Nitric oxide detoxification systems and cellular pathogenesis of Neisseria meningitidis

Nitric oxide is an important biological mediator of a variety of physiological processes and S nitrosylation, the addition of an NO moiety to the cysteine residue of a protein, is an established post-translational modification known to be involved in the regulation of a growing number of cellular pathways. The gram-negative bacteria, Neisseria meningitidis is capable of expressing a number of enzymes involved in partial denitrification including the nitric oxide reductase, NorB. This not only allows the bacteria to supplement their growth in the oxygen limited conditions associated with the nasopharnyx, but it also protects them from the antimicrobial effects associated with NO. Bacterial expression of NorB has a variety of consequences including enhanced bacterial survival in a macrophage cell model along with a reduction in total and nuclear levels of S nitrosothiol in an activated murine macrophage model. The work presented in this thesis was undertaken with the aim of establishing if the ability of the bacteria to detoxify NO and remove or prevent formation of S nitrosothiol would have physiological consequences with emphasis on cellular targets known to be regulated by S nitrosylation. A pre-stimulated murine macrophage model was established and the impact of bacterial infection on caspase-3 activity and cell death was investigated. Infection with wild-type bacteria led to a more rapid cell death, in the absence of caspase-3 activation, compared to cells infected with a ΔnorB mutant derivative. The transcription factor NFκB has been suggested to be regulated by S nitrosylation, and indeed the binding activity of the p65 subunit was shown to be negatively regulated in an iNOS and S nitrosylation dependent manner in a murine macrophage model in response to LPS and interferonγ (IFNγ) stimulation. Conversely, following an infection, modulation of NFκB binding activity appeared to be iNOS independent despite S-nitrosylation of NFκB occurring, suggesting an alternative mechanism of modulation that renders S nitrosylation superfluous. The ability of the bacteria to express NorB had no apparent impact on NFκB binding activity. The ubiquitous protein GAPDH was also investigated and its nuclear translocation was shown to be regulated by S nitrosylation. Infection with N. meningitidis was associated with a reduction in nuclear GAPDH translocation when compared to cells stimulated with LPS and IFNγ alone. Work was also commenced to establish if a reduction in total S-nitrosothiol as a result of bacterial NO detoxification could be observed in an in vivo mouse model. Total levels of S nitrosothiol and tri-iodide reactive species were unaffected by infection with Neisseria meningitidis either in the presence or absence of the norB gene at 4h post infection.