Lipid a remodeling modulates outer membrane vesicle biogenesis by Porphyromonas gingivalis

Outer membrane vesicles (OMVs) are small membrane enclosed sacs released from bacteria which serve as carriers of biomolecules that shape interactions with the surrounding environment. The periodontal pathogen, Porphyromonas gingivalis, is a prolific OMV producer. Here, we investigated how the structure of lipid A, a core outer membrane molecule, influences P. gingivalis OMV production, OMV-dependent TLR4 activation, and biofilm formation. We examined mutant strains of P. gingivalis 33277 deficient for enzymes that alter lipid A phosphorylation and acylation status. The lipid A C4'-phosphatase (lpxF)-deficient strain and strains bearing inactivating point mutations in the LpxF active site displayed markedly reduced OMV production relative to WT. In contrast, strains deficient for either the lipid A C1-phosphatase (lpxE) or the lipid A deacylase (PGN_1123; lpxZ) genes did not display alterations in OMV abundance compared to WT. These data indicate that lipid A C4'-phosphate removal is required for typical OMV formation. In addition, OMVs produced by ΔlpxF and ΔlpxZ strains, possessing only penta-acylated lipid A, stimulated robust TLR4 activation, whereas OMVs obtained from WT and ΔlpxE strains, containing predominantly tetra-acylated lipid A, did not. Hence, lipid A remodeling modulates the capacity of OMVs to engage host TLR4-dependent immunity. Finally, we demonstrate an inverse relationship between OMV abundance and biofilm density, with the ∆lpxF mutants forming denser biofilms than either WT, ΔlpxE, or ΔlpxZ strains. Therefore, OMVs may also contribute to pathogenesis by regulating biofilm formation and dispersal.IMPORTANCEPorphyromonas gingivalis is a bacterium strongly associated with periodontitis. P. gingivalis exports lipids, proteins, and other biomolecules that contribute to the bacterium's ability to persist in inflammatory conditions encountered during disease. These biomolecules are exported through several mechanisms, including via outer membrane vesicles (OMVs). Despite their ubiquity, the mechanisms that drive outer membrane vesicle production vary among bacteria and are not fully understood. In this study, we report that C4' dephosphorylation of lipid A, a major outer membrane molecule, is required for robust outer membrane vesicle production and biological function in P. gingivalis. This finding adds to the growing body of evidence that lipid A structure is an important factor in outer membrane vesicle biogenesis in diverse bacterial species.