Highly Sensitive In Vivo Imaging of Trypanosoma brucei Expressing "Red-Shifted" Luciferase
Highly Sensitive In Vivo Imaging of Trypanosoma brucei Expressing "Red-Shifted" Luciferase
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DOI：
10.1371/journal.pntd.0002571
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发表时间：
2013-11-01
影响因子：
3.8
通讯作者：
Taylor, Martin C.
中科院分区：
文献类型：
--
作者：
McLatchie, Alex P.;Burrell-Saward, Hollie;Taylor, Martin C.
背景：非洲人类锥虫病是由布氏锥虫复合体寄生虫感染引起的，威胁着撒哈拉以南非洲超过 7000 万人。新药的开发受到当前啮齿动物模型的局限性的阻碍，特别是对于第二阶段感染，这种感染一旦寄生虫进入中枢神经系统就会发生。表达萤火虫荧光素酶的病原体的生物发光成像（最大发射 562 nm）已在许多体内疾病模型中采用，以监测传播、药物治疗和免疫反应的作用。然而，检测波长低于 600 nm 的深部组织生物发光缺乏灵敏度，限制了体内成像在研究布氏锥虫和其他锥虫感染方面的广泛使用。 方法/主要发现：在这里，我们报告了一个系统，该系统可以在小鼠模型中检测不到 100 种生物发光的布氏锥虫寄生虫。作为记者，我们使用了密码子优化的红移荧光素酶 (PpyRE9H)，发射峰为 617 nm。将侧翼为上游 5'-变异表面糖蛋白非翻译区 (UTR) 和下游 3'-微管蛋白 UTR 的基因定向整合到 T. brucei 核糖体 DNA 基因座中后获得最大表达。在没有选择性药物的情况下，表达稳定至少 3 个月，并且与可检测的表型变化无关。可以实时监测寄生虫传播和药物疗效，并且可以轻松检测到脑部感染。体内灵敏度水平显着高于黄色萤火虫荧光素酶报告基因所达到的水平。结论/意义：此处描述的优化生物发光报告基因系将显着增强体内成像在小鼠模型中研究 II 期非洲锥虫病的应用。大大提高的敏感性为研究宿主-寄生虫关系提供了一个新的框架，特别是在中枢神经系统感染的情况下。它应该非常适合药物评价项目。
Background: Human African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids.Methodology/Principal findings: Here, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5'-variant surface glycoprotein untranslated region (UTR) and a downstream 3'-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter.Conclusions/Significance: The optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes.