He Chenmei, Cong Jiayu, Wang Ting, Xia Chuan
Department of Pathogen Biology and Microecology, College of Basic Medical Sciences, Dalian Medical University, Dalian, China.
Department of Bioengineering, College of Life Science and Technology, Jinan University, Guangzhou, China.
FASEB J. 2025 Jul 31;39(14):e70857. doi: 10.1096/fj.202500636R.
The type III secretion systems (T3SSs) of Salmonella enterica are encoded by genes located in the Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), which are essential for virulence. T3SSs mediate the translocation of effectors into the eukaryotic host cells, where the effectors alter cell signaling and manipulate cell functions. However, how these effectors interact with host cells is incompletely understood. Identification of the cellular binding partners could help us investigate the roles of the effectors. Here, using Salmonella SPI-2 T3SS effector SsPH2 as a bait, we performed a yeast two-hybrid screen and identified a LIM domain family protein LMO4 as a binding partner for SsPH2. This interaction was further confirmed by GST pull-down, coimmunoprecipitation, and immunofluorescence microscopy analysis. Interestingly, we determined that the expression of SsPH2 alters the subcellular localization of LMO4. Further, we revealed that the leucine-rich repeat (LRR) domain of SsPH2 and the two LIM domains of LMO4 are critical for the interaction. We demonstrated that SsPH2 mediates the Lysine 48 (K48)-linked poly-ubiquitination of LMO4 in vivo and in vitro. The Lysine 29 and Lysine 67 within the LIM domains were proven to be the major ubiquitination sites of LMO4. Furthermore, we determined that SsPH2 downregulates the levels of LMO4 by inducing the ubiquitination and proteasome-dependent degradation of LMO4. Importantly, the expression of SsPH2 destabilizes the IL-6 receptor component gp130, inhibiting the STAT3 activation. SsPH2 was also found to suppress cell migration while enhancing apoptosis. Overall, this work identifies LMO4 as a novel cellular target and ubiquitination substrate for Salmonella enterica effector SsPH2 and reveals new insights into the interplay between bacteria and the host cells.
肠炎沙门氏菌的III型分泌系统（T3SSs）由位于沙门氏菌致病岛1和2（SPI-1和SPI-2）中的基因编码，这些基因对毒力至关重要。T3SSs介导效应蛋白转运到真核宿主细胞中，在那里效应蛋白改变细胞信号传导并操纵细胞功能。然而，这些效应蛋白如何与宿主细胞相互作用尚不完全清楚。鉴定细胞结合伴侣有助于我们研究效应蛋白的作用。在此，我们以沙门氏菌SPI-2 T3SS效应蛋白SsPH2为诱饵，进行了酵母双杂交筛选，并鉴定出一种LIM结构域家族蛋白LMO4作为SsPH2的结合伴侣。通过谷胱甘肽S-转移酶（GST）下拉实验、免疫共沉淀和免疫荧光显微镜分析进一步证实了这种相互作用。有趣的是，我们确定SsPH2的表达改变了LMO4的亚细胞定位。此外，我们发现SsPH2的富含亮氨酸重复序列（LRR）结构域和LMO4的两个LIM结构域对这种相互作用至关重要。我们证明SsPH2在体内和体外介导LMO4的赖氨酸48（K48）连接的多聚泛素化。LIM结构域内的赖氨酸29和赖氨酸67被证明是LMO4的主要泛素化位点。此外，我们确定SsPH2通过诱导LMO4的泛素化和蛋白酶体依赖性降解来下调LMO4的水平。重要的是，SsPH2的表达使白细胞介素-6受体成分gp130不稳定，抑制信号转导和转录激活因子3（STAT3）的激活。还发现SsPH2抑制细胞迁移同时增强细胞凋亡。总体而言，这项工作将LMO4鉴定为肠炎沙门氏菌效应蛋白SsPH2的新型细胞靶点和泛素化底物，并揭示了细菌与宿主细胞相互作用的新见解。
