The roles of glutathione metabolism in growth and virulence of Listeria monocytogenes

摘要 单核细胞增多性李斯特菌是一种低G-C革兰氏阳性细菌，可在 免疫功能受损的个人、孕妇、新生儿和老年人。致病性李斯特菌使用 一种主要的转录调节因子，PrFA，以诱导其最重要的毒力基因。反过来，PRFA蛋白是 由谷胱甘肽直接激活，谷胱甘肽是一种含有半胱氨酸的三肽，也具有重要的抗氧化作用 在细菌和真核细胞中起解毒作用。 达到充分激活PRFA和毒力基因所需的细胞内谷胱甘肽水平 在感染过程中，李氏杆菌细胞需要合成谷胱甘肽或从胞浆中导入谷胱甘肽 宿主细胞，其中富含谷胱甘肽。令人惊讶的是，李斯特基谷胱甘肽的合成似乎贡献更大 与从真核细胞胞浆中摄取相比，对毒力更强。谷胱甘肽的摄取途径一直是 仅在几种细菌中发现；单核细胞增多性乳杆菌谷胱甘肽进口体(S)及其原因 它们在感染期间的明显低活性尚不清楚。 为了合成谷胱甘肽并生长，单核细胞增多性乳杆菌细胞必须获得半胱氨酸，这是一种必不可少的 氨基酸，或来自环境的相关化合物。有趣的是，李斯特菌细胞可以有效地转化为 外源谷胱甘肽转化为半胱氨酸。这种谷胱甘肽的降解，取决于未知的细胞外 或该途径的细胞内定位，可以降低细菌或宿主细胞中的代谢物水平，或两者兼而有之。 因此，严格控制谷胱甘肽的降解可能是李斯特菌毒力的关键一步。 感染。单核细胞增多性李斯特氏菌中谷胱甘肽到半胱氨酸的降解途径尚未确定，以及 李斯特菌基因组中没有编码已知的谷胱甘肽裂解酶的基因。因此， 一种新的谷胱甘肽降解酶存在于李斯特细胞中，其细胞定位未知。 完全缺乏关于谷胱甘肽摄取和降解途径的性质的信息， 它们的调节以及对谷胱甘肽水平和PRFA激活的贡献阻碍了我们对 不同李斯特菌生长条件下毒力基因是如何被诱导的。我们建议填补这一重要的 我们的知识差距，并确定参与摄取和 谷胱甘肽的降解。这些基因在各种生长条件下的表达模式将是 下定决心。相应途径对谷胱甘肽-李斯特氏池、PRFA-1表达的影响 依赖的毒力基因和小鼠感染模型中的毒力将是我们研究的主要目标。 单核细胞增多性乳杆菌是美国最致命的食源性病原体之一。这个项目将 使我们能够确定谷胱甘肽积累到需要的水平所需的代谢步骤 李斯特菌毒力。通过这样做，我们可能会发现潜在的治疗干预的新途径。类似 途径很可能存在于其他致病细菌中。

ABSTRACT Listeria monocytogenes is a low G+C Gram-positive bacterium that can cause severe disease in immunocompromised individuals, pregnant women, newborns, and the elderly. Pathogenic listerial strains use a master transcriptional regulator, PrfA, to induce its most important virulence genes. In turn, the PrfA protein is directly activated by glutathione, a cysteine-containing tripeptide, which also performs important antioxidation and detoxification functions in bacterial and eukaryotic cells. To reach the intracellular level of glutathione required for sufficient PrfA activation and virulence gene expression during infection, the listerial cells need either to synthesize glutathione or import it from the cytosol of host cells, which is rich in glutathione. Surprisingly, listerial glutathione synthesis appears to contribute more strongly to virulence than its uptake from the eukaryotic cytosol. Glutathione uptake pathways have been identified only in several bacterial species; the L. monocytogenes glutathione importer(s) and the reasons for their apparent low activity during infection are not known. To synthesize glutathione and just to grow, L. monocytogenes cells must obtain cysteine, an essential amino acid, or a related compound from the environment. Interestingly, listerial cells can convert efficiently exogeneous glutathione to cysteine. This glutathione degradation, depending on the yet unknown extracellular or intracellular localization of this pathway, can reduce the metabolite level in bacterial or host cells or both. Therefore, a tight regulation of glutathione degradation may be a critical step in listerial virulence during infection. The glutathione-to-cysteine degradation pathway in L. monocytogenes has not been identified, and none of the genes encoding known enzymes of glutathione cleavage are present in the listerial genome. Thus, a novel enzyme of glutathione degradation with an unknown cellular localization is present in listerial cells. The complete lack of information on the nature of the glutathione uptake and degradation pathways, their regulation, and contributions to the glutathione level and PrfA activation impedes our understanding of how virulence genes are induced under various conditions of listerial growth. We propose to fill this important gap in our knowledge and determine the L. monocytogenes genes that are involved in the uptake and degradation of glutathione. Expression patterns of these genes under various growth conditions will be determined. The impact of the corresponding pathways on the glutathione listerial pool, expression of PrfA- dependent virulence genes, and virulence in a mouse model of infection will be major targets of our research. L. monocytogenes is one of the deadliest foodborne pathogens in the United States. This project will allow us to identify metabolic steps required for the accumulation of glutathione at levels that are needed for listerial virulence. In doing so, we may uncover novel pathways for potential therapeutic intervention. Similar pathways are likely to exist in other pathogenic bacteria.