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: 8555935
• 负责人: Jose Ribeiro
• 金额: $ 50.89万
• 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/8555935
• 关键词: Academy; Affect; Aldehyde-Lyases; Anaplasma; Anti-Inflammatory Agents; Anti-Retroviral Agents; Antigens; Area; Arthropods; Binding; Biochemistry; Bioinformatics; Biological; Biological Assay; Blood; Bradykinin; Carbohydrates; Coagulation Process; Colitis; Collaborations; Collection; Crystallization; Cytoplasmic Tail; Data; Data Collection; Delayed Hypersensitivity; Disease Vectors; Environment; Erythrocytes; Eukaryotic Cell; Factor XII; Family member; Fibrinogen Receptors; Gene Expression Profile; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycosaminoglycans; Goals; Helicobacter; Hemostatic Agents; Immune response; Infection; Inflammation; Inflammatory Response; Insect Bites; Insect Vectors; Integrins; Laboratories; Leishmaniasis; Leukotrienes; Ligands; Methodology; Methods; Molecular; Molecular Mechanisms of Action; Organism; Pathway interactions; Peptides; Physiology; Plasmodium; Platelet aggregation; Process; Production; Protein Family; Proteins; Publishing; Recombinant Proteins; Recombinants; Research; Role; Saliva; Salivary; Salivary Proteins; Sand Flies; Schistosoma; Science; Sequence Analysis; Skin; Structure; Surface Plasmon Resonance; System; T-Lymphocyte; Techniques; Testing; Ticks; Toll-like receptors; Toxoplasma; Vaccines; Vector-transmitted infectious disease; Work; assay development; base; beamline; chemokine receptor; disease transmission; egg; feeding; fly; inhibitor/antagonist; mast cell; member; merozoite surface protein; microorganism; mouse model; novel; pathogen; prevent; protein protein interaction; 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.We have recently collaborated with Dr. Jesus Valenzuela to characterize During the 2012 fiscal year we have 1) determined the structures of two new salivary proteins and applied structural information to determine the mechanism of action of these proteins, 2) continued to produce recombinant proteins for use in an experimental saliva-based leishmaniasis vaccine, 3) published the structure and salivary function of a novel antiinflammatory salivary protein from a blood-feeding fly 4)Characterized an inhibitor of the contact activation pathway of coagulation from the sand fly, 5) Completed a collaboration with Louis Miller to determine molecular interactions in the Plasmodium invasion process. 6) Produced recombinant tick salivary proteins in a collaboration with Dr. Michalis Kotsyfakis of the Czech Academy of Sciences on the study of Anaplasma infection 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 and determined the structure of two new proteins 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 continued to produce salivary antigens from the saliva of Phlebotomous duboscqi in a recombinant system. 3)Proteins in the antigen-V protein family are widely distributed in the saliva of disease vectors, but they are not functionally well characterized. We have published a structural analysis and functionaal characterization of a leukotriene-binding member of this family from the blood feeding fly, Tabanus yao. The protein was also known to inhibits platelet aggregation by binding with the fibrinogen receptor integrin alphaIIbbetaIII. 4) 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. 5) I have collaborated with Louis Miller and members of his laboratory in a study of cellular protein-protein interactions of cytoplasmic domains of merozoite surface proteins and their roles in red blood cell invasion. We have completed a study using surface plasmon resonance to determine interaction of cytoplasmic domain peptides with the cellular proteins aldolase and glyceraldehyde-3-phosphate dehydrogenase.

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，以及从血吸虫卵中分离出明显的 T 细胞极化因子。我们最近与 Jesus Valenzuela 博士合作，表征了 在 2012 财年，我们 1) 确定了两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) 继续生产重组蛋白，用于基于唾液的实验性利什曼病疫苗，3 ) 发表了一种来自食血蝇的新型抗炎唾液蛋白的结构和唾液功能 4) 表征了白蛉凝血接触激活途径的抑制剂，5)与 Louis Miller 完成合作，确定疟原虫入侵过程中的分子相互作用。 6) 与捷克科学院的Michalis Kotsyfakis博士合作生产重组蜱唾液蛋白，研究无形体感染 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，结晶并确定了两种新蛋白的结构。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，我继续在重组系统中从 Phlebotomous duboscqi 的唾液中生产唾液抗原。 3）抗原-V蛋白家族中的蛋白质广泛分布于疾病载体的唾液中，但其功能尚未得到很好的表征。我们发表了来自吸血蝇 Tabanus yao 的该家族白三烯结合成员的结构分析和功能表征。该蛋白还可以通过与纤维蛋白原受体整合素 αIIbbetaIII 结合来抑制血小板聚集。 4）我们将来自 Phlebotomous duboscqi 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。 5) 我与 Louis Miller 及其实验室成员合作研究了裂殖子表面蛋白胞质域的细胞蛋白-蛋白相互作用及其在 红细胞侵袭。我们已经完成了一项研究，使用表面等离子共振来确定细胞质结构域肽与细胞蛋白醛缩酶和 3-磷酸甘油醛脱氢酶的相互作用。