Philippe Bulet

RNAseq analysis of the Drosophila genome has revealed further immune-induced genes. Two genes initially annotated as lncRNAs, CG44404 ( yulü ) and CG45045 ( shenshu ), are strongly induced. We report here that these two genes actually encode highly related secreted peptides found in the Sophophora subgenus of Drosophila species. We have generated single and double null mutants of these loci and found that the double mutant line did not display any enhanced susceptibility to an immune challenge with a panel of bacterial and fungal pathogens, except for Pseudomonas aeruginosa . We did not observe any increased P. aeruginosa burden in yulü-shenshu mutants, suggesting that the two peptides may not be required for resistance to infection. Rather, we find that they provide a level of protection against Outer Membrane Vesicles (OMVs) purified from either P. aeruginosa or Serratia marcescens culture supernatants. We have recently reported that S. marcescens OMVs induce the paralysis of flies through the induction of apoptosis in at least some neurons. Much of the virulence of these OMVs is mediated by the metalloprotease PrtA. While Yulü/Shenshu do not display any protease inhibition activity, the detection of an association between Yulü and the Drosophila complement thioester-containing protein 2 (Tep2) led to the finding that both Tep2 and Tep4 mutants are sensitive to the injection of PrtA while their overexpression significantly protects wild-type flies from the effects of this protease. Both Tep2 and Tep4 are able to inhibit the activity of PrtA in a thioester- and yulü/shenshu -dependent manner. Thus, these Teps may also function as α2-macroglobulins.

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 ten 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 can 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.

Crassostrea gigas big defensins (Cg-BigDefs) are a family of two-domain antimicrobial peptides with broad antibacterial activity. The C-terminal domain of Cg-BigDefs harbors a β-defensin-like structure, whereas the ancestral N-terminal domain adopts a globular structure. Here, we developed molecular tools to track the fine interactions of these two domains with Staphylococcus aureus and to gain insight into Cg-BigDef1 mechanisms of action. By using super-resolution microscopy and S. aureus mutants with specific deletions of cell wall components, we found that teichoic acids (TAs) play a key role in the Cg-BigDef1 interaction with S. aureus. A ΔtagO S. aureus mutant lacking cell wall teichoic acids (WTAs) exhibited increased resistance to Cg-BigDef1. Consistently, the binding of Cg-BigDef1 to S. aureus cell wall was significantly reduced in the ΔtagO mutant. In contrast, a ΔdltA S. aureus mutant unable to transfer d-alanine onto lipoteichoic acid (LTA) showed increased susceptibility to Cg-BigDef1 and lysed rapidly in contact with the peptide. Cg-BigDef1 bound to S. aureus cell wall. In addition, competitive binding with exogenously added LTA was sufficient to impair Cg-BigDef1 antimicrobial activity against S. aureus. These data suggest that TAs are conserved molecular motifs recognized by Cg-BigDef1. Finally, we found that Cg-BigDef1 interaction with S. aureus was mediated by its N-terminal domain, which enables the C-terminal β-defensin-like domain to interact with the bacterial cell wall. Altogether, our results identify TAs as important targets for Cg-BigDef1. This interaction appears to be mediated by the ancestral N-terminal domain characteristic of this peptide family.

