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 2016 fiscal year we have 1) Identified a putative salivary protein ancestor that circulates in the blood of mosquitoes and binds juvenile hormone, a hormone essential for development and reproduction, 2) Determined the molecular mechanism of action of albicin, an anticomplement protein from the malaria vector An. albimanus, and published this work. 3) Studied the mechanism of LJL143 a second complement inhibitor from the sand fly in collaboration with Dr. Valenzuela's lab. 4)Finished structural studies of an antiinflammatory protein from vector saliva that sequesters cysteinyl leukotriene compounds and published this work. 5) Completed a collaboration with Dr. Tovi Lehmann (NIAID) on analysis of cuticular hydrocarbons of Anopheles mosquitoes. 6) Continued a collaboration with Dr. Xueqing Xu (Southern Medical University, Guangzhou, China)to study the sodium channel blocking capabilities of a salivary protein from the rat flea, a vector of bubonic plague. 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 endoD7, 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. Moreover, the protein is found in adult males and females. Analysis of endoD7 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. We have crystallized the protein, collected diffraction data and obtained a molecular replacement solution for the N-terminal domain of the protein. I am in the process of using this solution to determine the entire structure. We are also continuing to study the binding properties of the protein and to evaluate the effect gene knockdown using double stranded RNA methods. 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 found that saliva of the malaria vector Anopheles albimanus contains albicin, a protein that inhibits activation of the classical pathway of complement. Over the past year, we completed this study by determining the molecular target of albicin and its mechanism of action. Using surface plasmon resonance and enzymatic assays we found that albicin acts by binding to the activated alternative C3 convertase complex. The protein does not bind well to the individual components, but binds with high affinity to the complex of C3b and factor Bb. Albicin stabilizes the structure of this complex and appears to induce oligomerization of it while completely blocking its activity. Blockade of this type could be useful for treatment of complement related diseases such as paroxysmal nocturnal hemoglobinuria. We have published this work in the Journal of Immunology. 3) In a project related to topic 2, I have worked to determine the target and mechanism of action of LJL143, a salivary inhibitor of the alternative pathway of complement from the sand fly Lutzomyia longipalpis. This inhibitor is bifunctional, having previously been shown to inhibit the activity of coagulation factor Xa. Using surface plasmon resonance and enzymatic assays, I found that LJL143 binds selectively to the unactivated form of the C3 convertase. When bound to the C3bB complex, LJL143 prevents the cleavage of factor B to factor Bb by factor D. While albicin inhibits the activity of the mature C3 convertase, LJL143 prevents activation of the immature form. 4) In the salivary gland of the Chagas' disease vector Rhodnius prolixus, the lipocalin protein family has been expanded and variant forms have been found that bind a variety of procoagulant and proinflammatory ligands. Over the past year we have finished the characterization and structure determination of a leukotriene binding lipocalin LTBP1. The structures revealed that the protein binds cysteinyl leukotrienes in a large binding pocket and stabilized the ligands through a conformational change mechanism. The work demonstrates that the vector uses multiple members of the lipocalin family to scavenge proinflammatory and prohemostatic ligands at the feeding site. We have published this work in ACS Chemical Biology. 5) I have completed my portion of a collaborative project with Dr. Tovi Lehmann of the LMVR involving the measurement of cuticular hydrocarbon content in field collected Anopheles coluzzii mosquitoes.
The aim of the study was to determine the importance of different factors including hydrocarbons in dessication resistance of the mosquitoes and their ability to survive conditions in the dry season Mali. This work was published in the Journal of Experimental Biology. 6) One likely function of the salivary secretions of blood feeders is to limit the sensation of pain in the host. With this in mind I have started a collaborative project with researchers in China to determine the activity of salivary components in modulating the function of various ion channels in nervous tissue. We have begun investigating a family of salivary proteins from the rat flea that are similar to toxins from other invertebrates. We have identified one small protein that acts as a blocker of the sodium channel NaV1.5. We have determined its structure and are characterizing its activity.
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