Distinct Bomanins at the Drosophila 55C locus function in resistance and resilience to infections

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Distinct Bomanins at the Drosophila 55C locus function in resistance and resilience to infections View ORCID Profile Yanyan Lou , View ORCID Profile Bo Zhang , View ORCID Profile Zhiyuan Zhang , View ORCID Profile Yingyi Pan , View ORCID Profile Jianwen Yang , View ORCID Profile Lu Li , View ORCID Profile Jianqiong Huang , View ORCID Profile Zihang Yuan , View ORCID Profile Samuel Liegeois , View ORCID Profile Philippe Bulet , View ORCID Profile Rui Xu , Li Zi , View ORCID Profile Dominique Ferrandon doi: https://doi.org/10.1101/2025.04.16.649162 Yanyan Lou 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China 2 Université de Strasbourg , Strasbourg, France 3 Modèles Insectes de l’Immunité Innée, UPR 9022 du CNRS , Strasbourg, France 4 Medical Laboratory of Shenzhen Luohu People’s Hospital, The Third Affiliated Hospital of Shenzhen University , Shenzhen, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yanyan Lou Bo Zhang 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bo Zhang Zhiyuan Zhang 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zhiyuan Zhang Yingyi Pan 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yingyi Pan Jianwen Yang 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China 2 Université de Strasbourg , Strasbourg, France 3 Modèles Insectes de l’Immunité Innée, UPR 9022 du CNRS , Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jianwen Yang Lu Li 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lu Li Jianqiong Huang 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China 2 Université de Strasbourg , Strasbourg, France 3 Modèles Insectes de l’Immunité Innée, UPR 9022 du CNRS , Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jianqiong Huang Zihang Yuan 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zihang Yuan Samuel Liegeois 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China 2 Université de Strasbourg , Strasbourg, France 3 Modèles Insectes de l’Immunité Innée, UPR 9022 du CNRS , Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Samuel Liegeois Philippe Bulet 5 Université Grenoble Alpes, Institute for Advanced Biosciences, Inserm U1209 , CNRS UMR 5309, Grenoble, France 6 Platform BioPark Archamps , Archamps, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Philippe Bulet Rui Xu 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China 2 Université de Strasbourg , Strasbourg, France 3 Modèles Insectes de l’Immunité Innée, UPR 9022 du CNRS , Strasbourg, France 7 Department of Pediatrics, The Affiliated Foshan Maternal and Children’s Hospital, Guangdong Medical University , Foshan, Guangdong, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rui Xu Li Zi 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dominique Ferrandon 1 Sino-French Hoffmann Institute, Guangzhou Medical University , Guangzhou, China 2 Université de Strasbourg , Strasbourg, France 3 Modèles Insectes de l’Immunité Innée, UPR 9022 du CNRS , Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dominique Ferrandon For correspondence: D.Ferrandon{at}ibmc-cnrs.unistra.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Host defense against many Gram-positive bacteria and fungal pathogens is mainly provided by the Toll-dependent systemic immune response in Drosophila . While antimicrobial peptides active against these categories of pathogens contribute only modestly to protection, Bomanin peptides are major effectors of the Toll pathway. Remarkably, flies deleted for the 55C locus that contains 10 Bomanin genes are as sensitive as Toll pathway mutant flies to these infections. Yet, the exact functions of single Bomanins in resistance or resilience to infections remain poorly characterized. Here, we have extensively studied the role of these Bomanin genes. BomT1 functions in resistance to Enterococcus faecalis while playing a role in resilience against Metarhizium robertsii infection, like BomS2. BomT1 and BomT2 prevent the dissemination of Candida albicans throughout the host, even though they are not sufficient to confer protection to immunodeficient flies against this pathogen in survival experiments. Furthermore, BomT1 and BomBc1 mutants are sensitive to an Aspergillus fumigatus ribotoxin. We conclude that 55C Bomanins have defined albeit sometimes overlapping roles in the different facets of host defense against infections. Introduction The major host defense against many Gram-positive bacteria, pathogenic yeasts and molds is mediated by the Toll pathway, which regulates one arm of the systemic humoral immune response ( Buchon et al , 2014 ; Ferrandon et al , 2007 ; Lemaitre & Hoffmann, 2007 ; Lindsay & Wasserman, 2014 ). This transmembrane receptor is activated by binding to the Spätzle cytokine, which is itself matured into an active Toll ligand by proteolytic cascades triggered upon sensing microbial cell wall components or the catalytic activity of proteases released by invading pathogenic microorganisms ( Liegeois & Ferrandon, 2022 ). Toll in turn activates a specific NF-κB intracellular signaling cascade that ultimately leads to the induction of expression of likely hundreds of genes, including those encoding antimicrobial peptide (AMP) genes ( De Gregorio et al , 2002 ; Irving et al , 2001 ; Troha et al , 2018 ). For instance, Drosomycin and Metchnikowin have been shown to have antifungal activity in vitro ( Fehlbaum et al , 1995 ; Levashina et al , 1995 ). Yet, even though AMPs play a significant role in the host defense against Gram-negative bacteria, they appear to be rather marginally required for protection against Gram-positive bacteria or fungi ( Cohen et al , 2020 ; Hanson et al , 2019 ). Remarkably, the deletion of 10 genes of the Bomanin family, members of which had initially been identified by mass-spectrometry analysis of the hemolymph of infected flies ( Uttenweiler-Joseph et al , 1998 ), phenocopied the susceptibility of Toll pathway mutants to these categories of pathogens ( Clemmons et al , 2015 ). The Bomanin family of 12 genes can be divided in three subgroups: short Bomanins (BomSs) that essentially contain a 16-amino-acid conserved Bomanin domain, tailed Bomanins (BomTs) for which the Bomanin domain is prolonged by a 15 to 82 amino-acid long extension, and bicipital Bomanins (BomBcs) that contain two Bomanin domains separated by a 43 to 103 amino-acid long linker. Functionally, Bomanins are required for a candicidal activity found in the hemolymph of flies and that may require a sufficiently strong expression of BomSs, without much specificity being involved within the distinct BomSs ( Lindsay et al , 2018 ). Of note, BomSs but not BomBcs require the activity of a Toll pathway-regulated protein, Bombardier (Bbd), which may be required for the secretion or stability of BomSs ( Lin et al , 2019 ). We have recently reported that an important component of host defense against the