Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila
Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila
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DOI：
10.1007/pl00008649
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发表时间：
2000-01-01
期刊：
影响因子：
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通讯作者：
Bubert, B
中科院分区：
文献类型：
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作者：
Köhler, R;Bubert, A;Bubert, B
将编码绿色荧光蛋白（GFP）的基因用作嗜肺军团菌中的报告基因。为了分析GFP在军团菌中的表达，构建了GFP与军团菌特异性mip（巨噬细胞感染增强剂）启动子（P-mip）和来自单核细胞增生李斯特菌的sod（超氧化物歧化酶）启动子（P-sod）的转录融合体。转化到强毒L.在嗜肺菌菌株JR 32中，用两种质粒检测到强GFP介导的荧光，尽管sod启动子与高10倍的强度相关。在L.用无启动子的GFP基因转化的嗜肺菌。比较了不同L.在其毒力特征上不同并用携带P-mip-gfp的质粒转化的嗜肺菌菌株显示GFP表达没有差异。以卡氏阿米巴原虫为宿主，用重组L.携带P-mip-gfp和P-sod-gfp融合体的嗜肺菌菌株表明，当细菌在细胞内复制时，mip启动子表达。GFP表达也被用来监测，在感染A. castellanii细胞，细胞内存活率，和宿主细胞杀伤的发生率，L。嗜肺菌菌株的毒力特性不同。如通过流式细胞术定量的，高毒力L.嗜肺菌Corby株对A. castellanii为费城菌株JR 32。使用无毒的费城衍生物25 D侵入，但没有观察到细胞内增殖。此外，我们还用流式细胞仪检测了细胞松弛素DI放线菌酮和甲胺对军团菌A. castellanii。总之，gfp似乎是一个方便的报告基因，其在军团菌中的表达可以在真实的时间，并允许在军团菌启动子活性的分析和感染过程的监测。
The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella pneumophila. To analyze GFP expression in Legionella, transcriptional fusions of gfp with the Legionella-specific mip (macrophage infectivity potentiator) promoter (P-mip) and the sod (superoxide dismutase) promoter (P-sod) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was associated with a 1 ten-fold higher intensity. No fluorescence was observed in L. pneumophila transformed with the promoterless gfp gene. Comparison of fluorescence yields between various L. pneumophila strains that differ in their virulence characteristics and were transformed with the P-mip-gfp carrying plasmid revealed no differences in GFP expression. Infection studies using Acanthamoeba bn castellanii as host and recombinant L. pneumophila strains carrying the P-mip-gfp and P-sod-gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly. GFP expression was also used to monitor, in infected A. castellanii cells, the intracellular survival of, and incidence of host-cell killing by, L. pneumophila strains that vary in their virulence properties. As quantified by flow cytometry the highly virulent L. pneumophila strain Corby was twice as infectious to A. castellanii as the Philadelphia strain JR 32. Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed. In addition, we examined by flow cytometry the influence of cytochalasin DI cycloheximide, and methylamine on the uptake of Legionella by A. castellanii. In conclusion, gfp appears to be a convenient reporter gene whose expression in Legionella can be followed in real time and allows analysis of promoter activities in Legionella and monitoring of the infection process.