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

媒介传播疾病：媒介-宿主相互作用的分子机制

• 批准号: 10014130
• 负责人: Jose Ribeiro
• 金额: $ 49.38万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014130
• 关键词: Adult; Aedes; Affect; Affinity; Agonist; Alternative Complement Pathway; Amino Acids; Anaphylatoxins; Anti-Bacterial Agents; Anticoagulants; Antimalarials; Arthropods; Bacterial Infections; Binding; Binding Proteins; Biochemistry; Bioinformatics; Biological; Biological Assay; Biological Metamorphosis; Blood; Calorimetry; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Coagulation Process; Collaborations; Collagen; Complement; Complement Activation; Complex; Cryoelectron Microscopy; Crystallization; Culicidae; DNA; Data; Detection; Development; Disease Vectors; EPHA3 gene; Eicosanoids; Environment; Enzymes; Eukaryotic Cell; Fat Body; Female; Fleas; Genetic Processes; Genetic Recombination; Goals; Hemocytes; Hemolymph; Hemorrhage; Hemostatic Agents; Hemostatic function; Hormones; Immune response; Immunity; Impairment; Inflammation; Inflammatory Response; Injections; Insect Hormones; Insect Vectors; Insecta; Juvenile Hormones; Knock-out; Leishmania; Leukotriene C4; Life; Ligand Binding; Ligands; Lutzomyia genus; Mediator of activation protein; Mefloquine; Methodology; Methods; Molecular; Molecular Mechanisms of Action; Organism; Parasites; Pathogenicity; Peptides; Phagocytosis; Pharmacologic Actions; Pharmacologic Substance; Phenotype; Phlebotomus; Physiology; Plasma; Platelet Activation; Platelet Aggregation Inhibitors; Preparation; Process; Production; Protein Engineering; Protein Family; Proteins; Publishing; Rattus; Recombinant Proteins; Reporting; Research; Roentgen Rays; Role; Saliva; Salivary; Salivary Glands; Salivary Proteins; Sand Flies; Schistosoma; Sequence Analysis; Serum; Site; Structural Protein; Structure; Surface Plasmon Resonance; System; Taxonomy; Techniques; Testing; Thrombin; Thromboxane A2; Ticks; Tissues; Toxoplasma; Vector-transmitted infectious disease; Work; antimicrobial; antimicrobial peptide; aptamer; assay development; biophysical analysis; complement system; disease transmission; doctoral student; egg; feeding; hormone binding protein; immunoregulation; inhibitor/antagonist; male; mating behavior; microorganism; mutant; pathogen; protein purification; protein structure; response; saliva function; small molecule; stem; transcriptome; transmission process; vector

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 2019 fiscal year we have 1) Continued our initial study of the role of Aedes aegypti hemolymph juvenile hormone binding protein in regulating hemocyte development and antibacterial immunity. 2) Continued the structural analysis of LJL143, a protein inhibitor of the alternative pathway of complement in the sand fly. We have been working on solution of the crystal structure of the inhibitor and the cryo EM structure of the complex of LJL143 with the C3bB proconvertase complex. 