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Allyl-isothiocyanate affects antimicrobial peptide expression and survival in Drosophila melanogaster

ABSTRACT: The global rise of antibiotic-resistant bacteria highlights the urgent need for alternative strategies to support host defense against infections. Bioactive plant-derived compounds, such as isothiocyanates, have gained attention due to their antimicrobial and immunomodulatory properties. Allyl-isothiocyanate (AITC), a hydrolysis product of glucosinolates found in Brassica vegetables, has demonstrated antimicrobial activity in vitro and the ability to modulate antimicrobial peptide (AMP) expression in cell culture models. However, it’s in vivo effects under metabolically challenging dietary conditions remain poorly understood. In this study, we investigated the impact of dietary AITC supplementation on immune responses and survival in Drosophila melanogaster exposed to a high-sucrose diet (HSD), a dietary condition known to impair metabolic health and immune function. Flies were fed an HSD with or without 0.25 mM AITC and subsequently subjected to oral infection with either Leuconostoc pseudomesenteroides or Pectobacterium carotovorum subsp. carotovorum, which preferentially activate the Toll and Imd signaling pathways, respectively. AMP expression was analyzed by qPCR and RNA sequencing, and physiological consequences were assessed by lifespan analysis. AITC did not affect food intake or basal AMP expression under non-infected conditions. HSD significantly reduced lifespan in both sexes, and AITC supplementation did not rescue this effect. Following infection, both HSD and AITC influenced survival in a sex- and pathogen-specific manner, with AITC frequently exacerbating mortality rather than improving outcomes. While selected AMPs, particularly Attacin D, were modulated in a context-dependent manner, RNA sequencing revealed no consistent transcriptional changes in core Toll or Imd pathway components. Overall, our findings indicate that dietary AITC does not enhance host defense under high-sugar conditions and that its effects on immunity and survival are strongly sex-specific. These results underscore the complexity of diet–immune interactions and caution against extrapolating in vitro antimicrobial effects to in vivo host protection.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE318172 | GEO | 2026/06/01

REPOSITORIES: GEO

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Project description:This project aims to define, by using a functional proteomic approach, the molecular partners associated with the canonical coupound of the IMD pathway. The Drosophila defense against pathogens largely relies on the activation of two major pathways, IMD and TOLL. The IMD pathway is activated mainly by Gram-negative bacteria, whereas the TOLL pathway responds predominantly to Gram-positive bacteria and fungi. The activation of this pathway leads to the rapid induction via NF-kB of numerous genes of the immune system, including various antimicrobial peptide genes. Recent evidence indicates that the IMD pathway is also involved in various other reactions in the absence of infection ("inflammatory-like" reactions). To gain a better understanding of the molecular machineries underlying the pleiotropic functions of this pathway, we have developed a proteomics analysis. Our first aim was to identify the proteins interacting with the 11 canonical members (PGRP-LC, PGRP-LE, IMD, BG4 (dFADD), DREDD, TAK1, TAB2, IAP2, IRD5 (DmIKKbeta), KEY (DmIKKgamma), and RELISH) previously shown to be involved in the activation of the IMD pathway. Each protein was fused N-terminally or C-terminally with a biotin tag and was stably expressed in S2 cells previously subjected to stable integration of the bacterial enzyme BirA to allow for biotinylation of the tags. In total, we established 22 stable transformant cell lines (11 genes x 2 tag locations), which were individually stimulated by heat-killed E.coli before harvest at 4 different time points (t=0 min, 10 min, 2 hour, and 8 hour). This procedure was followed by streptavidin-mediated affinity purification of 96 bait-protein complexes (22 transformants x 4 time points, plus 8 unstimulated controls without baits), on-bead trypsin digestions of the protein complexes and six LC-MS/MS analyses. We have identified 371 interactanting proteins in heat-killed E.coli stimulated Drosophila S2 cells, 90% of which have human orthologues. Comparative analysis of Gene Ontology (GO) annotation datasets from Human and Flies point to three significant common categories: (1) NuA4 Histone acetyltransferase complex, (2) SWI/SNF-type Chromatin remodeling complex, and (3) Transcription cofactor binding. Our Drosophila data point in particular to SUMOylation of the IkB-kinase homologue Ird5 in Drosophila, a level of regulation not described to date. Among numerous directions opened by our proteomics analysis, we have decided to first investigate this facet of the IMD pathway. We show nthat a highly conserved SUMOylation consensus site binds Drosophila SUMO (SMT3) in a challenge-dependent manner and that this process confers an increased level of activation of the antimicrobial peptide genes during bacterial challenge both in vitro and in flies. Our results demonstrate that SUMOylation of IKKbeta plays an important role in the induction of antimicrobial peptide genes. The MS analysis was performed on a FT ICR mass spectrometer (LTQ-FT Ultra, ThermoFisher Scientific, San Jose, CA) with the top 7 acquisition method: MS resolution 60,000, mass range 500-2000 Th, followed by 7 MS/MS (LTQ) on the 7 most intense peaks, with a dynamic exclusion for 90 s. The raw data were processed using Xcalibur 2.0.7 software. Each sample was first analyzed in triplicate then an exclusion list was added for three next runs. The database search was done on merged data using Mascot search engine (Matrix Science Mascot 2.3) on the 17D melanogaster databank (16535 sequences). Proteome Discoverer 1.3 (ThermoFisher Scientific) and Mascot were used to search the data and filter the results. The following parameters were used: up to 2 miss cleavages; MS tolerance 10 ppm; MSMS tolerance 1 Da; full tryptic peptides; partial modifications carbamidomethyla- tion (C), oxydation (M, H, W), Phosphorylation (Y).

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Allyl-isothiocyanate affects antimicrobial peptide expression and survival in Drosophila melanogaster

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