Molecular Insights into Hydrogen Peroxide-sensing Mechanism of the Metalloregulator MntR in Controlling Bacterial Resistance to Oxidative Stresses.

Manganese contributes to anti-oxidative stress particularly in catalase-devoid bacteria, and DtxR family metalloregulators, through sensing cellular Mn2+ content, regulate its homeostasis. Here, we show that metalloregulator MntR (So-MntR) functions dually as Mn2+ and H2 O2 sensors in mediating H2 O2 resistance by an oral streptococcus. H2 O2 disrupted So-MntR binding to Mn2+ transporter mntABC promoter and induced disulfide-linked dimerization of the protein. Mass spectrometry identified Cys-11/Cys-156 and Cys-11/Cys-11 disulfide-linked peptides in H2 O2 -treated So-MntR. Site mutagenesis of Cys-11 and Cys-156 and particularly Cys-11 abolished H2 O2 -induced disulfide-linked dimers and weakened H2 O2 damage on So-MntR binding, indicating that H2 O2 inactivates So-MntR via disulfide-linked dimerization. So-MntR C123S mutant was extremely sensitive to H2 O2 oxidization in dimerization/oligomerization, probably because the mutagenesis caused a conformational change that facilitates Cys-11/Cys-156 disulfide linkage. Intermolecular Cys-11/Cys-11 disulfide was detected in C123S/C156S double mutant. Redox Western blot detected So-MntR oligomers in air-exposed cells but remarkably decreased upon H2 O2 pulsing, suggesting a proteolysis of the disulfide-linked So-MntR oligomers. Remarkably, elevated C11S and C156S but much lower C123S proteins were detected in H2 O2 -pulsed cells, confirming Cys-11 and Cys-156 contributed to H2 O2 -induced oligomerization and degradation. Accordingly, in the C11S and C156S mutants, expression of mntABC and cellular Mn2+ decreased, but H2 O2 susceptibility increased. In the C123S mutant, increased mntABC expression, cellular Mn2+ content, and manganese-mediated H2 O2 survival were determined. Given the wide distribution of Cys-11 in streptococcal DtxR-like metalloregulators, the disclosed redox regulatory function and mechanism of So-MntR can be employed by the DtxR family proteins in bacterial resistance to oxidative stress.