Structural basis for immune cell binding of Fusobacterium nucleatum via the trimeric autotransporter adhesin CbpF

Abstract

Fusobacterium nucleatum (Fn), a commensal in the human oral cavity, is overrepresented in the colon microbiota of colorectal cancer (CRC) patients and is linked to tumor chemoresistance, metastasis, and a poor therapeutic prognosis. Fn produces numerous adhesins that mediate tumor colonization and downregulation of the host's antitumor immune response. One of these, the trimeric autotransporter adhesin (TAA) CEACAM binding protein of Fusobacterium (CbpF), targets CEACAM1 on T-cells and has been associated with immune evasion of Fn-colonized tumors. Whereas the role of CEACAM1 in homophilic and heterophilic cell interactions and immune evasion is well described, the mechanistic details of its interaction with fusobacterial CbpF remain unknown due to the lack of a high-resolution structure of the adhesin-receptor complex. Here, we present two structures of CbpF alone and in complex with CEACAM1, obtained by cryogenic electron microscopy and single particle analysis. They reveal that CbpF forms a stable homotrimeric complex whose N-terminal part of the extracellular domain comprises a 64 Å long β roll domain with a unique lateral loop extension. CEACAM1 binds to this loop with high affinity via its N-terminal IgV-like domain with a nanomolar dissociation constant as determined by surface plasmon resonance. This study provides a comprehensive structural description of a fusobacterial TAA, illustrates a yet undescribed CEACAM1 binding mode, and paves the way for rational drug design targeting Fn in CRC.

Keywords: bacterial autotransporter adhesin; cryogenic electron microscopy; host–microbiome interaction.

Fig. 1.

Structure of the F. nucleatum type Vc autotransporter adhesin CbpF. (A) Cryo-EM density of CbpF at 3.8 Å resolution. The map is colored in shades of red corresponding to the three protomers. N and C termini of the resolved parts are indicated. (B) Density map colored as in A with the three highest scoring AlphaFold2 models of the CbpF homotrimer fitted. The nonresolved parts after G274 are colored in shades of gray. Note the unstructured regions following after G274. (C) Inward-facing hydrophobic F, I, L, and V residues form the hydrophobic core of the β roll and hold the protomers together within the homotrimeric passenger domain. (D) Immediately C-terminal of the β roll, short α-helices and loops of the three CbpF protomers wrap around each other. (E) Surface representation of the CbpF β roll and the following α-helical domain (residues 25–274), colored by hydrophobicity (yellow: hydrophobic, cyan: hydrophilic). (F) Surface representation of the CbpF β roll and the following α-helical domain (residues 25–274), colored by Coulomb potential at pH 7.0 (red: negatively charged, −8 kT/e, blue: positively charged, 8 kT/e). The loop protruding from the β-sheet stack is highlighted (boxes in E and F) and shown in zoomed view in the Right panels in E and F.

Fig. 2.

Conservation of loop and comparison of CbpF structure with other Type Vc autotransporter adhesins. (A) Alignment of CbpF sequences from different Fusobacteria [Fn ATCC25586–the present structure, Fn ATCC23726, F. vincentii (Fv) ATCC49256, F. polymorphum (Fp) ATCC10953, and F. necrophorum (Fnec) 1_1_36S]. The section shows the region around the loop E142–Y149 in Fn ATCC25586, which is highlighted with pink dashed frame. (B) Comparison of the passenger domains of Fn CbpF with Y. enterocolitica YadA (PDB 1P9H), Moraxella catarrhalis UspA1 (PDB 3PR7 and 3NTN), Burkholderia pseudomallei BpaC (PDB 7O23), and Bartonella henselae BadA (PDB 3D9X). The lengths of the β roll domains (β-sheet stack and Trp ring domain for BadA) are indicated. (C) Structure alignment of one protomer of the five adhesins shown in B, with residues 142–149 from CbpF that form a loop protrusion outside the β roll highlighted. None of the other adhesins has a similar loop. Note that UspA1 (green) is also a CEACAM1 binding protein.

