunity responds to pathogens during early stages of infection in the mosquito which corresponds to ingestion of an infected bloodmeal and escape from the midgut to distal tissues (51). This association between IIS and JAK/STAT signaling is indicative that vector-competent insects have evolved immune mechanisms that are responsive to nutritional acquisition. While mosquitoes use an RNAidependent response during times of starvation, the bloodmeal provides the insulin needed to activate a JAK/STAT-dependent response during infection. While the ability to regulate immune responses based on nutritional status and how it impacts viral efficacy and transmission has yet to be established, it is plausible that insulin-mediated signaling could be targeted in future vector-control protocols. Its potential as a target depends on whether insulin's antiviral activity in the salivary glands is just as important as it is in midgut or fat body, critical digestive and immune organs (8, 9).

JAK/STAT signaling is an evolutionarily conserved immune response utilized in both insect and mammalian systems. Induction of JAK/STAT in mammals results in a Type I interferon (IFN) response against viral infection (52). The JAK/STAT pathway is similarly regulated by RIG-I-like receptor (RLR) signaling in mammalian systems (35, 53). This pathway is an important means of responding to WNV in both insects (10, 44) and mammals (52, 53). Because JAK/STAT is related to other regulatory processes like cell proliferation and differentiation [reviewed in (54)], researchers have also used the Drosophila model to study the pathway's role in a non-immunological context like cellular growth (47), differentiation (48), polarization (49), and oogenesis (55). Drosophila provide a unique system for studying JAK/STAT signaling at various levels of complexity in both an immune and regulatory context to provide clarity regarding the pathway's significance in the organism.

#### STING-MEDIATED IMMUNITY

Mediators of mammalian immunity have evolved from established signaling pathways that are present in invertebrate systems (35, 56). One such set of responses is stimulator of IFN genes (STING)-mediated immunity. Upon detection of viral nucleic acids in the cytosol, the DNA sensor cGAS metabolizes cyclic dinucleotides that bind to and activate STING (11), which induces phosphorylation and activation of various transcription factors like TBK1, IRF3, and STAT6 (19, 57). These transcription factors regulate the induction of Type I IFN responses through the secretion of IFN-α and β (11, 19, 57). STING-mediated immunity has been heavily studied within mammalian systems; however, it is only recently that STING and its role in innate immunity have been identified in insects and other invertebrates (17, 56, 58–60) (**Figure 2B**).

In Drosophila, STING signaling provides immunity against both bacterial and viral infections. During infection with Listeria monocytogenes, cyclic dinucleotides are produced which results in STING-mediated signaling and nuclear import of Relish, the fly ortholog of mammalian NF-κB (56). This immune response induces transcription and secretion of IMDcharacteristic antimicrobial peptides to reduce bacterial burden (56). STING-mediated antiviral immunity also occurs through Relish and IKKβ, which regulate expression of the antiviral factor Nazo (58). In the silkmoth, Bombyx mori, STING signaling activates antiviral activity through Dredd and IMD, leading to Relish signaling and induction of antimicrobial peptides against nucleopolyhedrovirus (NPV) (59). Other studies using the Drosophila system have further evaluated STING-mediated immunity by autophagy (17).

Autophagy is a cellular process in which intracellular structures and proteins are degraded in a lysosomaldependent manner [reviewed in (61)]. Because viruses are obligate intracellular pathogens, autophagy is an established antiviral response that is partially regulated by nutritional and STING-mediated signaling (62). While STING-mediated autophagy has been established in responding to numerous viruses in mammals, recent studies using Drosophila have demonstrated that insects can utilize autophagy to respond to ZIKV in neuronal tissues (17). Specifically, ZIKV infection results in a pro-inflammatory response in Drosophila brains which induces STING-mediated activation of autophagy and immunity (17). This form of antiviral immunity is indicative that neuronal protection against arboviruses is mediated through STING-mediated signaling. This study provides another example of the versatility provided in using the Drosophila system as the fly model is a viable means for studying antiviral immunity conserved between invertebrates and vertebrates.

Unlike the RNAi and JAK/STAT pathways, which are both regulated in part by insulin signaling, a direct link between IIS and STING has yet to be shown. However, since autophagy is partially regulated by nutritional status [reviewed in (63)] and STING has been previously shown to induce STAT6 in the mammalian system (19), it is plausible that STING-mediated immunity may also be partially regulated by insulin signaling. Since STING has only recently been discovered in the insect model, future research is needed to further evaluate how STING connects to other canonical immune and nutritional pathways and its involvement in vector competency.

## PROSPECTIVE

Both the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) agree that mosquito-borne arboviruses will be of great concern in the following years due to the expansion of mosquitoes' habitation range and activity into previously unexposed regions (64, 65). Research using Drosophila have permitted investigators to identify the key signaling events that occur during infection and develop more effective vector control protocols that target viral replication and likelihood of transmission. Recently, Drosophila have been used to identify ingestion of mammalian insulin as a key regulator in controlling WNV replication in the insect model. The genetic screen to identify insulin receptor was performed using Drosophila, and the role of insulin signaling was then validated in the mosquito model (10).

While Drosophila have been an invaluable tool in the study of arboviruses in place of the more relevant arthropod vector, there are limitations. For example, JAK/STAT signaling, while protective in the mosquito model against arboviruses such as WNV and DENV (10, 44, 66), may not be protective in Drosophila during SINV or vesicular stomatitis virus infections (12, 24). Because Drosophila are not the natural host for these arboviruses, there are limitations surrounding whether the responses observed are indeed what occurs in natural hosts. As such, work involving Drosophila must ensure that their findings are further evaluated in the more relevant model, whether that be the insect vector or human host.

Developing disease response protocols that aim at preventing transmission from arthropods to humans would be the most beneficial in terms of cost-efficiency and alleviating disease burden at a global scale. Research efforts also aim to identify novel therapeutics that are effective at treating humans postexposure. Because many of the immune pathways discussed here are also present in humans (**Figure 2**), future research aimed at identifying novel human-specific antiviral therapeutics could benefit from the use of Drosophila. The use of Drosophila to model host immunity at both the mammalian and vector level during arboviral infection has provided a greater depth of knowledge regarding which signaling pathways are involved during infection and how they can be targeted in the arthropods that transmit disease.

### AUTHOR CONTRIBUTIONS

CT wrote the first draft of the manuscript. AG revised the manuscript.

### FUNDING

Research in the Goodman lab was supported by NIH/NIAID Grant R01 AI139051 to AG. CT was supported by NIH/NIGMS pre-doctoral fellowship T32 GM008336.

### REFERENCES

controls mycobacterial infection. Nat Commun. (2017) 8:14642. doi: 10.1038/ncomms14642

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Trammell and Goodman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Bombardier Enables Delivery of Short-Form Bomanins in the Drosophila Toll Response

Samuel J. H. Lin, Amit Fulzele, Lianne B. Cohen, Eric J. Bennett and Steven A. Wasserman\*

*Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, San Diego, CA, United States*

Toll mediates a robust and effective innate immune response across vertebrates and invertebrates. In *Drosophila melanogaster*, activation of Toll by systemic infection drives the accumulation of a rich repertoire of immune effectors in hemolymph, including the recently characterized Bomanins, as well as the classical antimicrobial peptides (AMPs). Here we report the functional characterization of a Toll-induced hemolymph protein encoded by the *bombardier* (*CG18067*) gene. Using the CRISPR/Cas9 system to generate a precise deletion of the *bombardier* transcriptional unit, we found that Bombardier is required for Toll-mediated defense against fungi and Grampositive bacteria. Assaying cell-free hemolymph, we found that the Bomanin-dependent candidacidal activity is also dependent on Bombardier, but is independent of the antifungal AMPs Drosomycin and Metchnikowin. Using mass spectrometry, we demonstrated that deletion of *bombardier* results in the specific absence of short-form Bomanins from hemolymph. In addition, flies lacking Bombardier exhibited a defect in pathogen tolerance that we trace to an aberrant condition triggered by Toll activation. These results lead us to a model in which the presence of Bombardier in wild-type flies enables the proper folding, secretion, or intermolecular associations of short-form Bomanins, and the absence of Bombardier disrupts one or more of these steps, resulting in defects in both immune resistance and tolerance.

