Moffat J F, Edelstein P H, Regula D P, Cirillo J D, Tompkins L S
Department of Microbiology and Immunology, Stanford University School of Medicine, California 94305.
Mol Microbiol. 1994 Jun;12(5):693-705. doi: 10.1111/j.1365-2958.1994.tb01057.x.
To determine the effects, if any, of the Zn-metalloprotease on virulence of Legionella pneumophila infection, an isogenic mutant deficient in protease (encoded by the proA gene) was tested in an Acanthamoeba cell model, in guinea-pig macrophages, and in a guinea-pig pneumonia model. The cloned proA gene was completely inactivated by insertion of a kanamycin-resistance cassette into the protease gene of L. pneumophila AA100. This mutated gene was then introduced into the L. pneumophila chromosome by allelic exchange to form the isogenic ProA- mutant AA200. AA200 showed no difference in its ability to enter, survive, or grow in Acanthamoeba and explanted guinea-pig macrophages; neither light nor electron microscopy revealed morphological differences in the eukaryotic cells infected with the protease mutant or the wild-type strains. The proA gene was found to be expressed in L. pneumophila during intracellular growth in amoebae by measuring the light produced from a truncated luxC gene fusion with the proA promoter. Virulence of the protease mutant was attenuated when tested in a guinea-pig model of infection employing the intratracheal inoculation method. AA200 was slower to cause death, grew to lower numbers in the lungs, resulted in less necrotic debris and a larger macrophage infiltrate, and was more likely to be found in association with macrophage vacuoles than the parent strain. Although deletion of the protease was not sufficient to completely abolish virulence in a guinea-pig model, the mutation caused a delay in the lethal effects of L. pneumophila and attenuated the infection.
为了确定锌金属蛋白酶对嗜肺军团菌感染毒力的影响（若有影响），在棘阿米巴细胞模型、豚鼠巨噬细胞和豚鼠肺炎模型中对蛋白酶缺陷（由proA基因编码）的同基因突变体进行了测试。通过将卡那霉素抗性盒插入嗜肺军团菌AA100的蛋白酶基因，克隆的proA基因完全失活。然后通过等位基因交换将这个突变基因引入嗜肺军团菌染色体，形成同基因ProA-突变体AA200。AA200在进入、存活或在棘阿米巴和移出的豚鼠巨噬细胞中生长的能力上没有差异；光学显微镜和电子显微镜均未发现感染蛋白酶突变体或野生型菌株的真核细胞在形态上有差异。通过测量与proA启动子融合的截短luxC基因产生的光，发现proA基因在嗜肺军团菌于变形虫内细胞生长期间表达。当在采用气管内接种方法的豚鼠感染模型中进行测试时，蛋白酶突变体的毒力减弱。与亲本菌株相比，AA200导致死亡的速度较慢，在肺部生长的数量较少，产生的坏死碎片较少且巨噬细胞浸润较多，并且更有可能与巨噬细胞空泡相关联。虽然在豚鼠模型中缺失蛋白酶不足以完全消除毒力，但该突变导致嗜肺军团菌的致死作用延迟并减弱了感染。
