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: 8946415
• 负责人: Jose Ribeiro
• 金额: $ 48.09万
• 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/8946415
• 关键词: Affect; Anopheles Genus; Anti-Inflammatory Agents; Anti-Retroviral Agents; Anticoagulants; Antigens; Area; Arteriosclerosis; Arthropods; Binding; Binding Proteins; Binding Sites; Biochemistry; Bioinformatics; Biological; Biological Assay; Biology; Blood; Blood Vessels; Bradykinin; Brazil; Carbohydrates; Childhood; Clinical; Coagulation Process; Colitis; Collaborations; Collection; Complement; Complement Activation; Complement Inactivators; Complex; Crystallization; Culicidae; Data; Data Collection; Delayed Hypersensitivity; Disease Vectors; Environment; Eukaryotic Cell; Factor IXa; Factor XII; Gene Expression Profile; Glycosaminoglycans; Goals; Helicobacter; Hematological Disease; Hematology; Hemostatic Agents; Humpback Dolphins; Immune response; Immunology; Infection; Inflammation; Inflammatory; Inflammatory Response; Ingestion; Insect Bites; Insect Vectors; Insecta; Laboratories; Leishmaniasis; Ligands; Lutzomyia genus; Methodology; Methods; Molecular; Molecular Mechanisms of Action; Moscow; Organism; Pathway interactions; Patients; Peptide Hydrolases; Peptides; Physiology; Plasmodium; Polyphosphates; Postdoctoral Fellow; Process; Production; Protein Family; Proteins; Publishing; Recombinant Proteins; Recombinants; Research; Rhodnius; Role; Saliva; Salivary; Salivary Proteins; Sand Flies; Schistosoma; Sequence Analysis; Skin; Structure; Students; System; T-Lymphocyte; Techniques; Testing; Therapeutic; Thrombin; Thrombosis; Ticks; Toll-like receptors; Toxoplasma; Vaccines; Vector-transmitted infectious disease; Visit; Work; anticomplement; assay development; base; beamline; chemokine receptor; cysteinyl-leukotriene; disease transmission; egg; feeding; inhibitor/antagonist; mast cell; member; microorganism; mouse model; nitrophorin 2; novel; oncology; pathogen; prevent; protein structure; response; small molecule; transmission process; 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 2014 fiscal year we have 1) determined the structures of a new salivary protein and applied structural information to determine the mechanism of action of these proteins, 2) Continued to work with recombinant proteins contained in experimental leishmaniasis vaccines 3) Published the structure and function of a salivary protein that inhibits the hemostatic and inflammatory effects of the contact pathway of coagulation. 4)Characterized an inhibitor of the coagulation cascade protease thrombin that is derived from tick saliva. 5) Collaborated with Mikhail Panteleev of the Federal Research and Clinical Center of Pediatric Hematology, Immunology and Oncology, Moscow. The research used an inhibitor of FIXa from insect saliva to demonstrate the importance of microparticle activation of the contact pathway in many hematological disorders. 6) Continued studies on an antiinflammatory binding protein from the saliva of Rhodnius prolixus with Willy Jablonka, a post doc fellow in Dr. Ribeiro's laboratory. 7) Initiated a study on the characterization and identification of a complement inhibitor from the saliva new world Anopheles mosquitoes. 1) We continue our work on the crystallization of salivary proteins in the laboratory and now almost exclusively use remote data collection from the SER-CAT beamlines at Argonne National Laboratory for collection of diffraction data. We have produced recombinant protein, crystallized two proteins and determined the structure of one of them over the last year. We are also currently analyzing diffraction data on several additional novel proteins. 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 begun working on the crystallization and function of a potential antigen from Lutzomyia longipalpis 3)We have characterized members of the "SP-15" protein family from Phlebotomous duboscqi as inhibitors of the "contact" pathway of coagulation. These are major proteins in the saliva that act by binding to glycosaminoglycans secreted from mast cells in the skin. These carbohydrates serve as an activating matrix for coagulation factor XII. Inihbition of factor XII activation prevents the production of bradykinin in the skin thereby limiting inflammation in the area of the insect bite. In addition to identifying the mode of action of these inhibitors, we have determined the crystal structures of two forms and identified structural possible structural determinants for these activities. We have also shown that the protein blocks contact pathway activation by polyphosphate, a key endogenous activator. During the past year we published this work in Arteriosclerosis, Thrombosis and Vascular Biology. 4) Inhibitors of thrombin are important for blood feeding and are used as therapeutic anticoagulants. Along with Dr. Ribeiro and Willy Jablonka I identified a salivary peptide from the tick Hyalomma marginatum that inhibits thrombin at concentrations near 10 nanomolar. We have characterized the binding of the peptide, showed that it is cleavable by thrombin, and dissected its sequence in order to identify the essential sequences for binding, and the probable binding sites on thrombin. 5)I have produced recombinant nitrophorin 2, an inhibitor of coagulation factor IXa for a study to evaluate the importance of microparticle activation of the contact pathway in the propensity to thrombosis in patients with various hematological disorders. This work was in collaboration with Mikhail Panteleev and was published in Plos One. 6)Proteins in the lipocalin protein family are widely distributed in the saliva of disease vectors, but they are not functionally well characterized. I have determined the structure of a novel lipocalin from Rhodnius prolixus that we have shown binds cysteinyl leukotrienes. The structure of the protein-ligand complex shows large conformational changes 7) I initiated a study of an anti-complement protein in the saliva of Anopheles albinanus, a new-world vector of Plasmodium. Complement activation in response to feeding is well demonstrated, and is a known to make the ingestion of blood difficult. We find that old world anophelines do not do not have anticomplement activity in the saliva. We are in the process of isolating and identifying this factor. The project is being performed with Antonio Sousa, a visiting student from Brazil.

