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首页> 外文期刊>Infection and immunity >Identification of Legionella pneumophila mutants that have aberrant intracellular fates.
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Identification of Legionella pneumophila mutants that have aberrant intracellular fates.

机译：鉴定具有异常细胞内命运的嗜肺军团菌突变体。

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After uptake by macrophages, Legionella pneumiophila evades phagosome-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum. A collection of bacterial mutants defective for growth in macrophages were isolated, and the intracellular fate of each mutant strain was analyzed by fluorescence microscopy. To measure intracellular replication, bacteria inside macrophages were stained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI). Evasion of the endocytic pathway was quantified by immunofluorescence localization of lp120 [correction of IgpI20] (LAMP-1), a membrane protein of late endosomes and lysosomes, or by measuring colocalization of bacteria with a fluorescent tracer, Texas red-ovalbumin, preloaded into lysosomes. Replication vacuoles were quantified by immunofluorescence localization of BiP, an endoplasmic reticulum protein. By these approaches, four phenotypic groups of mutants were classified. One class formed replication vacuoles less efficiently than the wild type did; another formed replication vacuoles, but replication was abortive; in another class, most phagosomes containing bacteria acquired markers of the endocytic pathway but a minority formed replication vacuoles and the bacteria replicated; finally, a fourth class, the one most defective for intracellular growth, occupied vacuoles that acquired markers of the endocytic pathway.
机译：在被巨噬细胞摄取后，嗜肺军团菌逃避吞噬体-溶酶体融合，并在与内质网相关的隔室中复制。分离出在巨噬细胞中生长缺陷的细菌突变体的集合，并通过荧光显微镜分析每种突变体菌株的细胞内命运。为了测量细胞内复制，巨噬细胞内部的细菌用DNA染料4'，6-diamidino-2-phenylindole（DAPI）染色。通过对晚期内体和溶酶体的膜蛋白lp120的免疫荧光定位[IgpI20的校正]（LAMP-1）进行定量，或通过使用荧光示踪剂德克萨斯州红卵清蛋白测定细菌的共定位，定量了对内吞途径的逃避溶酶体。复制液泡通过BiP（一种内质网蛋白）的免疫荧光定位进行定量。通过这些方法，突变体分为四个表型组。一类形成复制空泡的效率比野生型低。另一个形成的复制空泡，但是复制是失败的。在另一类中，大多数含有细菌的吞噬体获得了内吞途径的标记，但少数形成复制液泡，细菌得以复制。最后，第四类是细胞内生长最有缺陷的第四类，它占据了获得内吞途径标记的液泡。

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