Wild fauna and flora are facing variable and challenging environmental disturbances. One of the animal groups that is most impacted by this, concerns pollinators. Pollinators face multiple threats, but the spread of anthropogenic chemicals (i.e. pesticides) form a major potential driver of these threats. WildPosh is a multi-actor, transdisciplinary project whose overarching mission and ambition are to significantly improve the evaluation of risk to pesticide exposure of wild pollinators, and enhance the sustainable health of pollinators and pollination services in Europe. As chemical exposure varies geographically, across cropping systems, inside the crop system and among pollinators, we will characterise exposure by doing fieldwork in 4 countries representing the four main climatic European regions, Mediterranean, Atlantic, Continental and Boreal climate in Germany, England, Estonia and Spain. We will also develop experiments in controlled conditions on different species of bees, syrphid flies, moths and butterflies, and collect in silico data on their traits and on toxicity of pesticides. With WildPosh, we aim to achieve the following objectives: 1. Determining the real-world agrochemical exposure profile of wild pollinators at landscape level, within and among sites; 2. Using integrated and controlled laboratory and semi-field experiments to characterise causal relationships between pesticides and pollinator health; 3. Building an open database on pollinator traits/distribution and chemicals to define exposure and toxicity scenarios by developing databases on ecological traits and the spatial distribution of pollinators in relation to their potential exposure to pesticide; 4. Proposing integrated systems-based risk assessment tools for risk assessment for wild pollinators; and 5. Driving policy and practice through interactive innovation, meeting the need for monitoring tools, novel and innovative screening protocols for practice and policymaker use.

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.

Fungal infections represent a significant global health concern, with a growing prevalence of antifungal drug resistance. Targeting glucosylceramides (GlcCer), which are functionally important glycosphingolipids (GSL) present in fungal membranes, represents a promising strategy for the development of antifungal drugs. GlcCer are associated with the antifungal activity of certain plant and insect defensins. The 44-residue ETD151 peptide, optimized from butterfly defensins, is active against several fungal pathogens. ETD151 has been shown to induce a multifaceted mechanism of action (MOA) in Botrytis cinerea , a multiresistant phytopathogenic fungus. However, the target has yet to be identified. Our findings demonstrate that the presence of GlcCer in membranes determines the susceptibility of Pichia pastoris and Candida albicans toward ETD151. To ascertain whether this is due to direct molecular recognition, we demonstrate that ETD151 selectively recognizes liposomes containing GlcCer from B. cinerea , which reveals a methylated-sphingoid base structure. The dissociation constant was estimated by microscale thermophoresis to be in the µM range. Finally, fluorescence microscopy revealed that ETD151 localizes preferentially at the surface of B. cinerea . Furthermore, the majority of prokaryotic cells do not contain GSL, which explains their resistance to ETD151. We investigated the susceptibility of Novosphingobium capsulatum , one of the rare GSL-containing bacteria, to ETD151. ETD151 demonstrated transient morphological changes and inhibitory growth activity (IC ₅₀ ~75 µM) with an affinity for the cell surface, emphasizing the critical importance of GSL as target. Understanding the MOA of ETD151 could pave the way for new perspectives in human health and crop protection.

Pressures on honey bee health have substantially increased both colony mortality and beekeepers’ costs for hive management across Europe. Although technological advances could offer cost-effective solutions to these challenges, there is little research into the incentives and barriers to technological adoption by beekeepers in Europe. Our study is the first to investigate beekeepers’ willingness to adopt the Bee Health Card, a molecular diagnostic tool developed within the PoshBee EU project which can rapidly assess bee health by monitoring molecular changes in bees. The Bee Health Card, based on MALDI BeeTyping®, is currently on level six of the Technology Readiness Level scale, meaning that the technology has been demonstrated in relevant environments. Using an on-line survey from seven European countries, we show that beekeepers recognise the potential for the tool to improve colony health, and that targeted economic incentives, such as subsidises, may help reduce cost being a barrier to the adoption and frequent use of the tool. Based on the description of the tool, 43% of beekeepers appear to be moderately confident in the effectiveness of the Bee Health Card. This confidence could increase if the tool was easy to use and not time consuming, and a higher confidence could also contribute to raising the probability of accepting extra costs linked to it. We estimate that, in the worst-case scenario, the cost per single use of the Bee Health Card should be between €47–90 across a range of European countries, depending on the labour and postage costs. However, the monetary benefits in terms of honey production could exceed this. In order to successfully tackle colony health issues, it is recommended using the BHC five times per year, from the end to the beginning of winter. Finally, we discuss the knowledge needs for assessing beekeeper health tools in future research.