The release of membrane vesicles (MVs), a conserved surface feature of Gram-negative bacteria, has been observed for many years, yet the mechanism by which MVs are released and their relevance in bacterial physiology, pathogenesis, and the generation of immune responses was largely unexplored. Through a quantitative, systematic approach, the studies presented here demonstrate that proteins within the Gram-negative envelope modulate MV production. The interaction of these envelope proteins with other structures within the membrane directly impact MV abundance, size, localization of release, and protein content, such that small MVs originate from the cell body and large MVs are derived from division septa. MV biogenesis occurs, therefore, in envelope regions where these important interconnections are temporarily less dense, allowing the release of the outer membrane in the form of an MV.;Modifications in MV character, such as protein content, occur due to disregulation of MV production by wild-type Salmonella typhimurium . As antigens known to be important during protective immunization are released in WT MVs, and MVs represent a new and promising approach for vaccination, we tested whether antigen content recognized by the innate and/or adaptive immune systems was altered in MVs derived from strains harboring envelope protein mutations. In addition, modification of lipid A structures in MVs, in order to reduce LPS toxicity but retain adjuvant properties, was also explored. Both the innate immune response, as measured by cytokine secretion and activation of Toll-like receptor signaling, and the adaptive immune response, characterized by B- and CD4+ T-cell antigen content, demonstrated quantifiable changes with both protein and LPS modification. Manipulation of these surface components, therefore, influenced the immunostimulatory properties of MVs derived from mutant strains, demonstrating the malleable nature of MVs and their promise as efficacious vaccines.
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