opportunistic fungal pathogen A. fumigatus is the protection against secreted mycotoxins, which is partially mediated by specific Bomanins ( Xu et al , 2023 ), in keeping with another study extending the concept to the defense against secreted virulence factors by another family of Toll pathway effectors, BaramicinA-derived peptides ( Huang et al , 2023 ). While the deletion of subsets of Bom genes revealed important information on the function of this family of effector peptides ( Clemmons et al ., 2015 ), little is known with respect to the function of individual Bom genes ( Chapman et al , 2020 ; Smith et al , 2023 ; Xu et al ., 2023 ). Here, we use complementary genetic strategies to implement the dissection of 55C Bomanin function in the host defense against a selected set of microbes representing distinct categories of pathogens. Results The 55C Bomanin locus is required in the host defense against several microbial infections We first checked that we could reproduce the data published previously when isogenizing the 55C deletions, Bom Δ55C and Bom Δleft , in our wild-type genetic background w [A5001]( Thibault et al , 2004 ) ( Fig. 1A ). As reported previously, we found that Bom Δ55C mutant flies were highly susceptible to challenges with Enterococcus faecalis and Candida glabrata (now called Nakaseomyces glabratus ). Bom Δleft mutant flies were either sensitive ( E. faecalis ) or behaved as wild-type flies ( C. glabrata ) as expected ( Fig. 1B-C ). We next tested additional pathogens. The two Bom deficiencies exhibited the same phenotype as C. glabrata after the injection of C. albicans , even though the two pathogenic yeasts are evolutionarily distant, the latter one being dimorphic ( Fig. 1D ). Whereas the Bom Δ55C line was as sensitive as the Toll pathway mutant line MyD88 , Bom Δleft displayed an intermediate sensitivity phenotype to an Aspergillus fumigatus challenge ( Fig. 1E ). As reported for the entomopathogenic fungus Beauveria bassiana , both Bom deficiency lines succumbed rapidly at the same rate to injected spores of a related fungus Metarhizium robertsii ( Fig. 1F ). In contrast, upon a natural infection with the same fungus, Bom Δleft mutants exhibited an intermediate susceptibility phenotype ( Fig. 1G ). Bom Δ55C mutant flies were remarkably at least as sensitive as MyD88 flies to these infections, in keeping with previous studies ( Clemmons et al ., 2015 ; Hanson et al ., 2019 ; Smith et al ., 2023 ). Download figure Open in new tab Figure 1. The 55C Bomanin locus is essential for host defense against multiple microbial infections (A) The chromosomal localization of the Bomanin gene cluster and the deletion intervals of the Bom Δ55C , Bom Δleft mutant strains. (B-G) Survival curve of wild-type w [A5001], MyD88 , Bom Δleft and Bom Δ55C flies with E. faecalis (B) , C. glabrata (C) , C. albicans (D) , A. fumigatus (E) , M. robertsii (F) injection and M. robertsii (G) natural infection. Note: (B-G) At least three independent experiments were performed, each experiment used biological triplicates of 20 flies; pooled data are shown. The data were analyzed between infected-mutant and - w [A5001] fly using the Log-Rank test; *** p<0.001; **** p<0.0001; ns, no significant difference. As the host defense encompasses both humoral and cellular immunity, we tested whether Bom Δ55C mutant flies were defective for the phagocytosis of live C. glabrata ( Liegeois et al , 2020 ). We did not observe any significant difference in the uptake of these live microorganisms ( Fig. EV1 ). Genetic strategies to dissect the different functions of the Bomanins encoded at the 55C locus The Bom genes located at the 55C locus are required for both resistance and resilience to infections. It is currently not known whether specific Bomanin genes are specific to host defense against a given pathogen, whether some are particularly involved in resistance or resilience, whether they may play redundant roles or are required for a “cocktail” effect. Here, we have implemented a dual genetic strategy to address these issues. First, we have attempted to study the loss-of-function sensitivity phenotype of single Bom genes at 55C after a challenge with either the Gram-positive bacterium E. faecalis , the pathogenic yeasts C. glabrata and C. albicans , the filamentous fungus A. fumigatus , or the entomopathogenic fungus M. robertsii in two infection models, injection and natural infection. We have generated deletions, knock-out or knock-in mutants using CRISPR-Cas9 technology for BomT1 , BomBc1 , BomS2 , BomT2 , and BomS5 (Fig. S1-S6). We expect to have generated null mutants for BomT1 , BomBc1 , BomS2 (two independent mutants), BomT2 (two deletion lines and one indel), and BomS5 . Of note, the deletion of BomS5 severely affects the expression of BomT2 (Fig. S6). In addition, we have also tested knock-down lines for BomT1 , BomBc1 , BomS4 , BomBc2 , and BomT2 by RNA interference (Fig. S7). We have failed to obtain mutant lines affecting the expression of BomS1 , BomS3 , and BomS6 . Second, we have initiated overexpression strategies to bypass any redundancy issues by overexpressing each gene from transgenes constructed for expression under the control of UAS enhancer sequences ( Brand & Perrimon, 1993 ). Bom genes were overexpressed first in a wild-type (Fig.S8) and second in a MyD88 immunodeficient background. However, upon checking the expression of the BomSs in the hemolymph by MALDI-TOF mass-spectrometry (MS)( Uttenweiler-Joseph et al ., 1998 ), we failed to observe the expected peaks in MyD88 flies (MS being semi-quantitative, it was difficult to assess the overexpression of the gene products in the context of an immune response in wild-type flies; in addition, no ectopic expression was detected in the absence of an immune challenge in flies overexpressing BomS genes). We reasoned that this effect might be caused by the absence of induction of other MyD88- dependent genes that might be required for their expression in the hemolymph. We therefore decided to test each Bom gene for a rescuing activity of the sensitivity of Bom Δ55C deficiency flies to various microbial challenges. Indeed, the role of Bombardier in the secretion or stability of BomS peptide was reported while this work was being pursued ( Lin et al ., 2019 ). As expected, we then did detect the expression of BomS peptide by MALDI-TOF MS (Fig. S9). The results we have obtained are summarized in Tables 1 and 2 (a key to understanding Table 1 is provided in Fig. EV2 ). In the following, we shall describe in more detail the most outstanding results we have obtained. View this table: View inline View popup Download powerpoint Table 1: Single overexpressed Bomanin genes may improve the protection against specific infections in distinct genetic backgrounds: a summary of 55C locus Bomanins Notes: the text in red ‘MyD88(+)’ in the table means that rescued BomS1 in MyD88 mutant background flies cannot rescue to C. glabrata infection but is actually more