A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation

Abstract

Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secreted effector as I ricinus serpin-2 (IRS-2), and we show that it primarily inhibits cathepsin G and chymase, while in higher molar excess, it affects thrombin activity as well. The inhibitory specificity was explained using the crystal structure, determined at a resolution of 1.8 Å. Moreover, we disclosed the ability of IRS-2 to inhibit cathepsin G-induced and thrombin-induced platelet aggregation. For the first time, an ectoparasite protein is shown to exhibit such pharmacological effects and target specificity. The stringent specificity and biological activities of IRS-2 combined with the knowledge of its structure can be the basis for the development of future pharmaceutical applications.

The Tick Salivary Protein Sialostatin L2 Inhibits Caspase-1-Mediated Inflammation during Anaplasma phagocytophilum Infection

“…Enzymatic assays were previously described (37,38). Briefly, recombinant sialostatin L2 and peptides were preincubated with cathepsin L (EMD Millipore) for 10 min before the addition of the corresponding substrates.…”

Ixodes ricinus Salivary Serpin IRS-2 Affects Th17 Differentiation via Inhibition of the Interleukin-6/STAT-3 Signaling Pathway

“…From this enumeration, it is apparent that tick serpins can be expected to play a role in tick feeding, suppressing both the antihemostatic and immune responses of the host. To date, only two I. ricinus serpins have been functionally characterized (12,14).…”

“…The expressed protein accumulated in inclusion bodies, which were separated. Refolded and concentrated IRS-2 was purified using a standard chromatographic method (fast protein liquid chromatography [FPLC]) (14,24). Lipopolysaccharide (LPS) contamination was removed by Arvys Proteins Company using the detergent-based method.…”

“…Both enzymes are part of the acute inflammatory response and are produced by activated neutrophils (cathepsin G) and mast cells (chymase). Moreover, IRS-2 is able to inhibit swelling and the migration of neutrophils into the inflamed tissue (14). The effects of IRS-2 on other cells of innate and acquired immunity have not been described so far.…”

Ixodes scapularistick saliva proteins sequentially secreted every 24h during blood feeding

“…For instance serpin EEC19556.1 in SCA (Fig 3B1) is 98% identical to AID54718.1, an inhibitor of trypsin and thrombin that also inhibited blood clotting and platelet aggregation [43]. Similarly I. ricinus serpin ABI94056, the homolog of I. scapularis serpin EEC14235.1 in this study (Fig 3B1 SCB) is an immunosuppressant, anti-inflammatory, and anti-hemostatic serpin [78][79][80]. In other studies, I. scapularis cystatin AAY66685.1 in this study (Fig 3B4 CCA) known as Sialostatin L2 and its close relative Sialostatin L have immuno-modulatory functions, and suppressed cytokine production in absence [81][82][83][84][85] or presence of B. burgdorferi [86].…”