Clementi Emily A, Marks Laura R, Roche-Håkansson Hazeline, Håkansson Anders P
Department of Microbiology and Immunology, University at Buffalo, State University of New York.
Department of Microbiology and Immunology, University at Buffalo, State University of New York; Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, State University of New York; New York State Center of Excellence in Bioinformatics and Life Sciences, University at Buffalo, State University of New York;
J Vis Exp. 2014 Feb 17(84):e51008. doi: 10.3791/51008.
Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca(2+), K(+), and H(+), respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.
膜去极化和离子通量是在生物系统中得到广泛研究的事件，因为它们能够深刻影响细胞功能，包括能量代谢和信号转导。虽然荧光和电生理方法，包括电极使用和膜片钳技术，已经在真核细胞中得到很好的发展，用于测量这些事件，但由于微生物体积小，加上细菌更复杂的外表面屏蔽了细胞膜，因此开发用于测量微生物中类似事件的方法更具挑战性。在我们对肺炎链球菌（肺炎球菌）死亡起始的研究中，我们想阐明膜事件的作用，包括极性、完整性和细胞内离子浓度的变化。在查阅文献时，我们发现相关研究非常少。其他研究人员监测了放射性同位素的摄取或平衡来测量离子通量和膜电位，并且有少数研究，主要是在革兰氏阴性菌中，使用羰花青或恶嗪荧光染料测量膜电位取得了一些成功，或者用离子敏感荧光指示剂染料的细胞渗透性乙酰氧基甲基（AM）酯形式加载细菌。因此，我们建立并优化了用于测量革兰氏阳性菌肺炎链球菌膜电位、破裂和离子转运的方案。我们开发了使用双恶嗪染料DiBAC4(3)和细胞不可渗透染料碘化丙啶分别测量膜去极化和破裂的方案，以及用比率染料Fura-2、PBFI和BCECF的AM酯最佳加载肺炎球菌的方法，以分别使用荧光检测微孔板读数仪检测细胞内Ca(2+)、K(+)和H(+)浓度的变化。这些方案是针对肺炎球菌的首创方案，并且这些染料中的大多数尚未在任何其他细菌物种中使用。虽然我们的方案已针对肺炎链球菌进行了优化，但我们相信这些方法应该为其他细菌物种的类似研究提供一个很好的起点。