susceptible to C. glabrata infection compared to MyD88 flies. ‘+’, the intensity of sensitivity. Of note, a key to decipher the indications provided in individual cells is provided in Fig. EV2 . BomT1 is required but not sufficient for resistance against E. faecalis When challenged with E. faecalis , the BomT1 knock-in (KI) null mutant flies succumbed at a rate that was almost as fast as MyD88 mutants ( Fig. 2A ). A similar, albeit milder, phenotype was observed when BomT1 was silenced ubiquitously only at the adult stage ( Fig. 2B ). Of note, the overexpression of BomT1 in wild-type flies did not protect them from E. faecalis ( Table 1 , Fig. S10A); however, it conferred a partial protection to Bom Δ55C deletion mutants, suggesting it may need to act in concert with another 55C Bomanin gene for protection to wild-type levels ( Fig. 2C ). We have so far failed to identify such a gene in our loss-of-function approach, even though one of the two null BomS2 mutant line displayed a sensitivity to E. faecalis ( Table 1 , Fig. EV3A ). We also noted that BomS1 overexpression also partially rescued the Bom Δ55C deletion sensitivity phenotype ( Table 1 , Fig. EV3B ). Unexpectedly, BomBc2 overexpression provided a degree of protection against E. faecalis in wild-type but not Bom Δ55C flies ( Table 1 , Fig. EV3C ), suggesting it may also act in concert with another 55C Bom gene, BomT1 being the best candidate. Download figure Open in new tab Figure 2. BomT1 partially mediates resistance to E. faecalis and resilience to M. robertsii infections (A-B) Survival curves of BomT1 KI mutant (A) or ubiquitous silencing of BomT1 at the adult stage (B) flies after E. faecalis (OD 600 =0.1, 4.6 nL/fly) injection. (C) survival curves of flies ubiquitously overexpressing BomT1 at the adult stage in Bom Δ55C background challenged by E. faecalis injection. (D) Bacterial load of BomT1 KI mutant, w [A5001] and MyD88 single flies post E. faecalis injection at 2h, 6h, and 16h. (E) Bacterial load upon death (BLUD) of BomT1 KI mutant, w [A5001] and MyD88 flies post E. faecalis injection. (F) Survival curves of BomT1 KI mutant and w [A5001] flies after M. robertsii (10^5 spores/mL, 5mL/group) natural infection. (G) Fungal load of BomT1 KI mutant, w [A5001] and MyD88 flies post M. robertsii natural infection at different time points. (H) Fungal load upon death (FLUD) of BomT1 KI single mutant flies, w [A5001] and MyD88 flies post M. robertsii natural infection. Note: each experiment has been performed more than three times and pooled data are presented. A, B, C, F : Each experiment used biological triplicates of 20 flies. D and G : The data are presented as means ± SEM and analyzed using the ANOVA (one-way) with Dunnett’s multiple comparisons test; E and H : The data are presented as means ± SD and analyzed using the Unpaired t-test. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; ns, no significant difference. We next investigated whether the E. faecalis burden was altered in BomT1 KI single flies. As shown in Fig. 2D , the bacterial load was higher than in wild-type flies from six hours onwards, implying that BomT1 is required for resistance. We also checked the bacterial load upon death (BLUD) of single flies and found that both MyD88 and BomT1 displayed an increased BLUD in the w [A5001] background ( Fig. 2E ), suggesting that more bacteria are needed to kill the BomT1 and MyD88 immuno-deficient flies. It is not clear however whether this finding reflects an increased resilience to E. faecalis infection. BomT1 may be involved in resilience to M. robertsii natural infection We found that BomT1 KI mutants were significantly more susceptible to M. robertsii natural infection, but not to the injection of its spores ( Fig. 2F , Table 2 ). Of note, in contrast to the E. faecalis sensitivity phenotype, BomT1 -silenced flies did not display any increased susceptibility to M. robertsii natural infection. This may reflect a hypomorphic effect of the RNAi approach, even though the silencing appeared rather strong at the transcript level (Fig. S7A). M. robertsii would also need to be more sensitive than E. faecalis to any remaining BomT1 in these silenced lines. In contrast to MyD88 flies, the fungal load in BomT1 single flies did not appear to vary at any time point ( Fig. 2G ). In terms of fungal load upon death (FLUD), there was no measured difference between wild-type and immunodeficient flies ( Fig. 2H ). Thus, our data are compatible with the hypothesis that BomT1 functions in resilience against M. robertsii natural infection. View this table: View inline View popup Download powerpoint Table 2: Loss-of-function Bomanin genes susceptibility to specific infections: a summary of 55C locus Bomanins Note: ns, not significant; sensitive, the mutants or single Bomanin knock-down flies are susceptible to the corresponding microorganism infection; ‘+’, the intensity of sensitivity. BomT2 indel corresponds to a CRISPR-Cas9 mutant that may still express a protein, which would lack the tail C-terminal to the Bomanin domain. Finally, BomT1 overexpression in wild-type but not in Bom Δ55C flies provided a moderate degree of protection against the entomopathogenic fungus, suggesting it may act in concert with another 55C Bom . In this respect, we discovered that the overexpression of BomBc1 , BomS4 , BomS2 or BomT2 protected flies from M. robertsii natural infection to the same extent than BomT1 overexpression in wild-type flies ( Table 1 ). Furthermore, BomBc2 , BomS3 , and especially BomS1 overexpression provided an even stronger level of protection ( Fig. EV3D-F , Table 1 ). BomS2 may be involved in resilience against injected M. robertsii In opposition to BomT1 , we found that the two BomS2 null mutants were sensitive to injected M. robertsii spores but not to a natural infection with the same pathogen ( Fig. 3A , Table 2 ). While we measured a somewhat increased fungal load at three days in BomS2 ΔKO6 mutant flies, we failed to reproduce this observation in BomS2 ΔKO36 flies as well as in BomS2 ΔKO6 / BomS2 ΔKO36 trans-heterozygous flies ( Fig. 3B-D ). Thus, BomS2 may be involved in resilience against this infectious challenge. Download figure Open in new tab Figure 3. BomS2 also partially mediates resilience to injected M. robertsii spores (A) Survival curves of two BomS2 null mutants, BomS2 ΔKO6 and BomS2 ΔKO36 after M. robertsii (10^7 spores/mL, 4.6nL/fly) injection. (B-D) Fungal load of BomS2 ΔKO (B) , BomS2 ΔKO36 (C) , and transheterozygous BomS2 ΔKO6 /BomS2 ΔKO36 mutant flies post M. robertsii injection at different time points (D) . (E) Survival experiments of BomS2- overexpressing flies in a wild-type background to M. robertsii injection. Note: each experiment has been performed more than three times and pooled data are displayed. A and E : Each experiment used biological triplicates of 20 flies. B , C and D : ANOVA, multiple comparisons, Dunnett’s multiple comparisons test. