The Salmonella Effector SspH2 Facilitates Spatially Selective Ubiquitination of NOD1 to Enhance Inflammatory Signaling

• Published in: Biochemistry (Easton) 2024-09, Vol.63 (18), p.2266-2279
• Main Authors: Delyea, Cole J., Forster, Malcolm D., Luo, Shu, Dubrule, Bradley E., Julien, Olivier, Bhavsar, Amit P.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; HEK293 Cells; Humans; Immunity, Innate; Inflammation - metabolism; Inflammation - microbiology; NF-kappa B - metabolism; Nod1 Signaling Adaptor Protein - genetics; Nod1 Signaling Adaptor Protein - metabolism; Nod2 Signaling Adaptor Protein - genetics; Nod2 Signaling Adaptor Protein - metabolism; Salmonella Infections - immunology; Salmonella Infections - metabolism; Salmonella Infections - microbiology; Salmonella typhimurium - metabolism; Signal Transduction; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitination
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/acs.biochem.4c00380

As part of its pathogenesis, Salmonella enterica serovar Typhimurium delivers effector proteins into host cells. One effector is SspH2, a member of the so-called novel E3 ubiquitin ligase family, that interacts with and enhances, NOD1 pro-inflammatory signaling, though the underlying mechanisms are unclear. Here, we report that SspH2 interacts with multiple members of the NLRC family to enhance pro-inflammatory signaling by targeted ubiquitination. We show that SspH2 modulates host innate immunity by interacting with both NOD1 and NOD2 in mammalian epithelial cell culture via the NF-κB pathway. Moreover, purified SspH2 and NOD1 directly interact, where NOD1 potentiates SspH2 E3 ubiquitin ligase activity. Mass spectrometry and mutational analyses identified four key lysine residues in NOD1 that are required for its enhanced activation by SspH2, but not its basal activity. These critical lysine residues are positioned in the same region of NOD1 and define a surface on the receptor that appears to be targeted by SspH2. Overall, this work provides evidence for post-translational modification of NOD1 by ubiquitin and uncovers a unique mechanism of spatially selective ubiquitination to enhance the activation of an archetypal NLR.