Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, United States of America.
PLoS Pathog. 2023 Jul 26;19(7):e1011526. doi: 10.1371/journal.ppat.1011526. eCollection 2023 Jul.
Mammalian cells synthesize the antioxidant glutathione (GSH) to shield cellular biomolecules from oxidative damage. Certain bacteria, including the gastric pathogen Helicobacter pylori, can perturb host GSH homeostasis. H. pylori infection significantly decreases GSH levels in host tissues, which has been attributed to the accumulation of reactive oxygen species in infected cells. However, the precise mechanism of H. pylori-induced GSH depletion remains unknown, and tools for studying this process during infection are limited. We developed an isotope-tracing approach to quantitatively monitor host-derived GSH in H. pylori-infected cells by mass spectrometry. Using this method, we determined that H. pylori catabolizes reduced GSH from gastric cells using γ-glutamyl transpeptidase (gGT), an enzyme that hydrolyzes GSH to glutamate and cysteinylglycine (Cys-Gly). gGT is an established virulence factor with immunomodulatory properties that is required for H. pylori colonization in vivo. We found that H. pylori internalizes Cys-Gly in a gGT-dependent manner and that Cys-Gly production during H. pylori infection is coupled to the depletion of intracellular GSH from infected cells. Consistent with bacterial catabolism of host GSH, levels of oxidized GSH did not increase during H. pylori infection, and exogenous antioxidants were unable to restore the GSH content of infected cells. Altogether, our results indicate that H. pylori-induced GSH depletion proceeds via an oxidation-independent mechanism driven by the bacterial enzyme gGT, which fortifies bacterial acquisition of nutrients from the host. Additionally, our work establishes a method for tracking the metabolic fate of host-derived GSH during infection.
哺乳动物细胞合成抗氧化剂谷胱甘肽 (GSH)，以保护细胞生物分子免受氧化损伤。某些细菌，包括胃病原体幽门螺杆菌，可以扰乱宿主 GSH 动态平衡。幽门螺杆菌感染会显著降低宿主组织中的 GSH 水平，这归因于感染细胞中活性氧物质的积累。然而，幽门螺杆菌诱导的 GSH 消耗的确切机制仍不清楚，并且用于研究感染过程中这一过程的工具也很有限。我们开发了一种同位素示踪方法，通过质谱法定量监测幽门螺杆菌感染细胞中宿主来源的 GSH。使用这种方法，我们确定幽门螺杆菌使用γ-谷氨酰转肽酶 (gGT) 从胃细胞中代谢还原型 GSH，gGT 是一种将 GSH 水解为谷氨酸和半胱氨酰甘氨酸 (Cys-Gly) 的酶。gGT 是一种具有免疫调节特性的已确立的毒力因子，是幽门螺杆菌在体内定植所必需的。我们发现幽门螺杆菌以 gGT 依赖的方式内化 Cys-Gly，并且在幽门螺杆菌感染期间 Cys-Gly 的产生与感染细胞中细胞内 GSH 的消耗相关。与细菌代谢宿主 GSH 一致，在幽门螺杆菌感染期间氧化型 GSH 的水平没有增加，并且外源性抗氧化剂无法恢复感染细胞的 GSH 含量。总之，我们的结果表明，幽门螺杆菌诱导的 GSH 消耗是通过细菌酶 gGT 驱动的氧化独立机制进行的，这加强了细菌从宿主获取营养的能力。此外，我们的工作建立了一种在感染过程中跟踪宿主来源的 GSH 代谢命运的方法。