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. The overexpression of BomS2 in wild-type but not in Bom Δ55C flies protected them from the effects of M. robertsii injection at two inoculum doses ( Fig. 3E , Table 1 ). Actually, we noted a similar level of protection conferred by the overexpression, also in adult wild-type flies, of BomBc1 or of BomS1 whereas that of BomS4 or BomS3 offered a more limited level of defense ( Fig. EV4 ). Of note, BomBc1 expresses distinct polymorphism isoforms in w 1118 and in Canton-S wild-type background (Fig. S2D). While the overexpression of the former enhanced the survival solely against injected M. robertsii , that of the latter did so only upon natural infection, an indication of the specificity of the roles of these two isoforms depending on the infection route of this pathogen ( Table 1 ). Unexpectedly, we detected that BomS2 ΔKO36 but not BomS2 ΔKO6 mutant flies displayed an increased sensitivity to E. faecalis ( Fig. EV3A , Table 2 ), possibly in keeping with the report that BomS2 silencing led to sensitivity to another, mild, Gram-positive bacterial pathogen, Lysinibacillus fusiformis ( Smith et al ., 2023 ). Bomanin-mediated host defenses against A. fumigatus, C. glabrata or C. albicans? We have not identified any Bom gene with a clear-cut sensitivity phenotype to any of these three pathogens using the set of mutants/RNAi lines we have tested. The sensitivity of one BomBc1 RNAi line to C. albicans was not confirmed in an independent line that appears as efficient in terms of silencing at the transcript level (Fig. S7B, S10B-C). Similarly, the susceptibility to a C. glabrata challenge of the BomT2 indel line, which may still express a protein lacking the BomT2 tail, was not observed with the two null mutant lines (Fig. S10D-E). We note that with respect to C. glabrata and A. fumigatus infections, the Bom Δ55C sensitivity phenotype can be rescued in general to a rather mild degree by the overexpression of a similar set of Bom genes, that is both BomT genes and BomS genes, with BomS1 overexpression being able to protect the Bom Δ55C deficiency flies only against A. fumigatus whereas BomS5 overexpression provided some defense to these flies only against C. glabrata . Interestingly, the overexpression of BomS1 in MyD88 flies made them more sensitive to a C. glabrata challenge (Fig. S11A). It has been previously reported that Bombardier is involved in resilience by protecting the flies from the noxious effects of BomS genes when not secreted/stabilized in the hemolymph. Thus, BomS1 may contribute to the Bbd resilience phenotype. We have noticed in this respect that the continuous overexpression throughout the life cycle of the fly of BomS1 led to a developmental phenotype characterized by Stubble-like bristles (Fig. S11B-D). In contrast to the two infections above, the overexpression of only one Bom gene, the w 1118 BomBc1 isoform, protected to a mild degree Bom Δ55C flies against C. albicans infection ( Fig. S10F). A role for Bomanins in preventing the dissemination of C. albicans throughout the fly body Survival assays may not reflect the full palette of Bomanin functions. We reasoned that one aspect of infection is the dissemination of the pathogen away from its initial infection site. As a bright GFP-expressing strain of C. albicans was available, we examined how C. albicans behaved within the fly once injected in the thorax. As expected (DF, unpublished observations), the pathogenic yeast remained mostly at the injection site in w [A5001], yet they managed to form hyphae. In striking contrast, we observed a dissemination of C. albicans throughout the body of Bom Δ55C flies, with hyphae detected in all three tagmata ( Fig. 4A ). Download figure Open in new tab Figure 4. 55C Bomanins prevent the dissemination of C. albicans throughout the fly body. (A) The conidia formation of infected flies with C. albicans . Red arrows: hyphae of C. albicans , yellow arrow: the melanized injection point. (B) Pooled data of C. albicans conidia formation in different flies at different fly sections. w [A5001]: wild-type flies. MyD88 : MyD88 mutant fly. Bom Δ55C (A5001) : Bom Δ55C backcrossed with w [A5001] fly. Bom Δ55C (yw): Bom Δ55C in initial yw background. BomS1, BomS2, BomS3, BomS4, BomS5, BomS6, BomT1, BomT2, BomBc1, BomBc2 : overexpressed BomS1, BomS2, BomS3, BomS4, BomS5, BomS6, BomT1, BomT2, BomBc1, BomBc2 flies individually in a Bom Δ55C background. BomT1 ΔKI : BomT1 mutant. BomBc1 KO : BomBc1 mutant, BomBc1 indel . Note: each group contained at least 20 flies. B : The data are analyzed using Fisher’s exact test. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Black *: comparison to Bom Δ55C ; Green *: comparison to w [A5001]. We next complemented the Bom Δ55C phenotype by overexpressing each 55C Bom gene one-by-one. Both BomT1 and BomT2 , and to a lesser extent BomBc1 prevented significantly the dissemination of C. albicans throughout the body of Bom Δ55C flies. Conversely, there was a trend for the BomBc1 indel mutant but not for the BomT1 KI mutant flies to allow some dissemination of C. albicans in an otherwise wild-type background ( Fig. 4B ). Implication of the 55C Bomanin cluster in the host defense against A. fumigatus mycotoxins We have previously reported that Bom Δ55C are sensitive to the injection of two mycotoxins secreted by A. fumigatus , namely the ribotoxin protein restrictocin and the family of fumitremorgin/verruculogen compounds (Fig. S12). We had also reported that A. fumigatus mutants lacking the locus encoding restrictocin or lacking the fumitremorgin biosynthetic cluster were only mildly less virulent than the wild-type fungus in MyD88 -immunodeficient flies. We have extended these observations to the Bom Δ55C and Bom Δleft fly lines. Whereas we still observed a reduced virulence in the Bom Δ55C line, as for MyD88 flies, we no longer measured a significant difference in the virulence of the mycotoxin-defective fungi as compared to wild-type fungi in the Bom Δleft line ( Fig. EV5A-D ). This suggests that the four genes remaining in Bom Δleft flies, namely BomS3 , BomT2 , BomS5 , and BomS6 are sufficient to protect the flies against restrictocin and verruculogen/fumitremorgins. We also tested directly the difference in sensitivity between Bom Δ55C and Bom Δleft after exposure to restrictocin, fumitremorgin B or verruculogen. Whereas verruculogen killed equally well both deletion lines, restrictocin and fumitremorgin B were not as toxic to the Bom Δleft as to the Bom Δ55C line ( Fig. 5A-C ), suggesting that the four remaining genes in Bom Δleft are not sufficient to confer protection against restrictocin and fumitremorgin B and that at least some of the six genes removed by this deficiency are required for host defense against these mycotoxins. We next injected restrictocin or verruculogen into BomT1 KI mutant and the BomBc1 indel mutant. We found that both mutants are as sensitive to restrictocin as MyD88 mutants, whereas none of them displayed any enhanced susceptibility to a verruculogen challenge ( Fig. 5D-G ). Download figure Open in new tab Figure 5. 