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 2010 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) finalized the study of a antiinflammatory-antiplatelt salivary protein from Anopheles gambiae saliva 4) began work on the expression of a T cell polarizing factor from the eggs of the parasite Schistosoma mansoni. 1) We continue our work on the crystallization of salivary proteins in the laboratory and are continuing to make data-collection visits to the Advanced Photon Source synchrotron facility at Argonne Natl. Laboratory as well as collecting data remotely from the lab in Rockville. We have produced recombinant protein, crystallized and determined the structure of two new proteins over the last year and have determined additional structures of another protein to evaluate various ligand complexes. We are also currently analyzing diffraction data on an additional two novel proteins. Proteins in the """"""""yellow"""""""" gene family are abundant in sand fly saliva and are important candidates for saliva-based leishmaniasis vaccines. In the saliva, they serve as antiinflammatory mediators and mediators of vascular tone. We have determined the X-ray crystal structure of this protein using single anomalous dispersion techniques, and have also determined the structure of the protein in complex with tryptamine, an analogg of serotonin. In a continuation of structural work begun last year, we determined the structure of the """"""""long-form"""""""" D7 protein from the malaria vector Anopheles stephensi in complex with an analog of thromboxane A2. This protein is a potent platelet aggregation inhibitor that acts by binding thromboxane A2 and prevents its interaction with platelet receptors. This is the first known structure of a protein binding a thomoboxane or thromboxane analog. Finally, we have determined the structure of a novel vasodilator from the saliva of Simuliium vittatum, a vector of filarial diseases. This protein has a structure unlike any known vasodilator, and probably acts by a novel mechanism. We are now working on a number of new crystallization projects including a platelet aggregation inhibitor from a horse fly that target integrin alphaIIb beta3, as well as a member of the D7 protein family from a Culex mosquito whose function is unknown. 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 dubosqi in a recombinant system. These proteins have been tested by Dr. Valenzuela's group as vaccine antigens in Rhesus monkeys. 3) After our isolation of the Th2 T-cell polarizing factor from the eggs of Schistosoma mansoni described in the last annual report, we are working on the recominant expression of an active form of this protein.
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