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: 7964621
• 负责人: Jose Ribeiro
• 金额: $ 60.87万
• 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/7964621
• 关键词: Aedes; Affect; Agonist; Anopheles Genus; Anopheles gambiae; Anti-Inflammatory Agents; Anti-Retroviral Agents; Antigens; Arthropods; Binding; Binding Proteins; Biochemistry; Biogenic Amines; Bioinformatics; Biological; Biological Assay; Black-legged Tick; Blood; C-terminal; CD4 Positive T Lymphocytes; Chagas Disease; Colitis; Collaborations; Complex; Cystatins; DIF factor; Data; Data Collection; Delayed Hypersensitivity; Disease Vectors; Eicosanoids; Environment; Eukaryotic Cell; Goals; Helicobacter; Hemostatic Agents; Immune response; Infection; Inflammatory; Insect Vectors; Laboratories; Leishmaniasis; Leukotrienes; Ligand Binding; Ligands; Lyme Disease; Macaca mulatta; Malaria; Methodology; Methods; Molecular; Molecular Mechanisms of Action; Mutagenesis; Norepinephrine; Organism; Parasites; Parasitic Diseases; Phenotype; Photons; Physiology; Pilot Projects; Platelet aggregation; Process; Production; Protein Binding; Protein Chemistry; Protein Family; Proteins; Recombinant Proteins; Recombinants; Relative (related person); Research; Rhodnius; Ribonucleases; Roentgen Rays; Role; Saliva; Salivary; Salivary Proteins; Sand Flies; Schistosoma; Schistosoma mansoni; Sequence Analysis; Serotonin; Skin; Source; Specificity; Structure; Synchrotrons; System; T-Lymphocyte; Techniques; Testing; Thromboxane A2; Thromboxane A2 Receptor; Ticks; Toll-like receptors; Toxoplasma; Vaccines; Vector-transmitted infectious disease; Visit; Work; assay development; base; chemokine receptor; disease transmission; egg; feeding; glycosylation; member; microorganism; mouse model; pathogen; protein structure; response; small molecule; success; transmission process; tryptamine analog; vaccine development; 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. Over this same period we have collaborated with Dr. Alan Sher's laboratory to characterize a number of pathogen-produced proteins involved in immune responses to infection. These projects included: The isolation of a T cell antigen from a Helicobacter species that is involved in the induction of colitis in a mouse model, the characterization of a chemokine receptor ligand from Toxoplasma which was evaluated for potential as an anti-retroviral agent, the isolation of a toll-like receptor ligand from Toxoplasma, and the isolation of an apparent T cell polarizing factor from the eggs of Schistosoma. During the 2009 fiscal year we have 1) determined the structures of four new salivary proteins and applied structural information to determine the mechanism of action of these proteins, 2) produced recombinant proteins for use in an experimental saliva-based leishmaniasis vaccine, 3) determined the mechanism of an antiinflammatory salivary protein from Anopheles gambiae saliva 4) finished the identification and characterization of a T cell polarizing factor from the eggs of the parasite Schistosoma mansoni. 1) We are now regularly crystallizing proteins in the laboratory and are making data-collection visits to the Advanced Photon Source synchrotron facility at Argonne Natl. Laboratory. We have produced recombinant protein, crystallized and determined the structures of a four new proteins over the last year and have determined additional structures of these proteins to evaluate various ligand complexes. The biogenic amine-binding protein from the Chagas' disease vector Rhodnius prolixus inhibits host infammatory responses and platelet aggregation by binding the biogenic amines serotonin and norepinephrine. We have determined the X-ray crystal structure of this protein using multiple anomalous dispersion techniques as well as the structure of the protein in complex with tryptamine, an analog of serotonin. We have also determined the structure of the "long-form" D7 protein from the malaria vector Anopheles stephensi. Structurally, the unique binding specificity of this protein apppears to relate to alterations in its C-terminal domain relative to the long D7 of Aedes aegypti. Finally, we have determined the structures of two members of the cystatin protein family, sialostatin and sialostatin 2, from the saliva of the Lyme disease vector Ixodes scapularis. These proteins have been found to profoundly affect the feeding success of ticks and to have potential imoportance as vaccine components. 2) Salivary components of vector sand flies have been shown to be useful as potential leishmaniasis vaccine components based on their ability to induce delayed hypersensitivity responses in host skin. As part of a vaccine development project directed by Jesus Valenzuela, I have produced three antigens from the saliva of Phlebotomous dubosqi in a recombinant system. These proteins will be tested for there ability to protect against infecttion in a pilot study in Rhesus monkeys. 3) Like the long D7 from Aedes aegypti, the D7 protein of Anopheles stephensi protein binds leukotrienes, but unlike the A. aegypti protein it does not bind biogenic amines. The A. stephensi protein also has the unique function of inhbiting platelet aggregation induced by the eicosanoid thromboxane A2. We have characterized this activity using a variety of thromboxane A2 receptor agonists and antagonists in platelet aggregation and ligand binding assays. 4) It was noted a number of years ago, that extracts of Schistosoma mansoni eggs induce polarization of CD4+ T cells toward a Th2 phenotype. The mechanism of Th2 polarization is not well understood and no specific factor inducing this differentiation has been isolated .In collaboration with Alan Sher and Dragana Jankovic of the Lab. of Parasitic Diseases, we have fractionated supernatants of egg cultures, and isolated an apparent single protein component, ribonuclease omega-1, which causes this effect. We are now working to understand the mechanism of action of this protein by developing a system for producing active recombinant protein. If successful, we will be able to use mutagenesis and protein chemistry to evaluate the importance of ribonuclease activity and glycosylation in the function of this agent.

