The purpose of this research is to investigate the molecular mechanisms of action of biologically active proteins from arthropod disease vectors and pathogenic microorganisms. We use biological and physical techniques to characterize and understand the modes of action of pharmacologically active components from the saliva of blood-feeding vector insects and ticks, as well as immunomodulatory components secreted by parasitic organisms such as Toxoplasma and Schistosoma. Proteins and small molecules found in the saliva of vectors inhibit the host hemostatic responses and are essential for the successful completion of a blood meal. Most vector borne diseases are transmitted during feeding, so elucidation of the physiology and biochemistry of this process is necessary for understanding disease transmission. Saliva has also been shown to have pronounced effects on host inflammatory and immune responses which persist after feeding and can dramatically alter the environment for the pathogen after transmission. Determining the specific role of salivary molecules in these processes is essential for the understanding their importance to pathogen survival after transmission Over the past several years we have identified the functions of numerous salivary molecules involved primarily in overcoming host hemostatic defenses. The raw material for these studies comes from the analyses of salivary transcriptomes produced in collaboration with Dr. Jose Ribeiro. Bioinformatic analysis of sequence data is used to predict function of salivary proteins. Candidate proteins are then expressed in bacterial or eukaryotic cell systems. The proteins are purified and assayed using a variety of methods. Functionally characterized proteins are then produced in larger quantity for structural and other biophysical studies. During the 2015 fiscal year we have 1) determined the structures of a new salivary protein and applied structural information to determine the mechanism of action of this protein, 2) Published work along with Dr. Valenzuela's lab on recombinant proteins contained in experimental leishmaniasis vaccines 3) Published the structure an antigenic protein produced by the hookworm. 4)Published a study of an inhibitor of the coagulation cascade protease thrombin that is derived from tick saliva. 5) Continued studies on an antiinflammatory binding protein from the saliva of Rhodnius prolixus with Willy Jablonka, a post doc fellow in Dr. Ribeiro's laboratory and determined the structures of a number of ligand complexes that were essential for elucidation of its mechanism. 6) Identified and characterized a complement inhibitor from the saliva new world Anopheles mosquitoes. 1) We continue our work on the crystallization of salivary proteins in the laboratory and now almost exclusively use remote data collection from the SER-CAT beamlines at Argonne National Laboratory for collection of diffraction data. We have produced recombinant protein, crystallized and determined the structure of a novel salivary protein from sand fly saliva over the last year. We are also currently analyzing diffraction data on several additional novel proteins. 2) Salivary components of vector sand flies have been shown to be useful as potential leishmaniasis vaccine components based on their ability to induce delayed hypersensitivity responses in host skin. As part of a vaccine development project directed by Jesus Valenzuela, I determined the structure of a salivary vaccine antigen which was included in a paper by Oliveira et al. in Science Translational Medicine. 3)The hookworm secretes a number of proteins belonging to the CAP superfamily that have not been functionally characterized, but are considered to be potential vaccine candidates for this organism. We obtained crystals, and I determined the X-ray crystal structure of one of these known as the hookworm platelet inhibitor. This resulted in a publication in Acta Crystallographica Section F. 4) Inhibitors of thrombin are important for blood feeding and are used as therapeutic anticoagulants. Along with Dr. Ribeiro and Willy Jablonka I identified a salivary peptide from the tick Hyalomma marginatum that inhibits thrombin at concentrations near 10 nanomolar. We have characterized the binding of the peptide, showed that it is cleavable by thrombin, and dissected its sequence in order to identify the essential sequences for binding, and the probable binding sites on thrombin. Recently, we published a paper on this work in Plos One. 5)I have produced recombinant nitrophorin 2, an inhibitor of coagulation factor IXa for a study to evaluate the importance of microparticle activation of the contact pathway in the propensity to thrombosis in patients with various hematological disorders. This work was in collaboration with Mikhail Panteleev and was published in Plos One. 6)Proteins in the lipocalin protein family are widely distributed in the saliva of disease vectors, but they are not functionally well characterized. I have determined the structure of a novel lipocalin from Rhodnius prolixus that we have shown binds cysteinyl leukotrienes. The structure of the protein-ligand complex shows large conformational changes and cleavage of the ligand. Ligand geometry suggests that the cleavage product is stabilized as carbocation. Crystallographic observation of this highly reactive ion bound to a protein is quite surprising, and possibly unprecedented. 7) I completed a study of an anti-complement protein in the saliva of Anopheles albinanus, a new-world vector of Plasmodium. Complement activation in response to feeding is well demonstrated, and is a known to make the ingestion of blood difficult. We find that old world anophelines do not do not have anticomplement activity in the saliva. We have isolated this protein using chromatographic methods, identified it and produced recombinant protein. The protein is highly active in inhibiting the alternative pathway of complement and could have implications for the susceptibility of plasmodium insect stages to complement and differences in susceptibility in the new and old world Anopheles species
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