Intracellular Macrophage Infections with E. coli under Nitrosative Stress.
Bio-protocol(2012)
摘要
() 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. , extraintestinal pathogenic (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 (Bokil 2011). This protocol details an intracellular murine macrophage-like cell infection, including modulation of the host nitrosative stress response, to model this host-pathogen interaction . 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 of strains or isogenic deletions.
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