FAD-I, a Fusobacterium nucleatum Cell Wall-Associated Diacylated Lipoprotein That Mediates Human Beta Defensin 2 Induction through Toll-Like Receptor-1/2 (TLR-1/2) and TLR-2/6

Abstract

We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ΔfadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.

FIG 1

Induction of hBD-2 mRNA by live bacterial cells (A) and cell wall preparations (10 μg/ml cell wall F. nucleatum [Fn] ATCC 25586, ATCC 23726, and ATCC 10953 (B). The data presented are means ± standard deviations (SD) of the results of three to six replicate experiments; *, significant (P < 0.05), and NS, not significant between the indicated experiments.

FIG 2

(A) Western immunoblot of FAD-I protein in whole-cell lysates of F. nucleatum ATCC 25586, ATCC 23726, ATCC 10953, ATCC 10953 ΔfadI, and ATCC 10953 ΔfadI/pFAD-I 23726 (ATCC 10953 ΔfadI with plasmid-based expression of FAD-I from ATCC 23726). For Western blotting, whole-cell lysates derived from 1.5 × 10⁷ cells of the different Fusobacterium strains at the mid-logarithmic growth phase were resolved in a 4 to 12% SDS-PAGE precast gel (NuPAGE, Invitrogen, USA) according to standard protocols, and anti-FAD-I antibody (1) was used to detect FAD-I. (B) Multiple-sequence alignment of FAD-I peptides from three F. nucleatum strains (ATCC 25586, ATCC 23726, and ATCC 10953). The Clustal W (54) Web-based service was used to align the sequences. The arrow indicates a conserved cysteine; the box at the N terminus indicates the signal peptide sequences; the dark-blue-shaded amino acids are common to all three proteins, while the amino acids shaded light blue or white indicate differences in sequences.

FIG 3

Induction of hBD-2 mRNA by cell wall preparations (10 μg/ml) of parent ATCC 10953, ATCC 10953 ΔfadI, ATCC 10953 ΔfadI/pFAD-I25586 (ATCC 10953 ΔfadI with plasmid-based expression of FAD-I from ATCC 25586), and ATCC 10953 ΔfadI/pFAD-I23726 (ATCC 10953 ΔfadI with plasmid-based expression of FAD-I from ATCC 23726) (A) and 25586 FnCW (10 μg/ml) (B) in the presence of anti-FAD-I antibody (1) or isotype control. The data presented are means ± SD of the results of three to six replicate experiments; *, significant (P < 0.05), and NS, not significant between the indicated treatments.

FIG 4

Comparison of hBD-2 induction and release from HOECs by rFAD-I, mature rFAD-I, and its signal peptide. HOECs were treated for 18 h with 10 μg/ml of the indicated peptides {rFAD-I, recombinant full-length FAD-I; Δ15 FAD-I, rFAD-I with the 15-amino-acid signal sequence deleted; Signal Peptide (SP), signal peptide only; [SP]+[Δ15 FAD-I], mixture of signal peptide and Δ15 FAD-I}. Fold changes in hBD-2 mRNA (A) and released peptide (B) compared to untreated HOECs were measured. The data presented are means ± SD of the results of six independent experiments; *, significant (P < 0.05) compared to untreated HOECs; #, significant (P < 0.05) compared to rFAD-I-treated cells.

FIG 5

(A and B) Tandem-mass-spectrometry chromatograms for identification of the lipid moiety in rFAD-I (A) and nFAD-I (B). The chromatogram shows the doubly charged peptide CANIDTGVDESK at m/z 914.53, where Cys modified by 576.4 Da corresponds to S-diacylglycerol cysteine. The asterisk indicates modification. (C and D) HOECs were treated for 18 h with 10 μg/ml of each of the peptides, as indicated [rFAD-I, recombinant FAD-I; rFAD-I (C16A), rFAD-I with the cysteine at position 16 mutated to alanine]. Fold changes in hBD-2 mRNA (C) and released peptide (D) compared to untreated HOECs were determined. The data presented are means ± SD of the results of six independent experiments; *, P < 0.05.

FIG 6

HOEC TLR-1/2 and TLR-2/6 interaction with rFAD-I. (A) HOECs were treated with either rFAD-I (10 μg/ml), Pam2Cys (a positive control for TLR-2/6; 20 ng/ml; Invivogen, USA), or Pam3Cys (a positive control for TLR-1/2; 20 ng/ml; Invivogen, USA) for 18 h in the presence or absence of anti-TLR antibodies (Invivogen, USA) as indicated. The antibodies were incubated with HOECs for 60 min prior to 18 h of incubation with the other reagents. HBD-2 mRNA fold changes compared to untreated HOECs were determined. The data presented are means ± SD of the results of three independent experiments; *, P < 0.05. (B and C) Flow cytometric analysis of TLR interaction with rFAD-I. HOECs were plated in 24-well clusters in duplicate. Upon attaining 70 to 80% confluence, the cells were harvested (0 h); left untreated (untreated); or incubated with either recombinant FAD-I (rFAD-I), Pam2Cys, or Pam3Cys for 30 min. Cells were harvested for surface staining (B) or intracellular detection (C) of TLRs by flow cytometry. (D to F) Flow cytometric analysis of TLR2 after rFAD-I and FITC-labeled rFAD-I challenge of cytochalasin D-treated HOECs. (D) Semiconfluent HOECs were harvested (0 h) and left untreated or treated with either dimethyl sulfoxide (DMSO), rFAD-I, rFAD-I plus CytD (Sigma, USA), FITC-labeled rFAD-I, or FITC-labeled rFAD-I plus CytD for 45 min. Cells were then harvested for TLR-2 surface staining and analyzed by flow cytometry. (E and F) The percentage of surface TLR2 (only)-expressing cells (E) or the percentage of surface TLR2⁺ rFAD-I–FITC double-positive cells (F) among HOECs harvested at 0 h, incubated with DMSO (DMSO), left untreated (untreated), or incubated with FITC-labeled rFAD-I [rFAD-I (FITC)] for 30 min in the presence or absence of 40 μM CytD was determined. The flow cytometric data presented are representative of two independent experiments.