Systemtic Identification And Functional Evaluation Of Interacting Proteins Of Salmonella Enterica Effectors

Salmonella entehca encodes two distinct type III secretion systems (T3SS) essential for virulence, encoded by genes located in the Salmonella pathogenicity islands1and2(SPI-1and SPI-2), respectively. These systems mediate the translocation of effectors into the eukaryotic host cell, where they alter cell signaling and manipulate host cell functions. However, the precise role of most effectors remains unknown. To better understand the molecular mechanism of Salmonella effectors interaction with host proteins, we made the following two approaches:1. Systematic yeast two-hybrid library screens for25Salmonella effectors (SsPH2^SseI、SpiC、seF、SseG、SlrP、SseJ、SopD2、SifB、SipC、ipB、SipA、SopD、 SipD、SptP、SopA、SopE2、SrfE、SrfA、SrfJ) were performed, partial of results were further confirmed by coimmunoprecipitation (Co-IP). This gave rise to321distinct protein-protein interactions. Together with literature-curated data, these results provide a comprehensive insight into the complexity of the host-bacteria protein interaction network involving in Salmonella invasion and introcellular replication, and a detailed pathway for these Salmonella effectors from gene to function is proposed.2. Using a yeast two-hybrid screen, we identified the LIM domain family protein LM04as a mammalian binding partner of the Salmonella effector SsPH2. The interaction was confirmed by GST pull-down and Co-IP assays. Using deletion mutants of both SsPH2and LMO4. we determined the LRR domain of SsPH2and the LIM domain of LM04were critical for the interaction. We further demonstrated that SsPH2was able to mediate ubiquitination of LM04both in vivo and in vitro, and this addition of poly-ubiquitin chains was neither lysine-48-linked nor lysine-63-linked. Furthermore,29lysine,67lysine located within the LIM domain were the major ubiquitination sites of LM04. We also discovered that stable expression of SsPH2in293T cells enhanced LM04protein expression, and this increase of protein level also depended on the SsPH2-mediated ubiquitination of LM04. Considering LM04acts as a scaffold for stabilization of the gpl30complex, which is a common receptor subunit of interleukin-6(IL-6), Hypothesis is that stable expression of SsPH2in293T cells reveals a positive regulation in IL-6signaling and results in a significant increase of transcriptional activity of Stat3(signal transducers and activators of transcription3). Identification of the ubiquitination substrates has been a major approach to understand the functions of ubiquitin ligases. Our findings assign a functional role to the Salmonella effector SsPH2as a binding partner and an E3ubiquitin ligase for mammalian LMO4. Through our project, we found some new clues which may facilitate future characterization of Salmonella effectors functions and functional mechanisms.