#### Keywords: Drosophila melanogaster, immunity, Toll, Bomanins, humoral

### INTRODUCTION

Innate immune pathways are found in plants, fungi, and animals and provide a rapid defense against a broad range of pathogens (1–3). In the fruit fly Drosophila melanogaster, the two major innate immune pathways are Toll and Imd (4–6). The Toll pathway is activated by Gram-positive bacteria with Lys-type peptidoglycan and by fungi, and is required for defense against these microbes (7–10). Conversely, the Imd pathway is activated by and plays a major role in survival against Gram-negative bacteria and Gram-positive bacteria with DAP-type peptidoglycan (11, 12). These pathways, which are both mediated by NF-κB transcription factors, are broadly conserved as initiators of innate immune responses. Activation of either pathway induces robust production of an array of immune molecules, including antimicrobial peptides (AMPs) (13–17).

Edited by:

*Susanna Valanne, Tampere University, Finland*

#### Reviewed by:

*Anni Kleino, University of Helsinki, Finland Brian P. Lazzaro, Pennsylvania State University (PSU), United States*

> \*Correspondence: *Steven A. Wasserman<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology*

Received: *18 October 2019* Accepted: *11 December 2019* Published: *10 January 2020*

#### Citation:

*Lin SJH, Fulzele A, Cohen LB, Bennett EJ and Wasserman SA (2020) Bombardier Enables Delivery of Short-Form Bomanins in the Drosophila Toll Response. Front. Immunol. 10:3040. doi: 10.3389/fimmu.2019.03040*

**58**

Lin et al. Bombardier Enables Bom Defense

AMPs are found in all kingdoms of life (18–22). These peptides have long been thought to play the principal effector role in innate immune defense due to their demonstrated in vitro antimicrobial activity and their marked upregulation after infection. However, recent research in D. melanogaster suggests that AMPs play a major role in Imd-mediated defense, but a relatively minor role in Toll-mediated immunity (23).

In contrast to the AMPs, the Drosophila-specific Bomanin peptides (Boms), which are highly induced after infection, are indispensable for resistance against pathogens controlled by the Toll pathway (24). Bom155C flies, which lack 10 of the 12 Bom genes, succumb to fungal and Gram-positive bacterial infections at rates indistinguishable from Toll-deficient flies (23, 24), suggesting that Boms rather than AMPs are the primary Toll effectors.

Bom peptides, like AMPs, are secreted from the fat body, the Drosophila immune organ, into the hemolymph, the Drosophila circulatory fluid. The family is comprised of three groups. The short-form peptides are 16–17 residues long and contain only the Bom motif. The tailed forms contain the Bom motif followed by a C-terminal tail. Finally, the bicipital forms consist of two Bom motifs connected by a linker region (24). Bom155C flies lack all six of the short-form Boms, two of the three tailed Boms, and two of the three bicipital Boms. High-level expression of short-form Boms is sufficient to rescue the sensitivity of Bom155C flies to C. glabrata infection (25). Furthermore, the absence of Toll-induced candidacidal activity in Bom155C hemolymph can be rescued by high-level expression of a short-form Bom (25). However, no in vitro antimicrobial activity has been observed with Bom peptides alone (25), suggesting that the Bomanins act in coordination with additional humoral effectors.

In this study, we demonstrate an essential role in Tollmediated humoral defense for a previously uncharacterized hemolymph protein, Bombardier (one that deploys Boms).

## MATERIALS AND METHODS CRISPR/Cas9 Deletion of bombardier

#### Locus

The bombardier gene (CG18067) was deleted using CRISPR/Cas9 technology according to established protocols (26). Briefly, a pair of gRNAs designed to delete the region 2R: 20,534,248– 20,536,154 were cloned into pU6-BbsI-chiRNA (Addgene plasmid #45946). Homology arms (1,017 bp left and 1,022 bp right) were cloned into pDsRed-attP (Addgene plasmid #51019). The plasmid pBS-Hsp70-Cas9 (Addgene plasmid #46294) was used as the Cas9 source. Constructs were injected into w 1118 embryos. F1 progeny were screened for DsRed eyes and homozygous lines were established. See **Supplemental Table 1** for gRNA and homology arm primer sequences.

### Toll Activation, Drosophila Infection, and Survival Analysis

Flies were raised at 25◦C on cornmeal molasses agar media1 . The w 1118 strain was used as the wild type. Microbial isolates, culture conditions, and conditions for infection for Enterococcus faecalis, Enterobacter cloacae, Fusarium oxysporum, and Candida glabrata were as described previously (24), except that C. glabrata was concentrated to OD600 = 100. Flies were incubated at 25◦C after live bacterial infection and at 29◦C after fungal infection. For heat-killed challenge, bacterial cultures were autoclaved and resuspended in 20% glycerol to OD600 = 10 for E. faecalis and OD600 = 300 for M. luteus. For both survival assays and hemolymph preparation, flies challenged with heat-killed bacteria were incubated at 29◦C.

#### Hemolymph Antimicrobial Assays

Candidacidal activity of hemolymph was assayed as described previously (25), except that hemolymph was prepared from groups of 30 flies and all activity assays were carried out for 1 h at room temperature. The number of colonies representing zero percent killing was set as the value obtained by assaying uninduced w 1118 hemolymph.

#### MALDI-TOF Analysis of Hemolymph

The Toll pathway was activated in flies using heat-killed M. luteus, then incubated at 29◦C for 24 h. Hemolymph was extracted as in Lindsay et al. (25), with slight modifications. Hemolymph extracted with glass capillaries from five male flies was pooled and transferred into 0.1% trifluoroacetic acid (TFA)/50% acetonitrile (ACN). One µl of each mixture was spotted on a Bruker MSP 96 ground steel plate, mixed 1:1 with a saturated solution of Universal MALDI matrix (Sigma-Aldrich) in 0.1% TFA/78% ACN, and air-dried. MALDI-TOF spectra were acquired using a Bruker Autoflex mass spectrometer. Data were collected from 1,500 to 10,000 m/z in positive linear mode, and 1,000–5,000 m/z in positive reflectron mode. Peptide calibration standard II (Bruker) was mixed with Universal MALDI matrix and used as an external calibration standard. At least ten independent samples were collected for each genotype. For peptide identification, peaks were matched to those of corresponding peaks in prior studies (13, 25). Representative spectra were visualized using R 3.3.2 and ggplot2 2.2.1 (27, 28).

#### Gene Expression Quantitation

The Toll pathway was activated with heat-killed M. luteus. Using TRIzol (Ambion), total RNA was extracted 18 h after Toll activation from four to six adult flies (2–5 days old). Next, cDNA was synthesized from 500 ng total RNA using the SuperScript II Reverse Transcriptase kit (Invitrogen). Quantitative RT-PCR was performed on an iQ5 cycler (BioRad) using iQ SYBR Green Supermix (BioRad). Quantification of mRNA levels was calculated relative to levels of the ribosomal protein gene rp49 using the Pfaffl method (29). Three independent replicates were completed. See **Supplemental Table 1** for qPCR primer sequences.

