Vector-Borne Diseases: Molecular Mechanisms in Vector-Host Interactions

National Institute Of Allergy And Infectious Diseases

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Abstract

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 past fiscal year we have 1) Completed our analysis the cryo em structure of the alternative proconvertase C3bB with the salivary inhibitor lufaxin and the structure of lufaxin coordinately bound to C3bB and coagulation factor Xa. 2) Determined the cryo EM structure of albicin, a mosquito salivary inhibitor in complex with the alternative C3 convertase of complement and contiaining Factor Bb, which was missing from our previously determed structure. 3) Prepared and isolated complexes of the tick salivary protein with human factor VIIa and factor X. Also prepared and isolated complexes containing these components and human tissue factor. We are currently analyzing these using cryo EM single particle analysis. 4) Continued to completion a study with Dr. Valenzuela and Dr. Tiago Serafim identifying IgM as the serum component that promotes aggregation of Leishmania parasites in the sand fly gut and promotes genetic recombination. 5) Completed a project with Stephen Lu and Dr. Ribeiro to determine the crystal structure and function of a serotonin binding protein from flea saliva that is descended from an acid phosphatase enzyme. 6) Initiated studies of the proteomics of the hemolymph of Aedes aegypti with the goal of studying immune responses. 1) 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. We have completed the cryo EM analysis of the alternative C3 convertase in complex with the salivary inhibitor lufaxin, revealing a novel mechanism where the inhibitor stabilizes the complex but prevents a conformational change that would allow activation. Lufaxin has been shown to inhibit coagulation factor Xa in addition to the alternative pathway of complement. Over the past year we have shown that the inhibtor can simultaneously bind to both the alternative convertase and factor Xa thereby blocking both complement activation and coagulation. We have also determined the cryo EM structure of the ternary complex containing complement and coagulation components. We collaborated with Dr. Robert Brodsky of the Dept. of Hematology at Johns Hopkins and Ivo Francischetti of the Dept. of Pathology to investigate the efficacy of this inhibitor in models of complementopathies where thrombosis is often involved. This work was published during the last year. 2) Along with our studies of lufaxin, we have continued a study of the structure and mechanism of inhibitors of the alternative pathway of complement from Anopheles mosquitoes. We have shown previously that these proteins target the alternative C3 convertase complex, C3bBb. Previously, we have determined the structure of an inhibited complex containing the mosquito inhibitor, albicin. The inhibitory mechanism is reminiscent of that of SCIN, the C3bBb inhibitory protein from Staphylococcus bacteria but the two proteins are unrelated evolutionarily. Albicin promotes formation of a dimeric form of the complex that is incapable of cleaving C3. Formation of the complex requires the presence of factor Bb which is formed by reacting C3b bound factor B with the protease factor D. Our initial structures showed albicin induced dimerization of the convertase complex but factor Bb had been lost during the sample preparation process. This year, by controlling the time course of complex formation we obtained a structure containing bound factor Bb. This allowed us to determine precisely the binding interactions of albicin with factor Bb and the mechanism of stabilization of the complex that is observed in biochemical experiments. 3) The Tissue factor-factor VII complex (extrinsic factor Xase) of coagulation is key for the activation of hemostasis and inflammation. The complex has not been well characterized from a structural standpoint due to its stability and lipid requirements. Ixolaris is an inhibitor of the complex from tick saliva that binds with factor VIIa and factor Xa or factor X while the two are interacting with membrane bound tissue factor. I have formed complexes of these components in solution and have been able to isolate and analyze their compositions. The stabilization due to bridging by ixolaris may be sufficient to determine the structure of the complex in solution by cryo EM. After isolation of the complex and preparation of EM grids our initial data show a promising particle distribution. We are currently working to obtain enough data for a full reconstruction. 4) The study with Drs. Valenzuela and Serafim on the human serum factor that causes clumping of Leishmania parasites in the sand fly gut and in vitro has been completed. Using protein isolation techniques and bioassay we have found that IgM "natural antibodies" are solely responsible for the clumping effect. After establishing this, it was shown that IgM antibodies in the blood meal promote genetic recombination of the parasites in vivo and in vitro. A paper concerning this work in revision at this time. 5) Blood-feeding insects contain proteins that bind small-molecule agonists of hemostasis and inflammation. Serotonin is a ubiquitous target of these proteins but the proteins themselves come from different protein families. We found that the rat flea produces large quantities of proteins in the acid phosphatase family that have mutations in the active site that render them inactive. We further showed that the proteins scavenge serotonin, histamine and cysteinyl leukotrienes. The structure of one of these proteins was determined by x-ray crystallography and the ligand binding sites for both categories of agonist were identified. A paper concerning this work is currently under review. 6) With Patricia Alvarenga, I am determining the proteome of Aedes aegypti hemolymph with the goal of studying the interactions of component proteins in response to infection by pathogens. The first proteome of uninfected males and females is comple