As a facultative aerobe with a high iron requirement and a highly active aerobic respiratory chain, Neisseria gonorrhoeae requires defense systems to respond to toxic oxygen species such as superoxide. The mechanism by which the majority of N. gonorrhoeae strains overcome the effects of superoxide is unclear, as only 20% of strains surveyed possess any superoxide dismutase (SOD) activity. All of the reported activity is SodB.
The hypothesis that this bacterium uses manganese, Mn(II), as a chemical quenching agent in a similar way to the already established process in Lactobacillus plantarum was proposed. It has been shown that supplementation of media with 100 µM Mn(II) considerably enhanced the resistance of this bacterium to oxidative killing by superoxide and hydrogen peroxide.
In contrast to previous studies, which suggested that some strains of N. gonorrhoeae might not contain a SOD, a sodB gene was identified by genome analysis and its presence in all strains examined was confirmed by Southern blotting, but no evidence for sodA or sodC was found. As a sodB mutant also showed Mn dependent resistance to oxidative killing, it is concluded that the Mn dependent protection was independent of SOD.
A search for putative Mn(II) uptake systems identified an ABC cassette-type system (MntABC) with a periplasmic-binding protein (MntC). This MntABC transporter belongs to a group of recently characterized ABC permeases, cluster IX. These are identified by the nature of the solute that is bound by their extracytoplasmic binding protein component. An mntC mutant was shown to have lowered accumulation of Mn(II). A series of in vitro killing assays, using either superoxide generators such as paraquat or hydrogen peroxide, and an in vivo neutrophil killing assay, demonstrated that the mntC mutant was more sensitive to oxidative killing than the wild-type cells. Therefore, N. gonorrhoeae has a Mn(II) transport system that is critical for resistance to oxidative stress.
Both Mn and MntC were demonstrated to play a regulatory role in gene expression. Mn dampened catalase induction by hydrogen peroxide and repressed pilin expression, the major virulence factor in N. gonorrhoeae, as shown by catalase enzymatic measurement and western blot, respectively. MntC may also act as an activator of both catalase and pilin production as shown in the analysis of the mntC mutant. In addition, the mntC mutant seemed to have a pleiotropic effect, i.e. MntC may be a global regulator.
It has been reported that Neisseria gonorrhoeae has a very high level of catalase activity, but the regulation of catalase expression has not been investigated extensively. In Escherichia coli and Salmonella typhimurium it has been demonstrated that OxyR is a positive regulator of hydrogen peroxide-inducible genes, including catalase. The oxyR gene from N. gonorrhoeae was cloned and used to complement an E. coli oxyR mutant, confirming its identity and function. In contrast to E. coli, the oxyR mutant in N. gonorrhoeae expressed nine-fold more catalase activity and was more resistant to hydrogen peroxide killing than the wild-type. These data are consistent with OxyR in N. gonorrhoeae acting as a repressor of catalase expression.
PsaA, an MntC homologue, is a 37-kDa pneumococcal lipoprotein which is part of an ABC Mn(II) transport complex in Streptococcus pneumoniae. psaA mutants have previously been shown to be significantly less virulent than the wild-type D39, but the mechanism underlying the attenuation has not been resolved. In this study, psaA and psaD mutants have been shown to be highly sensitive to oxidative stress, induced by superoxide and hydrogen peroxide, which might explain why they are less virulent than the wild-type. In addition, our investigations revealed altered expression of the key oxidative stress response enzymes SOD and NADH oxidase in psaA and psaD mutants, suggesting PsaA and PsaD may play an important role in regulation of oxidative stress responses and intracellular redox homeostasis.
Taken together, both MntC and PsaA are important in resistance to oxidative stress and play a regulatory role in antioxidant gene expression in N. gonorrhoeae (a Gram-negative organism) and S. pneumoniae (a Gram-positive organism). Therefore, this mechanism may be applied to other bacteria.