Frontiers in microbiology · 2026
Biochemical and functional characterization of Porphyromonas gingivalis HmuS protein reveals its participation in heme metabolism.
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Cierpisz Patryk, Śmiga Michał, Tracz Michał, da Silva Ronivaldo Rodrigues, DuBois Jennifer L, Olczak Teresa
Аннотация
BACKGROUND: Porphyromonas gingivalis lacks the complete heme biosynthesis pathway and does not utilize siderophores to acquire iron. The exact mechanism of iron extraction from heme in this bacterium is unknown. This study aimed to characterize the P. gingivalis HmuSPg protein and to compare its properties with those of the Bacteroides fragilis homologous protein.
METHODS: UV-visible spectroscopy was used to demonstrate heme binding and follow heme metabolism. Porphyrin levels in bacterial cells and pigment were determined using HPLC or LC-MS, and iron content using atomic absorption spectroscopy or the ferrozine-based method. The influence of the hmuSPg gene deletion on the expression of other genes was analyzed using RNA-seq, RT-qPCR, Western blotting, and enzymatic assay.
RESULTS: Although the HmuSPg contains a domain characteristic of cobaltochelatases and therefore may be assigned to the I class chelatase family, the protein exhibits distinct features. HmuSPg is a large protein composed of 6 domains and possesses, predicted by structural modeling, two potential heme-binding sites. In vitro, HmuSPg binds heme. NADH and the inner membrane fraction of P. gingivalis stimulate its in vitro engagement in heme metabolism. A significantly smaller amount of PPIX, determined in bacterial cells grown in liquid media and in the pigment deposited on the bacterial cells of the ΔhmuSPg mutant strain, suggests it may have lost the ability to release iron from heme and produce PPIX. Inactivation of the hmuSPg gene results in reduced biofilm formation and induces changes in the expression of genes involved in heme uptake and homeostasis.
CONCLUSION: HmuSPg may be used in heme metabolism under certain in vivo circumstances, where heme is still available, iron is efficiently sequestered by host proteins, and intracellular iron reserves are depleted.
METHODS: UV-visible spectroscopy was used to demonstrate heme binding and follow heme metabolism. Porphyrin levels in bacterial cells and pigment were determined using HPLC or LC-MS, and iron content using atomic absorption spectroscopy or the ferrozine-based method. The influence of the hmuSPg gene deletion on the expression of other genes was analyzed using RNA-seq, RT-qPCR, Western blotting, and enzymatic assay.
RESULTS: Although the HmuSPg contains a domain characteristic of cobaltochelatases and therefore may be assigned to the I class chelatase family, the protein exhibits distinct features. HmuSPg is a large protein composed of 6 domains and possesses, predicted by structural modeling, two potential heme-binding sites. In vitro, HmuSPg binds heme. NADH and the inner membrane fraction of P. gingivalis stimulate its in vitro engagement in heme metabolism. A significantly smaller amount of PPIX, determined in bacterial cells grown in liquid media and in the pigment deposited on the bacterial cells of the ΔhmuSPg mutant strain, suggests it may have lost the ability to release iron from heme and produce PPIX. Inactivation of the hmuSPg gene results in reduced biofilm formation and induces changes in the expression of genes involved in heme uptake and homeostasis.
CONCLUSION: HmuSPg may be used in heme metabolism under certain in vivo circumstances, where heme is still available, iron is efficiently sequestered by host proteins, and intracellular iron reserves are depleted.