Abstract
Porphyromonas gingivalis (Pg) is a prevalent pathogen that promotes human periodontal disease (PD) and exacerbates systemic comorbidities such as atherosclerosis, rheumatoid arthritis, and Alzheimer's disease. Pg produces nonphosphorylated tetra-acylated lipid A (NPLA) in its outer membrane (OM) that evades host Toll-like receptor 4 (TLR4), inflammasome pathways, and cationic peptides, enhancing bacterial survival. Here, we show that Pg also releases outer membrane vesicles (OMVs) that engage and divert host cell TLR4, inflammasome, and LL-37 responses away from the microbe. We determined that Pg OMVs are enriched for C4' monophosphoryl lipid A (C4'-MPLA), an established agonist for TLR4-TRIF-IFNβ and inflammasome-IL-1β responses. Comparisons of Pg 381 and Pg 33277 stationary phase cultures revealed higher OMV production by Pg 381, which correlates with its higher proinflammatory pathogenicity. The cationic peptide, polymyxin B (PMB), which selectively binds lipid A C4'-phosphate, reduces OMV-stimulated HEK cell TLR4 activation and THP-1 cell IL-1β production, confirming the proinflammatory role for OMV-C4'-MPLA. Similar to PMB, the host defense peptide, LL-37, inhibits OMV-C4'-MPLA-dependent HEK cell TLR4 activation. PMB and LL-37 also blocked OMV-C4'-MPLA-driven TLR4 activation in human umbilical vein endothelial cells. Finally, wild-type Pg-containing OM-NPLA is highly resistant to LL-37 antimicrobial activity, whereas the ΔlpxF mutant bacterium, retaining OM-C4'-MPLA, is killed by the peptide. In summary, Pg escapes host TLR4 signaling, inflammasome activation, and LL-37 interaction by retaining immunoevasive OM-NPLA. Moreover, Pg dispenses proinflammatory OMV-C4'-MPLA, which engages and redirects those host defenses. We suggest that OMV-C4'-MPLA triggers elevated IFNβ and IL-1β cytokines, which typify PD comorbidities, and drive PD-related alveolar bone loss.