Xue Juan, Huang Yuxuan, Zhang Hua, Hu Jiaqingzi, Pan Xing, Peng Ting, Lv Jun, Meng Kun, Li Shan
Institute of Infection and Immunity, Taihe Hospital, Hubei University of Medicine, Shiyan, China.
College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.
Front Microbiol. 2022 Jan 20;12:825743. doi: 10.3389/fmicb.2021.825743. eCollection 2021.
type III secretion system (T3SS) effector SseK3 is a glycosyltransferase delivered directly into the host cells to modify host protein substrates, thus manipulating host cellular signal transduction. Here, we identify and characterize the Arg-GlcNAcylation activity of SseK3 inside bacterial cells. Combining Arg-GlcNAc protein immunoprecipitation and mass spectrometry, we found that 60 bacterial proteins were GlcNAcylated during infection, especially the two-component signal transduction system regulatory protein PhoP. Moreover, the Arg-GlcNAcylation of PhoP by SseK3 was detected and , and four arginine residues, Arg65, Arg66, Arg118, and Arg215 were identified as the GlcNAcylation sites. Site-directed mutagenesis showed that the PhoP R215A change significantly reduced the DNA-binding ability and arginine to alanine change at all four sites (PhoP 4RA) completely eliminated the DNA-binding ability, suggesting that Arg215 is essential for the DNA-binding activity of PhoP and GlcNAcylation of PhoP affects this activity. Additionally, GlcNAcylation of PhoP negatively regulated the activity of PhoP and decreased the expression of its downstream genes. Overall, our work provides an example of the intra-bacterial activities of the T3SS effectors and increases our understanding of endogenous Arg-GlcNAcylation.
III型分泌系统（T3SS）效应蛋白SseK3是一种糖基转移酶，可直接递送至宿主细胞内以修饰宿主蛋白底物，从而操纵宿主细胞信号转导。在此，我们鉴定并表征了SseK3在细菌细胞内的精氨酸-N-乙酰葡糖胺化活性。结合精氨酸-N-乙酰葡糖胺化蛋白免疫沉淀和质谱分析，我们发现60种细菌蛋白在感染过程中发生了N-乙酰葡糖胺化，尤其是双组分信号转导系统调节蛋白PhoP。此外，检测到SseK3对PhoP的精氨酸-N-乙酰葡糖胺化作用，并鉴定出四个精氨酸残基Arg65、Arg66、Arg118和Arg215为N-乙酰葡糖胺化位点。定点诱变表明，PhoP的R215A变化显著降低了DNA结合能力，而所有四个位点的精氨酸到丙氨酸变化（PhoP 4RA）则完全消除了DNA结合能力，这表明Arg215对PhoP的DNA结合活性至关重要，且PhoP的N-乙酰葡糖胺化会影响该活性。此外，PhoP的N-乙酰葡糖胺化对PhoP的活性具有负调控作用，并降低其下游基因的表达。总体而言，我们的工作提供了一个T3SS效应蛋白细菌内活性的实例，并增进了我们对内源性精氨酸-N-乙酰葡糖胺化的理解。
