Streptococcus mutans surface alpha-enolase binds salivary mucin MG2 and human plasminogen

Abstract

Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis identified enolase as a cell surface component of Streptococcus mutans, which was confirmed by enzyme-linked immunosorbent assay, Western blotting, and transmission electron microscopy. Surface enolase was demonstrated to bind to human plasminogen and salivary mucin MG2. The results suggested a role for enolase in S. mutans attachment, clearance, or breach of the bloodstream barrier.

FIG. 1.

Plasminogen binding to S. mutans enolase. (A) Electrophoretically separated cell surface, cell wall, and cytoplasmic samples were incubated with rabbit anti-enolase antibody (Ab) (1:300) and anti-rabbit IgG-peroxidase (1:500). (B) Separated surface protein, cell wall, and cytoplasm samples were incubated with plasminogen (0.02 mg/ml), goat anti-plasminogen, and anti-goat IgG-peroxidase (1:500). (C) Surface protein, cell wall, and cytoplasm samples were incubated with blocking rabbit anti-enolase antibody (1:300) and centrifuged. The supernatants of those samples were isolated by SDS-PAGE and transferred to a PVDF membrane, which was incubated with plasminogen (0.02 mg/ml), goat anti-plasminogen, and anti-goat IgG-peroxidase (1:500). The binding activity of enolase with plasminogen was decreased by blocking rabbit anti-enolase antibody.

FIG. 2.

Enolase binding to salivary mucin MG2. (A) Saliva supernatant was separated by SDS-PAGE and transferred to a PVDF membrane. The left lane contained a salivary protein of about 180 kb, which interacted with purified enolase (0.05 mg/ml), rabbit anti-enolase antibody (Ab) (1:200), and anti-rabbit IgG conjugated with peroxidase (1:500). The right lane contained a salivary protein of about 180 kb, which interacted with rabbit anti-mucin MG2 (1:1,000) and anti-rabbit IgG conjugated with peroxidase (1:500). (B) Subcellular S. mutans samples were separated by SDS-PAGE and transferred to a PVDF membrane. Enolase (47 kDa) in surface protein, cell wall, cytoplasm, and whole-cell lysate samples was incubated with saliva supernatant (as a source of mucin MG2), rabbit anti-mucin MG2 antibody (1:1,000), and anti-rabbit IgG-peroxidase (1:500).

FIG. 3.

Localization of enolase in the subcellular fractions of S. mutans by ELISA. ELISA plates were coated with surface protein (SP), cell wall (CW), cytoplasm (Cyto), and cultural supernatant (CS) samples and incubated with rabbit anti-α-enolase or anti-γ-enolase antibody (1:500) and peroxidase-conjugated anti-rabbit IgG antibody (1:1,000). Following color development, the absorbance of each well was measured at 490 nm. The data were analyzed by Student's t test (independent samples with equal variances and two sides), and the P values of all groups were <0.05.

FIG. 4.

Ability of S. mutans enolase to bind to plasminogen and salivary mucins MG1 and MG2. The ability of enolase in surface protein (SP), cell wall (CW), and cytoplasm (Cyto) samples to bind to human plasminogen was detected by ELISA. An ELISA plate was coated with rabbit anti-enolase antibody (1:500) and incubated with sequentially subcellular samples of plasminogen (Pla; 0.02 mg/ml), goat anti-plasminogen antibody (1:1,000), and anti-goat IgG conjugated with peroxidase (1:1,000). In a similar experiment, proteins in the subcellular samples able to bind to human salivary mucins MG1 and MG2 were detected by ELISA. An ELISA plate was coated with subcellular samples (surface protein, cell wall, and cytoplasm samples), incubated sequentially with saliva supernatant (containing salivary mucins MG1 and MG2), rabbit anti-mucin MG1 (1:1,000) or anti-mucin MG2 antibodies (1:1,000), and anti-rabbit IgG conjugated with peroxidase (1:1,000). Following color development, the absorbance of each well was measured at 490 nm. The data were analyzed by Student's t test (independent samples with equal variances and two sides), and the P values of all groups were <0.05.