Antifungal chemical compounds identified using a C. elegans pathogenicity assay.
Antifungal chemical compounds identified using a C. elegans pathogenicity assay.
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DOI：
10.1371/journal.ppat.0030018
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发表时间：
2007-02
期刊：
影响因子：
6.7
通讯作者：
Mylonakis E
中科院分区：
文献类型：
--
作者：
Breger J;Fuchs BB;Aperis G;Moy TI;Ausubel FM;Mylonakis E
迫切需要开发新的抗真菌药物。一个简单的体内模型，评估化学化合物的文库，可以解决目前抗真菌发现的一些主要障碍。我们发现白色念珠菌，以及其他念珠菌种类，被秀丽隐杆线虫摄入，并在秀丽隐杆线虫肠道中建立持久的致命感染。重要的是，哺乳动物中念珠菌发病的关键成分，如丝的形成，也参与了线虫的杀死。我们设计了一种假丝酵母介导的秀丽隐杆线虫实验，该实验允许高通量体内筛选化学文库的抗真菌活性，同时同步筛选有毒化合物。该试验采用标准96孔板技术在液体培养基中进行，并允许研究非浮游形式的白色念珠菌。在对1,266种已知药物活性化合物的筛选中，发现15种（约1.2%）延长了白色念珠菌感染的线虫的存活时间，并抑制了白色念珠菌的体内丝化。在筛选中发现的两种化合物，咖啡酸苯乙酯（蜜蜂蜂胶的主要活性成分）和氟喹诺酮类药物依诺沙星在小鼠念珠菌病模型中显示出抗真菌活性。全动物秀丽隐杆线虫实验可能有助于研究白色念珠菌发病机制的分子基础，并鉴定出体外筛选真菌生长目标很可能无法鉴定出的抗真菌化合物。在筛选中发现的影响念珠菌体内毒力的化合物可以潜在地用作“探针化合物”，并且可能对其他真菌具有抗真菌活性。念珠菌属是医院感染的最重要原因之一，播散性念珠菌病的可归因死亡率仍然超过25%。因此，我们开发了一种液体培养基试验，使用模型线虫秀丽隐杆线虫作为假丝酵母菌感染的模式生物。蠕虫在固体培养基草坪上感染，然后转移到无病原体的液体培养基上。除非在孔中添加抗真菌化合物，否则大多数蠕虫会在3-4天内死亡。该模型与人类感染过程类似，因为念珠菌细胞能够产生细丝，这对人类感染过程至关重要。我们使用该病原体模型创建了一个半自动化的高通量筛选，使用秀丽隐杆线虫来评估许多类型的化合物的抗真菌效果。通过这个过程，我们已经确定了三种化合物，我们显示它们在秀丽隐杆线虫，体外和小鼠中具有不同程度的抗真菌活性。
There is an urgent need for the development of new antifungal agents. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery. We show that Candida albicans, as well as other Candida species, are ingested by Caenorhabditis elegans and establish a persistent lethal infection in the C. elegans intestinal track. Importantly, key components of Candida pathogenesis in mammals, such as filament formation, are also involved in nematode killing. We devised a Candida-mediated C. elegans assay that allows high-throughput in vivo screening of chemical libraries for antifungal activities, while synchronously screening against toxic compounds. The assay is performed in liquid media using standard 96-well plate technology and allows the study of C. albicans in non-planktonic form. A screen of 1,266 compounds with known pharmaceutical activities identified 15 (∼1.2%) that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Two compounds identified in the screen, caffeic acid phenethyl ester, a major active component of honeybee propolis, and the fluoroquinolone agent enoxacin exhibited antifungal activity in a murine model of candidiasis. The whole-animal C. elegans assay may help to study the molecular basis of C. albicans pathogenesis and identify antifungal compounds that most likely would not be identified by in vitro screens that target fungal growth. Compounds identified in the screen that affect the virulence of Candida in vivo can potentially be used as “probe compounds” and may have antifungal activity against other fungi. Candida spp. are among the most significant causes of nosocomial infections, and disseminated candidiasis continues to have an attributable mortality rate of over 25%. For this reason, we have developed a liquid media assay using the model nematode Caenorhabditis elegans as a model organism for Candida infection. The worms are infected on solid media lawns and then moved to pathogen-free liquid media. Unless antifungal compounds are added to the wells, the majority of worms die within 3–4 d. This model is similar to the infection process in humans, in that Candida cells are able to produce filaments, which are essential for the infection process in humans. We used this pathogen model to create a semi-automated, high-throughput screen using C. elegans to evaluate the antifungal effectiveness of many types of chemical compounds. Through this process, we have identified three compounds that we show have varying degrees of antifungal activity in C. elegans, in vitro, and in mice.
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