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 2018 fiscal year we have 1) Studied the role of Aedes aegypti hemolymph juvenile hormone binding protein in antimicrobial immunity. 2) Completed the study of the molecular mechanism of action of LJL143, an inhibitor of the alternative pathway of complement in the sand fly. 3) Continued structural and functional studies of mosquito salivary complement inhibitor. We have determined the crystal structures of the sg7 proteins from Anopheles freeborni and Anopheles stephensi, and shown that these are inhibitors of the alternative C3 convertase. A role for properdin in this inhibition was also established. 4)Continued our efforts to determine structure of the albicin-C3bBb complex by cryo electron microscopy. Began a study to determine the cryo EM structure of the alternative proconvertase C3bB in complex with LJL143. 5)Obtained diffraction quality crystals of LJL143 and began efforts at solving the X-ray crystal structure 6) In collaboration with Dr. Valenzuela and Dr. Tiago Serafim began a project to isolate an aggregation factor for Leishmania parasites from mammalian plasma. 1) Over the past decade we have shown that the D7 protein family in mosquito saliva functions by sequestering host-produced mediators of hemostasis and inflammation. Among these are the eicosanoids thromboxane A2 and leukotriene C4. Since these mediators are not known to function in the mosquito, the D7 proteins must be derived from ancestors with different function. I have identified mJHBP, a protein that shows sequence conservation between various genera of mosquitoes and is similar to salivary D7s but has changes in amino acid residues important for binding of vertebrate eicosanoid ligands. Analysis of various tissues and life stages showed that this protein is expressed in the fat body and directed to the blood of the insect. Analysis of mJHBP ligand binding using calorimetry showed that the protein binds the important insect hormone, juvenile hormone, with high selectivity. This hormone is essential for metamorphosis, egg development and male mating behavior. In collaboration with Zach Adelman and Eric Calvo we found that the protein is essential for haemocyte development and normal anti-microbial immunity. Adult females of an mJHBP CRISPR knockout show deficiencies in haemocyte proliferation and phagocytosis. This leads to an inability to control bacterial infections and a delay in the expression of antimicrobial peptides. Injection of wild-type protein reverses this phenotype and results normal ability to control bacterial infection. 2) Inhibition of the complement cascade is an important feature of saliva from blood feeding vectors. Activation of the complement system in host blood can result in the destruction of insect tissues and production of proinflammatory anaphylatoxins. In collaboration with Dr. Valenzuela, we have identified lufaxin, an inhibitor of the alternative pathway of complement in the saliva of the sand fly Lutzomyia longipalpis. Using surface plasmon resonance and reconstituted enzymatic systems, we have found that lufaxin specifically binds to the C3bB complex, and prevents its activation (cleavage) to C3bBb by factor D. It appears that the protein recognizes an open conformation of factor B that is recognized by the factor D protease. Inhibitors that bind to C3bBb, the mature form of the C3 convertase, have been described previously, but this is the first inhibitor described that specifically blocks the formation of the convertase. During this fiscal year we completed this study and published the results in Frontiers in Immunology. 3) We have determined the crystal structure of gsg7 proteins from Anopheles freeborni and An. stephensi. This inhibitor of the alternative pathway of complement binds to the activated C3 convertase, C3bBb. These structures will allow us to understand the molecular determinants of anti complement activity. We also found that variation in affinity of the inhibitor for the complex between species can reveal a requirement for properdin in the inhibitory mechanism. An freeborni and An stephensi inhibitors bind more weakly to the complex than the inhibitor from An albimanus. With the more weakly binding forms, additional stabilization by properdin greatly enhances the inhibitory activity. 4) In order to understand the mechanism of albicin, the complement inhibitor from An albimanus we are working to determine its structure in complex with C3bBb using cryo EM. I a collaboration with Dr. Natalia de Val from the FNLCR we have purified inhibited complexes and have obtained preliminary negative stained data that look very promising. We are now waiting to analyze these samples on the Krios Titan 300 kV electron microscope under cryo conditions. In a parallel study, we have also purified the inhibited complex of C3bB with LJL143 and are treating it in a similar manner. In this case we have also obtained promising negative stained images and are waiting for our spot for cryo analysis. 5) Related to section 4, we are working to determine the X-ray crystal structure of LJL143, which will then be used as part of the cryo EM structure of the inhibited C3bB complex. Purification of the protein from HEK 293 cell supernatants yielded over 10 mg /L of protein which produced crystals that diffracted beyond 2.5 angstroms resolution. The data are well behaved and the space group has been determined. We are currently working on methods to generate experimental phases. 6) Finally, I have begun a collaboration with Dr. Valenzuela and Dr. Serafim to isolate a component of mammalian plasma that promotes aggregation of Leishmania and appears to be of key importance in maintenance of its life cycle.
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