SseK3 Is a Salmonella Effector That Binds TRIM32 and Modulates the Host's NF-κB Signalling Activity

• Published in: PloS one Vol. 10; no. 9; p. e0138529
• Main Authors: Yang, Zhe, Soderholm, Amelia, Lung, Tania Wong Fok, Giogha, Cristina, Hill, Michelle M, Brown, Nathaniel F, Hartland, Elizabeth, Teasdale, Rohan D
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 22.09.2015; Public Library of Science (PLoS)
• Subjects: Activation; Amino acid sequence; Amino acids; Apoptosis; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cell Line, Tumor; Filaments; Genomes; Golgi apparatus; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; HeLa Cells; Host-Pathogen Interactions; Humans; Immunoblotting; Immunoglobulins; Immunology; Immunoprecipitation; Infections; Interleukin 8; Interleukin-8 - metabolism; Microscopy, Confocal; Mutation; Neoplasms - genetics; Neoplasms - metabolism; Neoplasms - microbiology; NF-kappa B - genetics; NF-kappa B - metabolism; NF-κB protein; Pathogens; Protein Binding; Proteins; RNA Interference; Salmonella; Salmonella typhimurium - genetics; Salmonella typhimurium - metabolism; Salmonella typhimurium - physiology; Signal Transduction; Studies; trans-Golgi Network - metabolism; Transcription Factors - genetics; Transcription Factors - metabolism; Tripartite Motif Proteins; Tumor necrosis factor; Ubiquitin; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases; Ubiquitination; Vacuoles; Queensland Australia; St Lucia; Victoria Australia
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0138529

Salmonella Typhimurium employs an array of type III secretion system effectors that facilitate intracellular survival and replication during infection. The Salmonella effector SseK3 was originally identified due to amino acid sequence similarity with NleB; an effector secreted by EPEC/EHEC that possesses N-acetylglucoasmine (GlcNAc) transferase activity and modifies death domain containing proteins to block extrinsic apoptosis. In this study, immunoprecipitation of SseK3 defined a novel molecular interaction between SseK3 and the host protein, TRIM32, an E3 ubiquitin ligase. The conserved DxD motif within SseK3, which is essential for the GlcNAc transferase activity of NleB, was required for TRIM32 binding and for the capacity of SseK3 to suppress TNF-stimulated activation of NF-κB pathway. However, we did not detect GlcNAc modification of TRIM32 by SseK3, nor did the SseK3-TRIM32 interaction impact on TRIM32 ubiquitination that is associated with its activation. In addition, lack of sseK3 in Salmonella had no effect on production of the NF-κB dependent cytokine, IL-8, in HeLa cells even though TRIM32 knockdown suppressed TNF-induced NF-κB activity. Ectopically expressed SseK3 partially co-localises with TRIM32 at the trans-Golgi network, but SseK3 is not recruited to Salmonella induced vacuoles or Salmonella induced filaments during Salmonella infection. Our study has identified a novel effector-host protein interaction and suggests that SseK3 may influence NF-κB activity. However, the lack of GlcNAc modification of TRIM32 suggests that SseK3 has further, as yet unidentified, host targets.