55C Bomanins confer protection against A. fumigatus mycotoxins (A-C) Survival curves of Bom Δ55C , Bom Δleft , MyD88, and w [A5001] flies after commercially-available mycotoxins, Verruculogen (5mg/ml, 4.6 nL/fly) (A), Restrictocin (5mg/ml, 2 nL/fly) (B), and Fumitremorgin B (5mg/ml, 2 nL/fly) (C) injection. (D-E) Survival curves of BomT1 KI flies after Restrictocin (D) and Verruculogen (E) injection. (F-G) Survival curves of BomBc1 KO flies after Restrictocin (F) and verruculogen (G) injection. Note: (A-G) Each experiment has been performed more than three times and pooled data are presented. Except for (E) and (G) , each experiment used biological triplicates of 20 flies. Finally, as a proxy to analyze the function of BomSs in the resilience to A. fumigatus mycotoxins, we tested Bbd mutants in which no BomSs are detectable in the hemolymph. We found Bbd mutant flies to be as sensitive to A. fumigatus as MyD88 flies (Fig. S13A). They were somewhat sensitive to restrictocin, highly sensitive to verruculogen and not susceptible to a gliotoxin challenge (Fig. S13B-D). Of note, we formally cannot a contribution of 55C BomTs to these phenotypes since it is unknown whether Bbd is required for the secretion/stability of BomTs in the hemolymph. We have previously documented the potential for BomS3 (restrictocin only) and especially BomS6 to rescue the sensitivity of Bom Δ55C flies to injected restrictocin or verruculogen ( Xu et al ., 2023 ). Discussion In this article, we report a genetic dissection of the 55C Bomanin locus using both loss-of-function and overexpression strategies. A major outcome of this study is the identification of BomT1 as being a major effector of host resistance against E. faecalis infection, albeit it likely functions together with other gene product(s) of the locus. Interestingly, the same gene appeared to be required in the host defense against M. robertsii natural infection; we failed to obtain evidence supporting a role for it in resistance against this infection. Likewise, we found that BomS2 but not BomS5 null mutants are required for protection against injected M. robertsii spores even though the fungal burden remained unaltered as compared to wild-type flies. We also report that two Bomanins are required for protection against a ribotoxin secreted by A. fumigatus , including BomT1 , implying that this Bom peptide is involved both in resistance and resilience to infections. Finally, we have discovered a disconnect between fungal dissemination and fungal lethality, with again BomTs appearing to prevent to some extent the dissemination of C. albicans hyphae. Thus, the functions of 55C Bomanins are complex and intertwined and involved in both the resistance and resilience facets of host defense. Limitations of the study Even though we have generated five null mutants at the 55C locus, we have failed to obtain any reagent affecting the expression of BomS1, BomS3 , and BomS6 ; we obtained data only by silencing gene expression for BomS4 and BomBc2 . As regards BomS4 , we have analyzed only one RNAi line, which however appears to be rather effective at silencing BomS4 transcripts. Yet, we cannot exclude a hypomorphic effect or a potential accumulation of the basally expressed BomS4 peptide prior to the silencing of the transcripts at the adult stage. Thus, because we did not find any BomS4 survival phenotype with the pathogens tested, off-target effects are not an issue; yet, a null mutant would be required to confirm this apparent absence of roles in the host defense against these bacterial and fungal pathogens. The deletion of one gene in a locus may affect the neighboring one, as appears to be the case for BomS5 for which BomT2 expression appears to be severely affected. As regards BomT1 , we have noted a reduced induction of BomS2 expression, which is located 5 kB away. It is unlikely that a second-site mutation in the KI mutant would also occur upon silencing BomT1 . We also note that BomT1 null mutants affect the survival to M. robertsii natural infection but not to that of injected spores whereas the BomS2 null phenotype is the converse. Of interest, this study confirms that distinct host defenses are relevant according to the route of infection of M. robertsii ( Wang, 2020 ). Notwithstanding, another limitation is that we have tested here only a limited panel of bacterial, yeast and filamentous fungal pathogens. Finally, overexpression experiments have to be interpreted with caution as the degree of overexpression of the peptides reached may not be physiologically relevant; results from this approach should be taken as indications of the potential abilities of the overexpressed gene. Nevertheless, we report in this study some specificity in the effects of overexpressed Bomanins, with some overlap in survival phenotypes to infections between some but not all short Bomanins, in contrast to a previous study on the redundant functions of BomS against C. glabrata ( Lindsay et al ., 2018 ). Role of BomT1 in the resistance to E. faecalis infection Clemmons et al ., had reported that the 55C locus is required for resistance against E. faecalis ( Clemmons et al ., 2015 ). We found that BomT1 mutants displayed a susceptibility to this bacterial pathogen that was almost as pronounced as that displayed by MyD88 , suggesting that it is a major mediator of resistance to E. faecalis since the bacterial burden is increased in this mutant. Yet, it may not act alone against this bacterium since we observed only a moderate rescue of the Bom Δ55C sensitivity phenotype by its overexpression. We note that Bom Δleft are also as susceptible to this infectious challenge, suggesting that the four-rightmost genes of the locus are not sufficient to mediate protection on their own. Interestingly, BomS1 overexpression also somewhat rescues Bom Δ55C flies and it would be interesting to test whether their joint overexpression in this immunodeficient context would bring the protection to wild-type levels. Unexpectedly, we found that BomBc2 provided a degree of protection against E. faecalis when overexpressed in wild-type but not Bom Δ55C flies, possibly reflecting a positive interaction with another 55C Bomanin gene product, possibly BomT1 . A BomBc2 null mutant will be required to clarify the function of this gene in host defense against infections. A weak phenotype of susceptibility to E. faecalis has previously been reported upon silencing BomBc1 but not BomT1 , BomS1 , and BomS4 ( Chapman et al ., 2020 ), in contrast to the results presented in our study ( Table 2 ). Our analysis indicates that specific Bomanins play a role in host defense against E. faecalis and thus are not compatible with the hypothesis that it is the overall amount of Bomanin peptides that is the relevant parameter for controlling microbial infections ( Clemmons et al ., 2015 ; Lindsay et al ., 2018 ). Involvement of 55C genes in resilience to