The Treponema denticola FhbB Protein Is a Dominant Early Antigen That Elicits FhbB Variant-Specific Antibodies That Block Factor H Binding and Cleavage by Dentilisin

Abstract

The Treponema denticola FhbB protein contributes to immune evasion by binding factor H (FH). Cleavage of FH by the T. denticola protease, dentilisin, may contribute to the local immune dysregulation that is characteristic of periodontal disease (PD). Although three FhbB phyletic types have been defined (FhbB1, FhbB2, and FhbB3), the in vivo expression patterns and antigenic heterogeneity of FhbB have not been assessed. Here, we demonstrate that FhbB is a dominant early antigen that elicits FhbB type-specific antibody (Ab) responses. Using the murine skin abscess model, we demonstrate that the presence or absence of FhbB or dentilisin significantly influences Ab responses to infection and skin abscess formation. Competitive binding analyses revealed that α-FhbB Ab can compete with FH for binding to T. denticola and block dentilisin-mediated FH cleavage. Lastly, we demonstrate that dentilisin cleavage sites reside within critical functional domains of FH, including the complement regulatory domain formed by CCPs 1 to 4. Analysis of the FH cleavage products revealed that they lack cofactor activity. The data presented here provide insight into the in vivo significance of dentilisin, FhbB and its antigenic diversity, and the potential impact of FH cleavage on the regulation of complement activation.

FIG 1

FhbB proteins elicit FhbB type-specific Ab responses. Cell lysates of T. denticola strains (indicated along the top) were screened by immunoblotting with α-FhbB1, α-FhbB2, or α-FhbB3 antiserum generated using different FhbB proteins (as indicated) or with serum harvested from mice with T. denticola-induced abscesses (α-Td 35405). Equivalent loading of cell lysates was verified by screening an identical immunoblot with α-FlaA antiserum (top blot). FlaA is a constitutively produced protein. Molecular mass standards are indicated in kilodaltons on the left. T. denticola strain 35405ΔfhbB, which does not produce FhbB, served as a negative control for these analyses.

FIG 2

FhbB is a dominant antigen during infection using the murine abscess model. BALB/c mice were inoculated with T. denticola strains 35405, 35405ΔfhbB, and 35405-CCE, and sera were recovered 10 or 20 days later. (A) Identical immunoblots of 35405, 35405ΔfhbB, and 35405-CCE cell lysates (lanes 1 to 3, respectively) were generated and screened with preimmune serum (Pre-IS) or with α-35405, α-35405-CCE, or α-35405ΔfhbB infection serum harvested 10 or 20 days postinoculation. A gel stain with Coomassie brilliant blue (CBB) is shown on the left. An FH affinity ligand binding immunoblot assay (FH-ALBI) was also conducted to verify that the ∼11-kDa protein bound FH. To verify that the ∼11-kDa FH binding protein was FhbB, a blot was screened with rat α-FhbB1₃₅₄₀₅ antiserum. Molecular mass standards are shown to the left in kilodaltons. (B) The abscess sizes in mice 10 days postinoculation with each strain were measured. The statistical significance was assessed using the a two-tailed Student t test. P values are indicated in the figure.

FIG 3

FhbB antisera block FH binding to recombinant FhbB proteins. Recombinant FhbB proteins (indicated above each panel) were immobilized in triplicate in ELISA plate wells. The B. hermsii FhbA FH binding protein (33) served as a negative control. After blocking, the wells were overlaid with serial dilutions of rat α-FhbB₃₅₄₀₅ (■), α-FhbB2_SP50 (●), α-FhbB3₃₃₅₂₁ (▲), or α-FhbB3₃₅₄₀₄ (◆) antisera (blocking Ab). The wells were washed, and 10% NHS was added as an FH source. FH binding (expressed as relative FH binding) was measured with α-FH antiserum as detailed in the text. The data shown indicate the means with the error bars indicating the standard deviations. The data presented are representative of three independent experiments. P values were determined using a two-tailed Student t test (**, P < 0.01; ***, P < 0.001).

FIG 4

α-FhbB antisera attenuates FH degradation by T. denticola. (A) Mid-log-phase T. denticola 35405 cells were incubated with purified human FH for 60 min in the presence of 0, 10.0, 1.0, or 0.5% α-FhbB1 antiserum. Strain 35405-CCE (with no α-FhbB1 antiserum) served as a negative control for FH degradation. Aliquots were removed (0 and 60 min), fractionated by SDS-PAGE, transferred to membranes, and screened with HRP-conjugated α-FH antiserum. Ab binding was detected using ABTS substrate. (B) T. denticola 35405 cells were incubated with FH and 1% α-FhbB antiserum (as indicated). After incubation for 0 or 60 min, FH degradation was assessed as in panel A. Molecular mass standards are indicated to the left of each panel in kilodaltons.

FIG 5

Dentilisin-generated FH cleavage products lack FH cofactor activity. (A) Schematic depicting the CCP organization of FH. The location of potential chymotrypsin cleavage sites (as identified using PEPTIDE cutter) are indicated below the schematic. The location of the sole dentilisin cleavage motif (P92/F93) is indicated above the schematic. The complement regulatory domain (CCP1-4) is shaded. The primary interaction site for FhbB (CCP7) is indicated. (B) Purified recombinant CCP1-4 was incubated with no cells, strain 35405-CCE, or strain 35405, and aliquots were removed over time (0 to 30 min; 37°C) as indicated. The samples were solubilized, fractionated by SDS-PAGE, transferred to membranes, and screened with α-FH antiserum as detailed in Materials and Methods. (C) Competence of the dentilisin-generated FH cleavage products to serve as cofactor for FI-mediated C3b cleavage. Strains 35405 and 35405-CCE were incubated with or without FH (as indicated), the cells were pelleted by centrifugation, and the supernatants were collected and incubated with C3b with or without FI (as indicated). The samples were solubilized, fractionated by SDS-PAGE, immunoblotted, screened with α-C3 antiserum, and Ab binding was detected as detailed in the text. The location of the characteristic 39-kDa C3dg band that is normally generated upon cleavage by FI is indicated. Molecular mass standards are indicated in panels B and C in kilodaltons.