Intracellular Assays to Monitor Survival and Growth of Yersinia pestis Within Macrophages.
Intracellular Assays to Monitor Survival and Growth of Yersinia pestis Within Macrophages.
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监测巨噬细胞内鼠疫耶尔森氏菌存活和生长的细胞内测定。
DOI：
10.1007/978-1-4939-9541-7_13
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Lawrenz,MatthewB
中科院分区：
文献类型：
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作者：
Pulsifer,AmandaR;VanCleave,TivaT;Lawrenz,MatthewB
鼠疫耶尔森氏菌能够在巨噬细胞内生存和复制，同时也能够生活在宿主的细胞外环境中。有助于更好地理解 howY 的测定。鼠疫菌在细胞内存活并破坏正常宿主抗菌防御，需要监测细胞内的能力。鼠疫的生存和复制。本章将描述监测细胞内存活和复制的三种不同测定方法，以及量化和呈现所获得数据的公式和方法。这些测定对于回答有关哪些细菌因子对细胞内存活很重要的众多问题至关重要。此外，这些测定经过修改后可用于其他感兴趣的细胞内病原体。第一个讨论的测定是传统的细菌计数测定，它通过经典的菌落形成单位 (CFU) 测定直接量化细菌数量。通过 CFU 测定来量化细菌负荷，可以区分细胞内/细胞相关细菌和细胞外细菌。然而，CFU 测定很费力，不允许对细菌生长进行直接动力学监测，并且难以适应高通量测定。使用荧光素酶监测细菌数量的生物发光生物报告仪可以对细菌生长进行简单的、基于读板器的实时动力学监测，并且可适用于高通量技术。最后，我们将描述使用荧光生物报告仪的活细胞显微镜，它可以监测单个细胞中的细菌复制，并可以在细胞内感染期间可视化细菌和宿主蛋白之间的相互作用。
Yersinia pestisis able to survive and replicate within macrophages, while also being able to live in the extracellular milieu of the host. Assays that facilitate better understanding of howY. pestissurvives intracellularly and subverts normal host antimicrobial defenses require the ability to monitor intracellularY. pestissurvival and replication. In this chapter three different assays for monitoring intracellular survival and replication will be described, along with the formulas and methods to quantify and present the acquired data. These assays are fundamental to answering a multitude of questions pertaining to which bacterial factors are important for intracellular survival. Additionally, these assays can be used, with modifications, for other intracellular pathogens of interest. The first assay discussed will be the conventional bacterial enumeration assay, which quantifies bacterial numbers directly through a classic colony forming units (CFU) assay. Quantifying bacterial burden through CFU determination allows for differentiation between intracellular/cell-associated bacteria and extracellular bacteria. However, CFU determination is laborious, does not allow for direct kinetic monitoring of bacterial growth, and is difficult to adapt to high throughput assays. Bioluminescence bioreporters that use luciferase to monitor bacterial numbers allow for simple, plate reader-based, real-time kinetic monitoring of bacterial growth that is amendable to high throughput techniques. Finally, we will describe live cell microscopy using fluorescent bioreporters, which allows for monitoring of bacterial replication in individual cells and the possibility to visualize interactions between bacterial and host proteins during intracellular infection.