Deubiquitinating enzyme OTUB1 is essential for intracellular control of Salmonella Typhimurium in macrophages during infection.

Objectives: The bacterium Salmonella Typhimurium (STm) causes non-typhoidal gastrointestinal tract disease upon ingestion of contaminated food or water. STm first invades intestinal epithelial cells and then infects basolaterally located macrophages. Macrophages play an important role in the control of the pathogen but also serve as a niche for intracellular bacterial growth. Invasion and replication within the macrophages trigger activation of pro-inflammatory signaling pathways, which are essential for anti-bacterial responses. These pathways are tightly regulated by posttranslational modifications including ubiquitination/deubiquitination. The deubiquitinating enzyme (DUB) OTUB1 is one such versatile DUB, which preferentially cleaves K48 ubiquitin chains from substrates or can bind to E2 ubiquitin-conjugating enzymes to prevent the ubiquitination of the substrate and thereby regulate signaling. The objective of this project is to study the role of OTUB1 in macrophages during Salmonella infection.

Methods: We generated conditional OTUB1-deficient mice (LysM-Cre OTUB1^fl/fl) with deletion of OTUB1 in macrophages. LysM-Cre OTUB1^fl/fl and control OTUB1^fl/fl mice were orally infected with SL1344 wild-type STm. For in vitro infection, OTUB1-deficient and -competent bone marrow-derived macrophages (BMDM) were used.

Results: Upon oral infection with STm, LysM-Cre OTUB1^fl/fl mice harbored higher bacterial load and showed increased inflammation in the spleen and liver compared to the OTUB1^fl/fl mice. In concurrence, OTUB1-deficient BMDMs were unable to control STm in vitro. The impaired control of Salmonella in-OTUB1-deficient BMDM was paralleled by impaired activation of protective pro-inflammatory NF-κB and MAPK signaling and decreased production of anti-bacterial nitric oxide (NO). Mechanistically, OTUB1 removes the K48-like ubiquitin chain, stabilizes the E2-conjugating enzymes UBC13 and UBCH5c, and thereby fosters NF-KB and MAPK-mediated production of antibacterial NO in macrophages.

Conclusions: These data identify OTUB1 as a critical regulator of UBC13 and UBCH5c mediated anti-STm response in macrophages.