title: A small molecule deubiquitinase inhibitor increases localization of inducible nitric oxide synthase to the macrophage phagosome and enhances bacterial killing.
abstract: Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Burkholder
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Perry
authors.forename: Christiane E
authors.initials: CE
authors.lastname: Wobus
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Donato
authors.forename: Hollis D
authors.initials: HD
authors.lastname: Showalter
authors.forename: Vaibhav
authors.initials: V
authors.lastname: Kapuria
authors.forename: Mary X D
authors.initials: MX
authors.lastname: O'Riordan
mesh_terms: D000818:Animals; D002460:Cell Line; D003487:Cyanoacrylates; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000502:physiology; D008089:Listeria monocytogenes / Q000502:physiology; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology*; D010588:Phagosomes / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology*; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D003487:Cyanoacrylates; D009570:Nitriles; D011480:Protease Inhibitors; D011725:Pyridines; D025801:Ubiquitin; C519751:degrasyn; D052247:Nitric Oxide Synthase Type II; D010450:Endopeptidases