| 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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| Expression and Purification of the Cas10-Csm Complex from Staphylococci
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Author:
Date:
2017-06-05
[Abstract] CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a class of prokaryotic immune systems that degrade foreign nucleic acids in a sequence-specific manner. These systems rely upon ribonucleoprotein complexes composed of Cas nucleases and small CRISPR RNAs (crRNAs). Staphylococcus epidermidis and Staphylococcus aureus are bacterial residents on human skin that are also leading causes of antibiotic resistant infections (Lowy, 1998; National Nosocomial Infections Surveillance, 2004; Otto, 2009). Many staphylococci possess Type III-A CRISPR-Cas systems (Marraffini and Sontheimer, 2008; Cao et al., 2016), which have been shown to prevent plasmid transfer and protect against viral predators (Goldberg et al., ...
[摘要] CRISPR-Cas（聚集的定期间隔的短回文重复-CRISPR相关蛋白）是一类以序列特异性方式降解外来核酸的原核免疫系统。这些系统依赖于由Cas核酸酶和小型CRISPR RNA（crRNA）组成的核糖核蛋白复合物。表皮葡萄球菌和金黄色葡萄球菌是人皮肤上的细菌居民，也是抗生素抗性感染的主要原因（Lowy，1998; National Nosocomial Infection Surveillance，2004; Otto，2009） 。许多葡萄球菌具有III型A CRISPR-Cas系统（Marraffini和Sontheimer，2008; Cao等人，2016），已被证明可预防质粒转移并防止病毒性捕食者（Goldberg这些生物体中，等等，2014; Hatoum-Aslan等人，2014; Samai等人，2015）。因此，在天然葡萄球菌背景中获得对这些系统的机械理解可以导致对影响这些病原体的进化和存活的因素的重要见解。 III-A型CRISPR-Cas系统编码称为Cas10-Csm的五亚单位效应复合物（Hatoum-Aslan等人，2013）。在这里，我们描述了一种用于表达和纯化Cas10-Csm的方法。表皮样背景或异源性S。金黄色葡萄球菌背景。该方法由两步纯化方案组成，包括Ni 2 + - ...
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