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 Mn content, regulate its homeostasis. Here, we show that metalloregulator MntR (So-MntR) functions dually as Mn and HO sensors in mediating HO resistance by an oral streptococcus. HO disrupted So-MntR binding to Mn transporter 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 HO-treated So-MntR. Site mutagenesis of Cys-11 and Cys-156 and particularly Cys-11 abolished HO-induced disulfide-linked dimers and weakened HO damage on So-MntR binding, indicating that HO inactivates So-MntR via disulfide-linked dimerization. So-MntR C123S mutant was extremely sensitive to HO 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 HO pulsing, suggesting a proteolysis of the disulfide-linked So-MntR oligomers. Remarkably, elevated C11S and C156S but much lower C123S proteins were detected in HO-pulsed cells, confirming Cys-11 and Cys-156 contributed to HO-induced oligomerization and degradation. Accordingly, in the C11S and C156S mutants, expression of and cellular Mn decreased, but HO susceptibility increased. In the C123S mutant, increased expression, cellular Mn content, and manganese-mediated HO 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.