A LEGIONELLA-PNEUMOPHILA GENE ENCODING A SPECIES-SPECIFIC SURFACE PROTEIN POTENTIATES INITIATION OF INTRACELLULAR INFECTION
A LEGIONELLA-PNEUMOPHILA GENE ENCODING A SPECIES-SPECIFIC SURFACE PROTEIN POTENTIATES INITIATION OF INTRACELLULAR INFECTION
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DOI：
10.1128/iai.57.4.1255-1262.1989
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发表时间：
1989-04-01
影响因子：
3.1
通讯作者：
ENGLEBERG, NC
中科院分区：
文献类型：
--
作者：
CIANCIOTTO, NP;EISENSTEIN, BI;ENGLEBERG, NC
为了在分子水平上研究军团病的发病机制，我们通过定向等位基因交换使编码嗜肺军团菌特异性24，000-道尔顿（Da）表面蛋白的基因突变。Southern杂交和免疫印迹分析表明，预测的DNA重排发生在L。嗜肺菌，具有24-kDa抗原表达的特异性缺失。与其同基因亲本相比，突变体感染转化的U937细胞（一种人巨噬细胞样细胞系）的能力显著受损;即，启动巨噬细胞单层感染所需的突变菌株的细菌接种物是CA。80-倍大于同基因亲本菌株。突变株重新克隆的24-kDa蛋白质基因的重新引入恢复完全感染性，表明感染性降低是由于该基因的突变。与亲本菌株相比，突变株的回收滴度约为1000 μ g/ml。 10-感染后不久，其细胞内生长速率降低了1倍，但在接下来的40小时内，其表现出相似的细胞内生长速率，表明突变体在其启动巨噬细胞感染的能力方面有缺陷，而不是在其细胞内复制的能力方面。当调理，突变株仍然是显着低于亲本菌株的感染性，尽管等效的巨噬细胞协会，这表明突变体不仅仅是缺少巨噬细胞附着的配体。突变体alcohol在灭活的人肺泡巨噬细胞中表现出降低的感染性，证明了用于分析该突变体表型的U937细胞模型的相关性。这些结果代表了克隆L. pneumophila基因是最佳细胞内感染所必需的;我们将该基因命名为mip，即巨噬细胞感染性增强剂。
To investigate the pathogenesis of Legionnaire''s disease at a molecular level, we mutated by directed allelic exchange a gene encoding a Legionella pneumophila-specific 24,000-dalton (Da) surface protein. Southern hybridization and immunoblot analyses demonstrated that the predicted DNA rearrangement occurred in L. pneumophila with a specific loss of 24-kDa antigen expression. Compared with its isogenic parent, the mutant was significantly impaired in its ability to infect transformed U937 cells, a human macrophagelike cell line; i.e., the bacterial inoculum of the mutant strain that was required to initiate infection of the macrophage monolayer was ca. 80-fold greater than that of the isogenic parent strain. The mutant strain regained full infectivity on reintroduction of a cloned 24-kDa protein gene, indicating that the reduced infectivity was due specifically to the mutation in that gene. Compared with the parent strain, the mutant strain was recovered at titers that were ca. 10-fold lower shortly after infection, but it exhibited a similar intracellular growth rate over the next 40 h, indicating that the mutant was defective in its ability to initiate macrophage infection rather than in its ability to replicate intracellularly. When opsonized, the mutant strain was still significantly less infectious than the parent strain, despite equivalent macrophage association, suggesting that the mutant was not merely missing a ligand for macrophage attachment. The mutant alsoi exhibited reduced infectivity in explanted human alveolar macrophages, demonstrating the relevance of the U937 cell model for analyzing this mutant phenotype. These results represent the first identification of a cloned L. pneumophila gene that is necessary for optimal intracellular infection; we disgnate this gene mip, for macrophage infectivity potentiator.