Neglectin, a salivary lipocalin-like from Rhodnius neglectus, scavenges pro-hemostatic and immune mediators to facilitate blood feeding

Triatomines, blood-feeding insects, are vectors of Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi. Rhodnius neglectus, native to the Brazilian Savanna, plays an important role in the sylvatic maintenance of T. cruzi. Triatomine saliva contains a diverse array of active biomolecules that counteract host hemostasis to facilitate blood feeding and parasite transmission. Survey of R. neglectus sialotranscriptome revealed a lipocalin-like similar to Triabin, a serine-protease inhibitor with a calycin fold known to form a non-covalent complex with thrombin. In this work, we characterized the biochemical and physiological functions of R. neglectus Triabin (Neglectin). Neglectin was expressed in mammalian cells (expi293) and purified by affinity and size-exclusion chromatography. Interestingly, Neglectin failed to inhibit blood clotting suggesting that it may have evolved a different salivary function in R. neglectus. Lipocalin-like proteins from triatomine species are known to bind to small molecule agonists of hemostasis. Hence, we used isothermal titration calorimetry and surface plasmon resonance to screen potential ligands for Neglectin. We found that Neglectin selectively binds to leukotrienes B4, C4, D4 and E4, as well as arachidonic acid with nanomolar affinities. Leukotrienes are released by mast cells and act quickly to induce pain, swelling, itching, and erythema. Neglectin also binds to platelet-activating factor, which is an ether phospholipid and acts as an agonist of platelet aggregation, and it is also involved in inflammatory and allergic reactions. Scavenging pro-hemostatic and immune mediators at the bite site suggests that Neglectin plays an important role in triatomine blood feeding.