Vibrio vulnificus secretes a broad-specificity metalloprotease capable of interfering with blood homeostasis through prothrombin activation and fibrinolysis

Abstract

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to the consumption of raw seafood. It secretes a metalloprotease that is associated with skin lesions and serious hemorrhagic complications. In this study, we purified and characterized an extracellular metalloprotease (designated as vEP) having prothrombin activation and fibrinolytic activities from V. vulnificus ATCC 29307. vEP could cleave various blood clotting-associated proteins such as prothrombin, plasminogen, fibrinogen, and factor Xa, and the cleavage could be stimulated by addition of 1 mM Mn2+ in the reaction. The cleavage of prothrombin produced active thrombin capable of converting fibrinogen to fibrin. The formation of active thrombin appeared to be transient, with further cleavage resulting in a loss of activity. The cleavage of plasminogen, however, did not produce an active plasmin. vEP could cleave all three major chains of fibrinogen without forming a clot. It could cleave fibrin polymer formed by thrombin as well as the cross-linked fibrin formed by factor XIIIa. In addition, vEP could also cleave plasma proteins such as bovine serum albumin and gamma globulin, and its broad specificity is reflected in the cleavage sites, which include Asp207-Phe208 and Thr272-Ala273 bonds in prothrombin and a Tyr80-Leu81 bond in plasminogen. Taken together, the data suggest that vEP is a broad-specificity protease that could function as a prothrombin activator and a fibrinolytic enzyme to interfere with blood homeostasis as part of the mechanism associated with the pathogenicity of V. vulnificus in humans and thereby facilitate the development of systemic infection.

FIG. 1.

SDS-PAGE analysis of purified vEP. vEP resolved from the Superdex 75 100/300 GL column was electrophoresed on 10% gel under reducing conditions. An arrow indicates the presence of the 48-kDa band, which has the same N terminus as the 36-kDa band.

FIG. 2.

Effect of temperatures on vEP activity. (A) vEP was incubated at various temperatures for 20 min, and the residual activity toward azocasein was measured as described in Materials and Methods. Enzyme activity from each sample was expressed as a percentage relative to that of sample incubated at 37°C. (B) SDS-PAGE analysis of vEP incubated at different temperatures in the absence or presence of 0.5% SDS or 1 mM Ni²⁺.

FIG. 3.

SDS-PAGE showing the substrate specificity of vEP. Corresponding proteins (10 μg each) as indicated were digested with 0.3 μg vEP in the presence or absence of 1 mM Mn²⁺ or Zn² and then separated on 12% gel. Prothrombin (A) was digested for 5 min at room temperature. Fibrinogen (B) was digested for 20 min at room temperature. Plasminogen (A), factor Xa (B), and BSA and gamma globulin (C) were digested for 60 min at 37°C. P1, P2, and P3 indicate fragments from which the N-terminal sequences have been determined.

FIG. 4.

Activation of prothrombin by vEP. (A) Thrombin activity was measured with thrombin-specific chromogenic substrate. Prothrombin (PT; 0.4 mg/ml) was activated by vEP (4 μg/ml) at room temperature, aliquots were withdrawn at different time intervals, and the reaction was stopped by the addition of NiCl₂ to 1 mM. The cleavage products were assayed for thrombin activity in the presence of 1 mM NiCl₂ and 0.4 mM of Boc-VPR-pNA at 37°C. (B) Thrombin activity was assayed by measuring the degree of fibrin formation. Prothrombin activated by vEP for 10 min was mixed with fibrinogen (0.5 mg/ml) in the presence of 1 mM NiCl₂, and the formation of fibrin polymer was monitored as an increase in turbidity at 350 nm. For controls, the reaction mixture contained only prothrombin (20 μg/ml) or vEP (0.2 μg/ml). Representative curves from each reaction performed two times in triplicate are shown.

FIG. 5.

(A) Effect of antithrombin III on the activity of vEP-activated prothrombin. Prothrombin (0.4 mg/ml) was activated by vEP (4 μg/ml) at room temperature for 10 min, and the reaction was terminated by addition of 1 mM NiCl₂. Aliquots were then assayed with Boc-VPR-pNA (0.4 mM) as a substrate in the presence or absence of antithrombin III. Data are the means ± standard errors from two separate experiments performed in triplicates. (B) Western blot analysis of vEP-activated prothrombin. vEP-activated prothrombin was subjected to SDS-PAGE under nonreducing conditions. The proteins were transferred to PVDF membrane and probed with antibody against thrombin. Lane 1, prothrombin; lane 2, thrombin; lane 3, vEP-activated prothrombin; lane 4, vEP only.

FIG. 6.

Fibrinolytic activity of vEP. (A) Fibrinolytic activity was assayed on fibrin plate. Samples were applied to the wells in the plate and allowed to incubate at room temperature overnight. (B) Fibrinolytic activity was measured by a decrease in the turbidity of fibrin polymer. Enzymes were applied as spots onto the fibrin polymer (catalyzed by thrombin with fibrinogen as substrate) and allowed to incubate at room temperature for 30 min. The decrease in turbidity was then measured at 350 nm. The data are the means ± standard error from two separate experiments performed in triplicate.

FIG. 7.

Cleavage of α- and γ-chain cross-links of fibrin by vEP, as analyzed by SDS-PAGE. Polymerization of fibrin was initiated by the addition of fibrinogen (1.3 mg/ml), thrombin (1.3 U/ml), and factor XIIIa (0.13 U/ml) and 1 mM CaCl₂ followed by incubation at room temperature for 1 h. The cross-linked fibrin was then digested with vEP (25 μg/ml) at room temperature for 30 min followed by SDS-PAGE analysis using 8% gel.