Neisseria meningitidis and Neisseria gonorrhoeae are the causative agents
of meningococcal disease and gonorrhoea respectively. Disease caused by these organisms is a significant health problem worldwide despite continual advances in treatment and vaccine development. Bacterial oxidative stress defences are often linked to bacterial virulence due to their ability to reduce the effectiveness of host antimicrobial systems. The pathogenic Neisseria are exposed to oxidative stress from both exogenous sources (as a result of host cell defences and host commensal organisms) and endogenous sources (as a result of their own respiratory processes), which has deleterious effects on the cell. Therefore, mechanisms for coping with oxidative stress are crucial for the survival of these organisms.
This thesis presents the identification and characterisation of a number of the oxidative stress defence mechanisms of N. gonorrhoeae strain 1291 and N. meningitidis strain MC58. This was primarily achieved by construction and analysis of mutant strains defective in specific oxidative sfress defences. Analysis of these mutant sfrains included investigation of their survival upon exposure to oxidative stress during in vitro paraquat, xanthine/xanthine oxidase and hydrogen peroxide oxidative killing assays. A model system of polymorphonuclear neufrophil (PMN) intracellular killing was also used to investigate the mutant strains.
An ABC-type manganese (Mn) transporter system was identified in N. meningitidis that is homologous to the N. gonorrhoeae Mn uptake system (MntABC) required for Mn-dependent resistance to oxidative stress. MntC, the periplasmic binding protein, was inactivated by allelic exchange in order to investigate the role of Mn in N. meningitidis oxidative defence. The mntC mutant sfrain was shown to be highly susceptible to in vitro oxidative killing. Supplementation of growth media with Mn did not enhance resistance to oxidative killing in N. meningitidis, in confrast to the situation previously
reported in N. gonorrhoeae. However, N. meningitidis is highly resistant to Mn compared to N. gonorrhoeae, suggesting that these species differ in their Mn homeostasis.
Superoxide dismutase (SOD) plays a key role in removal of toxic superoxide radicals and is almost ubiquitous in aerobic organisms. The role of the two SODs of N. meningitidis, the cytoplasmic SOD B and the periplasmic SOD C, in oxidative sfress defence was also investigated. The SOD B of N. meningitidis is highly active and analysis of a sodB mutant strain in oxidative stress assays revealed
that SOD B plays a role in protection against oxidative killing. This result is interesting in light of previous findings which indicate that the sodB homologue of N. gonorrhoeae is inactive and is not involved in oxidative stress defence. SOD C of N. meningitidis is also highly active. However, a sodC mutant strain was no more sensitive to in vitro oxidative killing than the wild type.
The cytochrome c peroxidase (Ccp) oiN. gonorrhoeae is involved in protection from hydrogen peroxide killing, as revealed by the decreased survival of ccp
and ccp/kat mutant strains of N. gonorrhoeae in in vitro hydrogen peroxide killing assays. The distribution of Ccp was investigated via PCR, Southern hybridisation and genome sequence analysis. The Ccp of N. gonorrhoeae was found to be widespread in this species and present in commensal Neisseria strains, but absent in all N. meningitidis strains tested.
A homologue of the yeast Sco (synthesis of cytochrome oxidase) protein was identified in N. gonorrhoeae and N. meningitidis, and found to be a novel
protein involved in oxidative stress defence. Members of the Scol/SenC family are required for the formation of the CUA centre of aaa-type cytochrome oxidases in several organisms. However, an aas-type cytochrome oxidase is absent in N. gonorrhoeae and N. meningitidis. The Neisseria sco mutant strains displayed normal cytochrome oxidase activity and growth rates under aerobic and anaerobic conditions. However, both N. gonorrhoeae and N. meningitidis sco mutant strains were highly sensitive to oxidative killing indicating that Sco is involved in protection against oxidative stress in these bacteria.
Accumulation of PMNs is characteristic of N. gonorrhoeae and N. meningitidis infection. Therefore, the role of the oxidative stress responses in survival of these organisms in human PMNs was examined using the oxidative stress defence mutant strains constructed during this thesis. PMNs generate substantial amounts of superoxide, hydrogen peroxide and hypochlorous acid as part of their oxygen-dependent bactericidal mechanism. However, the mutant strains studied did not display increased sensitivity to PMN killing. These findings indicate that the pathogenic Neisseria are highly adapted to PMN killing and/or are able to subvert the oxidative burst of PMNs. The results of this thesis have led to a better understanding of the oxidative stress response of
N. gonorrhoeae and N. meningitidis. This understanding may aid the future development of treatment and prevention strategies for disease caused by these organisms.