Fusobacterium nucleatum envelope protein FomA is immunogenic and binds to the salivary statherin-derived peptide

Abstract

We have previously shown that one of the minimal active regions of statherin, a human salivary protein, for binding to Fusobacterium nucleatum is a YQPVPE amino acid sequence. In this study, we identified the FomA protein of F. nucleatum, which is responsible for binding to the statherin-derived YQPVPE peptide. Overlay analysis showed that a 40-kDa protein of the F. nucleatum cell envelope (40-kDa CE) specifically bound to the YQPVPE peptide. The equilibrium association constant between the affinity-purified 40-kDa CE and the YQPVPE peptide was 4.30 x 10(6). Further, the purity and amino acid sequence analyses of the purified 40-kDa CE revealed approximately 98.7% (wt/wt) purity and a high degree of homology with FomA, a major porin protein of F. nucleatum. Thus, a FomA-deficient mutant failed to bind to the YQPVPE peptide. In addition, increased levels of a FomA-specific mucosal IgA antibody (Ab) and plasma IgG and IgA Abs were seen only in mice immunized nasally with cholera toxin (CT) and the purified 40-kDa FomA protein. Interestingly, saliva from mice that received FomA plus CT as a mucosal adjuvant nasally prevented in vitro binding of F. nucleatum to statherin-coated polyvinyl chloride plates. Taken together, these results suggest that induction of specific immunity to the 40-kDa FomA protein of F. nucleatum, which specifically binds to the statherin-derived peptide, may be an effective tool for preventing the formation of F. nucleatum biofilms in the oral cavity.

FIG. 1.

The YQPVPE peptide binds to the 40-kDa cell envelope (CE) protein of F. nucleatum. (A) The F. nucleatum (ATCC 25586) CEs that specifically bind to the YQPVPE peptide were determined by a ligand overlay assay. F. nucleatum whole CEs were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with the biotinylated YQPVPE peptide followed by horseradish peroxidase (HRP)-conjugated streptavidin for substrate development. Lane 1, molecular mass standard marker; lane 2, F. nucleatum whole CEs. (B and C) SDS-PAGE (B) and ligand overlay analysis (C) of F. nucleatum CEs purified by a YQPVPE peptide-coupled, CNBr-activated Sepharose 4B column. Lanes 1 and 3, molecular mass standard marker; lane 2, YQPVPE affinity column-purified F. nucleatum; lane 4, purified F. nucleatum CE incubated with the biotinylated YQPVPE peptide.

FIG. 2.

Biomolecular interactions of affinity column-purified F. nucleatum CEs and the YQPVPE peptide. The affinity of binding of the YQPVPE peptide to immobilized F. nucleatum CEs (A) or BSA (B) was estimated by the BIAcore system. HBSP buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% Tween 20 [pH 7.4]) was used as a running buffer, with a flow rate of 30 μl/min. The statherin peptide solution at various concentrations (1.3 μM to 20 μM) was monitored and injected over the 40-kDa CE (100 μg/ml) or BSA (100 μg/ml) on the CM5 sensor chip.

FIG. 3.

Homology search for the amino acid alignment of the 40-kDa F. nucleatum component. The 40-kDa CE of F. nucleatum was digested with V8 protease, and the amino-terminal sequences of the three cleaved fragments were determined using the BLASTp database program. The underlined sequences correspond to the identified amino-terminal sequences of F. nucleatum FomA and the 40-kDa CE of the F. nucleatum envelope obtained from the YQPVPE peptide-conjugated affinity column.

FIG. 4.

Construction and functional analysis of the ΔfomA mutant strain SN-3. (A) pFOMA151 contains an internal fragment of fomA and a kanamycin resistance gene (aphA3). SN-3 was produced by single-crossover recombination. (B) The ΔfomA mutant strain SN-3 and the wild-type strain of F. nucleatum (ATCC 25586) were subjected to PCR with forward primer fomABamF1 and reverse primer aphA3F2. (C) Dot blot assay for direct binding activity of ATCC 25586 or mutant strain SN-3 to the YQPVPE peptide. The dot blots show typical results for both the experimental (binding to the YQPVPE peptide) and control (binding to BSA) groups. The level of dot blot density for the control group was subtracted from the level of dot blot density for the experimental group in each experiment. The relative percentage of dot density for each was calculated relative to the dot density for wild-type F. nucleatum bound to the YQPVPE peptide, which was defined as 100%. The graph shows the average percentages of dot density for the binding of wild-type F. nucleatum (100%) or SN-3 (21%) to the YQPVPE peptide. The experiments were performed in triplicate on three separate occasions. Data are expressed as means ± standard deviations. The asterisk indicates a significant difference (P < 0.05) from the result for the wild-type strain.

FIG. 5.

FomA-specific immune responses in external secretions and mucosal lymphoid tissues. C57BL/6 mice were nasally immunized weekly for three consecutive weeks either with FomA protein (20 μg) plus cholera toxin (CT; 1 μg) as a mucosal adjuvant (filled bars) or with FomA only (open bars). (A) Seven days after the last immunization, the levels of FomA-specific IgA Abs in nasal washes and saliva were determined by FomA-specific ELISAs. Data are means ± SEMs (n = 15). Double asterisks indicate significant differences (P < 0.01) from results for control mice. (B and C) Seven days after the last immunization, mononuclear cells isolated from NPs, SMGs, and NALT were subjected to Ag-specific ELISPOT assays in order to determine the numbers of IgG and IgA AFCs. Mice immunized nasally with FomA alone were used as controls. Data are means ± SEMs (n = 15). Asterisks indicate significant differences (**, P < 0.01) from results for control mice.

FIG. 6.

Comparison of FomA-specific Ab responses in systemic lymphoid tissues of mice given FomA with (filled bars) or without (open bars) CT. Each mouse group was immunized nasally weekly for three consecutive weeks. (A) Seven days after the last immunization, FomA-specific IgM, IgG, IgA, and IgG subclass Ab responses in plasma were determined by Ag-specific ELISAs. (B) Seven days after the last immunization, mononuclear cells were isolated from spleens and CLNs and were then subjected to Ag-specific ELISPOT assays in order to determine the numbers of IgM, IgG, and IgA AFCs. Data are means ± SEMs (n = 15). Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) from results for control mice.

FIG. 7.

Effect of saliva on the formation of F. nucleatum biofilms on statherin-coated PVC plates. F. nucleatum (5 × 10⁷ bacteria/well) was preincubated with either KCl buffer alone (no inhibitor), saliva from naïve mice, saliva from mice immunized nasally either with FomA alone or with FomA plus CT, or YQPVPE peptide solution at 25°C for 3 h. Subsequently, each mixture, as well as a sample with no bacteria, was added to statherin-coated PVC plates and incubated at 25°C overnight. The resulting biofilm was stained with crystal violet and extracted with 95% ethanol. Biofilm formation was scored by measuring the absorbance at 595 nm. All assays were performed in triplicate, and means and standard deviations are shown. Asterisks indicate significant differences (**, P < 0.01) from results for control mice.