Molmeret Maelle, Alli O A Terry, Zink Steven, Flieger Antje, Cianciotto Nicholas P, Kwaik Yousef Abu
Department of Microbiology and Immunology, The University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084, USA.
Infect Immun. 2002 Jan;70(1):69-78. doi: 10.1128/IAI.70.1.69-78.2002.
The final step of the intracellular life cycle of Legionella pneumophila and other intracellular pathogens is their egress from the host cell after termination of intracellular replication. We have previously isolated five spontaneous mutants of L. pneumophila that replicate intracellularly similar to the wild-type strain but are defective in pore formation-mediated cytolysis and egress from mammalian and protozoan cells, and the mutants have been designated rib (release of intracellular bacteria). Here, we show that the rib mutants are not defective in the activity of enzymes secreted through the type II secretion system, including phospholipase A, lysophospholipase A, and monoacylglycerol lipase, although they are potential candidates for factors that lyse host cell membranes. In addition, the pilD and lspG mutants, which are defective in the type II secretion system, are not defective in the pore-forming toxin. We show that all five rib mutants have an identical point mutation (deletion) following a stretch of poly(T) in the icmT gene. Spontaneous revertants of the rib mutants, due to an insertion of a nucleotide following the poly(T) stretch in icmT, have been isolated and shown to have regained the wild-type phenotype. We constructed an icmT insertion mutant (AA100kmT) in the chromosome of the wild-type strain by allelic exchange. The AA100kmT mutant was as defective as the rib mutant in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. Both the rib mutant and the AA100kmT mutant were complemented by the icmT gene for their phenotypic defect. rtxA, a gene that is thought to have a minor role in pore formation, was not involved in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. We conclude that the icmT gene is essential for pore formation-mediated lysis of mammalian and protozoan cells and the subsequent bacterial egress.
嗜肺军团菌及其他细胞内病原体细胞内生命周期的最后一步是在细胞内复制终止后从宿主细胞中逸出。我们之前分离出了嗜肺军团菌的五个自发突变体，它们在细胞内的复制情况与野生型菌株相似，但在由孔形成介导的细胞溶解以及从哺乳动物和原生动物细胞中逸出方面存在缺陷，这些突变体被命名为rib（细胞内细菌释放）。在此，我们表明，尽管rib突变体是裂解宿主细胞膜的潜在因子候选，但它们在通过II型分泌系统分泌的酶（包括磷脂酶A、溶血磷脂酶A和单酰甘油脂肪酶）的活性方面并无缺陷。此外，在II型分泌系统中存在缺陷的pilD和lspG突变体在成孔毒素方面并无缺陷。我们发现所有五个rib突变体在icmT基因中一段聚（T）序列之后都有相同的点突变（缺失）。已分离出rib突变体的自发回复突变体，其原因是icmT基因中聚（T）序列之后插入了一个核苷酸，并且已证明其恢复了野生型表型。我们通过等位基因交换在野生型菌株的染色体中构建了一个icmT插入突变体（AA100kmT）。AA100kmT突变体在由孔形成介导的细胞溶解以及从哺乳动物和原生动物细胞中逸出方面与rib突变体一样存在缺陷。rib突变体和AA100kmT突变体的表型缺陷都通过icmT基因得到了互补。rtxA是一个被认为在孔形成中起次要作用的基因，它与由孔形成介导的细胞溶解以及从哺乳动物和原生动物细胞中逸出无关。我们得出结论，icmT基因对于由孔形成介导的哺乳动物和原生动物细胞裂解以及随后细菌的逸出至关重要。
