Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells, and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection. As P. gingivalis robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyper-inflammatory immune response from THP-1 macrophage-like cells with elevated TNF- α, IL-1β, and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF by SL-null P. gingivalis. No significant differences in gingipain activity were observed in our infection models and both strains produced OMVs of similar size. Using comparative 2-dimensional gel electrophoresis we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in P. gingivalis OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response.

Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells, and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection. As P. gingivalis robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyper-inflammatory immune response from THP-1 macrophage-like cells with elevated TNF-α, IL-1β, and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF by SL-null P. gingivalis. No significant differences in gingipain activity were observed in our infection models and both strains produced OMVs of similar size. Using comparative 2-dimensional gel electrophoresis we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in P. gingivalis OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response.