Molecular Definition Of Filarial And Related Nonfilarial Genes And Proteins

在过去的一年里，我们已经完全弥合了Loa Loa，W.bancrofti和S.stercoralis的基因组差距。我们现在有了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和转录组研究。 为了更好地了解强调蚊子来源的感染期幼虫(L3)在蜕皮后转变为哺乳动物适应的L3和L4，以及适应人类宿主的最初一周的发育程序，我们采用了一个允许L3发育和随后的蜕皮到L4的体外系统。通过9天的多次芯片和蛋白质组学评估，我们不仅确定了对L3/L4转换至关重要的基因/途径，而且通过药物抑制(半胱氨酸蛋白酶抑制)和RNAi(关键CPL的)，我们也证明了半胱氨酸蛋白酶在哺乳动物适应L3到L4的早期发育中所起的关键作用。 我们最近对明确的丝虫感染患者的血清、微丝虫的排泄/分泌产物(E/S)、沃氏原虫的所有阶段以及适当的对照进行了鸟枪质谱分析，以确定活动性感染的寄生虫来源的生物标志物。这导致了分子靶标的确定，这些分子靶标已经被用来配置定量免疫分析，以快速检测螺旋藻和Loa Loa的活动性感染。 我们已经利用信息学管道识别了广州管圆线虫、Loa Loa、五味子、斯特氏管圆线虫、克鲁兹线虫和各种血吸虫基因组中随机和/或散布的重复序列。我们已经为这些识别的目标中的每一个配置了定量聚合酶链式反应分析，并改进了验证分析中的检测极限。其中一些已被证明对在适当的患者样本中诊断这些感染是有用的。 从马来丝虫中鉴定出一种被我们称为马来丝虫IL-5受体结合蛋白(BmIL5RBP；又称Bm8757)的分子，它能竞争性地抑制人白介素5(IL-5)与其受体的结合。在表达和纯化重组BmIL5RBP并制备BmIL5RBP特异性兔抗体后，我们通过免疫组织化学和免疫电子显微镜对BmIL5RBP分子在马来丝虫上进行了定位。采用RNA干扰技术(RNAi)抑制BmIL5RBP基因和蛋白的表达。BmIL5RBP定位于马来丝虫的角质层，并在其排泄/分泌产物中释放。RNAi抑制BmIL5RBP基因的表达达33%，表面蛋白表达减少50%，并抑制BmIL5RBP在排泄/分泌产物中的释放。利用RNAi技术成功地下调了马来丝虫早期幼虫BmIL5RbPmRNA和蛋白的表达，为局部抑制人IL-5R提供了理论依据。这些发现提供了证据，证明寄生虫编码的IL-5R拮抗剂可能局部抑制重要宿主对嗜酸性粒细胞上IL-5的先天免疫激活。

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.