Thrombin-derived C-terminal fragments aggregate and scavenge bacteria and their proinflammatory products

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.

The role of full-length apoE in clearance of Gram-negative bacteria and their endotoxins

“…We also noticed that the bacteria treated with apoE aggregated, suggesting that apoE may be involved in the aggregation and clearance of toxic amyloid-β in the brain of patients with AD ( 52) and in the killing of microorganisms. The link between aggregation and antimicrobial activity is further supported by our previously published work on the host defense actions of proteolysed thrombin and its fragments, based on LPS-induced aggregation/scavenging and microbial killing (21,25).…”

“…molecular weight of the protein (Da) / (number of amino acids). All the experiments were performed at least three times (21,25).…”

Cell-Free DNA Promotes Thrombin Autolysis and Generation of Thrombin-Derived C-Terminal Fragments

“…Here, we show that the γ-thrombin like TCP fragment generated by autolysis is further proteolyzed by neutrophil elastase to 11-kDa TCP and low molecular weight ~3-kDa TCP ( Figure 4A ), which are present in wound fluids. The aggregation prone 11-kDa TCP forms amyloid-like structures with bacterial LPS and gram-negative bacteria thereby aiding in containment and robust clearance of invading bacteria and bacterial products ( 11 , 13 ). As previously reported, the smaller TCPs (~3 kDa) exert anti-endotoxic as well as anti-inflammatory effects in vitro and in vivo by neutralizing LPS and blocking LPS induced pro-inflammatory cytokine response ( 12 , 36 , 37 ).…”

Concentration- and pH-Dependent Oligomerization of the Thrombin-Derived C-Terminal Peptide TCP-25

“…In particular, 10 and 300 μM TCP-25 dissolved in 10 mM Tris at pH 7.4 or in 10 mM NaOAc at pH 5 were analyzed. After dissolving, 5 μL of each sample were adsorbed onto carbon-coated grids (Copper mesh, 400) for 60 s and stained with 7 μL of 2% uranyl acetate for 30 s. The grids were rendered hydrophilic via glow discharge at low air pressure [ 23 ]. Analysis was done on 10 view fields (magnification 4200×) of the mounted samples on the grid (pitch 62 μm) from three independent experiments.…”