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

基本信息

批准号：
10692025
负责人：
Thomas Nutman
金额：
$ 98.73万
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/10692025
关键词：
Angiostrongylus cantonensis Antibodies Ascaris Binding Binding Proteins Biological Assay Biological Markers Biological Process Brugia malayi Caspase Critical Pathways Culicidae Data Development Diagnosis Filaria bancrofti Gene Proteins Generations Genes Genome Genomics Goals Helminths Human Immunoassay Immunoelectron Microscopy Immunohistochemistry In Vitro Infection Informatics Interleukin-5 Intestinal Volvulus Knowledge Larva Life Cycle Stages Loa loa Mass Spectrum Analysis Membrane Proteins Messenger RNA Molecular Molecular Target Molting Nature Nematoda Organism Oryctolagus cuniculus Parasites Pathogenicity Patients Pharmacology Play Process Production Proteins Proteomics RNA Interference Recombinants Role Sampling Shotguns Strongyloides stercoralis System Therapeutic Intervention Time Trichocephalus trichiura Trypanosoma cruzi antagonist base detection limit diagnostic strategy eosinophil genomic data immune activation improved insight interleukin-5 receptor knock-down mRNA Expression parasitism programs protein expression rapid detection receptor telomere tool transcriptomics

项目摘要

In the past year we have closed fully genome gaps for Loa loa, W. bancrofti, and S. stercoralis. We now have telomere-to-telemere genomic data for these organisms. We have utilized the genomic data as the backdrop for performing a large number of proteomic and transcriptomic studies. To understand better the developmental programs that underscore the transition between the mosquito-derived infective stage larvae (L3) to mammalian adapted L3s and to L4s following a molt, and the initial week of adaptation to the human host, we adapted an in vitro system that allowed for L3 development and subsequent molting to the L4. Using microarray and proteomic assessments at multiple times through this 9 day process we have not only identified those genes/pathways that are critical for the L3/L4 transition but we have also demonstrated by both pharmacologic inhibition (cysteine protease inhibition) and RNAi (of the critical CPLs) the critical role played by cysteine proteases in the early development of mammalian adapted L3s to L4s. We have recently performed shotgun mass spectroscopy on both human sera of patients with defined filarial infections, excretory/secretory (E/S) products of Loa loa microfilariae, all stages of the O. vovlulus worm, and appropriate controls to identify parasite derived biomarkers of active infection. This has led to identification of molecular targets that have been used d to configure quantitative immunoassays for the rapid detection of active infection for O. volvulus and Loa loa. We have exploited informatic pipelines to identify tandomly and/or interspersed repeats within the genomes of Angiostrongylus cantonensis, Loa loa, Wuchereria bancrofti, Strongyloides stercoralis, T.cruzi, and the various Schistoma spp. We have configured qPCR assays for each of these identified targets and have improved the limits of detection in validated assays. Some of these have been shown to be useful for the diagnosis of these infections in appropriate patient samples. A molecule we termed Brugia malayi IL-5 receptor (IL-5R) binding protein (BmIL5Rbp; also known as Bm8757) was identified from B. malayi filarial worms and found to inhibit human interleukin-5 (IL-5) binding to its human receptor competitively. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference (RNAi) was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in its excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33%, reduced the surface protein expression by 50%, and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof of principle for the local inhibition of the human IL-5R. These findings provide evidence that a parasite-encoded IL-5R antagonist may locally inhibit a vital host innate immune activation of IL-5 on eosinophils.

在过去的一年中，我们缩小了Loa Loa，W。Bancrofti和S. Stercoralis的完全基因组差距。现在，我们拥有这些生物的端粒到telemere基因组数据。我们利用基因组数据作为进行大量蛋白质组学和转录组研究的背景。为了更好地理解强调蚊子衍生的感染性阶段幼虫（L3）对哺乳动物改编的L3和L4之间的过渡，以及对人类宿主的适应的最初一周，我们适应了一个体外系统，可以进行L3的开发，后来摩尔到L4。在这9天过程中，我们多次使用微阵列和蛋白质组学评估，我们不仅确定了那些对L3/L4过渡至关重要的基因/途径，而且我们还通过药理学抑制（半胱氨酸蛋白酶抑制）和RNAI（CPLS）（CPLS）（CPLS的关键作用）表现出了cysteine Protect in ll3 prodep n ll3 symamm insmamm sommamm sommamm sommamal n amamal nmamm s of n s ramamm insmamm insmamm insmamm insmamm insmamm insmamm insmamal s rabs insmall s rabs insmall s ryamal s ryabs。最近，我们对具有定义的丝状感染患者的两种人血清进行了shot弹枪质谱法，LOA LOA Microfilariae的排泄/分泌（E/S）产物，O. vovlulus蠕虫的所有阶段以及适当的控制措施，以识别活跃感染的寄生虫衍生的生物标志物。这导致鉴定了已用于配置定量免疫测定的分子靶标，以快速检测O. volvulus和Loa Loa的主动感染。我们已经利用了信息管道，以识别Agiostrongylus cantonensis，Loa Loa，Wuchereria bancrofti，strongyprofti，strongylodyloides stercoralis，t.cruzi，t.cruzi和各种Schistoma spp的基因组中的曲线和/或散布重复。我们已经为每个确定的目标配置了QPCR分析，并改善了经过验证的测定中的检测限制。其中一些已被证明可用于诊断适当的患者样品中这些感染。我们称为Brugia Malayi IL-5受体（IL-5R）结合蛋白（BMIL5RBP;也称为BM8757）的分子是从Malayi丝状蠕虫中鉴定出来的，并发现可以抑制人介毒物-5（IL-5）与其人类受体竞争的结合。在表达和纯化重组BMIL5RBP和BMIL5RBP特异性兔抗体的产生后，我们通过免疫组织化学和免疫电子显微镜将分子定位在马来虫蠕虫上。 RNA干扰（RNAi）用于抑制BMIL5RBP mRNA和蛋白质产生。 BMIL5RBP显示出定位于马来语的Brugia Malayi角质层，并将其排泄/分泌产品释放。 RNAi抑制BMIL5RBP mRNA的产生33％，将表面蛋白表达降低了50％，并抑制了排泄/分泌产物中BMIL5RBP的释放。 RNAi已成功地用于击倒Malayi早期幼虫阶段BMIL5RBP的mRNA和蛋白质表达，并为人类IL-5R的局部抑制提供了原理证明。这些发现提供了证据表明，寄生虫编码的IL-5R拮抗剂可能在局部抑制IL-5对嗜酸性粒细胞的宿主先天免疫激活。