| Phagolysosomal Trafficking Assay
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Author:
Date:
2014-07-05
[Abstract] Phagolysosomal trafficking is an important innate defense pathway that clears microbes by delivering them to lysosomes, the degradative compartment of the cell. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, subverts this host defense mechanism by arresting maturation of the phagosome. The ability of Mtb to arrest its delivery to the lysosome can be demonstrated by the prolonged co-localization of bacteria containing phagosomes/vacuole with early phagosomal markers [such as, Ras- related proteins in the brain 5 (Rab5) and Transferrin receptor (TfR)], and a failure to acquire late phagosomal and lysosomal markers (such as Rab7 and LAMP1) (Deretic and Fratti, 1999, Mehra et al., 2013). Here, a protocol is outlined for infection of macrophages with ...
[摘要] 吞噬溶酶体运输是重要的先天防御途径，通过将其递送至溶酶体，即细胞的降解区室来清除微生物。结核病的致病因子结核分枝杆菌（Mtb）通过阻滞吞噬体的成熟来破坏这种宿主防御机制。 Mtb阻止其递送至溶酶体的能力可以通过含有吞噬体/液泡的细菌与早期吞噬体标记物[例如，脑5中的Ras相关蛋白（Rab5）和转铁蛋白受体（TfR） ）]，以及未获得晚期吞噬体和溶酶体标记物（例如Rab7和LAMP1）（Deretic和Fratti，1999，Mehra等人，2013）。在这里，概述了用分枝杆菌物种感染巨噬细胞的方案，所述分枝杆菌物种如致病性Mtb疫苗菌株牛分枝杆菌 - 卡介苗（BCG）和快速分裂的非致病性耻垢分枝杆菌（Msmeg），然后间接免疫荧光显微镜观察宿主液泡标记。此后，通过使用数学工具处理细菌的二值图像来进行分枝杆菌和宿主液泡标记如TfR和LAMP1之间的共定位程度的自动定量。这导致直接在细菌/细菌簇周围的这些宿主标志物的平均荧光强度（MFI）的定量，相对于手动完成时具有增加的灵敏度。通过操纵宿主或病原体，该测定可用于评价细胞内运输的宿主或细菌决定簇。基本方法可以应用于研究其他细菌或颗粒状珠子的运输，尽管感染和吞噬体成熟的动力学将取决于吞噬性货物。数学分析工具可用于许多标准成像分析程序。然而，对于类似分析的任何适应性应由个体用户利用其成像和分析平台来确认。
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| Determination of Rifampicin-resistance Mutation Frequency and Analysis of Mutation Spectra in Mycobacteria
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Author:
Date:
2014-07-05
[Abstract] Understanding the genetic safeguarding mechanism of Mycobacterium tuberculosis (Mtb) may help us to explain i), how Mtb survive the genetic assaults elicited by both reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host macrophages and ii), why some strains of Mtb, e.g., Mtb strains from East Asian lineage and Beijing sublineage, exhibit high mutation rate and are more likely to acquire drug resistant mutations (e.g., rifampicin-resistance mutation) during infection. Mutation frequency analysis is a basic methods to study the genetic safeguarding mechanism. Moreover, to study the molecular mechanism of mutation, it is necessary to analyse the mutation spectrum (For example, oxidized cytosine may induce CG to TA mutation.). This ...
[摘要] 了解结核分枝杆菌（Mtb）的遗传保护机制可以帮助我们解释i）如何Mtb存活由活性氧（ROS）和活性氮（RNS）产生的遗传攻击 宿主巨噬细胞和ii），为什么一些来自东亚谱系和北京亚系的Mtb菌株（例如eg）具有高突变率，并且更可能获得耐药突变（例如，利福平 - 抗性突变）。 突变频率分析是研究遗传保护机制的基本方法。 此外，为了研究突变的分子机制，有必要分析突变谱（例如，氧化的胞嘧啶可能诱导CG到TA突变）。 该协议描述了确定耻骨分枝杆菌（Msm）和Mtb中的突变频率和了解突变谱的方法。
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| Analysis of Mycobacterial Protein Secretion
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Author:
Date:
2014-06-20
[Abstract] Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Analysis of proteins secreted by Mtb has been of interest to the field of tuberculosis research since certain secreted proteins interact with the host to promote virulence, while others may be important antigens or serve as biomarkers of infection. Here, we describe a protocol to prepare whole cell extracts (WCE) and short term culture filtrate (CF) from Mtb or the vaccine strain Mycobacterium bovis- bacillus Calmatte- Guérin (BCG) (Mehra et al., 2013). These are both slow growing mycobacteria, but the same basic procedure can easily be adapted to analyze secreted proteins from rapidly growing mycobacteria, such as Mycobacterium smegmatis (Msmeg), a non-pathogenic species commonly ...
[摘要] 结核分枝杆菌（Mtb）是结核病的致病因子。由Mtb分泌的蛋白质的分析已经对结核病研究领域感兴趣，因为某些分泌的蛋白质与宿主相互作用以促进毒力，而其他可能是重要的抗原或用作感染的生物标志物。在这里，我们描述了从Mtb或疫苗菌株牛分枝杆菌 - 卡介苗（BCG）制备全细胞提取物（WCE）和短期培养物滤液（CF）的方案（Mehra等人， et al。，2013）。这些都是缓慢生长的分枝杆菌，但是相同的基本程序可以容易地适于分析来自快速生长的分枝杆菌的分泌蛋白，例如耻垢分枝杆菌（Msmeg），其是实验室中常用的非致病物种。可以通过蛋白质印迹分析获得的级分，以检查感兴趣的蛋白质，或者如果抗体不可获得或通过质谱法检查整个分泌蛋白质组。所关注基因的遗传敲除突变体用作阴性对照。另外，应当在CF部分中评估胞质蛋白如分子伴侣GroEL或丙酮酸脱氢酶E2组分sucB（Rv2215/dlaT）的水平，以排除CF中的阳性信号是由于细菌裂解导致的可能性（参见图1）。通过改变菌株的生长条件，这种体外分泌测定法可用于检查改变分泌物组织的条件。我们感谢Magnus Stiegedal提供有关TCA（三氯乙酸）沉淀的有用提示。
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