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.We have recently collaborated with Dr. Jesus Valenzuela to characterize During the 2012 fiscal year we have 1) determined the structures of two new salivary proteins and applied structural information to determine the mechanism of action of these proteins, 2) continued to produce recombinant proteins for use in an experimental saliva-based leishmaniasis vaccine, 3) published the structure and salivary function of a novel antiinflammatory salivary protein from a blood-feeding fly 4)Characterized an inhibitor of the contact activation pathway of coagulation from the sand fly, 5) Completed a collaboration with Louis Miller to determine molecular interactions in the Plasmodium invasion process. 6) Produced recombinant tick salivary proteins in a collaboration with Dr. Michalis Kotsyfakis of the Czech Academy of Sciences on the study of Anaplasma infection 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 two new proteins 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 continued to produce salivary antigens from the saliva of Phlebotomous duboscqi in a recombinant system. 3)Proteins in the antigen-V protein family are widely distributed in the saliva of disease vectors, but they are not functionally well characterized. We have published a structural analysis and functionaal characterization of a leukotriene-binding member of this family from the blood feeding fly, Tabanus yao. The protein was also known to inhibits platelet aggregation by binding with the fibrinogen receptor integrin alphaIIbbetaIII. 4) 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. 5) I have collaborated with Louis Miller and members of his laboratory in a study of cellular protein-protein interactions of cytoplasmic domains of merozoite surface proteins and their roles in red blood cell invasion. We have completed a study using surface plasmon resonance to determine interaction of cytoplasmic domain peptides with the cellular proteins aldolase and glyceraldehyde-3-phosphate dehydrogenase.
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