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

谷胱甘肽代谢在单核细胞增生李斯特菌生长和毒力中的作用

• 批准号: 10671070
• 负责人: BORIS R BELITSKY
• 金额: $ 24.75万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-25 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10671070
• 关键词: Address; Affect; Amino Acids; Animals; Bacteria; Biochemical; Cells; Cysteine; Cytosol; Degradation Pathway; Dependence; Development; Disease; Drug Metabolic Detoxication; Elderly; Environment; Enzymes; Essential Amino Acids; Eukaryotic Cell; Gene Expression; Gene Expression Profile; Gene Silencing; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome; Glutathione; Glutathione Metabolism Pathway; Gram-Positive Bacteria; Growth; Human; Immunocompromised Host; In Vitro; Individual; Infection; Knowledge; Listeria monocytogenes; Maintenance; Metabolic; Metabolic Pathway; Metabolism; Modeling; Mus; Nature; Newborn Infant; Oral; Organism; Oxidation-Reduction; Pathogenesis; Pathogenicity; Pathway interactions; Peptides; Physiological; Pregnant Women; Proteins; Regulation; Research; Role; Signal Transduction; Source; Testing; Therapeutic Intervention; Toxic effect; United States; Virulence; Virulence Factors; Virulent; extracellular; foodborne; foodborne illness; foodborne infection; foodborne pathogen; mouse model; novel; novel therapeutics; oxidation; pathogen; pathogenic bacteria; response; transcription factor; transposon sequencing; uptake; virulence gene

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.

摘要 单核细胞增生李斯特菌是一种低G+C革兰氏阳性菌，可导致严重的疾病， 免疫功能低下的个体、孕妇、新生儿和老年人。病原体菌株使用 一个主要的转录调节因子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 以及细菌和真核细胞的解毒功能。 为了达到足够的PrfA激活和毒力基因所需的细胞内谷胱甘肽水平， 在感染过程中表达谷胱甘肽时，宿主细胞需要合成谷胱甘肽或从胞质中输入谷胱甘肽 富含谷胱甘肽的宿主细胞。令人惊讶的是，血浆谷胱甘肽的合成似乎贡献更多， 比从真核细胞胞质溶胶中摄取更强的毒力。谷氨酰胺摄取途径已被 仅在几种细菌中鉴定; L.单核细胞增生谷胱甘肽进口商（S）和原因 它们在感染过程中明显的低活性是未知的。 为了合成谷胱甘肽并生长，L。单核细胞增多症细胞必须获得半胱氨酸， 氨基酸或环境中的相关化合物。有趣的是，胰岛细胞可以有效地 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或两者中的代谢物水平。 因此，谷胱甘肽降解的严格调节可能是细菌毒力的关键步骤， 感染L.谷胱甘肽-半胱氨酸降解途径。单核细胞增多症尚未确定， 编码已知谷胱甘肽裂解酶的基因都不存在于拟南芥基因组中。因此，在本发明中， 一种新的谷胱甘肽降解酶存在于胰岛细胞中，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 毒力基因是如何在不同的菌丝生长条件下被诱导的。我们建议填补这一重要的 在我们的知识差距，并确定L。单核细胞增生基因参与摄取， 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被研究。 测定相应途径对谷胱甘肽过氧化物酶库、PrfA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。 L.单核细胞增多症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的代谢步骤， 病原体毒力。在这样做的过程中，我们可能会发现潜在的治疗干预的新途径。类似 其他致病菌中可能也存在这种途径。