Gibson, F. C. | Search bioRxiv

bioRxiv2026

Capsular K-antigen Coats Outer Membrane Vesicles of Porphyromonas gingivalis

Periodontal disease is an inflammatory disorder that arises from dysbiosis of the subgingival microbiota, with Porphyromonas gingivalis acting as a keystone pathogen in the shift from health to disease. P. gingivalis employs multiple strategies to subvert host immune defenses, and its capsular K-antigen serves as a key virulence determinant. Here, a pre-adsorbed antiserum (pAds106) was generated by removing nonspecific antibodies using cells from a K-antigen-null mutant (W83{Delta}PG0106), resulting in exceptional specificity for the P. gingivalis K1-antigen. Immunofluorescence analysis revealed that the K-antigen preferentially coats outer membrane vesicles (OMVs), rather than attaching to the bacterial cell surface. This localization was further confirmed by ELISAs of density gradient ultracentrifuge-purified OMVs, with background signal detected in OMVs derived from K-antigen-deficient strains, non-K1-strains, and other oral Bacteroidetes. K-antigen-coated OMVs exhibited higher hydrophilicity and elicited weaker inflammatory responses compared to K-antigen-deficient OMVs, consistent with previously reported properties of encapsulated strains. Importantly, the antiserum detected K-antigen-coated OMVs in subgingival plaque from periodontal patients, suggesting that K-antigen is actively produced at diseased sites. These findings revise the prevailing view that K-antigen solely encapsulates the bacterial cell body and suggest that K-antigen-coated OMVs produced by P. gingivalis play distinct roles in immune evasion during periodontal disease.

microbiology↗

bioRxiv2020

Sphingolipid containing outer membrane vesicles serve as a delivery vehicle to limit host immune response to Porphyromonas gingivalis

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-, IL-1{beta}, 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.

microbiology↗