Flow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages
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Author:
Date:
2018-02-20
[Abstract] Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb constitutively expressing mCherry and then metabolically label them with Bodipy-palmitate. Following the labeling procedure, we isolate Mtb-containing phagosomes on a sucrose cushion and disrupt the phagosomes with detergent. After extensive washes, the isolated bacteria are analyzed by flow cytometry to determine the level of Bodipy-palmitate signal associated with the bacteria. Using a Mtb mutant strain defective in fatty acid uptake in liquid culture we ...
[摘要] 结核分枝杆菌(Mtb)已经发展为从其宿主吸收脂肪酸。然而,直到最近,还没有可靠的方法来量化宿主细胞感染期间细菌对脂肪酸的摄取。在这里,我们描述了一种新的方法来量化细胞内杆菌对脂肪酸的摄取。我们用Mtb组成性表达mCherry感染巨噬细胞,然后用Bodipy-palmitate代谢标记它们。标记程序后,我们在蔗糖垫上分离含有Mtb的吞噬体,并用去污剂破坏吞噬体。大量洗涤后,通过流式细胞术分析分离的细菌以确定与细菌相关的Bodipy-棕榈酸酯信号的水平。使用液体培养物中脂肪酸摄取缺陷的Mtb突变株,我们确定该突变体在巨噬细胞感染期间同化比野生型菌株少10倍的Bodipy-棕榈酸酯。脂肪酸摄取的这种定量方法可用于进一步鉴定参与细胞内Mtb和可能的其他细菌的脂质摄取的途径。
【背景】结核分枝杆菌(Mtb)同化宿主来源的脂质(脂肪酸和胆固醇)的能力使得病原体能够在其宿主内存活(Russell等人,2010; Lovewell 等人,2016)。在小鼠感染期间和在人肺组织中,通过巨噬细胞内的Mtb上调胆固醇和脂肪酸代谢相关基因来支持该想法(Schnappinger等人,2003; Rachman等人,2006; Rohde等人,2007;Fontán等人,2008; Tailleux等人,2008; Homolka et al。,2010; Rohde et ...
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Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2
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Author:
Date:
2018-01-05
[Abstract] Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the de-protonated state at physiological pH rendering it susceptible to ROS-mediated oxidation. Direct and indirect techniques for detection of PTP oxidation have been developed (Karisch and Neel, 2013). To detect catalytically active PTPs, cell lysates are treated with iodoacetyl-polyethylene glycol-biotin (IAP-biotin), which irreversibly binds to reduced (S-) cysteine thiols. Irreversible oxidation of SHP-1 after treatment of cells with ...
[摘要] 细胞活性氧(ROS)对半胱氨酸依赖性蛋白酪氨酸磷酸酶(PTP)的氧化失活在调节多种细胞类型的信号转导中起关键作用。大多数PTP的磷酸酶活性取决于催化结构域内的“标记”半胱氨酸残基,其在生理pH下保持质子化状态,使其易受ROS介导的氧化。已经开发了用于检测PTP氧化的直接和间接技术(Karisch和Neel,2013)。为了检测催化活性的PTP,用碘乙酰 - 聚乙二醇 - 生物素(IAP-生物素)处理细胞裂解物,所述碘乙酰 - 聚乙二醇 - 生物素(IAP-生物素)不可逆地结合还原的(S-5)半胱氨酸硫醇。使用对磺酸(SO 3)特异性的抗体检测用过钒酸盐或H 2 O 2 2处理细胞后SHP-1的不可逆氧化, H)形式的PTP的保守的活性位点半胱氨酸。在该协议中,我们描述了用于检测造血PTP SHP的还原(S ; active)或不可逆氧化(SO 3 H;非活性)形式的方法-1,尽管这种方法适用于任何细胞类型中的任何半胱氨酸依赖性PTP。
【背景】活性氧(ROS)由细胞NADPH氧化酶和线粒体产生。大多数蛋白质酪氨酸磷酸酶(PTP)含有保守的催化半胱氨酸,其具有低的解离常数(pKa),其对ROS的氧化非常敏感(Rudyk和Eaton,2014)。 PTP的ROS失活在许多细胞类型中调节酪氨酸激酶介导的信号传导反应中起重要作用。在用ROS H 2 O ...
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