The FomA porin from Fusobacterium nucleatum is a Toll-like receptor 2 agonist with immune adjuvant activity

Abstract

Many bacterial components selectively activate immune and nonhematopoietic target cells via Toll-like receptor (TLR) signaling; modulation of such host responses defines the immune adjuvant properties of these bacterial products. For example, the outer membrane protein porins from Neisseria, Salmonella, and Shigella are known TLR2 agonists with established systemic and mucosal immune adjuvanticity. Early work indicated that the FomA porin from Fusobacterium nucleatum has immune adjuvant activity in mice. Using a purified recombinant FomA, we have verified its immune stimulatory properties and have defined a role for TLR2 signaling in its in vitro and in vivo activity. FomA induces interleukin 8 (IL-8) secretion and NF-κB-dependent luciferase activity in HEK cells expressing TLR2, IL-6 secretion, and cell surface upregulation of CD86 and major histocompatibility complex (MHC) II in primary B cells from wild-type mice, but it fails to activate cells from TLR2 knockout mice. Accordingly, the immune adjuvant activity of FomA is also TLR2 dependent. In a mouse model of immunization with ovalbumin (OVA), FomA induces enhanced production of OVA-specific IgM and IgG, including IgG1 and IgG2b antibodies, as well as enhanced secretion of IL-10 and IL-6, consistent with a Th2-type adjuvant effect. We also observe a moderate production of anti-FomA antibodies, suggesting that FomA is also immunogenic, a quality that is also TLR2 dependent. Therefore, modulation of host immune responses by FomA may be effective for targeting general host immunity not only to pathogens (as a novel TLR2 adjuvant) but also to F. nucleatum itself (as an antigen), expanding its use as a self-adjuvanted antigen in an immunization strategy against polymicrobial infections, including those by F. nucleatum.

Fig 1

FomA purification. (A) Representative chromatogram of FomA elution from gel filtration chromatography on Sephacryl S300 column. (B) SDS-PAGE and Coomassie staining of representative fractions from panel A. The FomA-containing fractions are indicated by the arrow. FomA was identified as a band of approximately 40 kDa in the major elution peak. (C) SDS-PAGE and silver staining of final FomA preparation. Lane 1, molecular weight standard. Lane 2, FomA, 2 μg total. Lane 3, E. coli LPS, 2 μg total.

Fig 2

TLR2-dependent cell activation by FomA. (A and B) TLR2-HEK cells and pcDNA-HEK cells (A) or TLR4-HEK cells and pcDNA-HEK cells (B) incubated with purified FomA (10 μg/ml), Pam₃CSK₄ (100 ng/ml), E. coli LPS (100 ng/ml), and TNF-α (20 ng/ml) for 18 h. NF-κB-dependent luciferase activity was measured and is expressed in arbitrary units ± SD normalized to nonstimulated cells. **, P < 0.0023 (for panel A) and P = 0.0075 (for panel B). (C and D) IL-8 secretion measured by ELISA of supernatants of TLR2-HEK cells and pcDNA-HEK cells (C) and TLR4-HEK cells and pcDNA-HEK cells (D) incubated as described above. IL-8 is expressed as pg/ml ± SD. ***, P = 0.0002; ****, P < 0.0001 (for panel C). **, P = 0.0014; ** P = 0.008 (for panel D). (E) IL-6 measured by ELISA of supernatant from purified splenic B cells from C57BL/6 mice (wt) and TLR2 KO mice stimulated with FomA, Pam₃CSK₄, and LPS as described above. IL-6 is expressed as pg/ml ± SD. ****, P < 0.0001.

Fig 3

Murine B cell activation in vitro. Purified splenic B cells from wt mice (A to F) and TLR2 KO mice (G to L) were incubated with 10 μg/ml of FomA (A, B, G, and H), 100 ng/ml of Pam₃CSK₄ (C, D, J, and I) or 100 ng/ml of E. coli LPS (E, F, K, and L) for 18 h. Surface upregulation of CD86 and MHC II was measured by flow cytometry. In all the histograms, the isotype control is indicated by the dotted line, cells incubated with medium alone are represented by the thin line, and stimulated cells are represented by the thick line.

Fig 4

Enhancement of antigen-specific antibody production by FomA. (A to D) wt mice immunized with ovalbumin (OVA) (10 μg) alone and in combination with FomA (10 μg/ml) or alum (200 μg). Preimmune sera (Pr) and sera after the final immunization (week 6) were examined by ELISA for quantification of OVA-specific total IgG and IgM levels. (A and B) Anti-OVA IgG (A) and anti-OVA IgM (B), expressed as μg/ml ± SD relative to mice immunized with OVA alone and OVA-FomA. ***, P = 0.0005 and 0.0001 by unpaired t test. (C and D) Anti-OVA IgG (C) and anti-OVA IgM (D), expressed as μg/ml ± SD relative to mice immunized with OVA alone and OVA-alum. ***, P = 0.0001; *, P = 0.04. (E to H) TLR2 KO mice were immunized as described above and OVA-specific IgG (E and G) and IgM (F and H) were quantified by ELISA. ****, P < 0.0001; **, P = 0.009.

Fig 5

Induction of antigen-specific IgG subclasses and cytokines by FomA. OVA-specific IgG subclasses measured in the sera of wt mice (A) and TLR2 KO mice (B) immunized with OVA alone or OVA-FomA by ELISA normalized to preimmune sera and expressed as mean titers ± SD. *, P = 0.03; ***, P = 0.0002. Sera were used at a 1:100 dilution. (C) Cytokine levels in sera from wt mice immunized with OVA alone or OVA-FomA measured by ELISA. The values were normalized to pooled preimmune sera and expressed as fold change ± SD. *, P = 0.015.

Fig 6

Immunogenicity of FomA. (A) Anti-FomA total IgG from wt mice immunized with FomA in the absence of additional adjuvants measured by ELISA. Preimmune sera (Pr) and sera after each boost (week 2, week 4, and week 6) are shown. Results are expressed in μg/ml ± SD. ****, P < 0.0001. (B) Anti-FomA total IgG from TLR2 KO mice as in panel A. ****, P < 0.0001. **, P = 0.004 for comparison of week 6 results in panels A and B. (C) Anti-FomA IgG subclasses in the sera from wt mice and TLR2 KO mice. Sera were used at a 1:100 dilution. Results are expressed as titers normalized to preimmune sera ± SD. *, P = 0.02; **, P = 0.006; *, P = 0.01 (left to right, respectively). (D) Western blot of FomA, detected as a band of ∼40 kDa by sera from a representative FomA-immunized mouse (week 6, 1:1,000 dilution).