Deficiency of deubiquitinating enzyme OTUB1 leads to impaired pathogen control in macrophages during Salmonella infection
SAGAR I. 1, BEYER S. 1, GOPALAKRISHNA N. 1, SCHLÜTER D. 1
1 Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany
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 OTUB1fl/fl) with deletion of OTUB1 in macrophages. LysM-Cre OTUB1fl/fl and control OTUB1fl/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 OTUB1fl/fl mice had higher pathogen load and inflammation in spleen and liver compared to the OTUB1fl/fl mice. We confirmed the OTUB1-dependent control of STm in vitro in infected BMDM. The impaired control of Salmonella in-OTUB1-deficient BMDM was paralleled by impaired activation of protective pro-inflammatory NF-κB and MAPK signaling. Mechanistically, OTUB1 augments NF-KB and MAPK activity by removing K48 ubiquitin chain and stabilizing the E2-conjugating enzymes UBC13 and UBCH5c, which are responsible for auto-ubiquitination and activation of TNF receptor-associated factor (TRAF)-6. Consequently, OTUB1 deficiency in macrophages leads to reduced production of cytokines and antibacterial molecules.
Conclusions: These data illustrate an important role of OTUB1 in the regulation of pro-inflammatory signaling and the intracellular control of STm in macrophages.