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

Abstract

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.

The Salmonella Effector SpvD Is a Cysteine Hydrolase with a Serovar-specific Polymorphism Influencing Catalytic Activity, Suppression of Immune Responses, and Bacterial Virulence

“…To investigate the influence of the amino acid side chain at position 161 and the requirement for the putative catalytic cysteine (Cys-73) on the anti-inflammatory activity of SpvD, HEK 293 cells were co-transfected with a reporter plasmid encoding luciferase under the control of an NF-κB promoter and a plasmid encoding either myc alone (pRK5 vector), myc-SpvD variants with alanine at position 154, or GFP-tagged positive control inhibitory proteins: Salmonella SseK3 (24) and NF-κB inhibitor α (IκBα) S32A/S36A mutant protein (25). Luciferase activity was measured 16 h after stimulation with TNFα or phorbol 12-myristate 13-acetate (PMA) and normalized to that of non-stimulated cells.…”

Salmonella effector SopD2 interferes with Rab34 function

“…Western immunoblotting using ECL and Odyssey infrared imaging system (LI-COR Biosciences) were performed as described previously (Yang et al, 2015). Western immunoblotting using ECL and Odyssey infrared imaging system (LI-COR Biosciences) were performed as described previously (Yang et al, 2015).…”

“…Salmonella infection was performed as previously described (Yang et al, 2015). Briefly, S. typhimurium (SL1344) was grown in LB broth overnight at 37 C with shaking, then subcultured at a dilution of 1:31 at 37 C with shaking for 3 h. The bacterial inoculum was subsequently prepared by pelleting at 13,500 g in a microfuge and resuspended in 1Â PBS.…”

“…Cell lysates were resuspended in SDS lysis buffer (100 mM Tris/HCL, pH 6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue, 200 nM dithiothreitol) and boiled at 95 C for 10 min. Western immunoblotting using ECL and Odyssey infrared imaging system (LI-COR Biosciences) were performed as described previously (Yang et al, 2015). Fluorescence intensities were detected by LI-COR Odyssey Infrared Imaging System (LI-COR).…”

Reducing the cost of semi-automated in-gel tryptic digestion and GeLC sample preparation for high-throughput proteomics

“…We have utilized this protocol for GeLC-MS/MS in several scientific studies [10][11][12][13][14][15][16][17][18]; here we report the results of a series of experiments conducted to determine the reproducibility, degree of contamination and time savings of the semi-automated protocols for IGD and GeLC. For these direct comparisons, the protocols were performed with the same incubation times, consumables and reagents.…”