Fig. 3.

Binding and affinity of F. nucleatum CbpF to human CEACAM1. (A) Representative micrographs that illustrate binding of nanodisc-embedded CbpF (Upper panels) in comparison to empty nanodiscs (Lower panels) to HEK293T cells transfected with a CEACAM1 expression plasmid. Magenta channel: protein labeled with AlexaFluor 647, green channel: fluorescence of CEACAM1-eGFP fusion protein. (Scale bar, 25 µm.) (B) Quantification of CbpF colocalization with cells as in A, using the mean integrated fluorescence signal of protein that colocalizes with the CEACAM1-eGFP fluorescence signal, determined from the indicated number of micrographs each. Data were visualized with the ggstatsplot package (34) and statistical analysis was performed using the ggsignif package in R (35). The mean is shown as red dot. The boxes represent the interquartile range, with the median shown as a horizontal line within the box. (C) Competition of CbpF cell binding by purified CEACAM1-ECD (1 or 2 µM) in solution. Data visualization and statistical analysis was carried out using the ggstatsplot package in R (34), with P_Holm-adj. values indicated. n.s.: not significant. The mean is shown as red dot. The boxes represent the interquartile range, with the median shown as a horizontal line within the box. Underlying representative micrographs are shown in SI Appendix, Fig. S5E. (D) SPR sensorgram showing binding of CbpF to immobilized CEACAM1/ECD. The concentrations of CbpF are indicated. The black solid lines show a global fit of the binding curves according to a 1:1 binding model and reveal a k_on of 8.13 ± 0.01 × 10⁴ M⁻¹ s⁻¹ and k_off of 5.1 ± 0.02 × 10⁻⁴ s⁻¹, resulting in a K_D of 6.2 nM.

Fig. 4.

Structure of the CbpF–CEACAM1 complex. (A) Cryo-EM density of the complex at 2.7 Å resolution. The map is colored in shades of red corresponding to the three CbpF protomers and in shades of blue corresponding to three CEACAM1 molecules, in which the IgV-like and IgC2-like A1 domains are resolved. (B) Model-to-map fit focused on one CEACAM1 bound to CbpF. Color code of the model as the maps in A. The density map is shown in transparent gray. (C) Model of the CbpF/CEACAM1 interface. Intermolecular H-bonds as obtained in ChimeraX are shown as dashed lines. Note that H92 from CbpF protomer n + 1 (dark red) contributes to the interface of CbpF protomer n (coral). (D) Cryo-EM density map of one CEACAM1 at 3.0 Å resolution (transparent gray), obtained by shifting the center of reconstruction of the complex map, with the fitted model including glycan moieties. (E and F) Surface hydrophobicity (E) and Coulomb potential at pH 7.0 (red: negatively charged, −8 kT/e, blue: positively charged, 8 kT/e) (F) of the interacting sites of CbpF (Left) and CEACAM1 (Right) from the complex. The views show the front views of the interacting sites. (G) Comparison of solvent-accessible surface areas (SASAs) of CbpF and CEACAM1 alone and in the complex. Residues with pronounced differences are labeled. (H) Overlay of CEACAM1-interacting loops of CbpF in the complex (black) with unbound CbpF (gray). (I and J) Comparison of SPR sensorgrams of CbpF wt and variants (I—loop deletion Δ142–149, J—point mutations) when they bind to immobilized CEACAM1/ECD. The concentrations of CbpF are indicated.

Fig. 5.

Model of CEACAM1-mediated adhesion of F. nucleatum to immune and cancer cells. F. nucleatum binds to and downregulates the activity of T-cells via CbpF, while the adhesins FadA and Fap2 mediate interaction with CRC cells in tumors. In addition, CbpF might also be able to form a ternary complex with CEACAM1 on T-cells and on cancer cells, facilitating both immune evasion and tumor colonization.