Antibodies with specificity to glycan motifs that decorate OMV cargo proteins

Abstract

Porphyromonas gingivalis is a major etiological agent of periodontal disease, and infections with this bacterium are associated with systemic pathologies, including atherosclerosis, rheumatoid arthritis, and Alzheimer's disease. P. gingivalis has a variety of immune evasion mechanisms and exhibits highly variable cell surface characteristics that are strain dependent, complicating the development of effective vaccines and therapeutics. Here, we show that a subset of immunoglobulin M (IgM) antibodies in antiserum raised against P. gingivalis strain W83 selectively recognize the outer membrane vesicles (OMVs). Pre-adsorption with a mutant strain lacking an OMV-specific lipoprotein (PG1881) that has been shown to be glycosylated significantly enhanced IgM specificity toward PG1881 and the OMVs. In addition, the IgM reactivity against the OMVs derived from a mutant lacking enzymes required for O-glycosylation was markedly reduced, indicating that the IgM targets the glycan motifs on proteins carried on OMVs. Importantly, the IgM exhibited specific recognition of OMVs from both P. gingivalis and Porphyromonas endodontalis, while showing low reactivity toward other genera belonging to the phylum Bacteroidetes. This study revealed a potential host evasion strategy and highlights the potential for utilizing O-glycans in vaccine development and OMV-targeted antibodies in therapeutic interventions to combat P. gingivalis infections.

Importance: O-glycosylation of cell surface proteins by bacteria is known to play a role in various functions including colonization and immune evasion. This study highlights the identification of IgM antibodies that specifically recognize O-glycosylated proteins that are selectively carried on outer membrane vesicles (OMVs). The findings suggest a potential host evasion mechanism and open new avenues for using OMVs in vaccine development and targeting O-glycans with antibodies as a therapeutic strategy against the subgingival pathobiont P. gingivalis.

Keywords: IgM antibodies; O-glycosylation; OMV; P. gingivalis; PG1881.

Fig 1

Immunofluorescence microscopy of P. gingivalis strain W83 and ATCC 33277 using anti-W83 antiserum. IgG antibodies in the antiserum were labeled by Alexa Fluor 647 (red), and secondary Alexa Fluor 488-labeled anti-IgM antibodies (green) were used to detect the IgM antibodies in the antiserum. IgM antibodies in the antiserum target OMV-like structures on both W83 and ATCC 33277 strains (BF, brightfield)

Fig 2

Pre-adsorbed IgM antibodies recognize OMVs of P. gingivalis. Western blot analysis of cell lysate (A) or OMV lysate (B) of W83 and the ΔPG1881 mutant using the pre-adsorbed antiserum as the primary antibodies and anti-IgM-HRP antibodies as the secondary antibodies to detect binding of IgM. (C) Immunofluorescence microscopy images of W83 and the corresponding ΔPG1881 mutant cells treated with the pre-adsorbed antiserum and then with anti-IgM-AF488 (green). The cells were also stained by the fluorescent nucleic acid stain SYTO85 (red) (WB, Western blot; CBS, Coomassie blue staining)

Fig 3

Pre-adsorbed IgM shows a significant reduction in binding to W83 cell lysates. ELISA using original antiserum (left panel) or pre-adsorbed antiserum (right panel) as the primary antibodies followed by either IgM-HRP or IgG-HRP as the secondary antibodies to detect the antigens on crude OMVs and cell lysates. The graphs are shown as the mean values ± standard deviations. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparison test. *P <0.05 compared to W83 cell lysates and ΔPG1881 cell lysates (left panel). ****P < 0.001 compared to W83-OMVs or W83 cell lysates (right panel).

Fig 4

Pre-adsorbed IgM showed a significant reduction in binding to purified OMVs from the O-glycosylation-deficient mutant. ELISA using the antiserum that was pre-adsorbed with W83ΔPG1881 mutant cells as the primary antibodies, followed by either IgM-HRP or IgG-HRP as the secondary antibodies to detect the antigens on purified OMVs from W83, ΔPG1881, PG1881 complementary strain (ΔPG1881 pT-C1881), and the O-glycosylation-deficient mutant (ΔPG1345-1346). Representative results from three biological replicates are shown. Representative results from three biological replicates are shown. The graphs are shown as the mean values ± standard deviations. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.001 compared to the W83-OMVs.

Fig 5

Pre-adsorbed IgM shows specific binding to crude OMVs from P. gingivalis and P. endodontalis. ELISA using PG1881 mutant cells pre-adsorbed antiserum as the primary antibodies and followed by either IgM-HRP or IgG-HRP as the secondary antibodies to detect the antigens on the crude OMVs from oral Bacteroidetes. The graphs are shown as the mean values ± standard deviations. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparison test. ****P < 0.001 compared to the Pg-OMVs group at the same concentration.