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

摘要 单核细胞增生李斯特氏菌是一种低 G C 革兰氏阳性细菌，可在人体中引起严重疾病。 免疫功能低下的个体、孕妇、新生儿和老年人。致病性李斯特菌菌株的用途 主要转录调节因子 PrfA，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，谷胱甘肽是一种含半胱氨酸的三肽，也具有重要的抗氧化作用 细菌和真核细胞中的解毒功能。 达到足够 PrfA 激活和毒力基因所需的细胞内谷胱甘肽水平 在感染期间表达，李斯特菌细胞需要合成谷胱甘肽或从细胞质中导入它 宿主细胞，富含谷胱甘肽。令人惊讶的是，李斯特菌谷胱甘肽合成似乎贡献更多 其毒力比从真核细胞质中摄取的毒力强。谷胱甘肽的摄取途径 仅在几种细菌物种中发现；单增李斯特氏菌谷胱甘肽输入者及其原因 它们在感染期间明显的低活性尚不清楚。 为了合成谷胱甘肽并生长，单核细胞增生李斯特氏菌细胞必须获得半胱氨酸，这是一种必需的物质 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 外源性谷胱甘肽转化为半胱氨酸。这种谷胱甘肽的降解，取决于尚不清楚的细胞外 或该途径的细胞内定位，可以降低细菌或宿主细胞或两者的代谢水平。 因此，严格调控谷胱甘肽降解可能是李斯特菌毒力的关键步骤。 感染。单核细胞增生利斯特氏菌中谷胱甘肽到半胱氨酸的降解途径尚未确定，并且 李斯特菌基因组中不存在编码已知谷胱甘肽裂解酶的基因。因此， 李斯特菌细胞中存在一种新的谷胱甘肽降解酶，其细胞定位未知。 完全缺乏有关谷胱甘肽摄取和降解途径性质的信息， 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 在李斯特菌生长的各种条件下如何诱导毒力基因。我们建议填补这一重要 我们的知识差距，并确定参与摄取和吸收的单核细胞增生李斯特氏菌基因 谷胱甘肽的降解。这些基因在不同生长条件下的表达模式将 确定。相应途径对谷胱甘肽李斯特菌池、PrfA-表达的影响 依赖毒力基因和小鼠感染模型的毒力将是我们研究的主要目标。 单核细胞增生李斯特菌是美国最致命的食源性病原体之一。该项目将 使我们能够确定谷胱甘肽积累所需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治疗干预的新途径。类似 其他致病菌中可能也存在这种途径。

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.