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 和成年雌性）马来丝虫并用已知的可溶性人类受体进行筛选，我们已经鉴定、克隆和表征了与人类 IL-5R、IL-10R 和 IL-13R 结合的不同分子。 我们称之为 Bm-IL5RBP 马来布鲁虫 IL5R 结合蛋白 (IL5Rbp) 的分子已以高水平表达，并保留了与人类受体结合的能力。 针对预测的免疫原性肽产生的抗体抑制 rBm-IL5BP 与其人类受体的结合，并已用于将 Bm-IL5RBP 定位（通过免疫 EM）至感染期幼虫的表面。 rBm-IL5BP本身并不延长人嗜酸性粒细胞的存活，但确实抑制人IL5延长嗜酸性粒细胞存活的能力。 马来芽孢杆菌 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-晶状体蛋白中心结构域，这是其他小热休克蛋白 (HSP) 的特征，但由于 .晶状体蛋白结构域两侧的短 N 端结构域和异常小的 C 端结构域似乎属于一类新型的小 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。与 huIL10 类似，rBmHSP12.6 在体外也增强了 MC/9 肥大细胞的生长和增殖，表明这种寄生虫主动合成可能调节人类免疫反应的分子。 有趣的是，我们还鉴定出了马来丝虫 L3 的一种分泌产物，它对人类嗜酸性粒细胞具有趋化作用。该分泌产物通过 G 偶联受体 CXCR3 发出信号。 我们使用分子方法明确地证明了原寄生虫曼森氏菌（Mp）含有内共生体沃尔巴克氏体。使用已知的引物扩增其他丝虫沃尔巴克氏体的 16S 核糖体 DNA，发现了相同的 1393bp 带，序列分析表明 Mp 沃尔巴克氏体 16S rDNA 具有单一共有序列，该序列与其他丝虫线虫的沃尔巴克氏体序列最相似。系统发育树状图，检查Mp Wolbachia 与其他 Wolbachia 16S rDNA 的关系表明沃尔巴克氏体追踪的 5S rRNA 与其寄生虫宿主几乎相同。从含 Mp 的全血中提取的蛋白质中也发现了沃尔巴克氏体表面蛋白 (WSP)。在 Mp 治疗试验之前，开发了一种 Mp 沃尔巴克氏体定量方法，不仅用于证明微丝蚴数量与沃尔巴克氏体拷贝数之间的关系，还用于证明抗生素消除 Mp 沃尔巴克氏体的效果。 作为众多工作人员组成的联盟的一部分，我们参与了去年发布的丝虫基因组注释草稿。 使用质谱与全阶段特异性胰蛋白酶消化串联完成了单独的蛋白质组分析。 我们不仅完成了成年男性、成年女性、微丝蚴和感染性幼虫的阶段特异性蛋白质组，而且还完成了布鲁氏菌沃尔巴克氏体蛋白质组和排泄/分泌蛋白质组。 这些分析提供了明确的证据，证明那些编码“假设蛋白质”的基因是真实的，并且还鉴定了所有预测开放阅读框中近 70% 的基因。此外，分泌组分析揭示了意想不到的分泌蛋白，其中许多具有宿主免疫调节特性。 针对丝虫感染和类圆线虫的保护性免疫的扩展方法正在进行中。 使用来自受辐射幼虫免疫的小鼠（对类圆线虫感染具有接近 100% 的保护）或对类圆线虫感染具有免疫力的人类的血清进行抗原亲和纯化，已鉴定出 6 个在大肠杆菌、酵母和杆状病毒中表达的候选物。 使用一种源自大肠杆菌的重组体进行测试，在小鼠模型中提供了 50% 的保护。 使用多重荧光素酶免疫沉淀测定 (LIPS) 已开发出针对类圆线虫、盘尾丝虫和罗阿罗阿虫感染的快速诊断方法。 由于 Loa Loa 感染需要更加定量的诊断，因此通过构建新的 Loa cDNA 文库和进行 EST 生成来鉴定新的诊断靶点。 5 种候选诊断已被鉴定并克隆到适合 LIPS 的载体中。