Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening
Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening
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DOI：
10.1128/jb.188.2.556-568.2006
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发表时间：
2006-01-01
影响因子：
3.2
通讯作者：
Goebel, W
中科院分区：
文献类型：
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作者：
Joseph, B;Przybilla, K;Goebel, W
单核增生李斯特菌成功地从细胞外环境过渡到细胞内环境需要对宿主环境中遇到的条件做出精确的适应性反应。尽管这种革兰氏阳性病原体的细胞内生活方式的许多关键步骤已被很好地描述，但我们对细胞质增殖所需因素的了解仍然相当有限。我们使用DNA微阵列和实时逆转录酶PCR分析来研究上皮细胞感染后细胞内单核增生乳杆菌的转录谱。当在脑心脏输注培养基中生长时，大约19%的基因相对于其转录水平差异表达至少1.6倍，包括编码碳和氮源摄取必需转运蛋白的基因、合成代谢途径相关因子、应激蛋白、转录调节因子和功能未知的蛋白质。为了验证细胞内基因表达谱的生物学相关性，采用插入复制诱变法构建了单核增生乳杆菌随机突变文库，并对细胞内生长缺陷菌株进行了筛选。通过结合这两种方法的结果，我们提供了证据，证明单核增生乳杆菌在上皮细胞复制过程中可以使用替代碳源，如磷酸化葡萄糖和甘油，也可以使用氮源，如乙醇胺，并且戊糖磷酸循环是宿主环境中糖代谢的主要途径，而不是糖酵解。此外，我们发现精氨酸、异亮氨酸、亮氨酸和缬氨酸的合成，以及一种特定的磷酸烯醇丙酮酸依赖的磷酸转移酶系统，在单核增生乳杆菌的细胞内生长中起着重要作用。
A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.