The Type III Secretion Effector SsPH2 of Salmonella enterica Targets LMO4 for Ubiquitination and Degradation

Abstract

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.