B-226. Neisseria meningitidis Depletes Endogenous S-Nitrosothiol in Activated Macrophages

J. R. Laver, T. M. Stevanin, M. E. Lee, R. C. Read;
Univ. of Sheffield, Sheffield, UNITED KINGDOM.

Background: S-nitrosylation is the coupling of Nitric Oxide (NO) with a reactive cysteine thiol to form an S-nitrosothiol (SNO) and is regulatory of numerous mammalian cellular functions. Macrophages produce NO and thus endogenous SNO via the inducible NO synthase (iNOS), which is expressed in response to infection. Neisseria meningitidis (Nm), the cause of meningococcal disease, is exposed to NO during natural colonisation and disease. The Nm genome includes the gene norB, encoding a nitric oxide reductase (NorB) that converts NO to nitrous oxide (N2O). NorB detoxifies NO and enhances survival of Nm within monocyte-derived macrophages (MDM). This work tested whether infection of activated macrophages with Nm is accompanied by depletion of endogenous host-cell SNO; as a consequence of NO metabolism by the organism. Methods: J774.2 murine macrophages were stimulated to produce SNO with exposure to lipopolysaccharide (LPS) and mouse interferon-γ (IFNγ) for 18 h. The cytoplasmic and nuclear SNO concentrations of Nm-infected, LPS/IFNγ-stimulated J774.2 murine macrophages were measured using tri-iodide dependent, ozone-based chemiluminescence. Results: NorB increased the rate of SNO decomposition in vitro. Cytoplasmic SNO concentration in activated J774.2 cells (139.661 + 52.24 pmol/mg) was significantly reduced during 4 h infection with log phase, wild type Nm (57.34 + 23.71 pmol/mg). This effect was ablated in cells infected with either heat-killed wild type bacteria (125.474 + 34.61 pmol/mg) or NorB-deficient mutant bacteria (116.062 + 37.28 pmol/mg). A mutant strain overexpressing the partial denitrification pathway (nsrR-) also significantly reduced SNO concentration (77.24 + 19.16 pmol/mg). A similar pattern was observed for macrophage nuclear SNO concentrations. Conclusion: NorB of Nm causes a significant reduction in the concentration of endogenously produced, host cell SNO. This works implies a novel role for nitric oxide detoxification in meningococcal pathogenesis, representing a hitherto unrecognized mechanism of meningococcal disease.