title: Intracellular Macrophage Infections withE. coliunder Nitrosative Stress.
abstract: Escherichia coli(E. coli) produces disseminated infections of the urinary tract, blood, and central nervous system where it encounters professional phagocytes such as macrophages, which utilize reactive nitrogen intermediates (RNI) to arrest bacteria.In vitro, extraintestinal pathogenicE. coli(ExPEC) can survive within bone marrow-derived macrophages for greater than 24 h post-infection within a LAMP1+ vesicular compartment, and ExPEC strains, in particular, are better adapted to intracellular macrophage survival than commensal strains (Bokilet al.,2011). This protocol details an intracellular murine macrophage-like cell infection, including modulation of the host nitrosative stress response, to model this host-pathogen interactionin vitro. To accomplish this, RAW 264.7 murine macrophage-like cells are pre-incubated with either L-arginine, an NO precursor, or IFNγ to yield a high nitric oxide (NO) physiological state, or L-NAME, an inducible NO synthase (iNOS)-specific inhibitor, to yield a low NO physiological state. This protocol has been successfully utilized to assess the contribution of a novel ExPEC regulator to intracellular survival and the nitrosative stress response during macrophage infections (Bateman and Seed, 2012), but can be adapted for use with a variety ofE. colistrains or isogenic deletions.
authors.affiliation: Department of Molecular Genetics and Microbiology Center for Microbial Pathogenesis, Department of Pediatrics, Duke University School of Medicine, Durham, NC, USA.
authors.forename: Stacey L
authors.initials: SL
authors.lastname: Bateman
authors.affiliation: Department of Molecular Genetics and Microbiology Center for Microbial Pathogenesis, Department of Pediatrics, Duke University School of Medicine, Durham, NC, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Seed