Characterization of the hemin-binding property of Porphyromonas endodontalis
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Porphyromonas endodontalis , found in the root canal of teeth, requires iron for growth. However, the mechanism of iron uptake in P. endodontalis remains unclear. The ability of bacteria to utilize heme compounds to acquire iron for growth has been reported in some pathogenic bacteria. In the present study, we analyzed the ability of P. endodontalis to obtain iron from heme compounds. Further, we investigated the hemin-binding characteristics of P. endodontalis and the relationship between hemin binding and Congo red binding. To confirm the bacterial growth in hemin-supplemented medium, iron was removed from the medium with an ironchelator, and hemin was supplemented to an iron-free medium. The hemin-binding characteristics of P. endodontalis were analyzed by incubating bacteria with hemin and measuring the optical density of the supernatant obtained via centrifugation, using hemin concentration standard curve. Although growth of P. endodontalis was not observed in the iron-depleted medium, it was observed in a hemin-supplemented medium. Further, hemin binding was dependent on the concentrations of hemin and bacteria. Hemin binding proceeded quickly in P. endodontalis, and the incubation temperature had no effect on this binding. Similar to hemin binding, Congo red binding of P. endodontalis was dependent on Congo red and bacterial concentrations. In addition, Congo red binding of P. endodontalis was inhibited by hemin prebinding. Hemin-agarose beads and SDS-PAGE were used to identify a 40-kDa protein that could be involved in hemin binding. The results showed that P. endodontalis could bind to and use hemin to obtain the iron required for growth.
Abstract
Porphyromonas endodontalis , found in the root canal of teeth, requires iron for growth. However, the mechanism of iron uptake in P. endodontalis remains unclear. The ability of bacteria to utilize heme compounds to acquire iron for growth has been reported in some pathogenic bacteria. In the present study, we analyzed the ability of P. endodontalis to obtain iron from heme compounds. Further, we investigated the hemin-binding characteristics of P. endodontalis and the relationship between hemin binding and Congo red binding. To confirm the bacterial growth in hemin-supplemented medium, iron was removed from the medium with an ironchelator, and hemin was supplemented to an iron-free medium. The hemin-binding characteristics of P. endodontalis were analyzed by incubating bacteria with hemin and measuring the optical density of the supernatant obtained via centrifugation, using hemin concentration standard curve. Although growth of P. endodontalis was not observed in the iron-depleted medium, it was observed in a hemin-supplemented medium. Further, hemin binding was dependent on the concentrations of hemin and bacteria. Hemin binding proceeded quickly in P. endodontalis, and the incubation temperature had no effect on this binding. Similar to hemin binding, Congo red binding of P. endodontalis was dependent on Congo red and bacterial concentrations. In addition, Congo red binding of P. endodontalis was inhibited by hemin prebinding. Hemin-agarose beads and SDS-PAGE were used to identify a 40-kDa protein that could be involved in hemin binding. The results showed that P. endodontalis could bind to and use hemin to obtain the iron required for growth.
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