利用荧光激活细胞分选技术分析持留菌生理学特性。
Analyzing Persister Physiology with Fluorescence-Activated Cell Sorting.
作者信息
Orman Mehmet A, Henry Theresa C, DeCoste Christina J, Brynildsen Mark P
机构信息
Department of Chemical and Biological Engineering, Princeton University, 205 Hoyt Laboratory, Princeton, NJ, 08544, USA.
Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
出版信息
Methods Mol Biol. 2016;1333:83-100. doi: 10.1007/978-1-4939-2854-5_8.
Bacterial persisters are phenotypic variants that exhibit an impressive ability to tolerate antibiotics. Persisters are hypothesized to cause relapse infections, and therefore, understanding their physiology may lead to novel therapeutics to treat recalcitrant infections. However, persisters have yet to be isolated due to their low abundance, transient nature, and similarity to the more highly abundant viable but non-culturable cells (VBNCs), resulting in limited knowledge of their phenotypic state. This technical hurdle has been addressed through the use of fluorescence-activated cell sorting (FACS) and quantification of persister levels in the resulting sorted fractions. These assays provide persister phenotype distributions, which can be compared to the phenotype distributions of the entire population, and can also be used to examine persister heterogeneity. Here, we describe two detailed protocols for analysis of persister physiology with FACS. One protocol assays the metabolic state of persisters using a fluorescent metabolic stain, whereas the other assays the growth state of persisters with use of a fluorescent protein.
细菌持留菌是一种表型变异体，具有惊人的抗生素耐受能力。据推测，持留菌会导致感染复发，因此，了解它们的生理学特性可能会带来治疗顽固性感染的新疗法。然而，由于持留菌丰度低、性质短暂，且与数量更多的活的但不可培养细胞（VBNCs）相似，尚未被分离出来，导致对其表型状态的了解有限。通过使用荧光激活细胞分选（FACS）和对分选后的组分中持留菌水平进行定量，解决了这一技术障碍。这些检测提供了持留菌表型分布，可与整个群体的表型分布进行比较，也可用于检查持留菌的异质性。在这里，我们描述了两种使用FACS分析持留菌生理学的详细方案。一种方案使用荧光代谢染料检测持留菌的代谢状态，而另一种方案则使用荧光蛋白检测持留菌的生长状态。
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