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。 同一时期，我们与 Alan Sher 博士的实验室合作，鉴定了许多与感染免疫反应有关的病原体产生的蛋白质。这些项目包括：从参与小鼠模型结肠炎诱导的螺杆菌属物种中分离 T 细胞抗原，从弓形虫中鉴定趋化因子受体配体，评估其作为抗逆转录病毒药物的潜力，从弓形虫中分离出 Toll 样受体配体，并从血吸虫卵中分离出明显的 T 细胞极化因子。 在 2009 财年，我们 1) 确定了四种新唾液蛋白的结构，并应用结构信息来确定这些蛋白的作用机制，2) 生产了用于实验性唾液利什曼病疫苗的重组蛋白，3) 确定冈比亚按蚊唾液抗炎唾液蛋白的机制4)完成了曼氏血吸虫卵中T细胞极化因子的鉴定和表征。 1) 我们现在定期在实验室中结晶蛋白质，并对阿贡国家实验室的先进光子源同步加速器设施进行数据收集访问。实验室。去年，我们生产了重组蛋白，结晶并确定了四种新蛋白的结构，并确定了这些蛋白的其他结构以评估各种配体复合物。来自恰加斯病载体 Rhodnius prolixus 的生物胺结合蛋白通过结合生物胺血清素和去甲肾上腺素来抑制宿主炎症反应和血小板聚集。我们使用多种异常分散技术确定了该蛋白质的 X 射线晶体结构，以及该蛋白质与色胺（血清素类似物）复合物的结构。我们还确定了来自疟疾载体斯氏按蚊的“长型”D7 蛋白的结构。从结构上看，该蛋白独特的结合特异性似乎与其 C 端结构域相对于埃及伊蚊长 D7 的改变有关。最后，我们从莱姆病媒介肩胛硬蜱的唾液中确定了半胱氨酸蛋白酶抑制剂蛋白家族的两个成员，即唾液酸抑素和唾液酸抑素2的结构。人们发现这些蛋白质对蜱的摄食成功率有深远的影响，并且作为疫苗成分具有潜在的重要性。 2) 媒介白蛉的唾液成分已被证明可用作潜在的利什曼病疫苗成分，因为它们能够在宿主皮肤中诱导迟发型超敏反应。作为 Jesus Valenzuela 指导的疫苗开发项目的一部分，我在重组系统中从 Phlebotomous dubosqi 的唾液中生产了三种抗原。这些蛋白质将在恒河猴的试点研究中测试其预防感染的能力。 3) 与埃及伊蚊的长 D7 一样，斯氏按蚊蛋白的 D7 蛋白结合白三烯，但与埃及伊蚊蛋白不同的是，它不结合生物胺。 A.stephensi蛋白还具有抑制由类二十烷酸血栓素A2诱导的血小板聚集的独特功能。我们在血小板聚集和配体结合测定中使用多种血栓素 A2 受体激动剂和拮抗剂来表征这种活性。 4) 多年前就有人指出，曼氏血吸虫卵的提取物会诱导 CD4+ T 细胞向 Th2 表型极化。 Th2 极化的机制尚不清楚，也没有分离出诱导这种分化的特定因素。与实验室的 Alan Sher 和 Dragana Jankovic 合作。在寄生虫病研究中，我们对鸡蛋培养物的上清液进行分级，并分离出一种明显的单一蛋白质成分，即核糖核酸酶 omega-1，它会导致这种效应。我们现在正致力于通过开发生产活性重组蛋白的系统来了解该蛋白的作用机制。如果成功，我们将能够使用诱变和蛋白质化学来评估核糖核酸酶活性和糖基化对该药物功能的重要性。