Leptospiral endostatin-like protein A is a bacterial cell surface receptor for human plasminogen

Abstract

The spirochete Leptospira interrogans is a highly invasive pathogen of worldwide public health importance. Studies from our laboratories and another have demonstrated that L. interrogans can acquire host plasminogen on its surface. Exogenous plasminogen activators can then convert bound plasminogen into the functionally active protease plasmin. In this study, we extend upon those observations and report that leptospiral endostatin-like protein A (LenA) binds human plasminogen in a dose-dependent manner. LenA-plasminogen interactions were significantly inhibited by the lysine analog xi-aminocaproic acid, suggesting that the lysine-binding sites on the amino-terminal kringle portion of the plasminogen molecule play a role in the binding. Previous studies have shown that LenA also binds complement regulator factor H and the extracellular matrix component laminin. Plasminogen competed with both factor H and laminin for binding to LenA, which suggests overlapping ligand-binding sites on the bacterial receptor. Finally, LenA-bound plasminogen could be converted to plasmin, which in turn degraded fibrinogen, suggesting that acquisition of host-derived plasmin by LenA may aid bacterial dissemination throughout host tissues.

FIG. 1.

Leptospira interrogans serovar Pomona strain JEN4 binds and processes plasminogen on its surface. (A) Intact leptospires preincubated with either plasminogen (PLG; 10 μg/ml), plasminogen (10 μg/ml) plus tranexamic acid (TA; 100 mM), or BSA alone were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with a monoclonal antibody to plasminogen (1:500; catalog no. sc-73708). (B) Intact leptospires were incubated with plasminogen (10 μg/ml) in the absence or presence of 100 mM tranexamic acid for 2 h at 30°C and washed twice, and the pellet was reconstituted in the reaction buffer. Fifty microliters of resuspended leptospires was transferred to each microwell, and plasminogen activator (uPA; 4 ng/well) was added. The plasmin activity was measured using a chromogenic substrate, d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride (Sub), and the optical density (OD) was measured at 405 nm. Plasmin conversion was inhibited by tranexamic acid or when uPA was not added to the reaction buffer.

FIG. 2.

LenA binds plasminogen. (A) ELISA results showing binding of immobilized LenA (10 μg/ml) to soluble plasminogen (10 μg/ml). Bound plasminogen was detected by sequential addition of an antiplasminogen (1:2,500; catalog no. NB600-930), HRP-conjugated protein G (1:5,000), and 3,3′,5,5′-tetramethyl benzidine substrate solution. The optical density was measured at 450 nm. Gelatin was used as a negative control. (B) ELISA results showing binding of increasing concentrations of LenA to immobilized plasminogen (10 μg/ml). Bound LenA was detected by a LenA-specific antiserum (1:250), followed by HRP-conjugated protein G and the substrate, as described for panel A. Results are representative of at least two independent experiments with 3 or more repeats. P values were calculated by Student's t test, assuming unequal variances.

FIG. 3.

Role of lysine residues in LenA-plasminogen interactions. (A) Binding of LenA (10 μg/ml) to immobilized plasminogen (10 μg/ml) in the absence or presence of ξ-aminocaproic acid (EACA; 10 mM) was analyzed by ELISA. (B) ELISA results showing binding of graded concentrations of LenA to the immobilized lysine binding domain of plasminogen. In both panels A and B, bound LenA was detected by sequential addition of a LenA-specific antiserum (1:250), HRP-conjugated protein G (1:5,000), and 3,3′,5,5′-tetramethyl benzidine substrate solution. The optical density was measured at 450 nm. BSA was used as a negative control. Results are representative of at least two independent experiments with 3 or more repeats. P values were calculated by Student's t test, assuming unequal variances.

FIG. 4.

Ionic interactions in LenA binding to plasminogen. Role of ionic interactions in LenA binding to plasminogen was analyzed by ELISA. ELISA plate wells were coated with plasminogen (10 μg/ml) and incubated with LenA (10 μg/ml) in the presence of increasing concentrations of NaCl (A) or heparin (B). In both panels A and B, bound LenA was detected using a LenA-specific antiserum (1:250), followed by incubation with HRP-conjugated protein G (1:5,000). Plates were developed using 3,3′,5,5′-tetramethyl benzidine substrate solution, and the optical density was measured at 450 nm. Results are representative of at least two independent experiments with 3 or more repeats. BSA was used as a negative control.

FIG. 5.

Plasminogen competes with factor H for binding to LenA. ELISA plate wells were coated with LenA (10 μg/ml) and incubated with plasminogen (0.125 μM) in the absence or presence of 0.185 μM (1:1.5), 0.25 μM (1:2), or 0.5 μM (1:4) factor H. (A) Plasminogen binding to LenA was detected by antiplasminogen (1:2,500; catalog no. NB600-930). (B) Factor H binding to immobilized LenA was detected using anti-factor H (1:2,500; catalog no. 341276). In both panels A and B, HRP-conjugated protein G (1:5,000) was used as the secondary antibody. Plates were developed with 3,3′,5,5′-tetramethyl benzidine substrate solution. The optical density was measured at 450 nm. An asterisk indicates a P value of less than 0.05.

FIG. 6.

Plasminogen and laminin compete for binding to LenA. ELISA plate wells were coated with LenA (10 μg/ml) and incubated with plasminogen (0.125 μM) in the absence or presence of 10 μg/ml, 20 μg/ml, or 40 μg/ml of laminin. (A) Plasminogen binding to LenA was detected by antiplasminogen (1:2,500; catalog no. NB600-930). (B) Laminin binding to immobilized LenA was detected using antilaminin (1:2,500; L9393). In both panels A and B, HRP-conjugated protein G (1:5,000) was used as the secondary antibody. Plates were developed with 3,3′,5,5′-tetramethyl benzidine substrate solution. The optical density was measured at 450 nm. An asterisk indicates a P value of less than 0.05.

FIG. 7.

LenA-bound plasminogen is processed to functionally active plasmin. (A) LenA (10 μg/ml), immobilized to microtiter plate wells, was incubated with plasminogen. After extensive washing, uPA was added and the plasmin activity was measured using a chromogenic substrate, d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. The optical density was measured at 405 nm. Presented data are representative of at least two independent experiments with 3 or more repeats. (B) LenA (10 μg/ml), immobilized to microtiter plate wells, was incubated with plasminogen (10 μg/ml). Subsequently, fibrinogen and uPA were added and incubated at 37°C for 24 h. The reaction mixtures were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a rabbit polyclonal fibrinogen antibody.