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: 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
• 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

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.

在过去的一年里，我们已经完全填补了Loa loa，W。bancrofti和S.粪虫我们现在有这些生物的端粒到端粒的基因组数据。我们利用基因组数据作为背景进行了大量的蛋白质组学和转录组学研究。 为了更好地理解强调蚊子衍生的感染期幼虫（L3）到哺乳动物适应的L3和蜕皮后到L4之间的过渡以及适应人类宿主的最初一周的发育程序，我们适应了允许L3发育和随后蜕皮到L4的体外系统。 通过这9天的过程多次使用微阵列和蛋白质组学评估，我们不仅鉴定了对L3/L4转变至关重要的那些基因/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（关键CPL）证明了半胱氨酸蛋白酶在哺乳动物适应性L3至L4的早期发育中所起的关键作用。 我们最近对确定的丝虫感染患者的血清、Loa loa微丝蚴的排泄/分泌（E/S）产物、O. vovlulus蠕虫和适当的对照以鉴定活性感染的寄生虫来源的生物标志物。 这导致了分子靶点的鉴定，这些分子靶点已被用于配置用于快速检测O.肠扭转和Loa loa。 我们已经利用信息管道来识别广州管圆线虫、Loa loa、班氏吴策线虫、粪类圆线虫、T.cruzi和各种血吸虫属的基因组内的重复序列。 我们已经为这些已识别的靶标中的每一种配置了qPCR检测试剂盒，并提高了经验证的检测试剂盒的检测限。 其中一些已被证明是有用的，这些感染的诊断在适当的患者样本。 从B中鉴定出我们称为马来丝虫IL-5受体（IL-5 R）结合蛋白（BmIL 5 Rbp;也称为Bm 8757）的分子。马来蠕虫并发现竞争性抑制人白细胞介素-5（IL-5）与其人受体的结合。在表达和纯化重组BmIL 5 Rbp并制备BmIL 5 Rbp特异性兔抗体后，我们将该分子定位于B。马来蠕虫通过免疫组织化学和免疫电镜。RNA干扰（RNAi）抑制BmIL 5 Rbp mRNA和蛋白的产生。BmIL 5 Rbp定位于马来丝虫的表皮，并在其排泄/分泌产物中释放。RNAi抑制BmIL 5 Rbp mRNA的表达33%，表面蛋白的表达减少50%，并抑制排泄/分泌产物中BmIL 5 Rbp的释放。利用RNAi技术成功地抑制了B早期幼虫BmIL 5 Rbp的mRNA和蛋白表达。malayi，并提供了局部抑制人IL-5 R的原理证明。这些发现提供了证据表明，寄生虫编码的IL-5 R拮抗剂可以局部抑制嗜酸性粒细胞上IL-5的重要宿主先天免疫激活。