Harding Clare R, Schroeder Gunnar N, Collins James W, Frankel Gad
Center for Molecular Bacteriology and Infection, Imperial College London.
J Vis Exp. 2013 Nov 22(81):e50964. doi: 10.3791/50964.
Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.
嗜肺军团菌是一种名为军团病的严重肺炎的病原体，是一种重要的人类病原体，可在肺泡巨噬细胞内感染并复制。其毒力取决于Dot/Icm IV型分泌系统（T4SS），该系统对于建立一个允许复制的液泡（称为含军团菌液泡，LCV）至关重要。嗜肺军团菌感染可在小鼠中建模，然而大多数小鼠品系不敏感，这促使人们寻找新的感染模型。我们最近发现，大蜡螟幼虫适合用于研究嗜肺军团菌感染。大蜡螟越来越多地被用作人类病原体的感染模型，并且几种细菌在昆虫模型和哺乳动物模型中的毒力之间存在良好的相关性。幼虫免疫防御的一个关键组成部分是血细胞，即专业吞噬细胞，它们摄取并破坏入侵者。嗜肺军团菌能够在这些细胞内感染、形成LCV并进行复制。在这里，我们展示了在大蜡螟模型中分析嗜肺军团菌毒力的实验方案，包括如何培养有感染力的嗜肺军团菌、用抑制剂预处理幼虫、感染幼虫以及如何提取感染细胞进行定量和免疫荧光显微镜检查。我们还描述了如何在竞争试验中量化细菌复制和适应性。这些方法允许快速筛选突变体，以确定嗜肺军团菌毒力中重要的因素，描述了一种有助于我们理解这种复杂病原体的新工具。
