Activation of Toll-like receptors by a secretory bacterial protease to induce an inflammatory response

Abstract

Vibrio vulnificus (V. vulnificus) is a pathogenic bacterium that is associated with septicemia and serious wound infection in susceptible individuals. Most of the fatal cases are caused by septic shock, which is resulted from various virulence factors including capsular polysaccharide, lipopolysaccharide and haemolysin secreted by the bacterium. A metalloprotease secreted by V. vulnificus has many biological activities and seems to induce vascular permeability and hemorrhagic reactions in animal models. In this laboratory, a broad-specificity extracellular metalloprotease designated to as vEP has been purified and characterized from V. vulnificus ATCC29307. vEP is capable of cleaving a variety of plasma proteins which are associated with blood clotting and also proteolytically activating prothrombin to active thrombin (Chang et al., 2005). The protease can be inhibited by its own N-terminal propeptide (Chang et al., 2007). In this study, the entire vEP-coding region was obtained by polymerase chain reaction (PCR), based on the sequence of vEP gene, cloned into a periplasmic-directed expression vector (pFLAG-ATS), and expressed in E. coli. The active recombinant vEP was purified from the periplasmic proteins of the transformant E. coli cells. The purified vEP enzyme appeared both as 45- and 35-kDa forms on SDS-polyacryamide gel. It has been shown that the intact 45-kDa protease could be autoprocessed to make 35-kDa in size by the removal of its C-terminal region. Two mutant proteases designated as DC99 and G202D were also expressed. In addition, the properties of C-terminal region were studied by comparing with those of the wild type vEP enzyme. One mutant protease DC99, which has a deletion of the C-terminal 99 amino acids from vEP showed a similar level of specific activity compared with that of wild type vEP. Another mutant protease G202D that has a substitution of glycine202 to aspartate200 exhibited approximately 58% level of specific activity, compared that of wild type vEP. The vEP-derived peptides (C-ter100 and N-ter139) were also purified and characterized. In this study, an additional role of vEP was also examined in terms of an inflammation-associated cytokine production in macrophage Raw 264.7 cells. vEP could mediate the production of pro-inflammatory cytokines such as TNF-a and IL-1b, in which NF-kB signaling pathway was activated through the degradation of IkB, as judged by ELISA, RT-PCR, real-time PCR, Western blotting, EMSA, and supershift assay. Among Toll-like receptors (TLRs), TLR2 and TLR4 could be activated by vEP, as judged by RT-PCR, immunoprecipitation, western blotting, and confocal imaging analysis. The results showed that vEP could induce an inflammatory response by activating TLR2 and TLR4, leading the translocation of NF-kB proteins into nucleus to activate the target genes including TNF-a gene. Increase in NO production and Cox-2 expression by vEP further supported the fact that vEP can act as an inflammatory inducer. In addition, a deletion mutant enzyme (called DC99) of vEP deficient in C-terminal 99 amino acids failed to show those kinds of responses at all. However, C-ter100 only was sufficient to produce TNF-a, suggesting that the C-domain of vEP may play a critical role in the induction of inflammatory response by activating the Toll-like receptors. It was also observed by conforcal imaging analysis that vEP could bind directly to TLR2 and TLR4 receptors through by its C-domain region. Taken together, the results obtained by the present study suggest that the production of extracellular protease(s) from the bacterium Vibrio can disturb blood homeostasis as well as induce an inflammation during the bacterial infection.