#### Hemolymph LC-MS

Flies were challenged with heat-killed M. luteus to activate the Toll pathway. Hemolymph was extracted from 100 to 110 each of w 1118 , 1bbd, and Bom155C flies using the same method as in the hemolymph antimicrobial assays, with 50– 60 flies processed per Zymo-Spin IC column (Zymo Research)

1http://blogs.cornell.edu/drosophila/dssc-cornmeal-recipe/

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and yielding a total of ∼10 µl hemolymph per genotype. Three independent biological replicates were processed for 1bbd and Bom155C , and two independent biological replicates were processed for w 1118. Extracted hemolymph was mixed 1:1 (vol/vol) with denaturing buffer (8 M Urea, 50 mM Tris, pH 7.8, 150 mM NaCl, protease and phosphatase inhibitors) and protein concentration was determined using a BCA assay. For each sample, 40 µg of hemolymph was diluted to 1 M urea using 50 mM ammonium bicarbonate and digested overnight with trypsin (Promega, V511A) at a 1:100 (trypsin:protein) ratio. After digestion, peptides were reduced with 1 mM dithiothreitol at room temperature for 30 min and then alkylated with 5 mM iodoacetamide at room temperature in the dark for 30 min. Formic acid was added to a 0.1% final concentration and peptides were desalted using the C18-Stage-Tip method and then vacuum dried. The dried peptides were reconstituted in 5% formic acid/5% acetonitrile and 1 µg of total peptide for each sample was loaded for MS analysis. Samples were run in technical triplicates on a Q-Exactive mass spectrometer with instrument and chromatography settings as described previously (30), except for the following modifications: the RAW files were analyzed using Andromeda/MaxQuant (version <IP_ADDRESS>) (31) with default settings (32) except the match between the run and LFQ quantitation settings was enabled for label free quantification. Data were searched against a concatenated targetdecoy database comprised of forward and reversed sequences from the unreviewed UniprotKB/Swiss-Prot FASTA Drosophila database (2019). A mass accuracy of 20 ppm was assigned for the first search and 4.5 ppm for the main search. The statistical analysis was calculated using the DEP analysis R-package (33).

#### Bacterial Load Quantification

Bacterial load upon death (BLUD) was obtained as in Duneau et al. (34), with slight modifications. Briefly, flies were infected with E. faecalis and vials were monitored every 30 min for newly dead flies. These flies were then individually homogenized with a pestle in 400 µl LB media. Homogenates were also prepared from individual live w 1118 flies 120 h postinfection (hpi). Homogenates were diluted serially in LB and spread on LB agar plates for incubation at 37◦C overnight. Colonies were counted manually and the number of viable bacteria per fly was calculated. Data were obtained from three independent experiments.

#### Data Analysis

GraphPad Prism 5 was used for statistical tests. Survival data were plotted as Kaplan-Meier curves and were analyzed using the Gehan-Breslow-Wilcoxon test to determine statistical significance. Statistical differences in candidacidal activity were calculated using one-way ANOVA followed by Tukey's test. Multiple Mann-Whitney U tests were used to calculate differences between BLUD samples (p = 0.0085 after Šidák correction for multiple comparisons, α = 0.05, k = 6). Spearman rank correlation was used to assess the relationship between BLUD and time of death.

#### RESULTS

### The bombardier Gene Is Specifically Required for Toll-Mediated Defense

The bombardier (bbd) gene contains a consensus Toll-responsive NF-κB binding site within its promoter region and is strongly expressed upon Toll activation by Gram-positive bacterial infection or other inducers (14, 17, 35, 36). The encoded protein is predicted to be secreted and to generate a mature protein of 222 amino acids with a coiled coil near its C-terminus (37, 38). Orthologs of Bombardier are found across the Drosophila genus, but in no other genera (39).

We began our analysis of the bombardier gene by generating a null mutant, using CRISPR/Cas9 to delete 1,906 bp encompassing the annotated transcriptional unit. Flies homozygous for this deletion (hereafter 1bbd) were viable and morphologically wild-type. Given that bombardier is Tollinducible, we assayed 1bbd flies for a potential loss-of-function phenotype in Toll-mediated immunity. Specifically, we infected adult 1bbd flies with various pathogens and then monitored survival. Two additional genotypes were used as controls: w 1118 flies, which served as the wild type, and Bom155C flies, which lack Toll-mediated humoral defenses due to deletion of the 10 of the 12 Bom genes (24).

As shown in **Figure 1**, we observed a marked immunodeficiency when 1bbd flies were challenged with representative species for the three classes of microbes against which Toll provides defense. With the yeast Candida glabrata, more than 90% of w 1118, but no 1bbd flies, survived 5 days after infection (**Figure 1A**). In the case of the filamentous fungus Fusarium oxysporum, 70% of w 1118 adults, but fewer than 20% of 1bbd adults, were alive 5 days post-infection (**Figure 1B**). Finally, with Enterococcus faecalis, a Gram-positive bacterium, 50% of wild-type flies, but no 1bbd flies, were alive 5 days after infection (**Figure 1C**).

The impairment of Toll-mediated defenses by deletion of bombardier was significant for all three pathogens (p < 0.0001). In the case of C. glabrata, the immunodeficiency of 1bbd phenocopied that observed for Bom155C flies (n.s., p > 0.05). In contrast, with either F. oxysporum or E. faecalis, the rate of death was greater for Bom155C than for 1bbd ( ∗∗∗p < 0.0001 for both infections). The 1bbd mutant thus displays a substantial, but not complete, loss of Toll-mediated defense.

The expression of bombardier is strongly induced by Toll, but not Imd activation (14). We therefore hypothesized that Imdmediated defenses would not require bombardier function. To test this prediction, we infected 1bbd flies with Enterobacter cloacae, a Gram-negative bacterium. In this experiment, 1bbd flies were as immunocompetent as w 1118 flies: more than 90% of both genotypes survived at least 5 days post-infection (**Figure 1D**). In contrast, 100% of RelE20 flies, which are deficient in Imd signaling (40), succumbed to infection within 1 day. Thus, bombardier functions in defense against a range of pathogens for which Toll mediates defense—yeast, filamentous fungi, and Lys-type Gram-positive bacteria—but not against Gram-negative bacteria, against which the Imd pathway is active.

FIGURE 1 | The *bombardier* gene is specifically required for Toll-mediated defense. (A–D) Survival curves of flies infected as indicated. The *w*1118 strain was the wild-type control; *Bom*155*C* and *RelE*20 were the susceptible controls (24, 40). Experiments were completed in triplicate with at least 25 flies per genotype in each replicate. Statistical significance was determined using the Gehan-Breslow-Wilcoxon test and 1*bbd* is shown relative to *w*1118 in black, relative to *Bom*155*C* in orange, and relative to *RelE*20 in red (\*\*\**p* < 0.0001; n.s., not significant, *p* > 0.05).

### The Candidacidal Activity of Hemolymph Requires Bombardier, but Neither Drosomycin Nor Metchnikowin

Next, we investigated the potential humoral role of Bombardier by preparing and assaying cell-free hemolymph. We have previously shown that hemolymph from wild-type flies exhibits a Toll-dependent and Bomanin-dependent candidacidal activity (25). However, we were also curious as to the identity of the active antifungal component. In particular, we considered the potential role of Metchnikowin (Mtk) and Drosomycin (Drs), two antimicrobial peptides (AMPs) that have documented antifungal activity in vitro and are strongly Toll-induced in vivo (14, 41, 42). We therefore took advantage of the recently described 1AMPs strain, which is deficient for Mtk and Drs, as well as all other induced AMPs other than the Cecropins (23). Extracting and assaying Toll-induced hemolymph, we found that hemolymph from 1AMPs flies had a killing activity against C. glabrata comparable to that of wild-type hemolymph (**Figure 2**). In contrast, we failed to detect any killing of C. glabrata by 1bbd hemolymph. We conclude that Boms and Bombardier, but neither Mtk nor Drs, are required for humoral defense against C. glabrata.

