Fernandez R C, Logan S M, Lee S H, Hoffman P S
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.
Infect Immun. 1996 Jun;64(6):1968-76. doi: 10.1128/iai.64.6.1968-1976.1996.
Legionella pneumophila 2064 was selectively radiolabelled in mouse L929 cells and human monocytes to identify proteins expressed early in the course of infection. Polypeptide profiles (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography) of adherent or intracellular bacteria indicated that a 60-kDa stress protein (Hsp60) was preferentially synthesized. Hsp60 synthesis was not induced by medium alone. The synthesis of many polypeptides, including OmpS (major outer membrane protein), diminished over the 1-h period postinfection. However, by 17 h postinfection OmpS and Hsp60 were the dominant proteins synthesized by 2064. To establish whether induction of Hsp60 was a correlate of virulence, an isogenic avirulent strain (2064M) of 2064 was isolated following selection on a nonpermissive medium. 2064M did not exhibit a stress response when adherent or intracellular in L929 cells or in human monocytes and failed to abrogate phagosome-lysosome fusion. When grown in vitro, 2064M exhibited no deficiencies in the heat shock response and its polypeptide profile resembled that of 2064. Immunogold electron microscopy was used to localize Hsp60 in L. pneumophila-infected L929 cells. There was an increase in the number of gold particles associated with phagosomes for phagosomes harboring single 2064 bacteria compared with those harboring 2064M. Moreover, by 1 h postinfection, a sixfold increase in the number of gold spheres associated with the membranes of phagosomes was observed for phagosomes harboring 2064 compared with those harboring 2064M. These studies indicate that virulent, but not NaCl-tolerant avirulent, strains of L. pneumophila respond to host-cell-associated environmental signals early in the course of infection. This response includes increased synthesis and possibly extracellular secretion of Hsp60 concomitant with repression of the expression of other genes, including ompS.
嗜肺军团菌2064在小鼠L929细胞和人单核细胞中被选择性地进行放射性标记，以鉴定在感染过程早期表达的蛋白质。贴壁或胞内细菌的多肽图谱（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和荧光自显影）表明，一种60 kDa的应激蛋白（热休克蛋白60，Hsp60）被优先合成。单独的培养基不会诱导Hsp60的合成。包括外膜主要蛋白（OmpS）在内的许多多肽的合成在感染后1小时内减少。然而，在感染后17小时，OmpS和Hsp60是2064合成的主要蛋白质。为了确定Hsp60的诱导是否与毒力相关，在非允许培养基上筛选后，分离出了2064的同基因无毒株（2064M）。当2064M在L929细胞或人单核细胞中贴壁或处于胞内时，未表现出应激反应，并且无法消除吞噬体-溶酶体融合。在体外培养时，2064M在热休克反应方面没有缺陷，其多肽图谱与2064相似。免疫金电子显微镜用于在嗜肺军团菌感染的L929细胞中定位Hsp60。与含有2064M的吞噬体相比，含有单个2064细菌的吞噬体中与吞噬体相关的金颗粒数量增加。此外，在感染后1小时，与含有2064M的吞噬体相比，含有2064的吞噬体中与吞噬体膜相关的金球数量增加了六倍。这些研究表明，嗜肺军团菌的有毒力但不耐NaCl的无毒株在感染过程早期对宿主细胞相关的环境信号有反应。这种反应包括Hsp60合成增加以及可能的细胞外分泌，同时伴随着包括ompS在内的其他基因表达的抑制。
