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: 7592363
• 负责人: Jose Ribeiro
• 金额: $ 85.97万
• 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/7592363
• 关键词: Affect; African; Amino Acid Sequence; Anti-Retroviral Agents; Antigen Presentation; Antigens; Arthropods; Binding; Binding Proteins; Biochemistry; Biogenic Amines; Bioinformatics; Biological; Biological Assay; Biosensor; Blood; CD4 Positive T Lymphocytes; Cathepsins; Cells; Colitis; Collaborations; Complex; Culicidae; Cysteine Protease; DIF factor; Data; Data Collection; Disease Vectors; Environment; Enzymes; Eukaryotic Cell; Evolution; Family; Farming environment; Fleas; Gene Expression Profile; Goals; Growth; Helicobacter; Hemostatic Agents; Histamine; Human; Immune response; Immune system; Immunization; Infection; Inflammation; Inflammatory; Insect Vectors; Interleukin-4; Investigation; Ixodes; Laboratories; Ligand Binding; Ligands; Lyme Disease; Malaria; Methodology; Methods; Molecular; Molecular Mechanisms of Action; Neurologic; Norepinephrine; Numbers; Organism; Parasites; Parasitic Diseases; Peptide Sequence Determination; Phenotype; Phosphoric Monoester Hydrolases; Photons; Physiology; Plague; Platelet aggregation; Population; Process; Production; Proteins; Purpose; RNA Interference; Rattus; Recombinant Proteins; Recombinants; Research; Roentgen Rays; Role; Saliva; Salivary; Salivary Proteins; Schistosoma; Schistosoma mansoni; Scorpions; Sequence Analysis; Serotonin; Source; Structural Protein; Structure; Synchrotrons; System; T-Lymphocyte; Techniques; Testing; Ticks; Toll-like receptors; Toxin; Toxoplasma; Vector-transmitted infectious disease; Visit; Work; Yellow Fever; Yersinia pestis; assay development; chemokine receptor; disease transmission; egg; feeding; in vitro Assay; member; microorganism; mouse model; novel; pathogen; practical application; programs; protein expression; protein structure; response; scale up; small molecule; transmission process; vector; vector mosquito

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 past fiscal year we have 1) integrated protein structural analysis as a major component of our program, 2) characterized the mechanism of a potent salivary immunomodulatory protein from a tick species, 3) Determined the repertoire of salivary proteins from the rat flea by analyzing its salivary transcriptome, and 4) isolated 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 two new proteins and have determined additional structures of these proteins to evaluate various ligand complexes. The "short form" D7 proteins found in saliva of the African malaria vector mosquito act to inhibit host infammatory responses and platelet aggregation by binding the biogenic amines serotonin, histamine and norepinephrine. We have determined the X-ray crystal structure of one of these proteins using multiple anomalous dispersion techniques as well as structures of the protein in complex with the ligands listed above. We have also determined the structure of the "long-form" D7 protein from the yellow fever mosquito which is a two-domain where one domain functions as a biogenic amine-binding protein while the function of the second domain is under investigation. The "long form" structure revealed a major confomational change that appears to be involved in the stabilization of bound ligands. As a potential practical application of this work, the short-form and long-form D7 proteins are being developed in collaboration with Dr. Loren Looger of HHMI Janelia Farms as biosensors for nerurotransmitters in neurological research. Along the same lines,we are working on the of two novel proteins serving as biogenic-amine binding proteins in thhe saliva of ticks. These proteins belong to the lipocalin protein familiy making them structurally unrelated to the mosquito D7 proteins. This information is being used to understand the process of blood feeding and the evolution of ticks. 2) Salivas of Ixodes (Lyme borreliosis vectors) ticks contain proteins aimed at supressing the immune system during the long feeding period of these species. We have examined the effects of two small proteins, sialostatin L and L2 which act to inhibit cathepsin type cysteine proteases. These molecules inhibit antigen presentation and inflammation. Supression of salivary expression of these proteins by RNAi results in profound growth inhibition of feeding ticks. We are examining the potential of immunization with this molecule to interfere with tick feeding and disease transmission. We are also pursuing structural studies with sialostatins L and L2 and their complexes with cathepsins. 3) The rat flea, Xenopsylla cheopis is a vector of the bubonic plague bacteium, Yersinia pestis. In order to study the biochemistry of blood feeding by this vector insect we have examined, in collaboration with Jose Ribeiro its entire repertoire of salivary proteins by sequencing and annotating its salivary transcriptome. Because this is the first member of its taxonomic order to be examined, a number of novel salivary molecules have been revealed, including a family related to scorpion toxins, and a family of phosphatase-like proteins which appear not to be active enzymes. 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 which causes this effect. This protein has been expressed in human HEK 293 cells, and the recombinant material shows activity as indicated by increased populations of IL-4 positive CD4+ T cells in an in-vitro assay. We are currenly scaling up the production of the recombinant protein for further studies.

