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. 2021 Aug 18;10(8):798.
doi: 10.3390/biology10080798.

Effect of Vibrio-Derived Extracellular Protease vEP-45 on the Blood Complement System

So Hyun Kwon  1 , Jung Eun Park  1   2 , Yeong Hee Cho  1   2 , Jung Sup Lee  1   2
Affiliations

Effect of Vibrio-Derived Extracellular Protease vEP-45 on the Blood Complement System

So Hyun Kwon et al. Biology (Basel). .

Abstract

Vibrio vulnificus is a pathogenic bacterium that can causes wound infections and fetal septicemia. We have reported that V. vulnificus ATCC29307 produces an extracellular zinc-metalloprotease (named vEP-45). Our previous results showed that vEP-45 can convert prothrombin to active thrombin and also activate the plasma kallikrein/kinin system. In this study, the effect of vEP-45 on the activation of the complement system was examined. We found that vEP-45 could proteolytically convert the key complement precursor molecules, including C3, C4, and C5, to their corresponding active forms (e.g., C3a, C3b, C4a, C4b, and C5a) in vitro cleavage assays. C5b production from C5 cleavage mediated by vEP-45 was not observed, whereas the level of C5a was increased in a dose-dependent manner compared to that of the non-treated control. The cleavage of the complement proteins in human plasma by vEP-45 was also confirmed via Western blotting. Furthermore, vEP-45 could convert C3 and C5 to active C3a and C5a as a proinflammatory mediator, while no cleavage of C4 was observed. These results suggest that vEP-45 can activate the complement system involved in innate immunity through an alternative pathway.

Keywords: C3a; C5a; Vibrio vulnificus; complement system; innate immunity; vEP-45 protease.

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Conflict of interest statement

The authors have declared no conflict of interest.

Figures

Figure 1
Figure 1
Cleavage of human complement proteins (C3, C4, and C5) by vEP-45 as determined by SDS-PAGE. (A,D,G) Human complement protein C3, C4, or C5 (each 5 µg) was incubated with vEP-45 (0.5, 2, 10, or 50 ng) for 10 min at 37 °C. Proteins from each sample were separated via SDS-PAGE and stained with Coomassie brilliant blue. The histograms show the production of (B) C3b α′ chain, (C) C3a, (E) C4b α′ chain, (F) C4a, (H) C5b α′ chain, and (I) C5a upon treatment with different concentrations of vEP-45, in which the values obtained using the control (Con; only C3b, C3a, C4b, C4a, C5b, and C5a) were considered as 100%. The uncropped SDS-polyacrylamide gel images can be found in Figure S1.
Figure 2
Figure 2
Cleavage of human complement proteins by vEP-45 as detected with Western blotting. (A,D,G) Human complement protein C3 (1 µg), C4 (0.5 µg), or C5 (0.5 µg) was incubated with vEP-45 (10, 25, 50, 100, 200, or 300 ng) for 3 min at 37 °C. Proteins from each sample were separated by SDS-PAGE; Western blotting was performed using anti-C3 antibody (A upper panel), anti-C3a antibody (A lower panel), anti-C4 antibody (D upper panel), anti-C4a antibody (D lower panel), anti-C5 antibody (G upper panel), or anti-C5a antibody (G lower panel). The histograms show the production of (B) C3b α′ chain, (C) C3a, (E) C4b α′ chain, (F) C4a, (H) C5b α′ chain, and (I) C5a upon treatment with different concentrations of vEP-45, in which the values obtained using control (Con; only C3b, C3a, C4b, C4a, C5b, and C5a) were regarded as 100%. The uncropped Western blot images can be found in Figure S2.
Figure 3
Figure 3
Cleavage of C3 and C5 by vEP-45 in human plasma. (A,D) Samples of human blood plasma (10%) were incubated with vEP-45 (0.5, 1, or 2 µg) for 3 min at 37 °C. Proteins from each sample were separated by SDS-PAGE; Western blotting was performed using anti-C3 antibody (A upper panel), anti-C3a antibody (A lower panel), anti-C5 antibody (D upper panel), or anti-C5a antibody (D lower panel). The histograms show the production of (B) C3b α′ chain, (C) C3a, (E) C5b α′ chain, and (F) C5a upon treatment with different concentrations of vEP-45, in which the values obtained from a control (Con; only C3b, C3a, C5b, and C5a) were considered as 100%. The uncropped Western Blot images can be found in Figure S3.
Figure 4
Figure 4
Effects of vEP-45 on the complement system during Vibrio infection.
See this image and copyright information in PMC

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