State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciences, 130112, Changchun, China.
Military Veterinary Institute, Academy of Military Medical Sciences, 130112, Changchun, China.
BMC Vet Res. 2021 Aug 30;17(1):289. doi: 10.1186/s12917-021-02993-9.
UTP-glucose-1-phosphoryl transferase (UGPase) catalyzes the synthesis of UDP-glucose, which is essential for generating the glycogen needed for the synthesis of bacterial lipopolysaccharide (LPS) and capsular polysaccharide, which play important roles in bacterial virulence. However, the molecular function of UGPase in Brucella is still unknown.
In this study, the ubiquitination modification of host immune-related protein in cells infected with UGPase-deleted or wild-type Brucella was analyzed using ubiquitination proteomics technology. The ubiquitination modification level and type of NF-κB Essential Modulator (NEMO or Ikbkg), a molecule necessary for NF-κB signal activation, was evaluated using Coimmunoprecipitation, Western blot, and dual-Luciferase Assay. We found 80 ubiquitin proteins were upregulated and 203 ubiquitin proteins were downregulated in cells infected with B. melitensis 16 M compared with those of B. melitensis UGPase-deleted strain (16 M-UGPase). Moreover, the ubiquitin-modified proteins were mostly enriched in the categories of regulation of kinase/NF-κB signaling and response to a bacterium, suggesting Brucella UGPase inhibits ubiquitin modification of related proteins in the host NF-κB signaling pathway. Further analysis showed that the ubiquitination levels of NEMO K63 (K63-Ub) and Met1 (Met1-Ub) were significantly increased in the 16 M-UGPase-infected cells compared with that of the 16 M-infected cells, further confirming that the ubiquitination levels of NF-κB signaling-related proteins were regulated by the bacterial UGPase. Besides, the expression level of IκBα was decreased, but the level of p-P65 was significantly increased in the 16 M-UGPase-infected cells compared with that of the 16 M- and mock-infected cells, demonstrating that B. melitensis UGPase can significantly inhibit the degradation of IκBα and the phosphorylation of p65, and thus suppressing the NF-κB pathway.
The results of this study showed that Brucella melitensis UGPase inhibits the activation of NF-κB by modulating the ubiquitination of NEMO, which will provide a new scientific basis for the study of immune mechanisms induced by Brucella.
尿苷二磷酸葡萄糖-1-磷酸转移酶（UGPase）催化 UDP-葡萄糖的合成，这对于生成细菌脂多糖（LPS）和荚膜多糖所需的糖原至关重要，而 LPS 和荚膜多糖在细菌毒力中起着重要作用。然而，UGPase 在布鲁氏菌中的分子功能尚不清楚。
本研究利用泛素蛋白组学技术分析了 UGPase 缺失或野生型布鲁氏菌感染细胞中宿主免疫相关蛋白的泛素化修饰。通过共免疫沉淀、Western blot 和双荧光素酶报告基因分析，评估了 NF-κB 信号激活所必需的分子 NF-κB 必需调节剂（NEMO 或 Ikbkg）的泛素化修饰水平和类型。与布鲁氏菌 16M-UGPase 缺失株相比，我们发现感染伯氏疏螺旋体 16M 的细胞中有 80 种泛素蛋白上调和 203 种泛素蛋白下调。此外，泛素修饰蛋白主要富集在激酶/NF-κB 信号调节和对细菌的反应类别中，这表明布鲁氏菌 UGPase 抑制宿主 NF-κB 信号通路中相关蛋白的泛素化修饰。进一步分析表明，与 16M 感染细胞相比，16M-UGPase 感染细胞中 NEMO K63（K63-Ub）和 Met1（Met1-Ub）的泛素化水平显著增加，进一步证实了 NF-κB 信号相关蛋白的泛素化水平受到细菌 UGPase 的调节。此外，与 16M 和 mock 感染细胞相比，16M-UGPase 感染细胞中 IκBα 的表达水平降低，但 p-P65 的水平显著升高，表明伯氏疏螺旋体 UGPase 可显著抑制 IκBα 的降解和 p65 的磷酸化，从而抑制 NF-κB 通路。
本研究结果表明，布鲁氏菌 UGPase 通过调节 NEMO 的泛素化来抑制 NF-κB 的激活，这将为布鲁氏菌诱导的免疫机制研究提供新的科学依据。