infections BomS2 behaves much as BomT1 mutants with no altered fungal burden after a challenge with M. robertsii spores thus arguing against a role in resistance. It is an open possibility that BomT1 and BomS2 rather function in resilience to secreted virulence factors. We have reported previously that some BaraA-derived peptides provide a degree of protection against Destruxin A, a toxic hexadepsipeptide secreted by the fungus ( Huang et al ., 2023 ). It remains unclear whether the 55C Bomanin locus is involved in resilience to Destruxin A as we have obtained contrasted results in our experiments. However, the number of secreted proteins, many of them proteases, and of secondary metabolites is so high that the two Bomanins may protect against other virulence factors ( Gao et al , 2011 ). Interestingly, some virulence factors able to inhibit the activation of the Toll pathway by either the GNBP3 sensor or the PSH protease bait have recently been identified ( Lu et al , 2024 ; Tang et al , 2025 ). However, as pointed earlier ( Clemmons et al ., 2015 ), the basal expression levels of many Bomanins in adult fat body or carcass is high, especially those of BomS1, BomBc2 , BomS2 , BomS3 , BomT2 , and BomS6 , which might be sufficient to provide a degree of protection despite the pathogen attack on the activation of the Toll pathway. Nevertheless, the strongest evidence we have obtained for a role of Bomanins in resilience against a mycotoxin is the sensitivity of both BomT1 and BomBc1 to a challenge by the A. fumigatus mycotoxin restrictocin (this work). Of note, a recent study has documented a role for BomS2 in the host defense against Lysinibacillus fusiformis , a mild Gram-positive pathogen. With respect to E. faecalis , we did observe a mutant phenotype for only one of two null mutants ( Fig. EV3A , Table 2 ). The dissemination of C. albicans within the Drosophila body may not be essential for its pathogenicity C. albicans is a dimorphic yeast able to form hyphae. The transition between the yeast to filamentous forms has been reported to be important for its virulence and it is believed that hyphae allow it to invade tissues thereby promoting its pathogenicity. Here, we find that Bom Δ55C mutants are highly permissive to the dissemination of the fungus, in keeping with its sensitivity to this infection. The rescue experiments of Bom Δ55C by single Bom gene overexpression revealed that BomT1 and BomT2 strongly prevent the dissemination of C. albicans within the fly yet do not allow it to better survive the infection. Since BomT1 or BomT2 overexpression did not protect Bom Δ55C mutants from C. albicans infection ( Table 1 ), these findings suggest that C. albicans can kill its host in the absence of a widespread dissemination. Thus, this pathogen may kill Bom Δ55C hosts through secreted virulence factors, a situation akin to that recently described for A. fumigatus infection ( Xu et al ., 2023 ). Yet, protection against these secreted virulence factors would necessarily be mediated by multiple 55C Bomanins since the overexpression of single 55C Bomanin genes did not protect Bom Δ55C flies from C. albicans infection ( Table 1 ). In contrast, the overexpression of a specific BomBc1 isoform also inhibited the dissemination of C. albicans and conversely the deletion of the BomBc1 locus nearly led to a significant dissemination of the fungus ( Fig. 4B ). Of note, we cannot totally exclude that we would have been able to detect with a high sensitivity any dissemination of C. albicans under the yeast form. Nevertheless, our data suggest that some long Bomanin peptides may interfere with filamentation. Interestingly, it has been reported that Daisho peptides, which are related to Bomanins ( Clemmons et al ., 2015 ), have been shown to bind to hyphae of the mold Fusarium oxysporum against which they are active ( Cohen et al ., 2020 ). This study reveals that distinct Bomanins have different activities in host defense against infections, ranging from antimicrobial activities against a prokaryote pathogen, E. faecalis , against eukaryotic pathogens including yeast and filamentous fungi and also provide protection against secreted virulence factors. Strikingly, BomS6 is able to protect flies from the action of a protein toxin, Restrictocin, and a secondary metabolite, verruculogen that targets a maxi-potassium channel by binding to a site within its transmembrane domain. At first sight, it is difficult to envision how this family of related peptides manages to function in such diverse facets of host defense. We would like to speculate here that there is a potential common target of this family of Toll pathway effectors, namely cytoplasmic membranes. Like several AMPs such as Cecropins, antimicrobial Bomanins such as BomT1 may interact with the bacterial membrane and possibly directly lead to its lysis, likely in association with other factors such as other Bomanins. A similar process may be at play as regards the fungicidal activity of BomS or action on hyphae of BomTs/BomBc1. With regard to the protection against mycotoxin, we note that a remarkable feature of Restrictocin is its ability to cross membranes. Future studies will tell whether BomS6 is able to modify the host cell permeability to the ribotoxin and whether the modification of the biophysical properties of neural membranes induces a conformational change in the Slowpoke receptor that would mask its verruculogen binding site, thereby allowing the host to recover from tremors induced by this neuromycotoxin ( Xu et al ., 2023 ). Material and methods Fly stocks and maintenance Fly incubation Flies were maintained under controlled environmental conditions (25°C, 65% RH) in standard incubators. The nutritional medium was prepared by combining the following components per production batch: 1.2 kg cornmeal (Priméal), 1.2 kg glucose (Tereos Syral), 1.5 kg yeast (Bio Springer), 90 g nipagin (VWR Chemicals) diluted into 350 mL ethanol (Sigma-Aldrich), 120 g agar-agar (Sobigel). Ultrapure water was added to obtain a 25 L batch of food. Indel mutants and RNAi lines The following mutant lines were used: the Bom Δ55C and Bom Δleft lines were a kind gift of Prof. Steven Wasserman. BomT2 Δtail and BomBc1 indel were generously provided by Prof. Jiyong Liu. The BomT1 KI line was contructed by Wellgenetics (Taipei, Taiwan). Other KO mutants were constructed by ourselves using CRISPR-Cas9 technology (see Supplementary Material). All mutant strains were isogenized in a w [A5001] background ( Thibault et al ., 2004 ). The following knockdown strains obtained from the Bloomington, Vienna, and Tsinghua stock centers were utilized: BomT1 (BL42617); BomBc1 (BL65901); BomBc2 (VDRC13926) and BomBc2 (BL61348); BomT2 (VDRC103059); BomS4 (THU5656). All the RNAi lines were crossed to a w; pUbi-Gal4, pTub-Gal80 ts (BDSC30140) driver lines at 18°C; hatched adults were placed at 29°C for 5 days to induce RNAi expression. Transgenic lines . The transgenic lines expressing single Bom genes of the 55C locus under the pUAS-hsp70 promoter control were generated (see Supplementary Material) and checked by sequencing ( Xu et al ., 2023 ). The transgenic flies were crossed to a w; pUbi-Gal4, pTub-Gal80 ts driver line, in a homozygous MyD88 , Bom Δ55C mutant or w A5001 background. The expression of the transgenes was checked by RT–qPCR and mass spectrometry analysis on collected hemolymph of single flies. Quantitative RT-PCR Total RNA was extracted from the whole-body of adult flies using TRIzol® reagent (Invitrogen), following the manufacturer’s protocol. For each biological replicate, five age- and sex-matched flies were pooled to minimize individual variability. RNA integrity was confirmed via NanoDrop. