Glyceraldehyde-3-phosphate dehydrogenase of Streptococcus pneumoniae is a surface-displayed plasminogen-binding protein

Abstract

The recruitment of plasminogen endows the bacterial cell surface of Streptococcus pneumoniae with proteolytic activity. In this study we demonstrate specific plasmin- and plasminogen-binding activity for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is located in the cytoplasm as well as on the surface of pneumococci. GAPDH exhibits a high affinity for plasmin and a significantly lower affinity for plasminogen.

FIG. 1.

Identification of the cellular distribution of the pneumococcal GAPDH. Immunoblot analysis, using anti-GAPDH antibodies, of the cell wall fraction (lane 1) and soluble fraction (lane 2) of S. pneumoniae R6x and of the soluble fraction of an S. pneumoniae eno internal deletion mutant expressing α-enolase with mutated plasminogen binding sites (lane 3).

FIG. 2.

Binding of pneumococcal GAPDH to plasminogen. Human plasminogen was immobilized in the different amounts indicated. GAPDH was purified under native conditions from the cytosolic compartment of the serotype 2 eno^int/del mutant pneumococci (ATCC 11733). Rows 1 to 3, binding of GAPDH applied in serial dilutions (1:2).

FIG. 3.

Immunoelectron microscopic visualization of GAPDH on the surface of S. pneumoniae ATCC 11733 (type 2) (A), unencapsulated R6x (B), and S. pneumoniae NCTC 10319 (type 35A) (C). (A to C) GAPDH was visualized on the bacterial cell surfaces of ultrathin sections of preembedding labeled samples by using anti-GAPDH antibodies and protein A-15-nm-diameter gold particle studies. (D) Visualization of unspecific binding of protein A-15-nm-diameter gold particles to type 2 pneumococci.

FIG. 4.

SPR measurements of GAPDH-plasminogen (A) and GAPDH-plasmin (B) interactions. Purified GAPDH protein was immobilized on a BIAcore CM5 sensor chip, and plasmin and plasminogen were used as the analyte. Binding kinetics and concentration dependence of binding of plasminogen and plasmin to GAPDH are shown as changes in plasmin resonance. Plasmin and plasminogen were used in concentrations of 500 (for plasminogen only), 250, 125, 62.5, 31.25, 15.625, 7.8, and 3.9 nM. The blank run was subtracted from the sensorgram. Pl and Plg, starts of injection; w, stop of injection; RU, relative units.