Vector-Borne Diseases: Biology Of Vector Host Relationship

媒介传播疾病：媒介宿主关系的生物学

基本信息

批准号：
7964402
负责人：
Jose Ribeiro
金额：
$ 142.04万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/7964402
关键词：
Active Sites Adult Anopheles Genus Anopheles gambiae Anti-Inflammatory Agents Anti-inflammatory Argasidae Arthropods Biochemical Bioinformatics Biological Biological Assay Biology Black-legged Tick Blood Blood Clot Blood Platelets Blood coagulation Cell Line Chemicals Coagulation Process Code Collaborations Complement Complementary DNA Complex Computer software Culicidae DNA Transposable Elements Dendritic Cells Disease Vectors Enzymes Eukaryotic Cell Evolution Family Female Future Gene Expression Profile Genes Genome Goals Greek Hand Helminths Hemorrhage Hemostatic Agents Hemostatic function Immune Immune response Immunosuppressive Agents Individual Inflammatory Injury Insecta Knowledge Lead Learning Legal patent Leukotrienes Lyme Disease Magic Malaria Maps Messenger RNA Methodology Methods Mining Modeling Molecular Molecular Biology Mycobacterium tuberculosis Nucleotides Organism Ornithodoros Paper Peptide Mapping Peptides Pharmacologic Substance Physiological Processes Platelet aggregation Process Property Protease Inhibitor Protein Family Proteins Publishing Reaction Recombinant Proteins Recombinants Reporting Research Saliva Salivary Salivary Glands Salivary Proteins Screening procedure Serotonin Simuliidae Simulium genus Site South American Stomoxys calcitrans System Techniques Ticks Upper arm Vaccines Vasodilator Agents Vector-transmitted infectious disease Work Writing Yellow Fever cDNA Library cancer therapy cutinase design expression cloning feeding fly in vitro testing in vivo insight interest novel pathogen prevent programs protein aminoacid sequence success sucking transmission process vaccine development vasoconstriction vector vector mosquito

