Faucher Sébastien P, Mueller Catherine A, Shuman Howard A
Department of Microbiology and Immunology, Columbia University Medical Center New York, NY, USA.
Front Microbiol. 2011 Apr 4;2:60. doi: 10.3389/fmicb.2011.00060. eCollection 2011.
Legionella pneumophila is the causative agent of Legionnaires' disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot type IVB secretion system, which is used to translocate more than 150 effector proteins into host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called selective capture of transcribed sequences to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to l-arginine, l-histidine, and l-proline as well as many transport systems involved in amino acid and iron uptake. Genes involved in catabolism of glycerol are also induced during intracellular growth, suggesting that glycerol could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.
嗜肺军团菌是军团病的病原体，这是一种急性肺部感染。嗜肺军团菌能够在吞噬性原生动物（如卡氏棘阿米巴）和哺乳动物专职吞噬细胞中感染并繁殖。最著名的嗜肺军团菌毒力决定因素是Icm/Dot IVB型分泌系统，该系统用于将150多种效应蛋白转运到宿主细胞中。虽然已经研究了军团菌对卡氏棘阿米巴细胞内环境的转录反应，但对于军团菌在人类巨噬细胞内的转录反应了解较少。在本研究中，监测了嗜肺军团菌在富含AYE肉汤的指数期和指数后期以及感染人类培养巨噬细胞期间的转录组。这是通过微阵列和一种称为转录序列选择性捕获的RNA扩增程序来完成的，以检测来自少量细胞内细菌的少量mRNA。细胞内诱导的基因包括参与导致L-精氨酸、L-组氨酸和L-脯氨酸的氨基酸生物合成途径的基因，以及许多参与氨基酸和铁摄取的转运系统。参与甘油分解代谢 的基因在细胞内生长期间也被诱导，这表明甘油可以用作碳源。与在富含肉汤中生长的对照细菌相比，编码Icm/Dot系统的基因在细胞内没有差异表达，但编码几种转运效应蛋白的基因被强烈诱导。此外，我们利用转录组数据根据其表达模式预测了以前未被识别的Icm/Dot效应基因，并证实了三个候选基因的转运。这项研究全面展示了嗜肺军团菌如何应对人类巨噬细胞的细胞内环境。