### Short-Form Bom Peptides Are Specifically Absent From 1bbd Hemolymph

MALDI-TOF provides a robust tool for characterizing small (<5,000 MW) peptides present in hemolymph after Toll activation. As shown in **Figures 3A,B**, such a readout includes the aforementioned AMPs (Mtk and Drs), several short-form Boms (BomS1, S2, S3, and S6; see **Supplemental Table 2** for updated Bomanin nomenclature), and other induced peptides (e.g., IM4). We have previously shown that deleting the 55C Bom gene cluster removes the peaks attributable to the shortform Boms, while leaving the remaining signals unaffected (25). Remarkably, analysis of 1bbd hemolymph yielded a similar pattern. As shown in **Figures 3C,D**, the short-form Boms that were readily detectable in the wild type—S1, S2, S3, and S6—were absent in 1bbd hemolymph, whereas the remaining peptides, including Mtk, Drs, and IM4, displayed a wild-type profile.

Although 1bbd disrupts the accumulation of short-form Bom peptides in hemolymph, this effect does not reflect a disruption in transcription or stability of the corresponding Bom mRNAs: robust induction of Toll-regulated genes, including genes of short-form Boms, was readily detectable with qRT-PCR (**Supplemental Figure 1**).

each point representing one replicate. One-way ANOVA was calculated followed by Tukey's test. Significance is shown relative to the null hypothesis of 0% killing (\*\*\**p* < 0.0001; n.s., not significant, *p* > 0.05). Error bars represent the 95% confidence interval.

Because proteins such as Bombardier and bicipital Boms are too large to be detected by our MALDI-TOF protocol, we used LC-MS to further characterize the relationship between Bombardier and the Boms in hemolymph. For these studies, we prepared Toll-induced hemolymph from three genotypes: w 1118 , 1bbd, and Bom155C . In wild-type hemolymph, we readily detected Bombardier protein (**Figure 4**), consistent with the presence of a canonical secretion signal sequence in the Bombardier coding sequence. Bombardier, like the Boms, is thus secreted into hemolymph upon Toll induction. We also detected all three bicipital Boms—BomBc1, BomBc2, and BomBc3. The LC-MS studies thus complemented the MALDI-TOF studies, with bicipital Boms detected by the former and short-form Boms by the latter (tailed Boms are not detected by either protocol). Next, we assayed 1bbd hemolymph. As expected, Bombardier was not detected. However, the three bicipital Boms were present at comparable levels in wild-type and 1bbd hemolymph (see **Figure 4**). Combined with the MALDI-TOF studies, these results demonstrate that 1bbd blocks accumulation in hemolymph of short-form, but not bicipital, Boms. Lastly, we analyzed hemolymph from Bom155C flies, which lack 10 of the 12 Bom genes. As expected, the products of the two deleted bicipital genes (BomBc1 and BomBc2) were absent, whereas the product of the remaining bicipital gene (BomBc3) was present at wild-type levels (see **Figure 4**). Turning our attention to Bombardier, we observed no effect of the 55C Bom deletion. Thus, Bombardier is required for the presence of short-form Boms in hemolymph, but the 55C Boms are not required for the presence of Bombardier.

#### Bombardier Mediates Both Infection Resistance and Tolerance

The 1bbd survival phenotype could be due to an inability to control pathogen growth—a defect in resistance—or an inability to endure infection—a defect in tolerance. Because flies lacking Bombardier demonstrate an increased susceptibility to infection and decreased levels of known resistance factors, the shortform Boms, it seemed likely that 1bbd flies, like Bom155C flies, have a defect in infection resistance. In exploring this hypothesis, we found that the model recently developed by Duneau et al. provided a useful framework (34). Following infection of an individual fly, there are two stereotypic outcomes: either the pathogen replicates, reaches a lethal burden, and the fly dies; or the pathogen is controlled at a level below the lethal burden and the fly survives with a persistent infection. Variation in survival curves for different pathogens and fly genotypes reflects variation in both the time required to reach lethal burden and in the fraction of flies that are able to control the infection before it reaches such a threshold. In cases where a fraction of flies control infection, group survival typically drops after infection and then reaches a plateau (23).

The survival curve for 1bbd flies infected with E. faecalis does not plateau (see **Figure 1C**). Instead, it exhibits a profile that we hypothesize reflects two phases of death. In the first phase, extending roughly 2 days post-infection, some 1bbd flies reach a lethal burden of E. faecalis and die, as reflected in a sharp decline in survival; the remainder control the infection. In the second phase, from 2.5 days onward, those flies with a persistent infection die at a reduced but steady rate, due to a defect in tolerance. If this hypothesis is correct, flies dying in the first phase should have a bacterial load upon death (BLUD) comparable to that of wild-type flies dying from infection. Furthermore, those dying in the second phase should have a much lower pathogen burden, comparable to that of wild-type survivors with a persistent infection.

To test our predictions regarding pathogen burden, we measured the BLUD of individual flies after infection with live E. faecalis and divided the data into two time intervals (**Figure 5**). For the earlier interval (dead flies obtained between 17 and 51.5 hpi), both Bom155C and 1bbd bacterial loads upon death were not significantly different from w 1118 (**Figure 5**, red, p > 0.05). For the later time interval (flies obtained between 68 and 120.5 hpi), 1bbd flies perished at significantly lower bacterial loads compared to that of 1bbd flies which died earlier (**Figure 5**, 1bbd early compared to 1bbd late, p < 0.0001), indicating that these two groups die from distinct causes. Importantly, late-death 1bbd flies perished at significantly lower bacterial loads than those of w 1118 suffering early deaths (p < 0.0001), demonstrating that 1bbd flies have a defect in tolerance.

Together, the survival curve and BLUD data offer strong support for our two-phase-model: 1bbd flies died early in infection with high bacterial loads, due to a defect in resistance, and died later with lower bacterial loads, reflecting a deficiency in tolerance. However, we note that the bacterial loads of

1bbd flies dying in the later phase were still significantly greater than those of w 1118 flies alive 120 hpi (**Figure 5**, 1bbd late compared to live w 1118 , p < 0.0001). This indicates that the later-death 1bbd group has not completely controlled infection compared to the live w 1118 flies, and suggests that both resistance and tolerance contribute to the later 1bbd fly deaths. Although we cannot rule out a minor resurgence in bacterial proliferation preceding late death of bbd flies, we note that BLUD and time of death were not significantly correlated for these flies (**Supplemental Figure 2**, Spearman correlation test, r = −0.2654, p = 0.1564).

#### Immune Activation, Specifically Bom Expression, Is Deleterious in the Absence of Bombardier

What is the nature of the tolerance defect we observed in 1bbd flies? More specifically, is their health impaired by an excessive or toxic immune response, or is death due to another class of impaired tolerance (43)? To distinguish between these explanations, we assayed the effect of activating the immune response in 1bbd flies in the absence of infection.

When 1bbd flies were challenged with heat-killed E. faecalis, we observed a decrease in survival that first was apparent 3 days post-challenge followed by a steady decline in the number of live flies in the following days (**Figure 6A**), consistent with the timing of the late-phase deaths (see **Figure 5**). Overall, the death rate was slower than that of live infection, but the extent of killing was similar between heat-killed and live E. faecalis: fewer than 20% of flies survived (compare **Figures 1C**, **6A**). In contrast, no effect on survival was observed upon challenge of either w 1118 or Bom155C flies with heat-killed E. faecalis: >95% flies survived seven or more days post-challenge.

The effect of immune stimulation on 1bbd survival was not specific to E. faecalis. When we repeated the challenge experiments with heat-killed Micrococcus luteus, which activates the Toll response [see **Supplemental Figure 1**, as well as (25, 44)], the effect on 1bbd survival was again marked: 5 days after challenge, fewer than 5% of 1bbd flies were alive, compared to

survival of >95% of w 1118 and 85% of Bom155C flies over the same period of time (**Figure 6B**).

