Periplasmic crowding and peptidoglycan hydrolase activity as drivers of outer membrane vesiculation in Acinetobacter baumannii

Acinetobacter baumannii produces outer membrane vesicles (OMVs) to alleviate envelope stress, though the mechanisms remain poorly understood. To induce periplasmic accumulation of misfolded proteins and trigger stress, a degP mutant is exposed to elevated temperatures. Periplasmic crowding-induced OMV production is demonstrated using fluorescence recovery after photobleaching, where green fluorescent protein is targeted to the periplasm via the DegP signal peptide. OMV proteomics and western blotting reveal accumulation of OmpA and LPS in OMVs. Quantification using lipophilic dye and electron microscopy shows increased OMV production and larger vesicle sizes in degP mutants at elevated temperatures, despite normal growth. Deletion of the lytic transglycosylase mltB abolishes OMV formation in the degP mutant. Interestingly, the surA mutant, characterized by increased outer membrane permeability but impaired OMV production, exhibits enhanced OMV protrusions upon mltB overexpression. These results indicate that peptidoglycan hydrolysis is a key step in OMV biogenesis under periplasmic crowding stress, linking cell wall remodeling to vesicle formation.