Molecular basis of H2O2 resistance mediated by Streptococcal Dpr. Demonstration of the functional involvement of the putative ferroxidase center by site-directed mutagenesis in Streptococcus suis.
H(2)O(2) is an unavoidable cytotoxic by-product of aerobic life. Dpr, a recently discovered member of the Dps protein family, provides a means for catalase-negative bacteria to tolerate H(2)O(2). Potentially, Dpr could bind free intracellular iron and thus inhibit the Fenton chemistry-catalyzed formation of toxic hydroxyl radicals (H(2)O(2) + Fe(2+) --> (.)OH + (-)OH + Fe(3+)). We explored the in vivo function of Dpr in the catalase- and NADH peroxidase-negative pig and human pathogen Streptococcus suis. We show that: (i) a Dpr allelic exchange knockout mutant was hypersensitive ( approximately 10(6)-fold) to H(2)O(2), (ii) Dpr incorporated iron in vivo, (iii) a putative ferroxidase center was present in Dpr, (iv) single amino acid substitutions D74A or E78A to the putative ferroxidase center abolished the in vivo iron incorporation, and (v) the H(2)O(2) hypersensitive phenotype was complemented by wild-type Dpr or by a membrane-permeating iron chelator, but not by the site-mutated forms of Dpr. These results demonstrate that the putative ferroxidase center of Dpr is functionally active in iron incorporation and that the H(2)O(2) resistance is mediated by Dpr in vivo by its iron binding activity.
Pulliainen
Arto Tapio
AT
Department of Medical Biochemistry and Molecular Biology, University of Turku, Finland. endonukleaasi@hotmail.com
Haataja
Sauli
S
Kähkönen
Sanni
S
Finne
Jukka
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002570:Ceruloplasmin / Q000502:physiology*; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D005798:Genes, Bacterial; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007502:Iron Chelating Agents / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D017011:Streptococcus suis / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C414750:Dpr protein, Streptococcus; D007502:Iron Chelating Agents; D006861:Hydrogen Peroxide; D002570:Ceruloplasmin