Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping.
Expanding the toolbox for Trypanosoma cruzi: A parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping.
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DOI：
10.1371/journal.pntd.0006388
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发表时间：
2018-04
影响因子：
3.8
通讯作者：
Taylor MC
中科院分区：
文献类型：
--
作者：
Costa FC;Francisco AF;Jayawardhana S;Calderano SG;Lewis MD;Olmo F;Beneke T;Gluenz E;Sunter J;Dean S;Kelly JM;Taylor MC
克氏锥虫感染导致南美锥虫病，这是整个拉丁美洲的一个主要公共卫生问题。没有疫苗，唯一的药物有严重的副作用。我们对寄生虫生物学和疾病发病机制的理解有限，阻碍了产生新疗法的努力。研究受到疾病的复杂性，感染的长期性以及在慢性阶段几乎无法检测到寄生虫的事实的影响。此外，还对T. cruzi生物学受到适用于这种寄生虫的基因操作技术的有限灵活性的限制。在这里，我们描述了两项技术创新，这将使寄生虫在疾病进展中的作用得到更好的评估。首先，我们生成了一个T。cruzi报道菌株，其表达包含红移荧光素酶和绿色荧光蛋白结构域的融合蛋白。生物发光允许在单个动物内跟踪感染的动力学，并在切除的组织中精确定位特定的感染灶。然后，荧光可用于可视化组织切片中的单个寄生虫，以在细胞水平上研究宿主-寄生虫相互作用。使用这种策略，我们已经能够定期发现慢性感染的小鼠组织内的单个寄生虫首次。第二个进展是将精简的CRISPR/Cas9功能整合到这种报告菌株中，可以使用基于PCR的方法促进基因组编辑，而不需要DNA克隆。该系统允许在可以快速评估体内表型的背景中快速产生无效突变体和荧光标记的寄生虫。这里所描述的技术将有多种应用研究方面的T。cruzi生物学和查加斯病的发病机制以前无法用常规方法。这些试剂和细胞系是作为社区资源生成的，可应要求免费提供。5-8拉丁美洲有200万人感染了单细胞寄生虫克氏锥虫，这是南美锥虫病的病原体。其中，约三分之一将发展为慢性疾病病理，导致残疾和过早死亡。只有两种药物可用，这两种药物都有严重的副作用。我们对这种感染的慢性阶段知之甚少，因为寄生虫负担极低，动物模型也有局限性。在这里，我们描述了一个T。Cruzi报道菌株，其已被遗传修饰以表达既是生物发光的又是荧光的融合蛋白。这些寄生虫可以在整个感染过程中进行监测，并在感染小鼠的组织切片中鉴定出单个寄生虫。这使我们能够第一次在感染的慢性阶段在细胞水平上分析宿主-寄生虫相互作用。我们还将CRISPR/Cas9基因组编辑系统的简化版本整合到报告菌株中。我们通过在鞭毛附着蛋白GP 72中产生无效突变体，通过用红色荧光蛋白替换绿色荧光蛋白，以及通过标记内源性蛋白DNA拓扑异构酶1A，证明了该系统的实用性。这个T cruzi报告基因将显著增强我们揭示慢性恰加斯病的发病机制和免疫学的能力。
Infection with Trypanosoma cruzi causes Chagas disease, a major public health problem throughout Latin America. There is no vaccine and the only drugs have severe side effects. Efforts to generate new therapies are hampered by limitations in our understanding of parasite biology and disease pathogenesis. Studies are compromised by the complexity of the disease, the long-term nature of the infection, and the fact that parasites are barely detectable during the chronic stage. In addition, functional dissection of T. cruzi biology has been restricted by the limited flexibility of the genetic manipulation technology applicable to this parasite. Here, we describe two technical innovations, which will allow the role of the parasite in disease progression to be better assessed. First, we generated a T. cruzi reporter strain that expresses a fusion protein comprising red-shifted luciferase and green fluorescent protein domains. Bioluminescence allows the kinetics of infection to be followed within a single animal, and specific foci of infection to be pinpointed in excised tissues. Fluorescence can then be used to visualise individual parasites in tissue sections to study host-parasite interactions at a cellular level. Using this strategy, we have been routinely able to find individual parasites within chronically infected murine tissues for the first time. The second advance is the incorporation of a streamlined CRISPR/Cas9 functionality into this reporter strain that can facilitate genome editing using a PCR-based approach that does not require DNA cloning. This system allows the rapid generation of null mutants and fluorescently tagged parasites in a background where the in vivo phenotype can be rapidly assessed. The techniques described here will have multiple applications for studying aspects of T. cruzi biology and Chagas disease pathogenesis previously inaccessible to conventional approaches. The reagents and cell lines have been generated as a community resource and are freely available on request. 5–8 million people in Latin America are infected with the single-cell parasite Trypanosoma cruzi, the causative agent of Chagas disease. Of these, approximately one-third will develop chronic disease pathology, leading to disability and premature death. Only two drugs are available, both of which can have severe side effects. We know relatively little about the chronic phase of this infection because the parasite burden is extremely low, and animal models have limitations. Here, we describe a T. cruzi reporter strain that has been genetically modified to express a fusion protein which is both bioluminescent and fluorescent. These parasites can be monitored throughout the infection, and individual parasites identified in tissue sections from infected mice. This allows us for the first time to analyse host-parasite interactions at a cellular level in the chronic phase of infection. We have also incorporated a streamlined version of the CRISPR/Cas9 genome editing system into the reporter strain. We demonstrated the utility of this system by generating null mutants in the flagellar attachment protein GP72, by replacing the green fluorescent protein with a red fluorescent protein, and by tagging the endogenous protein DNA topoisomerase 1A. This T. cruzi reporter line will significantly enhance our ability to unravel the pathogenesis and immunology of chronic Chagas disease.
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免疫主导锥虫表面糖蛋白的删除破坏鞭毛 - 细胞粘附。
DOI：
10.1083/jcb.122.1.149
发表时间：
1993-07
期刊：
The Journal of cell biology
影响因子：
--
作者：
Cooper R;de Jesus AR;Cross GA
通讯作者：
Cross GA
影响因子：
1.5
作者：
Elias, MCQB;Marques-Porto, R;Schenkman, S
通讯作者：
Schenkman, S
影响因子：
3.4
作者：
Lewis, Michael D.;Francisco, Amanda Fortes;Kelly, John M.
通讯作者：
Kelly, John M.
影响因子：
3.8
作者：
McLatchie, Alex P.;Burrell-Saward, Hollie;Taylor, Martin C.
通讯作者：
Taylor, Martin C.
影响因子：
2.9
作者：
Branchini, BR;Southworth, TL;Roda, A
通讯作者：
Roda, A