Human oral and gut microbiomes are crucial for maintenance of homeostasis in the human body. Porphyromonas gingivalis, the key etiologic agent of chronic periodontitis, can cause dysbiosis in the mouth and gut, which results in local and systemic infectious inflammatory diseases. Our previous work resulted in extensive biochemical and functional characterization of one of the major P. gingivalis heme acquisition systems (Hmu), with the leading role played by the HmuY hemophore-like protein. We continued our studies on the homologous heme acquisition protein (Bvu) expressed by Bacteroides vulgatus, the dominant species of the gut microbiome. Results from spectrophotometric experiments showed that Bvu binds heme preferentially under reducing conditions using Met145 and Met172 as heme iron-coordinating ligands. Bvu captures heme bound to human serum albumin and only under reducing conditions. Importantly, HmuY is able to sequester heme complexed to Bvu. This is the first study demonstrating that B. vulgatus expresses a heme-binding hemophore-like protein, thus increasing the number of members of a novel HmuY-like family. Data gained in this study confirm the importance of HmuY in the context of P. gingivalis survival in regard to its ability to cause dysbiosis also in the gut microbiome.

Iron and heme are essential nutrients for all branches of life. Pathogenic members of the Bacteroidota phylum, including Porphyromonas gingivalis, do not synthesize heme and rely on host hemoproteins for heme as a source of iron and protoporphyrin IX. P. gingivalis is the main pathogen responsible for dysbiosis in the oral microbiome and the initiation and progression of chronic periodontitis. It utilizes Hmu and Hus systems for heme uptake, including HmuY and HusA hemophore-like proteins and their cognate HmuR and HusB TonB-dependent outer membrane heme receptors. Although the mechanisms of heme uptake are relatively well characterized in P. gingivalis, little is known about the importance of heme uptake systems in heme and iron homeostasis and virulence. Therefore, this work aimed to investigate these mechanisms in detail. We characterized the P. gingivalis double mutant strain deficient in functional hmuY and hmuR or husA and husB genes. Global gene expression and phenotypic analyses revealed that the Hmu system significantly influences heme homeostasis, confirming its main role in heme supply. Both systems, particularly the Hus system, affect the virulence of P. gingivalis. Our results demonstrate the diverse role of Hmu and Hus systems in P. gingivalis heme and iron homeostasis and virulence.