As both M. luteus and E. faecalis induce the Toll pathway, Toll activation could be the key factor in 1bbd mortality. To address this hypothesis, 1bbd flies were crossed with MyD88kra1 (Tolldeficient) flies to generate the MyD88kra1 1bbd double mutant, and the resulting flies were challenged with heat-killed E. faecalis and M. luteus. Unlike 1bbd flies, MyD88kra1 1bbd flies survived challenge with Toll activators (**Figures 7A,B**). Because blocking the Toll pathway with MyD88kra1 rescues the 1bbd phenotype triggered by heat-killed bacteria (p < 0.0001 compared to 1bbd, p > 0.05 compared to MyD88kra1 for both heat-killed bacteria), we conclude that Toll activation underlies the death of 1bbd flies in the absence of infection.

As described above, Bom genes are transcribed in 1bbd flies (**Supplemental Figure 1**), but short-form Bom peptides do not appear in hemolymph (**Figure 3**). This suggests a mislocalization of these peptides, perhaps in an unprocessed or misfolded state. Given that short-form Bom genes are among the most abundantly transcribed genes after infection (17, 36), such mislocalized or misfolded Boms could rapidly accumulate to high levels in 1bbd flies. Could this explain the death of 1bbd flies upon immune stimulation? To address this question, we generated Bom155C 1bbd double mutants and assayed the effect of immune induction alongside both Bom155C and 1bbd flies (**Figures 7A,B**). The result was unequivocal: introducing Bom155C , which deletes all of the short-form Boms, eliminated the effect of 1bbd on survival following immune stimulation (p < 0.0001 compared to 1bbd, p > 0.05 compared to Bom155C for both heat-killed bacteria). The fact that Bom155C is epistatic to 1bbd demonstrates that Toll-driven expression of Bom genes is specifically responsible for the death of immune-stimulated 1bbd flies.

#### DISCUSSION

The results presented in this study identify a key factor that regulates humoral and Bom-mediated defense in Drosophila. We demonstrate that 1bbd flies are defective in resistance to pathogens controlled by the Toll pathway. The results support the hypothesis that this defect results from the absence of shortform Boms in 1bbd hemolymph. Absence of Boms is sufficient to cause a defect in resistance (24) and 1bbd hemolymph appears to be lacking the short-form Boms but no other component, save Bombardier itself. Furthermore, 1bbd phenocopies Bom155C with regard to survival after C. glabrata infection, and resistance to C. glabrata can be restored in Bom155C flies by expression of short-form Boms (25). Finally, 1bbd hemolymph lacks candidacidal activity, which is dependent on short-form Bom peptides (25) and which we show here does not require Drs or Mtk.

For pathogens other than C. glabrata, the effect of deleting Bombardier is less severe than that of deleting the ten Bom genes clustered at 55C. Our mass spectrometry data suggest an explanation. Whereas, short-form Boms are absent from 1bbd hemolymph, bicipital Boms are present. (Tailed Boms were not detected with either mass spectrometry method.) Therefore, we postulate that the bicipital Boms, which are not required for resistance to C. glabrata (25), are functional against other pathogens. This would explain why 1bbd flies are more resistant than Bom155C flies upon infection with E. faecalis or F. oxysporum (**Figure 1**). In this regard, we note that Bombardier and all three forms of Bom proteins—short, tailed, and bicipital are found across the Drosophila genus, supporting the notion that all three classes of Boms are immunoprotective and therefore maintained across the Drosophila genus.

It might appear that our discovery of Bombardier was serendipitous, given our role in defining the Bomanin gene family (24, 25). In hindsight, however, the link was forged in our approach. We selected CG18067 from the most strongly inducible Toll-regulated loci, a group that also includes eight of the Bomanin genes. Next, we engineered a CRISPR/Cas9 deletion of CG18067 and assayed this knockout with the identical set of pathogens that we had used for the Bom155C deletion, screening for loss of survival upon infection. Having examined a gene that is as strongly induced as the Bomanins, present in the same range of species as the Bomanins, and with a spectrum of loss-of-function phenotypes similar to that of the Bomanins, it is not particularly surprising that we would find ourselves studying a gene that affects the Bomanins.

#### Bombardier Function and Structure

What is the function of Bombardier? Deleting the gene results in the absence of short-form Boms from hemolymph, an effect

we find is at the level of protein. Other mature immune peptides are present at normal levels in the hemolymph, and there is thus no general defect in translation, secretion, or processing. Based on these findings, we propose that Bombardier normally functions either to chaperone short Boms as they are secreted from the fat body into the hemolymph or, alternatively, to protect the Boms from misfolding or aggregation while in the hemolymph. We further hypothesize that it is the ectopic localization or aberrant form of short-form Boms in 1bbd flies that generates morbidity upon Toll pathway activation. In support of this idea, we showed that Bom expression underlies the lethality observed in 1bbd flies (**Figure 7**). Whether the short-form Boms physically interact with Bombardier, perhaps in the context of a larger antimicrobial complex, is currently unknown.

Activation of Toll-like receptor (TLR) signaling is important for innate immunity, but induction of the pathway can lead to autoimmune disorders and chronic inflammatory disease (45–48). Here we report an autoimmune activity driven by Toll-induced Bom expression in flies lacking a downstream pathway component, Bombardier. To what extent this parallel can be exploited in the context of understanding autoimmune disorders promises to be a significant focus for future investigation.

#### DATA AVAILABILITY STATEMENT

The LC-MS datasets generated for this study can be accessed on the MassIVE data repository using the accession identifier: MSV000084509.

#### AUTHOR CONTRIBUTIONS

SL and SW: conceptualization, writing—original draft, and visualization. SL, AF, and EB: methodology. SL and AF: formal analysis. SL, AF, and LC: investigation. SL, AF, LC, EB, and SW: writing—review and editing. EB and SW: supervision and funding acquisition.

#### FUNDING

This work was supported by National Institutes of Health (NIH) grant R01 GM050545 (to SW), NIH grant GM119132 (to EB), and NIH predoctoral training grant T32 GM008666 (to SL). The funders had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript.

#### REFERENCES

#### ACKNOWLEDGMENTS

We thank Scott Lindsay for a wealth of helpful discussions and technical assistance; Andres Mauricio Caraballo-Rodriguez and the Pieter Dorrestein Lab for guidance on and access to MALDI-TOF mass spectrometry equipment; Scott Rifkin for advice on statistical approaches; Emily Troemel, Bill McGinnis, Scott Lindsay, and Roland Liu for comments on the manuscript; and the laboratories of Bill McGinnis, Valentino Gantz, Ethan Bier, and James Posakony for access to equipment.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.03040/full#supplementary-material

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Lin, Fulzele, Cohen, Bennett and Wasserman. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Daisho Peptides Mediate *Drosophila* Defense Against a Subset of Filamentous Fungi

Lianne B. Cohen, Scott A. Lindsay, Yangyang Xu, Samuel J. H. Lin and Steven A. Wasserman\*

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, United States

Fungal infections, widespread throughout the world, affect a broad range of life forms, including agriculturally relevant plants, humans, and insects. In defending against fungal infections, the fruit fly Drosophila melanogaster employs the Toll pathway to induce a large number of immune peptides. Some have been investigated, such as the antimicrobial peptides (AMPs) and Bomanins (Boms); many, however, remain uncharacterized. Here, we examine the role in innate immunity of two related peptides, Daisho1 and Daisho2 (formerly IM4 and IM14, respectively), found in hemolymph following Toll pathway activation. By generating a CRISPR/Cas9 knockout of both genes, 1daisho, we find that the Daisho peptides are required for defense against a subset of filamentous fungi, including Fusarium oxysporum, but not other Toll-inducible pathogens, such as Enterococcus faecalis and Candida glabrata. Analysis of null alleles and transgenes revealed that the two daisho genes are each required for defense, although their functions partially overlap. Generating and assaying a genomic epitope-tagged Daisho2 construct, we detected interaction in vitro of Daisho2 peptide in hemolymph with the hyphae of F. oxysporum. Together, these results identify the Daisho peptides as a new class of innate immune effectors with humoral activity against a select set of filamentous fungi.