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。 在过去的财政年度中，我们 1) 综合蛋白质结构分析作为我们计划的主要组成部分，2) 表征了蜱种有效唾液免疫调节蛋白的机制，3) 通过以下方法确定了大鼠跳蚤唾液蛋白的全部成分：分析其唾液转录组，4) 从寄生虫曼氏血吸虫卵中分离出 T 细胞极化因子。 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。我们生产了重组蛋白，结晶并确定了两种新蛋白的结构，并确定了这些蛋白的其他结构以评估各种配体复合物。在非洲疟疾媒介蚊子的唾液中发现的“短形式”D7 蛋白通过结合生物胺血清素、组胺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其中一种蛋白质的 X 射线晶体结构，以及与上面列出的配体复合的蛋白质的结构。我们还确定了来自黄热病蚊子的“长型”D7 蛋白的结构，该蛋白是一个双结构域，其中一个结构域充当生物胺结合蛋白，而第二个结构域的功能正在研究中。 “长形式”结构揭示了一个主要的构象变化，该变化似乎与结合配体的稳定有关。作为这项工作的潜在实际应用，短型和长型 D7 蛋白正在与 HHMI Janelia Farms 的 Loren Looger 博士合作开发，作为神经学研究中神经递质的生物传感器。同样，我们正在研究蜱唾液中两种作为生物胺结合蛋白的新型蛋白质。这些蛋白质属于脂质运载蛋白家族，因此在结构上与蚊子 D7 蛋白质无关。这些信息被用来了解蜱虫的吸血过程和进化。 2) 硬蜱（莱姆疏螺旋体病载体）的唾液中含有旨在在这些物种的长期进食期间抑制免疫系统的蛋白质。我们研究了两种小蛋白（唾液酸抑素 L 和 L2）的作用，它们可抑制组织蛋白酶型半胱氨酸蛋白酶。这些分子抑制抗原呈递和炎症。通过 RNAi 抑制这些蛋白质的唾液表达会导致进食蜱的生长受到严重抑制。我们正在研究用这种分子进行免疫以干扰蜱虫进食和疾病传播的潜力。我们还在进行唾液酸抑素 L 和 L2 及其与组织蛋白酶复合物的结构研究。 3) 鼠蚤（Xenopsylla cheopis）是腺鼠疫细菌（鼠疫耶尔森氏菌）的传播媒介。为了研究这种媒介昆虫吸血的生物化学，我们与 Jose Ribeiro 合作，通过对其唾液转录组进行测序和注释，检查了其全部唾液蛋白。因为这是其分类学中第一个被检查的成员，所以已经揭示了许多新的唾液分子，包括与蝎子毒素相关的家族，以及似乎不是活性酶的磷酸酶样蛋白家族。 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。在寄生虫病的研究中，我们对鸡蛋培养物的上清液进行了分级，并分离出了导致这种效应的明显单一蛋白质成分。该蛋白已在人 HEK 293 细胞中表达，并且体外测定中 IL-4 阳性 CD4+ T 细胞数量的增加表明重组材料显示出活性。我们目前正在扩大重组蛋白的生产以进行进一步研究。