Gao L Y, Harb O S, Kwaik Y A
Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington 40536-0084, USA.
Infect Immun. 1998 Mar;66(3):883-92. doi: 10.1128/IAI.66.3.883-892.1998.
We have recently shown that many mutants of Legionella pneumophila exhibit similar defective phenotypes within both U937 human-derived macrophages and the protozoan host Acanthamoeba (L.-Y. Gao, O. S. Harb, and Y. Abu Kwaik, Infect. Immun. 65:4738-4746, 1997). These observations have suggested that many of the mechanisms utilized by L. pneumophila to parasitize mammalian and protozoan cells are similar, but our data have not excluded the possibility that there are unique mechanisms utilized by L. pneumophila to survive and replicate within macrophages but not protozoa. To examine this possibility, we screened a bank of 5,280 miniTn10::kan transposon insertion mutants of L. pneumophila for potential mutants that exhibited defective phenotypes of cytopathogenicity and intracellular replication within macrophage-like U937 cells but not within Acanthamoeba polyphaga. We identified 32 mutants with various degrees of defects in cytopathogenicity, intracellular survival, and replication within human macrophages, and most of the mutants exhibited wild-type phenotypes within protozoa. Six of the mutants exhibited mild defects in protozoa. The defective loci were designated mil (for macrophage-specific infectivity loci). Based on their intracellular growth defects within macrophages, the mil mutants were grouped into five phenotypic groups. Groups I to III included the mutants that were severely defective in macrophages, while members of the other two groups exhibited a modestly defective phenotype within macrophages. The growth kinetics of many mutants belonging to groups I to III were also examined, and these were shown to have a similar defective phenotype in peripheral blood monocytes and a wild-type phenotype within another protozoan host, Hartmannella vermiformis. Transmission electron microscopy of A. polyphaga infected by three of the mil mutants belonging to groups I and II showed that they were similar to the parent strain in their capacity to recruit the rough endoplasmic reticulum (RER) around the phagosome. In contrast, infection of macrophages showed that the three mutants failed to recruit the RER around the phagosome during early stages of the infection. None of the mil mutants was resistant to NaCl, and the dot or icm NaCl(r) mutants are severely defective within mammalian and protozoan cells. Our data indicated that in addition to differences in mechanisms of uptake of L. pneumophila by macrophages and protozoa, there were also genetic loci required for L. pneumophila to parasitize mammalian but not protozoan cells. We hypothesize that L. pneumophila has evolved as a protozoan parasite in the environment but has acquired loci specific for intracellular replication within macrophages. Alternatively, ecological coevolution with protozoa has allowed L. pneumophila to possess multiple redundant mechanisms to parasitize protozoa and that some of these mechanisms do not function within macrophages.
我们最近发现，嗜肺军团菌的许多突变体在人源U937巨噬细胞和原生动物宿主棘阿米巴中均表现出相似的缺陷表型（L.-Y. Gao、O. S. Harb和Y. Abu Kwaik，《感染与免疫》65:4738 - 4746，1997年）。这些观察结果表明，嗜肺军团菌用于寄生哺乳动物细胞和原生动物细胞的许多机制是相似的，但我们的数据并未排除嗜肺军团菌存在独特机制以在巨噬细胞而非原生动物中存活和复制的可能性。为了研究这种可能性，我们筛选了一组5280个嗜肺军团菌miniTn10::kan转座子插入突变体，寻找在巨噬细胞样U937细胞中表现出细胞致病性和细胞内复制缺陷表型，但在多食棘阿米巴中未表现出该表型的潜在突变体。我们鉴定出32个在人巨噬细胞的细胞致病性、细胞内存活和复制方面存在不同程度缺陷的突变体，并且大多数突变体在原生动物中表现出野生型表型。其中6个突变体在原生动物中表现出轻微缺陷。这些缺陷位点被命名为mil（巨噬细胞特异性感染位点）。基于它们在巨噬细胞内的生长缺陷，mil突变体被分为五个表型组。第一组到第三组包括在巨噬细胞中严重缺陷的突变体，而另外两组的成员在巨噬细胞中表现出中等程度的缺陷表型。我们还研究了许多属于第一组到第三组的突变体的生长动力学，结果表明它们在外周血单核细胞中具有相似的缺陷表型，而在另一种原生动物宿主蠕虫哈特曼氏阿米巴中表现出野生型表型。对属于第一组和第二组的3个mil突变体感染的多食棘阿米巴进行透射电子显微镜观察发现，它们在吞噬体周围募集糙面内质网（RER）的能力与亲本菌株相似。相比之下，对巨噬细胞的感染显示，这3个突变体在感染早期未能在吞噬体周围募集RER。没有一个mil突变体对NaCl有抗性，而dot或icm NaCl(r)突变体在哺乳动物细胞和原生动物细胞中存在严重缺陷。我们的数据表明，除了巨噬细胞和原生动物摄取嗜肺军团菌的机制存在差异外，嗜肺军团菌寄生哺乳动物细胞而非原生动物细胞还需要一些基因位点。我们推测，嗜肺军团菌在环境中已进化为一种原生动物寄生虫，但获得了在巨噬细胞内进行细胞内复制的特异性位点。或者，与原生动物的生态共同进化使嗜肺军团菌拥有多种冗余机制来寄生原生动物，并且其中一些机制在巨噬细胞中不起作用。
