Molmeret Maëlle, Bitar Dina M, Han Lihui, Kwaik Yousef Abu
Department of Microbiology and Immunology, Room 316, University of Louisville College of Medicine, 319 Abraham Flexner Way 55A, Louisville, KY 40202, USA.
Infect Immun. 2004 Jul;72(7):4040-51. doi: 10.1128/IAI.72.7.4040-4051.2004.
Although the early stages of intracellular infection by Legionella pneumophila are well established at the ultrastructural level, a detailed ultrastructural analysis of late stages of intracellular replication has never been done. Here we show that the membrane of the L. pneumophila-containing phagosome (LCP) is intact for up to 8 h postinfection of macrophages and Acanthamoeba polyphaga. At 12 h, 71 and 74% of the LCPs are disrupted within macrophages and A. polyphaga, respectively, while the plasma membrane remains intact. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic. At 18 h, 77% of infected macrophages and 32% of infected A. polyphaga amoebae harbor cytoplasmic bacteria. At 24 h, 99 and 78% of infected macrophages and amoebae, respectively, contain cytoplasmic bacteria. On the basis of lysosomal acid phosphatase staining of infected macrophages and A. polyphaga, the lysosomal enzyme is present among the bacteria when host vesicles are dispersed among bacteria. Our data indicate that bacterial replication proceeds despite physical disruption of the phagosomal membrane. We also show that an lspG mutant that is defective in the type II secretion system and therefore does not secrete the hydrolytic enzymes metalloprotease, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A is as efficient as the wild-type strain in disruption of the LCP. Therefore, L. pneumophila disrupts the phagosomal membrane and becomes cytoplasmic at the last stages of infection in both macrophages and A. polyphaga. Lysosomal elements, mitochondria, cytoplasmic vesicles, and amorphous material are all dispersed among the bacteria, after phagosomal disruption, within both human macrophages and A. polyphaga. The disruption of the LCP is independent of the hydrolytic enzymes exported by the type II secretion system.
尽管嗜肺军团菌细胞内感染的早期阶段在超微结构水平上已得到充分证实，但对细胞内复制后期的详细超微结构分析从未进行过。在此我们表明，在巨噬细胞和多食棘阿米巴感染后长达8小时，含嗜肺军团菌的吞噬体（LCP）膜保持完整。在12小时时，分别有71%和74%的LCP在巨噬细胞和多食棘阿米巴中被破坏，而质膜保持完整。在感染后18小时和24小时，线粒体、溶酶体、囊泡和无定形物质等细胞质成分分散在细菌之间，这些细菌被认为处于细胞质中。在18小时时，77%的受感染巨噬细胞和32%的受感染多食棘阿米巴含有细胞质细菌。在24小时时，分别有99%和78%的受感染巨噬细胞和阿米巴含有细胞质细菌。基于对受感染巨噬细胞和多食棘阿米巴的溶酶体酸性磷酸酶染色，当宿主囊泡分散在细菌之间时，溶酶体酶存在于细菌之中。我们的数据表明，尽管吞噬体膜发生了物理破坏，细菌仍能进行复制。我们还表明，II型分泌系统存在缺陷、因此不分泌水解酶金属蛋白酶、对硝基苯酚磷酸胆碱水解酶、脂肪酶、磷脂酶A和溶血磷脂酶A的lspG突变体，在破坏LCP方面与野生型菌株一样有效。因此，嗜肺军团菌在巨噬细胞和多食棘阿米巴感染的最后阶段破坏吞噬体膜并进入细胞质。在吞噬体破坏后，溶酶体成分、线粒体、细胞质囊泡和无定形物质在人类巨噬细胞和多食棘阿米巴的细菌之间均有分散。LCP的破坏与II型分泌系统输出的水解酶无关。
