| The discovery of RitR (Repressor of Iron Transport Regulator), the first iron regulatory protein identified in the pathogen Streptococcus pneumoniae, and a characterization of its action are described. RitR is an atypical two-component orphan response regulator that is co-transcribed with gnd (6-phosphogluconate dehydrogenase), and possesses an unusual asparagine substitution at its canonical phosphorylatable aspartate position. In a ritR(-) mutant vs. ritR(+) wild-type murine infection model comparison, ritR was shown to be necessary for lung, but not systemic infection, and can therefore be considered as a tissue-specific regulatory element important for disease progression. A model is proposed by which RitR: (1) represses iron uptake at high oxygen concentrations when S. pneumoniae colonizes the upper and lower respiratory tract, and (2) is instrumental in the regulation of iron and oxidative stress in these environments.; By DNA footprinting, RitR was shown to repress iron uptake by directly binding to the promoter of the ATP-binding cassette (ABC) transporter piu (Pneumococcal Iron Uptake). These data revealed a RitR-binding motif containing an invariant "ATTA" core. The binding motif was used to search in silico for additional RitR target sequences. Conversely, RitR was associated with the activation of several genes involved with DNA repair, iron storage, and iron toxicity remediation. A comparison between ritR(-) mutant and wild-type cells in which streptonigrin killing assays and plasma mass spectrometry were used, RitR was shown to control uptake of both hemin and of ferrous iron. In addition, the ritR(-) mutant was more susceptible to peroxide-induced oxidative stress compared to wild-type cells, owing to a higher intracellular iron concentration.; gnd-ritR expression was found to be mediated through transcriptional rather than post-translational phosphoryl transfer to aspartate, the latter of which represents the most commonly reported response of two-component regulators. Both hemin and protoporphyrin IX repressed gnd-ritR production, whereas iron was able to reverse this repression. A model is proposed according to which transcriptional control of the gnd-ritR operon in S. pneumoniae is instrumental in maintaining iron homeostasis, avoiding iron-related toxicity, and remediating genotoxicity due to iron-catalyzed synthesis of reactive iron intermediates. |
