Membrane Tethering of Honeybee Antimicrobial Peptides in <i>Drosophila</i> 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. Herein, it is demonstrated that glycosylphosphatidylinositol-mediated membrane tethering of honeybee defensin1 (Def1) in Drosophila melanogasterenhances its antimicrobial efficacy by <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mo>≈</mml:mo></mml:mrow> <mml:annotation>$$\approx$$</mml:annotation></mml:semantics> </mml:math> 100-fold compared to secreted or untethered forms, while preserving physiological and behavioral integrity under baseline conditions. Using a genetically engineered Drosophila model, three Def1 variants are expressed: native (Def1), secreted (s-Def1), and membrane-tethered (t-Def1). Flies expressing t-Def1 exhibit superior bacterial clearance of Pseudomonas aeruginosa and show improved survival postinfection, 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 exacerbates intestinal barrier dysfunction, as evidenced by elevated Smurf phenotype incidence, highlighting a trade-off between antimicrobial potency and epithelial vulnerability. This 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.