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 2014 财年，我们 1) 确定了一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) 继续研究实验性利什曼病疫苗中包含的重组蛋白，3) 发布了结构和唾液蛋白的功能，抑制凝血接触途径的止血和炎症作用。 4) 表征了源自蜱唾液的凝血级联蛋白酶凝血酶抑制剂。 5) 与莫斯科儿科血液学、免疫学和肿瘤学联邦研究和临床中心的 Mikhail Panteleev 合作。该研究使用昆虫唾液中的 FIXa 抑制剂来证明微粒激活接触途径在许多血液疾病中的重要性。 6) 与Ribeiro 博士实验室的博士后Willy Jablonka 继续研究来自Rhodnius prolixus 唾液的抗炎结合蛋白。 7) 启动了新世界按蚊唾液补体抑制剂的表征和鉴定研究。 1) 我们继续在实验室进行唾液蛋白结晶工作，现在几乎完全使用来自阿贡国家实验室 SER-CAT 光束线的远程数据收集来收集衍射数据。去年我们生产了重组蛋白，结晶了两种蛋白并确定了其中一种的结构。我们目前还在分析其他几种新型蛋白质的衍射数据。 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，我已开始研究 Lutzomyia longipalpis 潜在抗原的结晶和功能 3)我们将来自杜博斯奇静脉的“SP-15”蛋白家族成员鉴定为凝血“接触”途径的抑制剂。这些是唾液中的主要蛋白质，通过与皮肤肥大细胞分泌的糖胺聚糖结合而发挥作用。这些碳水化合物充当凝血因子 XII 的激活基质。抑制因子 XII 激活可防止皮肤中缓激肽的产生，从而限制昆虫叮咬区域的炎症。除了确定这些抑制剂的作用模式外，我们还确定了两种形式的晶体结构，并确定了这些活性的可能结构决定因素。我们还表明，该蛋白质可阻断多磷酸盐（一种关键的内源性激活剂）的接触途径激活。去年，我们在《动脉硬化、血栓形成和血管生物学》上发表了这项工作。 4) 凝血酶抑制剂对于血液喂养很重要，并用作治疗性抗凝剂。我与 Ribeiro 博士和 Willy Jablonka 一起从蜱虫 Hyalomma marginatum 中鉴定出一种唾液肽，它可以在浓度接近 10 纳摩尔时抑制凝血酶。我们表征了该肽的结合，表明它可被凝血酶切割，并剖析了其序列，以确定结合的必需序列以及凝血酶上可能的结合位点。 5)我已经生产了重组硝基蛋白2，一种凝血因子IXa的抑制剂，用于一项研究，以评估接触途径的微粒激活对于患有各种血液疾病的患者的血栓形成倾向的重要性。这项工作是与 Mikhail Panteleev 合作的，并发表在 Plos One 上。 6）脂质运载蛋白家族中的蛋白质广泛分布于疾病媒介的唾液中，但其功能尚未得到很好的表征。我已经确定了来自 Rhodnius prolixus 的一种新型脂质运载蛋白的结构，我们已经证明它与半胱氨酰白三烯结合。蛋白质-配体复合物的结构显示出较大的构象变化 7) 我发起了一项针对白按蚊唾液中抗补体蛋白的研究，白按蚊是一种新的疟原虫载体。补体对进食的反应已被充分证明，并且众所周知，补体激活会使血液的摄入变得困难。我们发现旧大陆按蚊的唾液中并非没有抗补体活性。我们正在分离和识别这个因素。该项目是与来自巴西的访问学生 Antonio Sousa 一起执行的。