J. Biol. Chem. 2020 295: 3417-. doi:10.1074/jbc.RA120.012741
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.
This work was supported by grants from the Swedish Research Council (projects 2012-1883 and 2017-02341), the Welander-Finsen, Crafoord, Österlund, Johanssons, and Söderberg Foundations, the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation, The Swedish Government Funds for Clinical Research (ALF), and the medical faculty of Lund University. We thank Ann-Charlotte Strömdahl and Christina Diersing for excellent technical assistance with the THP-1 cell line. We acknowledge the Lund
University Bioimaging Centrum (LBIC) for access to confocal and electron microscopy facilities.