3) Completed structural and functional analysis of a platelet aggregation inhibitor protein from the saliva of Phlebotomus sand flies. 4) Identified an extremely potent inhibitor of thrombin from saliva of the rat flea. 5) Collaborated with Dr. Long and her PhD student Erin Coonahan in the characterization of DNA aptamers selected to bind antimalarial drugs. 6) In collaboration with Dr. Valenzuela and Dr. Tiago Serafim isolated a blood serum constituent that promotes aggregation of Leishmania parasites in the sand fly gut and may act to promote recombination. 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. In collaboration with Zach Adelman and Eric Calvo we found that the protein is essential for haemocyte development and normal anti-microbial immunity. Adult females of an mJHBP CRISPR knockout show deficiencies in haemocyte proliferation and phagocytosis. This leads to an inability to control bacterial infections and a delay in the expression of antimicrobial peptides. Injection of wild-type protein reverses this phenotype and results normal ability to control bacterial infection. We have further shown that ligand binding is essential for activity of the protein by designing a panel of site-directed mutants in which the binding of juvenile hormone is impaired or completely eliminated. Injection of these mutant proteins into knockout mosquitoes showed that only proteins with hormone binding capability were able to restore normal hemocyte development and immune responses to the deficient line. 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. To continue this we are working to determine the X-ray crystal structure of LJL143, which will then be used to determine the cryo EM structure of the inhibited C3bB complex. Purification of the protein from HEK 293 cell supernatants yielded over 10 mg /L of protein which produced diffracting crystals. In collaboration with Dr. Natalia De Val at NCI Frederick I have been working to determine the cryo-EM structure of the inhibited C3bB complex. We have determined the structure of the C3bB complex and are working to collect usable data from preparations including the inhibitor. 3) In order to feed normally, blood-feeding insects need to inhibit the hemostatic system of the host. Platelet activation is an early hemostatic response to wounding that acts to stem the loss of blood. Wounding by vector mouthparts leads to platelet activation through contact with exposed collagen, leading to release of secondary agonists which amplify the activation response. We found that D7 proteins from Phlebotomus sand flies bind the secondary agonist thromboxane A2 causing inhibition of activation. We also determined the crystal structure of this protein and explored its evolutionary relationships with related proteins in mosquito saliva. This work was published in Scientific Reports. 