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the iScript™ cDNA Synthesis Kit (Bio-Rad), with random hexamer primers, according to the manufacturer’s instructions. The primers used in quantitative PCR were listed in Table S5. Mass spectrometry Single fly hemolymph was collected from individual flies and immediately transferred to 2 µL of 0.1% trifluoroacetic acid (TFA) solution on ice to prevent protein degradation and maintain sample integrity. The 4-hydroxy-α-cyanocinnamic acid (4-HCCA) sandwich spotting matrix method was employed for sample loading. Two distinct matrix solutions were prepared: Matrix 1: A saturated solution of 4-HCCA (20 mg/mL) in 100% acetone. Matrix 2: A solution containing 20 mg/mL 4-HCCA in a 2:1 acetonitrile/0.1% TFA mixture. For each hemolymph sample from a single fly, 0.6 µL of the sample was loaded onto the MALDI target plate. The sample was directly applied to a dried bed of 0.5 µL of Matrix 1 and the sample was overlaid with 0.4 µL of Matrix 2 to ensure optimal crystallization and ionization efficiency during MS analysis. After air drying under ambient condition, the prepared samples were analyzed using a MALDI-TOF mass spectrometer. The instrument was calibrated with standard peptide/protein mixtures to ensure accurate mass measurements. Data acquisition was performed in positive ion mode over a mass range of 800–20,000 Da, with a laser intensity optimized for the detection of small peptides and proteins present in the hemolymph. Microbial culture and infection Enterococcus faecalis . E. faecalis strains ( ATCC 19433 ) were maintained on Luria-Bertani agar (LBA) plates stored at 4°C for at most a month. For experimental replicates, single colonies were isolated under sterile conditions and inoculated into 5 mL LB broth. Cultures were incubated at 37°C with shaking (220 rpm) for 6-8 hours to reach mid-logarithmic phase (OD 600 = 0.6-0.8, measured via Nanodrop spectrophotometer). Cells were collected by centrifugation (7,500 rpm, 2 min, 4°C) and resuspended in phosphate-buffered saline (PBS). This washing procedure was repeated three times to remove residual culture media. Final bacterial suspensions were adjusted to OD 600 =0.1 concentration, ensuring experimental consistency across biological replicates. 4.6 nL of standardized bacterial suspension was delivered per fly via thoracic microinjection Candida albicans ( CAM15.4 ) and C. glabrata ( ATCC2001 ) were maintained on yeast extract peptone dextrose (YPD) after two rounds on agar plates at 29°C. Single colonies were taken using a sterile tungsten needle for infections, 3-5 days old adult flies were anesthetized and pricked in the thorax region with the needle containing fungal cells (colony pricking assay). Mock-infected controls were performed using needles dipped into sterile PBS. Metarhizium robertsii ( 2575 ) was propagated on Potato Dextrose Agar (PDA; BD Difco™) plates at 25°C for 7-14 days to ensure optimal sporulation. Spores were collected by gently scraping plate surfaces with PBST (PBS + 0.01% Tween-20) for injection experiments or sterile ddH 2 O + 0.01% Tween-20 for natural infection assays. Suspensions were filtered through Miracloth to remove mycelial debris and quantified using a hemocytometer. For injection experiments, 4.6 nL of standardized conidial suspension (10^7 spores/mL) was delivered per fly via thoracic microinjection. For natural infection, 5 mL of diluted conidial suspension (10^5 spores/mL) was applied to groups of 20 flies. Aspergillus fumigatus . A. fumigatus strain maintenance and infection procedures were performed as previously described ( Xu et al ., 2023 ). Micrococcus luteus. M. luteus was cultivated in Luria-Bertani Broth at 37°C under aerobic conditions for 24 hours. Bacterial cells were pelleted by centrifugation at 3,000 rpm for 10 minutes, followed by two successive washing cycles in phosphate-buffered saline (PBS). The pellet was resuspended in 1 mL PBS after each centrifugation step to ensure complete removal of culture medium components. Needles were dipped in a concentrated pellet prior to pricking flies. Bacterial/ fungal load in single flies Individual infected flies were processed for microbial enumeration using a modified protocol as follows: single flies were transferred to 1.5 mL microcentrifuge tubes containing 20 µL phosphate-buffered saline with Tween-20 (PBST) and two 3 mm stainless steel beads. Samples were homogenized by mechanical disruption using a Mixer Mill (Retsch MM 300) at 30 Hz for 2 min. Homogenates were subjected to serial dilutions (10⁻¹ to 10⁻⁵) in PBST based on pre-determined time-point-specific infection kinetics. Aliquots (50 µL) of each dilution were spread-plated in duplicate onto potato dextrose agar (PDA) plates for fungi or LBA ones for bacteria, with one fly equivalent per plate. Plates were incubated at organism-specific temperatures (29°C for fungi, 37°C for bacteria) in a humidified incubator. Fungal colonies were enumerated after 48 h incubation, while bacterial colonies were counted after 24 h. The limit of detection was established at 10 CFU/fly based on the lowest dilution yielding ≥30 colonies per plate. Flies died in 30 minutes after injection were collected for Bacteria load upon death (BLUD) or fungi load upon death (FLUD) analysis. Mycotoxins preparation and injection Mycotoxin standard solutions were prepared as described previously ( Xu et al ., 2023 ). Commercially available mycotoxin powders (Restrictocin in PBS, verruculogen in dimethyl sulfoxide (DMSO), and fumitremorgin B in DMSO) were dissolved to generate stock solutions. Working concentrations were prepared by serial dilution in PBS or DMSO. Stock solutions were stored at −20°C in amber vials protected from light, while fresh working solutions were prepared daily. Survival tests 5-7 day-old adults were utilized to ensure uniform physiological maturity. Flies were housed in vials containing standard cornmeal-agar medium at a density of 20 individuals per vial, with three biological replicates maintained for each experimental condition. During infection studies, daily survival rates were monitored by transferring surviving flies to fresh vials and recording mortality. In vivo phagocytosis Phagocytosis of Bom Δ55C mutant flies was measured