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. Over this same period we have collaborated with Dr. Alan Sher's laboratory to characterize a number of pathogen-produced proteins involved in immune responses to infection. These projects included: The isolation of a T cell antigen from a Helicobacter species that is involved in the induction of colitis in a mouse model, the characterization of a chemokine receptor ligand from Toxoplasma which was evaluated for potential as an anti-retroviral agent, the isolation of a toll-like receptor ligand from Toxoplasma, and the isolation of an apparent T cell polarizing factor from the eggs of Schistosoma. During the 2014 fiscal year we have 1) determined the structures of a new salivary protein and applied structural information to determine the mechanism of action of these proteins, 2) Continued to work with recombinant proteins contained in experimental leishmaniasis vaccines 3) Published the structure and function of a salivary protein that inhibits the hemostatic and inflammatory effects of the contact pathway of coagulation. 4)Characterized an inhibitor of the coagulation cascade protease thrombin that is derived from tick saliva. 5) Collaborated with Mikhail Panteleev of the Federal Research and Clinical Center of Pediatric Hematology, Immunology and Oncology, Moscow. The research used an inhibitor of FIXa from insect saliva to demonstrate the importance of microparticle activation of the contact pathway in many hematological disorders. 6) 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. 7) Initiated a study on the characterization and identification of 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 two proteins and determined the structure of one of them 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 have begun working on the crystallization and function of a potential antigen from Lutzomyia longipalpis 3)We have characterized members of the """"""""SP-15"""""""" protein family from Phlebotomous duboscqi as inhibitors of the """"""""contact"""""""" pathway of coagulation. These are major proteins in the saliva that act by binding to glycosaminoglycans secreted from mast cells in the skin. These carbohydrates serve as an activating matrix for coagulation factor XII. Inihbition of factor XII activation prevents the production of bradykinin in the skin thereby limiting inflammation in the area of the insect bite. In addition to identifying the mode of action of these inhibitors, we have determined the crystal structures of two forms and identified structural possible structural determinants for these activities. We have also shown that the protein blocks contact pathway activation by polyphosphate, a key endogenous activator. During the past year we published this work in Arteriosclerosis, Thrombosis and Vascular Biology. 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. 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 7) I initiated 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 are in the process of isolating and identifying this factor. The project is being performed with Antonio Sousa, a visiting student from Brazil.
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