4)In addition to the inhibition of platelet activation, 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 inhibits the enzyme with a Ki value of less than 100 picomolar. Additionally, the inhibitor is a relatively small peptide of only 36 amino acids that may have pharmaceutical potential in addition to being the first salivary anticoagulant identified from the Siphonaptera, the taxonomic order of fleas. 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. 6)Finally, 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 and appears to be of key importance in the poorly understood process of genetic recombination in this important parasite.

本研究的目的是研究来自节肢动物疾病媒介和病原微生物的生物活性蛋白的分子作用机制。我们利用生物和物理技术来表征和了解来自吸血媒介昆虫和蜱唾液的药理活性成分的作用方式，以及弓形虫和血吸虫等寄生生物分泌的免疫调节成分。 载体唾液中发现的蛋白质和小分子会抑制宿主的止血反应，对于成功完成血粉至关重要。大多数媒介传播疾病是在喂养过程中传播的，因此阐明该过程的生理学和生物化学对于了解疾病传播是必要的。唾液还被证明对宿主炎症和免疫反应有显着影响，这些反应在进食后持续存在，并且可以在传播后显着改变病原体的环境。确定唾液分子在这些过程中的具体作用对于了解它们对传播后病原体存活的重要性至关重要 在过去的几年中，我们已经确定了许多唾液分子的功能，这些分子主要涉及克服宿主的止血防御。这些研究的原材料来自与 Jose Ribeiro 博士合作进行的唾液转录组分析。序列数据的生物信息分析用于预测唾液蛋白的功能。然后候选蛋白在细菌或真核细胞系统中表达。使用多种方法纯化和分析蛋白质。然后大量生产具有功能特征的蛋白质，用于结构和其他生物物理研究。 在2019财年，我们1)继续对埃及伊蚊血淋巴保幼激素结合蛋白在调节血细胞发育和抗菌免疫中的作用进行初步研究。 2)继续对白蛉补体旁路途径蛋白抑制剂LJL143进行结构分析。我们一直致力于解决抑制剂的晶体结构以及LJL143与C3bB原转化酶复合物的复合物的冷冻电镜结构。 3) 完成了白蛉唾液中血小板聚集抑制蛋白的结构和功能分析。 4) 从大鼠跳蚤唾液中鉴定出一种极其有效的凝血酶抑制剂。 5) 与 Long 博士和她的博士生 Erin Coonahan 合作，对选择用于结合抗疟药物的 DNA 适体进行表征。 6) 与 Valenzuela 博士和 Tiago Serafim 博士合作，分离出一种血清成分，该成分可促进白蛉肠道中利什曼原虫寄生虫的聚集，并可能促进重组。 1) 在过去的十年中，我们已经证明，蚊子唾液中的 D7 蛋白家族通过隔离宿主产生的止血和炎症介质来发挥作用。其中包括类二十烷酸血栓素 A2 和白三烯 C4。由于这些介质在蚊子中的功能尚不清楚，因此 D7 蛋白必定源自具有不同功能的祖先。我已经鉴定出 mJHBP，这种蛋白质在不同属的蚊子之间显示出序列保守性，与唾液 D7 相似，但在对脊椎动物类二十烷酸配体的结合很重要的氨基酸残基上发生了变化。对各种组织和生命阶段的分析表明，这种蛋白质在脂肪体中表达，并定向到昆虫的血液。使用量热法对 mJHBP 配体结合进行分析表明，该蛋白质以高选择性结合重要的昆虫激素、保幼激素。这种激素对于变态、卵子发育和雄性交配行为至关重要。通过与 Zach Adelman 和 Eric Calvo 的合作，我们发现该蛋白质对于血细胞发育和正常的抗微生物免疫至关重要。 mJHBP CRISPR 敲除的成年雌性显示出血细胞增殖和吞噬作用的缺陷。这导致无法控制细菌感染和抗菌肽表达的延迟。注射野生型蛋白可逆转这种表型，并产生控制细菌感染的正常能力。我们通过设计一组定点突变体进一步证明配体结合对于蛋白质的活性至关重要，其中保幼激素的结合被削弱或完全消除。将这些突变蛋白注射到基因敲除蚊子体内表明，只有具有激素结合能力的蛋白质才能恢复正常的血细胞发育和对缺陷系的免疫反应。 2) 补体级联的抑制是血液喂养载体唾液的一个重要特征。宿主血液中补体系统的激活可导致昆虫组织的破坏和促炎性过敏毒素的产生。我们与 Valenzuela 博士合作，在白蛉 Lutzomyia longipalpis 的唾液中发现了 lufaxin，一种补体替代途径的抑制剂。为了继续这一工作，我们正在努力确定 LJL143 的 X 射线晶体结构，然后将其用于确定受抑制的 C3bB 复合物的冷冻电镜结构。从 HEK 293 细胞上清液中纯化蛋白质，产生超过 10 mg/L 的蛋白质，并产生衍射晶体。我与 NCI Frederick 的 Natalia De Val 博士合作，一直致力于确定受抑制的 C3bB 复合物的冷冻电镜结构。我们已经确定了 C3bB 复合物的结构，并正在努力从包括抑制剂在内的制剂中收集可用数据。 3）吸血昆虫为了正常进食，需要抑制宿主的止血系统。血小板激活是对受伤的早期止血反应，可阻止失血。载体口器造成的伤害会通过与暴露的胶原蛋白接触而导致血小板活化，从而导致次级激动剂的释放，从而放大活化反应。我们发现来自白蛉的 D7 蛋白与二级激动剂血栓素 A2 结合，从而抑制激活。我们还确定了这种蛋白质的晶体结构，并探讨了其与蚊子唾液中相关蛋白质的进化关系。这项工作发表在《科学报告》上。 4）媒介昆虫除了抑制血小板活化外，还必须抑制凝血级联才能正常进食。从大鼠跳蚤唾液腺的转录组中，我们鉴定出了一种有效的凝血酶抑制剂，其抑制该酶的 Ki 值小于 100 皮摩尔。此外，该抑制剂是一种相对较小的肽，仅含 36 个氨基酸，除了是从跳蚤分类目 Siphonaptera 中鉴定出的第一种唾液抗凝剂之外，还可能具有药物潜力。 5) 我与Long博士和Erin Coonahan合作，利用表面等离子共振分析抗疟药物哌喹和甲氟喹与Erin选择的DNA适体的结合亲和力，目标是开发血液中这些物质的实用检测系统。 6）最后，我与 Valenzuela 博士和 Serafim 博士合作，分离出哺乳动物血浆中的一种成分，该成分可促进利什曼原虫在白蛉肠道中聚集，并且似乎在这一重要的基因重组过程中发挥着至关重要的作用。寄生虫。