Abstract
Thrombin-derived C-terminal peptides (TCPs), including a major 11-kDa fragment (TCP96), are produced through cleavage by human neutrophil elastase and aggregate lipopolysaccharide (LPS) and the Gram-negative bacterium Escherichia coli. However, the physiological roles of TCP96 in controlling bacterial infections and reducing LPS-induced inflammation are unclear. Here, using various biophysical methods, in silico molecular modeling, microbiological and cellular assays, and animal models, we examined the structural features and functional roles of recombinant TCP96 (rTCP96) in the aggregation of multiple bacteria and the Toll-like receptor (TLR) agonists they produce. We found that rTCP96 aggregates both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa, and their cell-wall components LPS, lipid A, and lipoteichoic acid (LTA). The Gram-negative bacteria E. coli and P. aeruginosa were particularly sensitive to aggregation-induced bacterial permeabilization and killing. As a proof of concept, we show that rTCP96 reduces LPS-induced NF-κB activation in human monocytes, as well as in mouse models of LPS-induced subcutaneous inflammation. Moreover, in a mouse model of subcutaneous inoculation with P. aeruginosa, rTCP96 reduced bacterial levels. Together, these results link TCP-mediated aggregation of endotoxins and bacteria in vitro to attenuation of inflammation and bacterial levels in vivo.
Highlights
Thrombin-derived C-terminal peptides (TCPs), including a major 11-kDa fragment (TCP96), are produced through cleavage by human neutrophil elastase and aggregate lipopolysaccharide (LPS) and the Gram-negative bacterium Escherichia coli
The results showed that recombinant TCP96 (rTCP96) reduced the levels of the Gram-negative strains E. coli and P. aeruginosa by 100 –1000 – fold, whereas the reduction of the Gram-positive S. aureus and E. faecalis was, albeit statistically significant, less marked (Fig. 2)
Compatible with the results using the live/dead assay (Fig. 3A and Fig. S1) were observed (Fig. S2). These results show that rTCP96 can induce aggregation and permeabilization of various Gram-negative and Gram-positive bacteria, leading to bacterial killing
Summary
TCP96 represents an HNE-generated fragment, which is nine amino acids shorter (from the N terminus) than the B4 chain of ␥-thrombin (Fig. 1) [15]. We measured a significant increase of -sheet structure/aggregation by detecting thioflavin T1 (ThT) fluorescence in the samples of rTCP96 (5 M), which were treated with LPS (E. coli) in the concentration range from 10 to 500 g/ml in Tris buffer, pH 7.4 (Fig. 4B). We performed the ThT assay to determine the increase of -sheet structure in rTCP96 treated with the TLR ligands LPS (E. coli), LPS (P. aeruginosa), lipid A (E. coli), LTA (S. aureus), and PGN (S. aureus). A significant increase of hydrodynamic radius, compatible with the observed interactions between rTCP96 and the TLR ligands, was detected in the presence of LPS (E. coli), LPS (P. aeruginosa), lipid A (E. coli), and LTA (S. aureus) but not in the presence of PGN (S. aureus) (Fig. 4D). An increase of -sheet structure of rTCP96 was detected by CD measurements in the samples incubated with the two LPS forms, lipid A, and LTA (S. aureus). No significant reduction of S. aureus was observed (Fig. 8, D and E)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
