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 of a potent antithrombin peptide from the flea. 2) Improved resolution and finalized analysis of the cryo em structure of the alternative proconvertase C3bB with the salivary inhibitor lufaxin. 3) Determined the structure of lufaxin coordinately bound to C3bB and coagulation factor Xa. 4) Determined the cryo EM structure of albicin, a mosquito salivary inhibitor in complex with the alternative C3 convertase of complement. 5) Completed a study of the structural and functional variability of salivary D7 proteins among the subgenera of Anopheles mosquitoes and continued work on the structure and function of a hemolymph D7-like protein that binds serotonin. 6) Completed 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. 7) Determined with Stephen Lu and Dr. Ribeiro the crystal structure and function of a serotonin binding protein from flea saliva that is descended from an acid phosphatase enzyme. 8) Continued a collaboration with Dr. Ackerman on eNOS-globin interactions. 1) Vector insects must inhibit the coagulation cascade in order to feed normally. From the transcriptome of the rat flea salivary gland, we have identified a potent inhibitor of thrombin (XC43) that binds to the enzyme with a Ki value of less than 10 picomolar. We are have completed our characterization of this peptide, along with a crystal structure showing that despite binding in a substrate-like manner, this peptide is extremely resistant to cleavage by the protease. Normally, this level of resistance includes stabilization of a larger inhibitory protein with a number of disulfide bonds. XC43 appears to be stabilized primarily through interactions with subsites surrounding the cleavage site thereby preventing product release. This work was published in the Journal of Biological Chemistry 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. 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. 3) 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 are collaborating 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. 4) Along with our studies of lufaxin, we have completed 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. Over the past year, 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. We have also performed experiments using gel filtration chromatography and surface plasmon resonance to further characterize the mechanism. 5) With Patricia Alvarenga and Dr. Ribeiro, I have completed a project to examine the structural diversity of the D7 protein family in Anopheles mosquitoes. We previously had found that the major long form D7 proteins from An. stephensi had lost the ability to bind serotonin that we see in Ae. aegypti. Here we show that this a feature only of the subgenus Cellia. In the subgenera Nyssorhynchus and Anopheles biogenic amines are bound by these proteins suggesting that this is the ancestral condition. This work has been published in Insect Biochemistry and Molecular Biology 6) 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. This work has been published in pre print form in BioRXiv. 7) 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. 8) I am collaborating with Dr. Ackerman in a study to characterize physically the binding of globins with the endothelial nitric oxide synthase