following our previously published protocol (Liégeois et al, 2020). Statistical analysis and reproducibility All survival data were analyzed using GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA). Survival curves were compared using the log-rank (Mantel-Cox) test unless otherwise specified in the figure legends. For quantitative analyses of microbial burden, gene expression levels, and GFP fluorescence intensity, unpaired, non-parametric Mann-Whitney statistical tests or Student t-test were employed as appropriate; dissemination data were anlyzed using Fisher’s exact test. Significance values: *P < 0.05; **P < 0.01; ***P < 0.0003; ****P < 0.0001; ns, not significant. A list of reagents is available as Supplementary Table 1. Data availability This study includes no data deposited in external repositories. Author contributions Yanyan Lou : Conceptualization; resources; formal analysis; validation; investigation; methodology; writing – original draft; writing – review and editing. Bo Zhang : Conceptualization; resources; formal analysis; investigation; methodology; writing – original draft; writing – review and editing. Zhiyuan Zhang, Yingyi Pan, Jianwen Yang, Lu Li, Jianqiong Huang : Resources; investigation; writing – review and editing. Zihang Yuan : investigation; writing – original draft; writing – review and editing. Samuel Liégeois : resources; formal analysis; validation; investigation; methodology. Philippe Bulet : Resources; formal analysis; validation; investigation; methodology; performed and analyzed the mass spectrometry analysis; Rui Xu : funding acquisition; designed, analyzed the mycotoxins experiments. Zi Li : Conceptualization; resources; funding acquisition. Dominique Ferrandon : Conceptualization; resources; supervision; funding acquisition; validation; methodology; writing – original draft; project administration; writing – review and editing. Disclosure and competing interests statement The authors declare that they have no conflict of interest. Expanded view figure legends Download figure Open in new tab Figure EV1: Bomanins at 55C locus are not involved in phagocytosis. Complementary to Fig. 1 6,000 live yeast cells of C. glabrata were injected into w [A5001] wild-type flies and Bom Δ55C flies (Δ55C) and their phagocytic index was monitored 2 hours after injection. Mann-Whitney test. Download figure Open in new tab Figure EV2: A key annotation of every single cell for Table 1 . Complementary to Table 1 . Download figure Open in new tab Figure EV3: Overexpression of BomS1, BomBc2, or BomS3 enhances resistance to E. faecalis or M. robertsii . Complementary to Fig. 2 . (A) Survival curves of BomS2 ΔKO36 null mutant flies after E. faecalis infection. (B) Survival curves of BomS1- overexpressing flies in a Bom Δ55C background after E. faecalis injection. (C) Survival experiments of BomBc2 -overexpression flies in a wild-type background after E. faecalis injection. (D) Survival curves of BomS1- overexpressing flies in a wild-type background against M. robertsii natural infection. (E-F) Survival curves of BomBc2 (E) , BomS3 (F) overexpressing flies in a wild-type background after M. robertsii natural infection. Note: (A-F) Each experiment has been performed more than three times. Each experiment used biological triplicates of 20 flies. Pooled data are shown. Download figure Open in new tab Figure EV4: Overexpression of BomBc1, BomS1, BomS3 , and BomS4 in a wild type genetic background can protect them from injected M. robertsii spores. Complementary to Fig. 3 . (A-D) Survival curves of BomBc1 (A), BomS1 (B), BomS3 (C), and BomS4 (D) - overexpressing flies in a wild-type background after M. robertsii injection. Note: (A-D) Each experiment has been performed more than three times. Each experiment used biological triplicates of 20 flies. Pooled data are displayed. Download figure Open in new tab Figure EV5: Mycotoxins mutants of A. fumigatus are as virulent as wild-type fungi when injected into Bom Δleft flies. Complementary to Fig. 5 . (A-D) Survival curves of w [A5001] (A) , MyD88 (B) , Bom Δ55C (C) , and Bom Δleft (D) flies to A. fumigatus ΔftmA (fumitremorgin/verruculogen pathway inactivated), A. fumigatus Δaspf1 (restrictocin mutant), and A. fumigatus genetic background controls ( A. fumigatus CEA17 or A. fumigatus A1160 ) injection. Note: (A-D) Each experiment has been performed more than three times. Each experiment used biological triplicates of 20 flies. Pooled data are exhibited. Acknowledgements We thank Anne Beauvais and Jean-Paul Latge for the A. fumigatus strain used in this study, Bruno Lemaitre, Jiyong Liu, Steven Wasserman, and the Guangzhou Drosophila Resource Center for fly stocks. Stocks obtained from the Bloomington Drosophila Stock Center, Vienna, and Tsinghua stock centers were also used in this study. We gratefully acknowledge the contributions of Miriam Yamba for expert technical help. YL was respectively partially funded through the Sino-Foreign cooperative graduate education project of Guangzhou Medical University, the International Training Plan for young outstanding scientific research talents of Guangdong Province, and Overseas Postdoctoral Talent Support Program of Guangdong Province. This work was supported by the Association Platform BioPark of Archamps on its Research & Development budget (PB), from the National Natural Science Foundation of China Project (#32370931), the China High-end Foreign Talent Program, and the 111 Project (#D18010; China) to DF. Funding Association Platform Biopark Archamps, , Research and Devlopment budget National Natural Science Foundation of China, https://ror.org/01h0zpd94 , 32370931 China High End Foreign Talent Program, , 111 Project, , D18010 References ↵ Brand AH , Perrimon N ( 1993 ) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes . 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Share Distinct Bomanins at the Drosophila 55C locus function in resistance and resilience to infections Yanyan Lou , Bo Zhang , Zhiyuan Zhang , Yingyi Pan , Jianwen Yang , Lu Li , Jianqiong Huang , Zihang Yuan , Samuel Liegeois , Philippe Bulet , Rui Xu , Li Zi , Dominique Ferrandon bioRxiv 2025.04.16.649162; doi: https://doi.org/10.1101/2025.04.16.649162 Share This Article: Copy Citation Tools Distinct Bomanins at the Drosophila 55C locus function in resistance and resilience to infections Yanyan Lou , Bo Zhang , Zhiyuan Zhang , Yingyi Pan , Jianwen Yang , Lu Li , Jianqiong Huang , Zihang Yuan , Samuel Liegeois , Philippe Bulet , Rui Xu , Li Zi , Dominique Ferrandon bioRxiv 2025.04.16.649162; doi: https://doi.org/10.1101/2025.04.16.649162 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Immunology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41911) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13371) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22482) Immunology (17728) Microbiology (40363) Molecular Biology (17163) Neuroscience (88536) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)