项目摘要

In our search for understanding the evolution of blood sucking by arthropods, we realize the methodology used has drastically changed in the past few years. Traditionally, the research process first identified a biological activity in saliva or salivary gland homogenates of a particular organism, and then proceeded to isolate that activity as a relatively pure entity to allow its molecular identification. In the case the activity derived from a protein, peptide fingerprinting allowed the design and use of nucleotide probes to clone the coding mRNA (in the form of a cDNA) and final identification of the peptide sequence; the clone also allowed the manufacture of recombinant protein for further studies. Nowadays, the process has reverted. cDNA libraries are constructed from salivary glands of blood sucking arthropods and mass sequenced. Bioinformatic analysis reveals the salivary transcriptome of these organisms, which contains many unique protein families with unknown properties. We then proceed to select clones for expression, bioassay screening and characterization. Accordingly, there are two processes used in our lab, first, the construction and analysis of salivary gland cDNA libraries, and second, the recombinant expression and characterization of these proteins. We have also been developing bioinformatic capabilities in the form of specific software to help direct our studies. Sialotranscriptome discovery projects: Because host hemostasis (the physiological process that prevents blood loss, consisting of platelet aggregation, blood clotting and vasoconstriction) is a complex and redundant phenomenon, the salivary glands of blood sucking arthropods consist of a magic potion with diverse chemicals that in a redundant way counteract host mechanisms to prevent blood loss, allowing the fast acquisition of a meal. Salivary transcriptome made in the past few years indicate that the magic potion consists of 70-100 different proteins in the case of mosquitoes, for example, to over 400 in the case of ticks (Ticks feed for several days and have to disarm host immune reactions, in addition to the hemostatic system). Transcriptome studies also show that the salivary proteins of blood sucking arthropods are at a very fast pace of evolution, perhaps explaining why every genera studied so far has several unique protein families. Indeed there are unique proteins found at the subgenus level. Given we can now describe in detail the sialotranscriptome (from the Greek word sialo = saliva) of a single organism, we can ask now what is the universe of salivary proteins associated to blood feeding, the so called sialoverse. There are near 19,000 species of blood sucking arthropods in 500 different genera. If we find (minimally) 5 novel protein families per genus (within the 70-500 proteins in each sialome), there are at least 2,500 novel proteins to be discovered, each one with an interesting pharmacological property. We have so far explored less than a dozen genera of blood sucking arthropods, and it is our goal to extend sialotranscriptome discovery to map this pharmacological mine for future studies, and in the process learn the paths taken by genomes in their evolution to blood feeding, and identify proteins with pharmacological and vaccine potential. In the current fiscal year (2009), we produced a total of 14 papers and one patent application. Five of the papers describe sialomes, including the sialome of the black fly Simulium vittatum (1), the first so far produced for this family of flies, of the South American malaria vector Anopheles darlingi (2), of larval An. gambiae mosquitoes (3), and of the stable fly, Stomoxys calcitrans (4), the first from the fly suborder Brachycera, and of a soft tick Ornithodoros coriaceus (5). Several new protein families were discovered in the sialomes of the black fly and the stable fly that awaits functional characterization. The An. darlingi sialome allowed understanding of the fast evolution of mosquito salivary proteins, including the differentiation of families between the sub families anopheline and culicine. The sialome of larval mosquitoes, which do not blood feed, allow for comparison with those of the adult female and identification of unique proteins that are associated with blood feeding. Finally, the sialome of O. coriaceus allowed for the identification of families of proteins common to soft ticks and determination of the fast divergence of these protein families. Functional sialomic studies: We advanced our knowledge regarding the function of several salivary proteins, as well as discovering novel salivary properties. One of the most abundant proteins of the mosquito vector of Yellow Fever was crystallized and its function revealed as a protein scavenger of serotonin and inflammatory leukotrienes (6). A salivary cysteinyl protease inhibitor of the Lyme disease vector, Ixodes scapularis, was shown to inhibit dendritic cells and to have immunosuppressive activity (7). The gSG6 protein of An. gambiae has been immunologically characterized as a good marker of vector exposure (8). A review article on the analysis of tick salivary proteins was also written (9). Expertise capabilities spin off: Our bioinformatic capability lead to collaboration with diverse studies, leading to the publishing of software to analyze transcriptomes (10), which is being widely used; for helping annotating secreted proteins of parasitic worms (11); to identify immune-related genes in the malaria vector Anopheles gambiae (12), and to model the evolution of transposable elements in mosquitoes (13). Our collaboration with Dr. Warwick Britton, lead to the identification in the genome of Mycobacterium tuberculosis of a family of cutinase proteins that has vaccine potential, reported in 2008. Now we report biochemical properties of these recombinant enzymes and enzyme active site identification (14). A patent application has been submitted for the use of a tick anticlotting agent as a suppressor of metastasis for cancer treatment (15).

