Molecular interactions of Porphyromonas gingivalis fimbriae with host proteins: kinetic analyses based on surface plasmon resonance

Abstract

Fimbriae of Porphyromonas gingivalis are thought to play an important role in the colonization and invasion of periodontal tissues. In this study, we analyzed the interactions of P. gingivalis fimbriae with human hemoglobin, fibrinogen, and salivary components (i.e., proline-rich protein [PRP], proline-rich glycoprotein [PRG], and statherin) based on surface plasmon resonance (SPR) spectroscopy with a biomolecular interaction analyzing system (BIAcore). The real-time observation showed that the fimbriae interacted more quickly with hemoglobin and PRG than with other proteins and more intensely with fibrinogen. The significant association constant (ka) values obtained by BIAcore demonstrated that the interactions between fimbriae and these host proteins are specific. These estimated Ka values were not too different; however, the Ka values for hemoglobin (2.43 x 10(6)) and fibrinogen (2.16 x 10(6)) were statistically greater than those for the salivary proteins (1.48 x 10(6) to 1.63 x 10(6)). The Ka value of anti-fimbriae immunoglobulin G for fimbriae was estimated to be 1. 22 x 10(7), which was 6.55-fold higher than the mean Ka value of the host proteins. Peptide PRP-C, a potent inhibitor of PRP-fimbriae interaction, dramatically inhibited fimbrial association to PRP and PRG and was also inhibitory against other host proteins by BIAcore. The binding of fimbriae to these proteins was also evaluated by other methods with hydroxyapatite beads or polystyrene microtiter plates. The estimated binding abilities differed considerably, depending on the assay method that was used. It was noted that the binding capacity of PRP was strongly diminished by immobilization on a polystyrene surface. Taken together, these findings suggest that P. gingivalis fimbriae possess a strong ability to interact with the host proteins which promote bacterial adherence to the oral cavity and that SPR spectroscopy is a useful method for analyzing specific protein-fimbriae interactions.

FIG. 1

Estimation of fimbrial binding affinity to the host proteins by the BIAcore system. The same molar amount of each host protein was immobilized on the matrix of the chip. Fimbriae were injected at flow rate 10 μl/min for 240 s. (A) The binding ability of fimbriae to each host protein was monitored and presented as a sensogram (plotted as RU versus time). For kinetic studies, fimbriae with increasing concentrations (a, 16 μM; b, 8 μM; c, 4 μM; d, 2 μM; e, 1 μM) were injected over the sensor chip. (B) Kinetic analysis of fimbrial binding to host proteins. A plot of dRU/dt versus RU (slope) was calculated from the sensograms, and then the slopes at different concentrations were replotted against the concentrations of fimbriae. The different angles of the obtained linear lines represent the variation of the affinities, giving the k_as from equation 1. (C) k_dis (1/s) was determined directly from the linear lines of the plots at the dissociation phase. A ln(Rt₁/Rt_n) plot was calculated [Rt₁ is the RU at the initial phase of dissociation (t₁), and Rt_n is the RU at time t_n] and replotted against the times. The angles of the regression linear lines represent resistibility to dissociation.

FIG. 2

Demonstration of the effect of peptide PRP-C by the BIAcore system. Fimbriae (4 μM) were injected with or without simultaneous addition of each peptide (400 μM of peptide PRP-C, SM 15, or A1) to the sensory chip of the BIAcore system as described in the text.

FIG. 3

Binding of P. gingivalis fimbriae to the host proteins on an apatitic or a polystyrene surface. (A) Binding abilities of fimbriae to host proteins were assayed by using HA beads. ¹²⁵I-labeled fimbriae (0.5 nmol) was added to a tube containing host protein-coated HA beads and incubated at RT for 1 h. ¹²⁵I-labeled fimbriae (0.5 nmol) added (□); ¹²⁵I-labeled fimbriae (0.5 nmol) plus peptide PRP-C (50 nmol) added (■); fimbriae (0.5 nmol) plus nonlabeled fimbriae (25 nmol) added to obtain the nonspecific binding level (). The specific binding level was calculated by subtracting the nonspecific binding level. Significant differences (P < 0.01) were observed among fimbrial binding levels (□) to host proteins in the absence of peptide PRP-C. (B) Binding assay performed with polystyrene surface. The wells of 96-well microtiter plates were coated with host proteins or BSA (100 μl of 0.1 mg/ml in PBS) at 37°C for 2 h. An aliquot of fimbriae and, if necessary, peptide PRP-C as an inhibitor was added to the wells and then incubated at RT for 1 h. Rabbit anti-fimbriae immunoglobulin G (1:1,000) was added to detect the amount of bound fimbriae. □, 12.2 pmol of added fimbriae; ■, 24.4 pmol of added fimbriae; , 12.2 pmol of fimbriae plus 100 pmol of added peptide PRP-C; ▩, 12.2 pmol of fimbriae plus 1 nmol of added peptide PRP-C. Significant differences (P < 0.01) were observed among fimbrial binding levels (■) to host proteins in the absence of peptide PRP-C and between PRP and BSA and between fibrinogen and hemoglobin. All assays were performed in triplicate, on three separate occasions.