High-throughput nano-biofilm microarray for antifungal drug discovery.
High-throughput nano-biofilm microarray for antifungal drug discovery.
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DOI：
10.1128/mbio.00331-13
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发表时间：
2013-06-25
期刊：
影响因子：
6.4
通讯作者：
Ramasubramanian AK
中科院分区：
文献类型：
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作者：
Srinivasan A;Leung KP;Lopez-Ribot JL;Ramasubramanian AK
微米和纳米技术已经从根本上改变了生物学研究从基因组学到组织工程，相对例外的微生物细胞培养，这仍然主要是在微量滴定板和培养皿中进行。在这里，我们提出了纳米级的机会致病真菌白念珠菌微阵列平台上的文化。微阵列由1，200个单独培养物组成，所述单独培养物为30 nl包封在惰性藻酸盐基质中的白色念珠菌生物膜（“纳米生物膜”）。我们证明，这些纳米生物膜是类似于传统的宏观生物膜在其形态，建筑，生长和表型特征。我们还证明，纳米生物膜微阵列是一个强大的和有效的工具，用于加速药物发现过程：（i）组合筛选对28种抗真菌化合物的集合中的免疫抑制剂FK 506（他克莫司）的存在下，确定了6种药物，显示协同抗真菌活性，和（ii）筛选对NCI挑战集小分子库确定了三个迄今未知的命中。这种基于细胞的微阵列平台允许微生物细胞培养的小型化，并与其他高通量筛选技术完全兼容。微生物通常仍然生长在培养皿，试管和锥形瓶中，尽管微型化的最新进展使其他相关研究领域受益，包括基因组学和蛋白质组学。小规模培养微生物在减少时间、成本和试剂使用方面特别有价值。本文介绍了发展，表征和应用纳米培养的机会致病真菌，白色念珠菌。尽管体积减少了2,000倍以上，但从纳米级培养物获得的生长特征和药物反应概况与行业标准相当。该平台还能够快速识别对白色念珠菌生物膜有效的新候选药物，这是医院获得性感染死亡的主要原因。
Micro- and nanoscale technologies have radically transformed biological research from genomics to tissue engineering, with the relative exception of microbial cell culture, which is still largely performed in microtiter plates and petri dishes. Here, we present nanoscale culture of the opportunistic fungal pathogen Candida albicans on a microarray platform. The microarray consists of 1,200 individual cultures of 30 nl of C. albicans biofilms (“nano-biofilms”) encapsulated in an inert alginate matrix. We demonstrate that these nano-biofilms are similar to conventional macroscopic biofilms in their morphological, architectural, growth, and phenotypic characteristics. We also demonstrate that the nano-biofilm microarray is a robust and efficient tool for accelerating the drug discovery process: (i) combinatorial screening against a collection of 28 antifungal compounds in the presence of immunosuppressant FK506 (tacrolimus) identified six drugs that showed synergistic antifungal activity, and (ii) screening against the NCI challenge set small-molecule library identified three heretofore-unknown hits. This cell-based microarray platform allows for miniaturization of microbial cell culture and is fully compatible with other high-throughput screening technologies. Microorganisms are typically still grown in petri dishes, test tubes, and Erlenmeyer flasks in spite of the latest advances in miniaturization that have benefitted other allied research fields, including genomics and proteomics. Culturing microorganisms in small scale can be particularly valuable in cutting down time, cost, and reagent usage. This paper describes the development, characterization, and application of nanoscale culture of an opportunistic fungal pathogen, Candida albicans. Despite a more than 2,000-fold reduction in volume, the growth characteristics and drug response profiles obtained from the nanoscale cultures were comparable to the industry standards. The platform also enabled rapid identification of new drug candidates that were effective against C. albicans biofilms, which are a major cause of mortality in hospital-acquired infections.