#### *Edited by:*

Dan Hultmark, Umeå University, Sweden

#### *Reviewed by:*

Ulrich Theopold, Stockholm University, Sweden Hai-peng Liu, Xiamen University, China

#### *\*Correspondence:*

Steven A. Wasserman<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology

*Received:* 18 October 2019 *Accepted:* 06 January 2020 *Published:* 23 January 2020

#### *Citation:*

Cohen LB, Lindsay SA, Xu Y, Lin SJH and Wasserman SA (2020) The Daisho Peptides Mediate Drosophila Defense Against a Subset of Filamentous Fungi. Front. Immunol. 11:9. doi: 10.3389/fimmu.2020.00009 Keywords: innate immunity, toll, *Drosophila*, humoral, antifungal

#### INTRODUCTION

Fungal infections have a devastating impact on a wide range of organisms. They are destructive to agricultural plants around the world, including rice, wheat, and tomatoes (1). Additionally, fungi infect more than one million humans annually (2). Existing antifungal treatments are limited, with only one new class of drugs, echinocandins, developed in the past 15 years. Furthermore, extensive usage of limited classes of related antifungals has led to the increasingly frequent appearance of drug-resistant fungi (2). An enhanced understanding of naturally occurring antifungal defenses is thus of tremendous potential benefit.

The fruit fly Drosophila melanogaster is a robust model for fungal infections, replicating many features of murine fungal infections (3, 4). In the wild, flies have been found to be infected with a number of filamentous fungi, including Beauveria, Metarhizium, and Fusarium species (5, 6). In combatting these infections, flies rely on the Toll innate immune pathway (7, 8). Toll provides defense against not only filamentous fungi, but also yeasts and those Gram-positive bacteria that produce a cell wall containing Lys-type peptidoglycan (8–10). A second innate immune pathway,

**68**

defined by the Imd receptor, provides defense against Gramnegative bacteria and the limited number of Gram-positive bacteria that produce a cell wall containing DAP-type peptidoglycan (11, 12).

Systemic activation of Toll signaling induces a broad set of genes first identified by microarray analysis and mass spectroscopy (13–16). Many of the induced innate immune genes are transcribed in the fly fat body, with the protein products secreted into the hemolymph. These include antimicrobial peptides (AMPs), the Bomanin peptides, and a number of uncharacterized peptides.

 In contrast, the Bomanin family of peptides (Boms) are required for defense against both classes of pathogens (21). Boms, which are Drosophila-specific, are readily detected in hemolymph following Toll activation. Here we describe the functional characterization of additional immune effectors, the Daisho peptides, which appear in hemolymph following systemic infection and are required for defense against a subset of filamentous fungi.

### MATERIALS AND METHODS

#### Fly Husbandry and Strain Generation

Flies were raised at 25◦C on cornmeal molasses agar media1 . The w 1118 strain was used as the wild type. MyD88− flies were MyD88kra1 , and imd− flies were imdshadok .

As described in Results, the genes for the immune induced peptides IM4 and IM14 have been given the designations daisho1 and daisho2, respectively. The null allele 1daisho, deleting both genes, as well as the individual gene deletions, 1dso1 and 1dso2, were generated using CRISPR/Cas9 technology, applying methods described previously (22). Pairs of guide RNAs that targeted Cas9 to delete the region 2R: 20,868,460–20,870,480 for 1daisho), 2R: 20,868,783–20,869,392 for 1dso1, and 2R: 20,870,332–20,870,728 for 1dso2 were cloned into the pU6- BbsI-chiRNA vector (Addgene plasmid # 45946). Homology arms of ∼1 kb were cloned into pHD-DsRed (Addgene plasmid # 51434). Cas9 was provided by plasmid pBS-Hsp70-Cas9 (Addgene plasmid #46294). Constructs were based on target sequences in the w 1118 strain and injected into w 1118. See **Table S1** for primer sequences.

The FLAG epitope tag was cloned between the signal sequence and mature peptide of Dso2 in the context of the pHD-DsRed homologous repair template. This FLAG-Dso2 construct was introduced at the dso2 genomic locus using the 1dso2 guide RNAs.

Plasmids expressing dso1 or dso2 transcripts from the pBomS3 promoter were made using methods previously described (23). Briefly, the BomS3 gene promoter was placed 5′ to the ORF encoding either Dso1 or Dso2. These constructs were then each integrated via ΦC31-mediated transgenesis at an attP landing site located at 86Fb on the D. melanogaster third chromosome (BDSC stock #24749). The transgenes were crossed into the 1dso1 and 1dso2 backgrounds and homozygous stocks were derived. An empty vector control was also introduced at the 86Fb attP landing site.

#### Microbial Cultures

For survival experiments, microbes were cultured as follows. Enterococcus faecalis NCTC 775 (ATCC 19433) and Enterobacter cloacae were grown overnight at 37◦C in LB media and concentrated to an OD600 of 10 in 20% glycerol. Candida glabrata CBS 138 [ATCC 2001] was grown overnight in YPD media at 37◦C and concentrated to an OD600 of 100 in PBS, 0.1% Tween. All filamentous fungi were grown on malt extract agar plates at 29◦C until sporulation was observed (10–15 days). Fungal material was then strained through glass wool with sterile water to collect spores, which were concentrated in 20% glycerol and stored at −80◦C before being used at the following concentrations (in spores/ml): Aspergillus flavus (sequenced strain): 5 × 109 ; A. fumigatus AF293 (FGSC# A1100): 6 × 109 ; A. parasiticus Nor-1 mutant (NRRL #6111): 3 × 109 ; Botrytis cinerea (B05.10): 3 × 109 ; Fusarium graminearum (NRRL #5883): 8 × 108 ; F. oxysporum f. sp. lycopersici 4287 (FGSC #9935): 3 × 108 ; F. verticillioides (FGSC #7415): 3 × 109 ; Neurospora crassa: 1 × 109 .

For the induction of the Toll response, heat-killed Micrococcus luteus was prepared as previously described (23).

#### Survival Assays

Groups of 20–25 adult male flies aged 2–7 days were collected and stabbed with a needle dipped in a suspension of bacteria, yeast, or fungal spores. Where needed, MyD88− or Bom155C flies were used as controls immunodeficient for the Toll-mediated response. Flies infected with E. faecalis were incubated at 25◦C; all other infected flies were incubated at 29◦C. Fly deaths were recorded at least twice per day for the duration of each experiment. Any deaths that occurred within the first 6 h were set aside to exclude from the data any deaths due to traumatic injury. The experiment was repeated three times and results combined. Statistical analyses were performed using the Gehan-Breslow-Wilcoxon test.

#### MALDI-TOF

After Toll induction with heat-killed M. luteus, flies were incubated at 29◦C for 24 h, after which hemolymph was collected via capillary as previously described (23). Hemolymph in 0.1% trifluoroacetic acid/50% acetonitrile was mixed 1:1 with Universal Matrix (Sigma-Aldrich). Samples were then dried onto a Bruker MSP 96 ground steel plate. Spectra were collected from 1,500 to 10,000 m/z for linear mode, and 1,000– 5,000 m/z for reflectron mode, both with positive polarization. Peptide calibration standard II (Bruker) was used as an external calibration standard. For each genotype, at least five independent samples were collected. Representative spectra are shown. Peaks were identified via corresponding m/z values from previous studies (13, 16). Spectra were visualized using R 3.3.2 and ggplot2 2.2.1 (24, 25).

1http://blogs.cornell.edu/drosophila/dssc-cornmeal-recipe/

#### Quantitation of Pathogen Load

Pathogen load in infected flies was measured by qRT-PCR of fungal RNA (26, 27). Adult male flies, 2–7 days old, were stabbed with a needle dipped in F. verticillioides at 3 × 109 spores/ml. Flies were then incubated at 29◦C. Groups of 5–6 flies were collected at the stated times and frozen in liquid nitrogen. Total RNA was isolated with TRIzol (Ambion) and cDNA was made via SuperScript RT II (Invitrogen). EF1A was selected as a proxy gene for fungal load based on its stable expression (28). Measurements by qRT-PCR were performed on the iQ5 cycler (BioRad) with iQ SYBR Green Supermix (BioRad) using the primers listed below. Values were normalized to fly mRNA based on expression of the rp49 gene.

