Capsular polysaccharide-mediated protein loading onto extracellular membrane vesicles of Shewanella vesiculosa HM13

Bacterial extracellular membrane vesicles (EMVs) play crucial physiological roles, including intercellular communication and nutrient acquisition, which depend on the functions of their cargo molecules. However, the molecular mechanisms governing selective cargo loading onto EMVs remain poorly understood. Here, we investigated the role of capsular polysaccharide (CPS) in the association of P49, the major cargo protein of EMVs from Shewanella vesiculosa HM13, with EMVs. Deleting genes neighboring the P49 gene led to a loss of EMV-associated CPS and a defect in P49 loading. In vitro binding assays using P49-free EMVs and purified P49 revealed that exogenously added P49 specifically binds to CPS-containing EMVs, but not to CPS-deficient EMVs. Furthermore, gel-filtration analysis demonstrated that purified P49 directly binds to CPS extracted from cells. Transmission electron microscopy revealed that P49 constitutes peripheral nanoparticles on EMV surfaces, which were absent in CPS-deficient mutants. These findings identify CPS as a key factor required for P49 association with EMVs and support a model in which CPS functions as a scaffold for cargo protein loading. This P49-CPS system provides a basis for future mechanistic studies of P49 loading onto EMVs and may contribute to the development of post-vesiculation protein display strategies using bacterial EMVs.