在我们寻求了解节肢动物吸血进化的过程中，我们意识到所使用的方法在过去几年中发生了巨大变化。传统上，研究过程首先鉴定特定生物体的唾液或唾液腺匀浆中的生物活性，然后将该活性分离为相对纯净的实体，以进行分子鉴定。在活性源自蛋白质的情况下，肽指纹分析允许设计和使用核苷酸探针来克隆编码mRNA（以cDNA的形式）并最终鉴定肽序列；该克隆还可以制造重组蛋白以供进一步研究。如今，这一过程已经恢复。从吸血节肢动物的唾液腺构建 cDNA 文库并进行质量测序。生物信息分析揭示了这些生物体的唾液转录组，其中包含许多具有未知特性的独特蛋白质家族。然后我们继续选择克隆进行表达、生物测定筛选和表征。因此，我们实验室使用两个过程，第一，唾液腺cDNA文库的构建和分析，第二，这些蛋白质的重组表达和表征。我们还一直在以特定软件的形式开发生物信息能力，以帮助指导我们的研究。唾液酸转录组发现项目：由于宿主止血（防止失血的生理过程，包括血小板聚集、血液凝固和血管收缩）是一种复杂而多余的现象，吸血节肢动物的唾液腺由一种含有多种化学物质的神奇药水组成，这些化学物质以多余的方式抵消防止失血的宿主机制，允许快速获取食物。过去几年的唾液转录组表明，对于蚊子来说，这种神奇的药水由 70-100 种不同的蛋白质组成，例如，对于蜱虫来说，有超过 400 种不同的蛋白质（蜱虫会进食数天，并且必须解除宿主的免疫反应），除了止血系统）。转录组研究还表明，吸血节肢动物的唾液蛋白进化速度非常快，这或许可以解释为什么迄今为止研究的每个属都有几个独特的蛋白质家族。事实上，在亚属水平上发现了独特的蛋白质。鉴于我们现在可以详细描述单个生物体的 sialotranscriptome（来自希腊语 sialo = 唾液），我们现在可以问与血液喂养相关的唾液蛋白宇宙是什么，即所谓的 sialoverse。吸血节肢动物有 500 个不同属，近 19,000 种。如果我们（至少）在每个属中发现 5 个新蛋白质家族（每个 sialome 中的 70-500 个蛋白质），则至少有 2,500 个新蛋白质有待发现，每个蛋白质都具有有趣的药理学特性。到目前为止，我们已经探索了不到十几个吸血节肢动物，我们的目标是扩展唾液转录组的发现，为未来的研究绘制这个药理学矿藏，并在此过程中了解基因组在进化到吸血方面所采取的路径，并鉴定具有药理学和疫苗潜力的蛋白质。在本财年（2009），我们总共发表了 14 篇论文和一项专利申请。其中五篇论文描述了唾液酸组，包括黑蝇 Simulium vittatum (1) 的唾液酸组，这是迄今为止为该蝇科生产的第一个、南美疟疾媒介达林按蚊 (Anopheles darlingi) 幼虫的唾液酸组 (2)。冈比亚蚊子 (3)、稳定蝇 Stomoxys calcitrans (4)（第一个来自短角蝇亚目的蝇）和软蜱 Ornithodoros coriaceus (5)。在黑蝇和稳定蝇的唾液酸中发现了几个新的蛋白质家族，等待功能表征。安. darlingi sialome 有助于了解蚊子唾液蛋白的快速进化，包括按蚊亚科和蚊子亚科之间的分化。不吸血的幼虫蚊子的唾液酸组可以与成年雌性蚊子的唾液酸组进行比较，并鉴定与吸血相关的独特蛋白质。最后，O. coriaceus 的唾液酸组允许鉴定软蜱常见的蛋白质家族并确定这些蛋白质家族的快速分化。功能唾液酸研究：我们增进了对几种唾液蛋白功能的了解，并发现了新的唾液特性。黄热病蚊子载体中最丰富的蛋白质之一被结晶，其功能被揭示为血清素和炎症性白三烯的蛋白质清除剂 (6)。莱姆病媒介肩胛硬蜱的唾液半胱氨酰蛋白酶抑制剂可抑制树突状细胞并具有免疫抑制活性 (7)。 An 的 gSG6 蛋白。冈比亚已被免疫学鉴定为媒介暴露的良好标记 (8)。还撰写了一篇关于蜱唾液蛋白分析的评论文章 (9)。专业能力衍生：我们的生物信息能力导致与各种研究的合作，导致转录组分析软件的发布 (10)，并得到广泛使用；用于帮助注释寄生虫的分泌蛋白 (11)；鉴定疟疾病媒冈比亚按蚊 (Anopheles gambiae) 中的免疫相关基因 (12)，并模拟蚊子中转座元件的进化 (13)。我们与 Warwick Britton 博士合作，在 2008 年报道了结核分枝杆菌基因组中具有疫苗潜力的角质酶蛋白家族的鉴定。现在我们报告这些重组酶的生化特性和酶活性位点鉴定 (14) 。已提交一项专利申请，内容是使用蜱抗凝血剂作为癌症治疗的转移抑制剂 (15)。