Isolation of Mycobacterium tuberculosis mutants defective in the arrest of phagosome maturation
Isolation of Mycobacterium tuberculosis mutants defective in the arrest of phagosome maturation
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DOI：
10.1073/pnas.0401657101
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发表时间：
2004-09-14
影响因子：
11.1
通讯作者：
Russell, DG
中科院分区：
文献类型：
--
作者：
Pethe, K;Swenson, DL;Russell, DG
结核分枝杆菌存在于其宿主的吞噬细胞内。它通过阻止其吞噬体的正常成熟来确保其持续存活，所述吞噬体保留在巨噬细胞的早期内体系统内。虽然个别细菌组分已被证明可以调节吞噬体生物发生，但在活的完整细菌中起作用的机制仍然难以捉摸。我们已经开发了一种遗传筛选，有利于分离缺陷突变体，在逮捕他们的吞噬体的成熟。将巨噬细胞与被追赶到溶酶体中的铁-葡聚糖一起孵育。随后用M.转座子诱变细菌文库中的结核病。经过四轮富集，分离的大多数突变体不能防止其吞噬体酸化，并且细胞内存活减弱。受影响的基因的功能范围从那些没有已知的同源物到推定的转运蛋白和脂质合成酶。需要对这些细菌进行进一步的鉴定。除了阐明M.对于结核病，这种筛选可能适用于限制其吞噬体成熟的其他病原体。
Mycobacterium tuberculosis resides within the phagocytes of its host. It ensures its continued survival through arresting the normal maturation of its phagosome, which is retained within the early endosomal system of the macrophage. Although individual bacterial components have been shown to modulate phagosome biogenesis, the mechanism(s) active in live, intact bacteria remain elusive. We have developed a genetic screen that facilitates the isolation of mutants defective in arresting the maturation of their phagosomes. Macrophages were incubated with iron-dextran that was chased into lysosomes. The cells were subsequently infected with M. tuberculosis from a library of transposon-mutagenized bacteria. After four rounds of enrichment, the majority of mutants isolated were unable to prevent acidification of their phagosomes and were attenuated for intracellular survival. The genes affected range in function from those with no known homologues to putative transporters and lipid synthesis enzymes. Further characterization of these bacteria is needed. In addition to clarifying the processes active in modulation of phagosome biogenesis by M. tuberculosis, this screen may be applicable to other pathogens that restrict the maturation of their phagosome.