Antibiotic-induced protein mislocalization drives hypervesiculation in Salmonella

The incorporation of cytosolic proteins into bacterial outer membrane vesicles (OMVs) has remained mechanistically unresolved, as these proteins are normally excluded from vesiculation sites by the inner membrane (IM) barrier. Here, we show that antibiotic-induced envelope perturbation compromises this spatial barrier, enabling an aberrant interaction between proteins that are ordinarily segregated across cellular compartments and thereby promoting MV formation in Salmonella. Exposure to non-lethal cephalexin (LEX) induced robust hypervesiculation, accompanied by inhibition of the essential division penicillin binding protein (PBP) 3 and accumulation of the PBP5. Under envelope perturbing conditions, PBP5 accumulated at lateral sites associated with MV emergence and promoted periplasmic crowding independently of its catalytic activity. A cytosolic acyl carrier protein (ACP) emerged as a PBP5 interacting partner under LEX exposure, coinciding with cryogenic electron microscopy detected IM perturbation and aberrant ACP mislocalization. Formation of the PBP5 ACP complex promoted PBP5 accumulation, resulting in periplasmic crowding, progressive weakening of outer membrane peptidoglycan tethering, and ultimately MV formation. Together, these findings uncover a mechanism whereby stress-induced disruption of subcellular compartmentalization enables a non-canonical interaction between normally segregated proteins, driving hypervesiculation in Salmonella.