Molecular Definition Of Filarial And Related Nonfilarial Genes And Proteins

丝虫及相关非丝虫基因和蛋白质的分子定义

• 批准号: 7732463
• 负责人: Thomas B Nutman
• 金额: $ 17.03万
• 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/7732463
• 关键词: Adult; Affinity; Antibiotics; Antibodies; Antigens; Baculoviruses; Binding; Biological Assay; Brugia; Brugia malayi; C-terminal; CXC chemokine receptor 3; Characteristics; Class; Consensus Sequence; Crystallins; Diagnosis; Diagnostic; Digestion; Dose; Drug Delivery Systems; Escherichia coli; Expressed Sequence Tags; Female; Gene Proteins; Generations; Genes; Genome; Growth; Heat shock proteins; Helminths; Human; IL5 gene; Immune response; Immunity; In Vitro; Infection; Interleukin-10; Interleukin-5; Larva; Libraries; Loa; Loa loa; Localized; Luciferases; Mansonella; Membrane Proteins; Methods; Microfilaria; Molecular; Mus; N-terminal; Numbers; Onchocerca; Open Reading Frames; PAWR protein; Parasites; Peptides; Phage Display; Property; Proteins; Proteome; Proteomics; Publishing; Recombinant DNA; Recombinants; Ribosomal DNA; Ribosomal RNA; Sequence Analysis; Serum; Shotguns; Signal Transduction; Staging; Strongyloides; Surface; Testing; Vaccines; Whole Blood; Wolbachia; Yeasts; alpha-Crystallins; cDNA Library; eosinophil; immunogenic; interleukin-13 receptor; male; mast cell; mouse model; novel; novel diagnostics; receptor; receptor coupling; treatment trial; vector

Using phage display libraries (from infective larvae L3 and adult females) Brugia malayi and screens with known soluble human receptors, we have identified, cloned and characterized distinct molecules that bind to the human IL-5R, IL-10R, and IL-13R. The molecule we term, Bm-IL5RBP Brugia malayi IL5Rbinding protein(IL5Rbp) has been expressed at high levels in a manner that retains its ability to bind to the human receptor. Antibodies raised to predicted immunogenic peptides inhibit the binding of rBm-IL5BP to its human receptor and have been used to localize (by immuno-EM) Bm-IL5RBP to the surface of the infective stage larvae. The rBm-IL5BP does not itself prolong the survival of human eosinophils, but does inhibit the ability of human IL5 to prolong eosinophil survival. B. malayi HSP12.6 (BmHSP12.6)has been identified and characterized as a molecule that binds to the human IL-10R. Structural analyses of BmHSP12.6 showed that it has a highly conserved alpha-crystallin central domain that is characteristic of other small heat shock proteins (HSPs) but because of . short N-terminal domain and an unusually small C-terminal domain flanking the crystallin domain appears to belong to a novel class of small HSPs. Recombinant BmHSP12.6 binds to huIL10R in a dose dependent fashion and inhibits the binding of human IL-10 (huIL10) to its receptor. rBmHSP12.6 also enhanced the growth and proliferation of MC/9 mast cells in vitro similar to huIL10, suggesting that this parasite actively synthesizes a moleclule that may modulate the human immune response. Interestingly, we have also identified a secreted product from Brugia malayi L3 that is chemotactic for human eosinophils. This secreted products signals through the G-coupled receptor CXCR3. We used a molecular approach to show definitively that the fialrial parasite, Mansonella perstans (Mp) contains the endosymbiont, Wolbachia. Using primers known to amplify the 16S ribosomal DNA of other filarial Wolbachiae, an identical 1393bp band was found that on sequence analysis demonstrated a single consensus sequence for Mp Wolbachia 16S rDNA that was most similar to Wolbachia sequences from other filarial nematodes.Phylogenetic dendrograms, examining the relationship of the Mp Wolbachia to other Wolbachia 16S rDNA, showed that the Wolbachia tracked almost identically to the 5S rRNA of their parasite host. Wolbachia surface protein (WSP) was also demonstrated in protein extracted from Mp-containing whole blood. In advance of a treatment trial of Mp, a method for the quantitation of Mp Wolbachia was developed and used to demonstrate not only a relationship between microfilarial numbers and Wolbachia copy numbers, but also to demonstrate the effect of antibiotic on ridding Mp of Wolbachia. As part of a consortium of many workers, we have been a part of the annotation of the filarial genome published in draft from this past year. A separate proteomic analysis has also been completed using mass spectrospcopy in tandem with whole stage-specific tryptic digestion. Not only has the stage specific proteomes been completed for adult males, adult females, microfilariae, and infective larvae, but we have also completed the Brugia Wolbachia proteome and the excretory/secretory proteome. These analyses have provided clear evidence that those genes encoding "hypothetical proteins" are real and have also identified close to 70% of all predicted open reading frames. Further the secretome analyses have revealed unexpected secreted proteins many of which have host-immunomodulatory properties. An expanded approach to protective immunity in both filarial infections and in strongyloides is underway. Antigens affinity purified using sera from either mice immunized with irradiated larvae that show close to 100% protection to challenge infection with S. strongyloides or humans demonstrating immunity to Ss infection have identified 6 candidates that have been expressed in E. coli, yeast and baculovirus. Testing with one E. coli-derived recombinant has provided 50% protection in a mouse model. Rapid diagnostics have been developed for Strongyloides, Onchocerca and Loa loa infections using mutiplexed luciferase immunoprecitation assays (LIPS). Because Loa loa infections are in need of a more quantitative diagnostic, identification of new diagnostic targets have been undertaken by constructing new Loa cDNA libraries and performing EST generation. 5 candidate diagnostics have been identified and cloned into vectors suitable for LIPS.