Primers: Fv\_EF1A\_F1: GGCTTTCACTGACTACCCTCC TCT, Fv\_EF1A\_R1: ACTTCTCGACGGCCTTGATGACAC, rp49\_F1: CAAGGGTATCGACAACAG, rp49\_R1: CTTGTT CGATCCGTAACC.

#### Peptide Gel Electrophoresis and Immunoblotting

Hemolymph samples were collected via the Zymo-Spin IC column method (23) from 30 male flies aged 2–7 days that had been induced with heat-killed M. luteus and incubated for 24 h at 29◦C. Samples were run on a SDS-tricine, 18% separating/10% spacer/4% stacking, acrylamide gel2 . Protein samples were then transferred to a PDVF membrane, blocked with 5% milk in TBST and stained with primary α-FLAG M2 (Sigma) (1:500) and secondary sheep α-mouse HRP (Amersham Biosciences) (1:1,000). The immunoblot was then treated with West Pico PLUS substrate (Thermo Scientific) and exposed to film.

#### Peptide Hyphal Binding and Immunofluorescence

The immunostaining protocol was adapted from Luo et al. (29). F. oxysporum was grown in 5 ml malt extract broth from a starting concentration of 2.9 × 105 spores/ml. After overnight shaking at room temperature, fungal hyphae were collected by centrifugation at 1,000 g for 10 min and resuspended in PBS. Hemolymph was collected via the Zymo-Spin IC column method (23) from 420 male flies that had been induced with heat-killed M. luteus 24 h prior and incubated at 29◦C, yielding ∼35 µl cell-free hemolymph. Next, aliquots of 200 µl hyphae and 35 µl hemolymph were shaken at room temperature for 30 min. The samples were washed three times with PBS before fixation with 4% formaldehyde for 1 h. After washing another three times with PBS, samples were blocked for 1 h with 5% BSA. Samples were then incubated with α-FLAG antibody (1:200) overnight at 4◦C. After washing with PBS, samples were stained for 2 h with donkey α-mouse Alexa555 (1:400) and DAPI (1:200) and then washed and mounted on slides. Samples were imaged with a Ti2 Widefield microscope (Nikon) and analyzed with the NISelements software and OMERO.

#### RESULTS

#### Generation of Flies Null for the *daisho* Gene Pair

Pioneering mass spectrometry experiments by Bulet et al. identified two dozen peptide IMs (immune-induced molecules) that accumulate in Drosophila hemolymph upon induction of the innate immune response, principally the Toll pathway (13, 16). Among these, the Bomanins have been found to play an essential role against a broad range of pathogens (21, 23) while several, including the 15 aa long IM4 and 24 aa long IM14, have unknown functions. Based on our demonstration of defensive functions for these peptides, we have renamed them Daisho1 and Daisho2, for daisho, the Japanese term for a matched pair of samurai swords, one short and one long.

The Daisho peptides are closely related to one another and occupy adjacent positions in the genome, where they are divergently transcribed (**Figure S1**). As shown in **Figure 1A**, the sequence of amidated mature Daisho1 (Dso1) has 67% identity with the corresponding region of the mature Daisho2 (Dso2) peptide. Like the Bomanins, the daisho genes are widespread among the Drosophila genus, but not identified elsewhere. To investigate the potential role of the daisho genes in innate immunity, we used CRISPR/Cas9 technology to delete both genes. The 2.0 kb deleted region includes the entire dso1 gene, the upstream region for both genes, and the first exon of dso2 (including the start codon). Flies homozygous for the 1daisho1,2 deletion, hereafter 1daisho, were viable and fertile.

With the 1daisho stock in hand, we carried out MALDI-TOF studies of hemolymph (**Figures 1B–E**). As described above, following Toll activation wild-type hemolymph displays robust expression of immune peptides, including the Daisho peptides, Bomanins, and AMPs. The signals from Dso1 and Dso2 were ablated in 1daisho, as evidenced by the loss of signal at 1,722 mass/charge (m/z) (Dso1) and 2,694 m/z (Dso2). Furthermore, the spectra of induced 1daisho hemolymph was wild-type for all previously identified peaks other than Dso1 and Dso2, including the Bomanins and AMPs, Metchnikowin (Mtk), and Drosomycin (Drs). The absence of Dso1 and Dso2 thus did not detectably alter the accumulation or modification of other Toll-induced peptides in the hemolymph.

In addition to previously identified peaks, 1daisho hemolymph contained one previously unseen signal. The 1,724 m/z signal of this peak, readily apparent in reflectron mode, is identical to that predicted for the BomS5 amidated peptide, previously known as CG15065 (**Figure 1E**). This signal had not been detected previously because in the wild type it lies in the shoulder of the robust Dso1(IM4) peak. Its existence in Toll-induced hemolymph was expected, however, on the basis of microarray and RNAseq data demonstrating strong Toll-activated induction of the BomS5 locus (15, 30).

#### The *daisho* Genes Are Specifically Required for Defense Against *F. oxysporum*

We next turned to a functional assay to determine whether the absence of the Daisho peptides impaired survival following systemic infection. Because the Toll pathway responds to and

2https://molbio.mgh.harvard.edu/szostakweb/protocols/protein\_page/index.html

protects against infection by many Gram-positive bacteria and fungi, we focused on these classes of pathogens. We stabbed adult flies with a needle dipped in a suspension of bacteria, yeast, or fungal spores and then monitored survival. We used w 1118 flies as our wild-type, i.e., immunocompetent, control and Bom155C flies, which lack the 10-gene Bom cluster, as an immunodeficient control (21).

For a number of the pathogens tested, 1daisho flies behaved identically to the wild type. Roughly 50% of both wild-type and 1daisho flies survived 6 or more days following infection with the Gram-positive bacteria Enterococcus faecalis, whereas 100% of Bom155C flies died within 2 days (**Figure 2A**). Likewise, wildtype and 1daisho flies survived a week or longer after infection with the yeast Candida glabrata, whereas Bom155C flies died in 4 days or fewer (**Figure 2B**). We also found no effect of 1daisho on immune defenses mediated by the Imd pathway: wild-type, 1daisho, and Bom155C flies all survived infection with the Gramnegative bacteria Enterobacter cloacae, whereas, control imd- flies died within 1 day (**Figure 2C**).

For one pathogen in the initial test set, the filamentous fungus Fusarium oxysporum, deletion of the daisho genes had a marked effect on survival (**Figure 2D**): 50% of flies homozygous for 1daisho died within 4 days of infection. In contrast, <70% of wild-type flies survived 7 or more days post-infection. Thus, loss of the Daisho peptides disrupts defense against F. oxysporum, but not other tested pathogens. Interestingly, loss of the Daisho peptides did not impact survival peptides did not impact survival as severely as did loss of the Boms, which led to 50% death after 2 days, very similar to complete loss of Toll signaling (21).

### 1*daisho* Flies Are Susceptible to Some but Not All Filamentous Fungi

We next investigated whether the susceptibility of 1daisho flies to F. oxysporum reflected a general susceptibility to filamentous fungi. For these studies, we focused on filamentous fungi for which flies deficient for Toll signaling, and thus for induction of Daisho1, Daisho2, and other Toll effectors, exhibit a significantly decreased survival relative to wild type (**Figure 3**). The control fly strains in each case were w 1118 (wild type) and kra-1 (MyD88−), a loss-of-function allele for an essential mediator of Toll signaling (31).

