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

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

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 2020 fiscal year we have 1) Completed our initial study of the role of Aedes aegypti hemolymph juvenile hormone binding protein in regulating hemocyte development and antibacterial immunity. 2) Determined the structure of LJL143, a protein inhibitor of the alternative pathway of complement in the sand fly in complex with the C3bB proconvertase complex of the complement pathway. We determined the crystal structure of the inhibitor and docked it into the complex. 3) Completed structural and functional analysis of inhibitors of the alternative pathway of complement from saliva of mosquitoes. 4) Continued our characterization of potent inhibitor of thrombin from saliva of the rat flea including in vivo studies and determination of the crystal structure of the thrombin-inhibitor complex. 5) Completed publication of a collaborative study with Dr. Long and her PhD student Erin Coonahan on the characterization of DNA aptamers selected to bind antimalarial drugs. 6) Continued a study in collaboration with Dr. Valenzuela and Dr. Tiago Serafim on a substance that promotes aggregation of Leishmania parasites in the sand fly gut. 7) Began a collaboration with Dr. Ackerman on eNOS-globin. 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. After describing effects of this protein on innate immunity of mosquitoes, we are trying to understand its effects on gene expression and any binding partners that may be involved. The study has also been expanded to another member of the same gene family that is found in Aedes aegypti. We have found that like the juvenile hormone binding protein, this protein is present in the hemolymph. The protein has been shown to tightly bind serotonin leading us to hypothesize that it is involved in serotonin function in the hemolymph. Among other things, circulating serotonin is involve in hemocyte maturation and the ability of hemocytes to phagocytose particles. 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. This protein is unusual in that it binds the C3bB proconvertase complex and prevents its activation by factor D. We have isolated the inhibited complex and determined its structure using cryo electron microscopy in collaboration with Dr. Natalia de Val at the Center for Molecular Microscopy in Frederick MD. We were able to place all components of C3bB at about 4.5 angstroms resolution. To complete this structural analysis we had to determine the X-ray crystal structure of the novel inhibitor lufaxin. This year we completed the structure and were able to accurately dock it into the cryo em map of inhibited C3bB. We have demonstrated formation of this complex in solution using gel filtration chromatography and surface plasmon resonance. The protein was found to bind to both C3b and factor B components, and through induction of conformational changes, stabilize their interaction while preventing activation of the enzymatic activity of the complex. We are completing this project by determining the cryo em structure of the lufaxin-inhibited cobra venom factor-factor B complex, hopefully at higher resolution. 3) 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. We have determined their structures and found that they represent a novel mosquito protein family. We have completed this study and this manuscript has been published in the Journal of Biological Chemistry 4)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 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. The manuscript has been submitted. 5) I have collaborated with Dr. Long and Erin Coonahan to use surface plasmon resonance to analyze the binding affinity of the antimalarial drugs piperaquine and mefloquine for DNA aptamers selected by Erin with the goal of developing a practical detection system for these in the blood. The manuscript on this project was published this year in Science Translational Medicine 6) I have collaborated with Dr. Valenzuela and Dr. Serafim to isolate a component of mammalian plasma that promotes aggregation of Leishmania in the sand fly gut. We have identified this component and we are now identifying its molecular target on the Leishmania cell. 7) I am collaborating with Dr. Ackerman in a study to characterize physically the binding of globins with the endothelial nitric oxid