Membrane Tethering of Honeybee Antimicrobial Peptides in Drosophila Enhances Pathogen Defense at the Cost of Stress-Induced Host Vulnerability

Antimicrobial peptides (AMPs) represent a promising alternative to conventional antibiotics in combating multidrug-resistant pathogens, yet their clinical translation is hindered by proteolytic instability, cytotoxicity, and poor bioavailability. Here, we demonstrate that glycosylphosphatidylinositol (GPI)-mediated membrane tethering of honeybee defensin1 (Def1) in Drosophila melanogaster enhances its antimicrobial efficacy by ∼100-fold compared to secreted or untethered forms, while preserving physiological and behavioral integrity under baseline conditions. Using a genetically engineered Drosophila model, we expressed three Def1 variants: native (Def1), secreted (s-Def1), and membrane-tethered (t-Def1). Flies expressing t-Def1 exhibited superior bacterial clearance of Pseudomonas aeruginosa and improved survival post-infection, with no adverse effects on locomotion, courtship, or sleep architecture. However, under stress paradigms—including sleep deprivation and dextran sulfate sodium (DSS)-induced gut injury—t-Def1 exacerbated intestinal barrier dysfunction, as evidenced by elevated Smurf phenotype incidence, highlighting a trade-off between antimicrobial potency and epithelial vulnerability. Our work establishes Drosophila as a powerful platform for dissecting AMP mechanisms and engineering spatially targeted therapies, offering translational insights for pollinator health and human infectious disease management. These results advocate for iterative refinement of membrane-anchoring strategies to balance therapeutic efficacy with host safety, advancing the development of next-generation AMPs with minimized off-target effects.