As shown in **Figure 3**, susceptibility of 1daisho flies to the filamentous fungi species varied. Survival was significantly less than wild-type for F. verticillioides and F. graminearum (panels A, B), two Fusarium species closely related to F. oxysporum. In the case of F. graminearum, survival of 1daisho flies was intermediate between that of wild-type and MyD88− flies, a pattern very similar to that observed with F. oxysporum, where 1daisho survival falls between wild type and Bom155C, which behaves similarly to MyD88− (21). In contrast, 1daisho flies displayed a much greater immune impairment upon infection with F. verticillioides than with F. oxysporum, dying to a comparable extent and at a similar rate as the MyD88− control (compare **Figures 2D**, **3A**).

Variation in survival was also observed among Aspergillus species. The survival curves of 1daisho infected with either A. parasiticus or A. flavus largely tracked with MyD88− (panels C, D). Upon A. fumigatus infection, however, 1daisho flies survived at least twice as long as MyD88− flies (**Figure 3E**).

For some filamentous fungi, loss of Daisho1 and Daisho2 did not affect survival. For example, 80% of wild-type and 1daisho flies survived for at least 7 days after infection with Botrytis cinerea, whereas >50% of MyD88− flies died after 2 days (**Figure 3F**). Likewise, wild-type and 1daisho flies survived Neurospora crassa infection for 6 days or more, but 50% of MyD88− flies died after 3 days (**Figure 3G**). Overall, we find that the Daisho peptides play a vital role in survival after infection with certain species of filamentous fungi, but are not important for infections with others.

#### *daisho1* and *daisho2* Are Each Required for Defense

Daisho1 and Daisho2 are highly similar in sequence and expression pattern. Are they functionally redundant? To address this question, we explored the function of each individual locus. We again used CRISPR/Cas9, generating deletions that removed the entire coding sequence for either daisho1 or daisho2. The 5' endpoints of each deletion were chosen to lie within 100 bp of the transcriptional start site, minimizing potential disruption of elements in the regulatory region separating the two genes (**Figure S1**). For both deletions, MALDI-TOF analysis of induced hemolymph confirmed loss of the deleted gene product but no other peptides, indicating that either Daisho1 or Daisho2 can be stably expressed in the absence of the other (**Figure 4**).

To test the effect on defense of deleting dso1 or dso2, we stabbed adults with F. verticillioides spores, for which 1daisho flies have a reduced survival. Deleting either the dso1 or dso2 gene resulted in susceptibility to F. verticillioides markedly different from wild-type and comparable to that of the double deletion (**Figure 5**). Thus, Daisho1 and Daisho2 each act in defense against F. verticillioides infection.

Since deletion of either dso1 or dso2 had as severe an effect on survival as the double mutant, it was possible that each gene has a specific and distinct function in antifungal defense. Alternatively, survival might depend only on total dosage for the two genes, with loss of either dropping expression below the threshold required. To distinguish between these models, we generated transgenes placing each ORF under control of pBomS3, shown previously to be strongly Toll-responsive promoter (23), and then

highlighting loss of Dso1 (A) and Dso2 (B) in deletion mutants.

assayed the transgenes for rescue of 1dso1 or 1dso2. As shown in **Table 1**, pBomS3-driven dso1 rescued 1dso1, improving the median survival from 46 to 93 h (p < 0.0001). The same was true of pBomS3-driven dso2 in the 1dso2 background (p < 0.0001) (see **Figure S2** for full survival curves). Flies expressing the empty vector construct at the same chromosomal location did not show any increase in survival (**Figure S3**).

Having confirmed the activity of the two constructs, we expressed each in a background deficient for the other. dso2 expression significantly improved survival of 1dso1 flies,

TABLE 1 | Median survival in hours of dso1 and dso2 deletion mutations rescued by homotypic and heterotypic transgenes.

Data derived from *Figure S2*. n.a., not applicable.

increasing median survival from 46 to 78 h (p < 0.0001). Similarly, dso1 expressed in a 1dso2 background improved median survival from 46 to 55 h (p = 0.0005). Nevertheless, rescue was incomplete. The median survival of dso2 expressed in 1dso1 background (78 h) did not reach median survival of 1dso1 rescued with dso1 (93 h) (n.s., p = 0.09). Furthermore, dso1 did not rescue survival of 1dso2 (55 h) to the same level as dso2 (93 h) (p < 0.0001). The data thus indicate that the two loci encode functions that are neither fully distinct nor fully redundant.

#### Deleting *daisho1* and *daisho2* Results in an Elevated Pathogen Load in Infected Flies

To investigate whether Daisho1 and Daisho2 affect pathogen growth during infection, we measured fungal load after infection in 1daisho and wild-type flies. After stabbing adult males with F. verticillioides, groups of 5–6 infected flies were collected and RNA was extracted. Fungal EF1A transcript levels were measured as a proxy for pathogen load and normalized to the fly reference gene rp49. Directly after infection (2 h), there was no significant difference between 1daisho and wild-type flies by Mann-Whitney U test (**Figure S4**). By the next day, however, 1daisho flies had a pathogen load roughly 10-fold greater than wild-type (p = 0.0317).

#### FLAG-Dso2 Binds to *F. oxysporum* Hyphae

We next tagged Dso2, the larger of the two peptides, with the FLAG epitope, using CRISPR/Cas9 to introduce the tag at the amino-terminus of the endogenously expressed mature peptide. Immunoblot analysis of induced hemolymph from FLAG-Dso2 flies revealed a single band detectable with α-FLAG antibody (**Figure 6A**). MALDI-TOF analysis of hemolymph confirmed the loss of the Dso2 peak at 2,694 m/z and the appearance of a peak with an m/z ratio of 3,689, the value expected for FLAG-Dso2 (**Figure 6B**).

Having confirmed that FLAG-Dso2 peptide is stably expressed, we next assayed its activity in providing antifungal defense. Specifically, flies homozygous for FLAG-dso2 at the dso2 locus were infected with F. verticillioides and their survival was compared to both wild-type flies and 1dso2 flies. Survival of FLAG-Dso2 flies was not wild-type, but was significantly better than that of flies lacking the dso2 gene (**Figure S5**). We conclude that the FLAG-Dso2 peptide is active in providing defense against F. verticillioides infection.

Next, we assayed FLAG-tagged Dso2 peptide in hemolymph for its ability to bind fungus. We collected hemolymph from Toll induced flies, incubated it with hyphae from F. oxysporum, and fixed samples. The majority (>80%) of F. oxysporum hyphae had no visible signal when stained with α-FLAG antibody (**Figure 7A**). Among the remaining hyphae, we observed a variety of staining patterns, including, but not limited to, signals concentrated in the regions between nuclei (**Figure 7B**), extending across greater fractions of hyphae (**Figure 7C**) or spanning the length of hyphae (**Figure 7D**). In parallel experiments with untagged wild-type hemolymph, no signal was detected (**Figures 7E–H**). We conclude that Daisho2 peptide in hemolymph can bind to F. oxysporum hyphae.

In summary, our results demonstrate that the pair of immuneinduced peptides, Daisho1 and Daisho2, mediate Toll-induced defense against specific filamentous fungi, most likely via a humoral effect on fungal hyphae.

#### DISCUSSION

#### Role of the Daisho Peptides in Antifungal Defense

In this study we found that the related peptides Daisho1 and Daisho2 are required in D. melanogaster for defense against a subset of filamentous fungi. We have also demonstrated that the two peptides have partially overlapping functions. Survival data reveal a dependence on the overall level of Dso1 and Dso2, with each peptide able to partially compensate for the absence of the other. Furthermore, each peptide accumulates in the absence of the other.

The Daisho peptides lack known motifs of defined function. As noted previously (21), there is a similarity in size and sequence between Dso1 and Dso2 and the Bomanin peptides. There are, however noteworthy differences, including the presence of a CxxC motif in the Bomanins and the broader requirement for the Bomanins in Toll-mediated defense.