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 2009 fiscal year we have 1) determined the structures of four new salivary proteins and applied structural information to determine the mechanism of action of these proteins, 2) produced recombinant proteins for use in an experimental saliva-based leishmaniasis vaccine, 3) determined the mechanism of an antiinflammatory salivary protein from Anopheles gambiae saliva 4) finished the identification and characterization of a T cell polarizing factor from the eggs of the parasite Schistosoma mansoni. 1) We are now regularly crystallizing proteins in the laboratory and are making data-collection visits to the Advanced Photon Source synchrotron facility at Argonne Natl. Laboratory. We have produced recombinant protein, crystallized and determined the structures of a four new proteins over the last year and have determined additional structures of these proteins to evaluate various ligand complexes. The biogenic amine-binding protein from the Chagas'disease vector Rhodnius prolixus inhibits host infammatory responses and platelet aggregation by binding the biogenic amines serotonin and norepinephrine. We have determined the X-ray crystal structure of this protein using multiple anomalous dispersion techniques as well as the structure of the protein in complex with tryptamine, an analog of serotonin. We have also determined the structure of the """"""""long-form"""""""" D7 protein from the malaria vector Anopheles stephensi. Structurally, the unique binding specificity of this protein apppears to relate to alterations in its C-terminal domain relative to the long D7 of Aedes aegypti. Finally, we have determined the structures of two members of the cystatin protein family, sialostatin and sialostatin 2, from the saliva of the Lyme disease vector Ixodes scapularis. These proteins have been found to profoundly affect the feeding success of ticks and to have potential imoportance as vaccine components. 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 produced three antigens from the saliva of Phlebotomous dubosqi in a recombinant system. These proteins will be tested for there ability to protect against infecttion in a pilot study in Rhesus monkeys. 3) Like the long D7 from Aedes aegypti, the D7 protein of Anopheles stephensi protein binds leukotrienes, but unlike the A. aegypti protein it does not bind biogenic amines. The A. stephensi protein also has the unique function of inhbiting platelet aggregation induced by the eicosanoid thromboxane A2. We have characterized this activity using a variety of thromboxane A2 receptor agonists and antagonists in platelet aggregation and ligand binding assays. 4) It was noted a number of years ago, that extracts of Schistosoma mansoni eggs induce polarization of CD4+ T cells toward a Th2 phenotype. The mechanism of Th2 polarization is not well understood and no specific factor inducing this differentiation has been isolated .In collaboration with Alan Sher and Dragana Jankovic of the Lab. of Parasitic Diseases, we have fractionated supernatants of egg cultures, and isolated an apparent single protein component, ribonuclease omega-1, which causes this effect. We are now working to understand the mechanism of action of this protein by developing a system for producing active recombinant protein. If successful, we will be able to use mutagenesis and protein chemistry to evaluate the importance of ribonuclease activity and glycosylation in the function of this agent.
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