使用噬菌体展示文库（来自感染性幼虫L3和成年女性）Brugia Malayi和带有已知可溶性人体受体的筛选，我们已经鉴定出，克隆和表征与人IL-5R，IL-10R和IL-13R结合的不同分子。 我们称为BM-IL5RBP MALAYI IL5RBINDING蛋白（IL5RBP）的分子以高水平表达，以保持其与人体受体结合的能力。 提出的预测免疫原性肽的抗体抑制了RBM-IL5BP与其人体受体的结合，并已用于（通过免疫EM）BM-IL5RBP与感染性阶段幼虫的表面进行定位。 RBM-IL5BP本身并不能延长人嗜酸性粒细胞的存活，而是抑制了人IL5延长嗜酸性粒细胞生存的能力。 B. Malayi HSP12.6（BMHSP12.6）已被鉴定并表征为与人IL-10R结合的分子。 BMHSP12.6的结构分析表明，它具有高度保守的α-晶状体中心结构域，该结构域是其他小型热休克蛋白（HSP）的特征，但由于。短的N末端结构域和侧面侧翼的异常小的C末端结构域似乎属于一类新型的小型HSP。重组BMHSP12.6以剂量依赖性方式与HUIL10R结合，并抑制人IL-10（HUIL10）与其受体的结合。 RBMHSP12.6还增强了与Huil10相似的MC/9肥大细胞的生长和增殖，这表明该寄生虫会积极合成一个可能调节人类免疫反应的分子。 有趣的是，我们还确定了Brugia Malayi L3的分泌产品，该产品对人嗜酸性粒细胞是趋化性的。该分泌的产品通过G偶联受体CXCR3发出信号。 我们使用了一种分子方法来确定地表明，fialrial寄生虫曼森氏菌（MP）包含沃尔巴奇的内共生体。使用已知的引物来扩增其他丝状wolbachiae的16S核糖体DNA，发现一个相同的1393bp频段发现，在序列分析中，MP Wolbachia 16S rDNA的单个共识序列与其他丝状细胞生成的wolbachia序列最相似Wolbachia 16s rDNA表明，沃尔巴奇（Wolbachia）几乎跟踪到其寄生虫宿主的5s rRNA。从含MP的全血中提取的蛋白质中也证明了Wolbachia表面蛋白（WSP）。在对MP的治疗试验之前，开发了MP Wolbachia的定量方法，并用来证明微手至沃尔巴氏菌拷贝数之间的关系，还可以证明抗生素对沃尔巴基亚骑士MP的影响。 作为许多工人财团的一部分，我们一直是过去一年中发表在草稿中的丝状基因组注释的一部分。 还使用质量光谱镜与整个阶段特异性的胰蛋白酶消化同时完成了单独的蛋白质组学分析。 不仅针对成年男性，成年女性，微毛虫和感染性幼虫完成了阶段的特异性蛋白质组织，而且我们还完成了Brugia Wolbachia蛋白质组和排泄/分泌蛋白质组。 这些分析提供了明确的证据，表明那些编码“假设蛋白”的基因是真实的，并且还确定了所有预测的开放式阅读框架的近70％。此外，分泌组的分析揭示了意外的分泌蛋白，其中许多具有宿主免疫调节特性。 在丝状感染和强层中，正在扩大保护性免疫的方法。 抗原亲和力使用来自用辐照幼虫免疫的两只小鼠的血清纯化，这些幼虫显示出接近100％的保护，以挑战强链链球菌或人类对SS感染的免疫，已经鉴定了6种在大肠杆菌，酵母菌和杆菌病毒中表达的候选者。 用一个大肠杆菌的重组测试在小鼠模型中提供了50％的保护。 使用静态的荧光素酶免疫沉淀试验（LIPS）开发了针对强层，OnChocerca和LOA LOA感染的快速诊断。 由于LOA LOA感染需要更定量的诊断，因此已经通过构建新的LOA cDNA库并进行EST Generation来实现了新的诊断目标的识别。 5个候选诊断已被鉴定并克隆到适合嘴唇的矢量中。