Molecular Definition Of Filarial And Related Nonfilarial Genes And Proteins

在完成了Loa Loa、W.bancrofti和(最近)O.volverus的基因组后，我们利用基因组数据作为进行大量蛋白质组和转录组研究的背景。我们已经完成了几乎所有主要哺乳动物阶段的大规模蛋白质组学和转录组学鉴定，结果鉴定了超过85%-90%的从稻瘟病菌基因组/推测的蛋白质组预测的产物。分析还获得了沃尔巴克氏菌的大部分蛋白质组，沃尔巴克氏菌是草鱼的专性内共生体。寄生虫性别和阶段特异性蛋白的表达确定了与寄生虫分化相关的那些途径。 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。 我们通过评估在阿苯达唑(ALB)治疗前以及治疗后3周和3个月从肯尼亚5个农村居民收集的粪便，研究了土壤传播蠕虫在改变肠道微生物群中所起的作用。有趣的是，与年龄匹配的对照组相比，无论是蛔虫还是美洲坏死菌的存在都没有显著改变微生物群的整体多样性，尽管在ALB治疗和清除土传蠕虫(STH)后，由梭状芽胞杆菌构成的微生物群的比例显著增加(而由肠杆菌属构成的比例减少)。治疗后丰富度也有显著下降，我们的数据表明，通过驱虫清除STH改变了肠道微生物区系。 我们已经生成了一个296兆碱基(Mb)的参考质量基因组，由17902个蛋白质编码基因组成，这些基因来自一条具有代表性的蛔虫，来自肯尼亚农村的60个人类宿主，那里的养猪业很少。值得注意的是，大多数人类分离株(63/68)拥有线粒体基因组，这些基因组更接近猪蛔虫而不是蛔虫。比较系统基因组学分析发现了1100多万个核编码的SNPs，但只有两种不同的遗传类型在所分析的基因组中发生了重组。核基因组具有广泛的杂合性，所有样品都以遗传马赛克的形式存在，并具有类猪链霉菌或类地线虫的遗传模式，支持高度杂交的蛔虫物种遗传复合体。由于似乎不存在人为传播这些杂交蠕虫的障碍，因此有必要采取一种单一健康的方法来控制人类蛔虫病的传播。 已经在盘尾丝虫病患者的血清中发现了卷曲棘球绦虫的microRNA，并发现了基于miRNA多态性的地区差异。

Having completed the genomes of Loa loa, W. bancrofti, and (most recently) O.volvulus, we have utilized the genomic data as the backdrop for performing a large number of proteomic and transcriptomic studies. We have completed a large-scale proteomic and transcriptomic characterization of almost all the major mammalian stages of O. volvulus, resulting in the identification of more than 85-90% of the products predicted from the O. volvulus genome/putative proteome. The analysis also yielded much of the proteome of Wolbachia, the obligate endosymbiont of O. volvulus. Parasite sex- and stage-specific protein expression identified those pathways related to parasite differentiation. 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 examined the role played by soil transmitted helminths in altering the intestinal microbiome by assessing stools collected from residents of 5 rural Kenyan villages prior to and 3 weeks and 3 months following albendazole (ALB) therapy. Interestingly,, the presence of neither Ascaris lumbricoides nor Necator americanus infection significantly altered the overall diversity of the microbiota in comparison with age-matched controls, although Following ALB therapy and clearance of soil-transmitted helminths (STH), there were significant increases in the proportion of the microbiota made up by Clostridiales ( and reductions in the proportion made up by Enterobacteriales. There was also a significant posttreatment decrease in richness, Our data suggest that clearance of STH through deworming alters the gut microbiota. We have generated a 296 megabase (Mb) reference quality genome comprised of 17902 protein-coding genes derived from a single, representative Ascaris worm collected from 60 human hosts in Kenyan villages where pig husbandry is rare. Notably, the majority of human isolates (63/68) possessed mitochondrial genomes that clustered closer to the pig parasite Ascaris suum than to A. lumbricoides. Comparative phylogenomic analyses identified over 11 million nuclear-encoded SNPs but just two distinct genetic types that had recombined across the genomes analysed. The nuclear genomes had extensive heterozygosity and all samples existed as genetic mosaics with either A. suum-like or A. lumbricoides-like inheritance patterns supporting a highly interbred Ascaris species genetic complex. As no barriers appear to exist for anthroponotic transmission of these hybrid worms, a one-health approach to control the spread of human ascariasis will be necessary. MicroRNA for O. volvulus have been identified in the sera of patients with onchocerciasis and regional differences based on miRNA polymorphisms have been identified.