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. 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 year we have 1) Completed single particle cryo EM analysis of complexes of the tick salivary inhibitor ixolaris with human tissue factor, coagulation factor VIIa and coagulation factor X. This is being done in collaboration with Ivo Francischetti. The structure shows a unique binding mechanism for a two-headed Kunitz inhibitor, explains the substrate selectivity difference between ixolaris and endogenous tissue factor pathway inhibitor and provides an experimental structure of the tissue factor-factor VIIa-factor X coagulation complex. 2) Completed a study with Patricia Alvarenga on the discovery of a hemolymph serotonin binding protein from the mosquito Aedes aegypti. 3) Continued study to determine the mechanism of a blackfly complement inhibitor, sicpin, with Eric Calvo. 4) Completed a study showing that binding of platelet factor-4 antibody complexes with the platelet collagen receptor GPVI activate platelets and may play a role in heparin-induced thromobcytopenia. 1) 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. We have determined a structure for the complex that contains all four components. Using a combination of this structure and structure prediction with Alphafold 3 we have generated a model which shows the factor X binding site, the factor VIIa binding site and how the host coagulation components are arranged in 3D space. This work was done in collaboration with Ivo Francischetti and a manuscript on this is in preparation. 2) With Patricia Alvarenga and Jose Ribeiro, I have completed a study on the discovery of a hemolymph serotonin binding protein from the mosquito Aedes aegypti. This is a relative of the salivary protein known as D7 which modulates host hemostatic responses by binding serotonin. We have shown that this hemolymph binds serotonin with high affinity but does not bind other biogenic amines. It is most closely related to a protein from Anopheles mosquitoes which also binds serotonin. We also have collected evidence that this Anopheles protein does not occur in the saliva. The study shows that serotonin binding by this group of mosquito proteins may function in any number of endogenous functions including immunity and that these hemolymph proteins may be evolutionary progenitors of serotonin binding proteins in saliva. A manuscript on this work is in preparation. 3) With Eric Calvo I am performing some mechanistic studies of a complement inhibitor from the black fly. Sicpin, a salivary inhibitor blocks both the classical and alternative pathways and targets the complement component C3. We have verified using gel filtration chromatography that a complex containing C3 and sicpin is formed. The target for the inhibitor has been verified as C3 and not C3b or C3(H2o) A manuscript on this work is in preparation. 4) With Ivo Francischetti I have completed a study showing that binding of platelet factor-4 antibody complexes with the platelet collagen receptor GPVI activates platelets and may play a role in heparin-induced thromobcytopenia (HIT). We have used a combination of cellular assays and measurements of protein-protein interactions to show that the complex of platelet factor 4 and a monoclonal antibody that is known to induce HIT in animal models causes platelets to aggregate and binds to surfaces of the extracellular domain of glycoprotein VI (GPVI). PF4-mAb complexes are known to induce HIT but the targets of these complexes are not well understood. This work has been published in the Journal of Thombosis and Hemostasis.