Wiater L A, Dunn K, Maxfield F R, Shuman H A
Departments of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Infect Immun. 1998 Sep;66(9):4450-60. doi: 10.1128/IAI.66.9.4450-4460.1998.
During infection, the Legionnaires' disease bacterium, Legionella pneumophila, survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes. In order to understand the molecular basis of this organism's ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication. These mutations define a set of 20 genes (19 icm [for intracellular multiplication] genes and dotA [for defect in organelle trafficking]). In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L. pneumophila or one of several mutants defective in different icm genes or dotA. By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophila colocalized with host lysosomes prelabeled with rhodamine-dextran. Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection. These results indicate that several, and possibly all, of the icm and dotA gene products act at an early step during phagosome establishment to determine whether L. pneumophila-containing phagosomes will fuse with lysosomes. Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication.
在感染过程中，军团菌病细菌嗜肺军团菌在一个接近中性pH值且不与宿主溶酶体融合的特殊吞噬体内存活并繁殖。为了了解这种生物体控制其细胞内命运能力的分子基础，我们分离并鉴定了一组转座子产生的突变体，这些突变体无法杀死巨噬细胞，随后发现它们在细胞内繁殖方面存在缺陷。这些突变定义了一组20个基因（19个icm[细胞内繁殖]基因和dotA[细胞器运输缺陷]基因）。在本报告中，我们描述了一种吞噬体-溶酶体融合（PLF）的定量测定方法，以及它在测量感染了野生型嗜肺军团菌或不同icm基因或dotA缺陷的几种突变体之一的细胞中PLF水平的应用。通过使用定量共聚焦荧光显微镜，可以通过确定荧光素标记的嗜肺军团菌与罗丹明-葡聚糖预标记的宿主溶酶体共定位的程度，逐个细菌地对PLF进行评分。值得注意的是，所研究的六个基因中的突变在感染后30分钟内就导致了PLF的最大水平。这些结果表明，几个，甚至可能所有的icm和dotA基因产物在吞噬体形成的早期阶段起作用，以确定含有嗜肺军团菌的吞噬体是否会与溶酶体融合。虽然不能排除，但这些基因产物的后续活性对于成功的细胞内复制可能不是必需的。
