Porphyromonas gingivalis HmuY and Bacteroides vulgatus Bvu—A Novel Competitive Heme Acquisition Strategy

Abstract

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.

Unique Properties of Heme Binding of the Porphyromonas gingivalis HmuY Hemophore-like Protein Result from the Evolutionary Adaptation of the Protein Structure

“…HmuY is the first and best characterized representative of a novel family of bacterial hemophore-like proteins, distinct from a family of classical secreted hemophores [ 16 , 30 ]. Previously, we performed detailed phylogenetic analyses [ 24 , 25 , 26 , 27 , 28 ], which demonstrated that although the proteins forming this unique family are phylogenetically related and structurally similar, differences in amino acid sequences ( Figure 1 ) could result in different structure of heme-binding pockets ( Figure 2 ). The independent evolution of heme binding resulted in different mechanisms of heme coordination in HmuY (two histidine residues) compared to its homologs characterized so far (two methionine residues).…”

“…Our theoretical and experimental analyses identified homologs of P. gingivalis HmuY in other bacteria [ 24 , 25 , 26 , 27 , 28 ]. In general, the identity of amino acid sequences of these proteins is rather low.…”

“…In general, the identity of amino acid sequences of these proteins is rather low. Although three-dimensional protein structures of members of the HmuY family identified in the phylum Bacteroidetes are similar overall, significant differences exist in regions corresponding to the heme-binding pocket [ 26 , 27 , 28 , 29 ]. This finding was confirmed by our crystallographic studies revealing a unique protein structure of the P. gingivalis HmuY protein in both the apoform (PDB ID: 6EWM) [ 26 ] and in the complex with heme (PDB ID: 3H8T) [ 29 ].…”

“…HmuY family proteins function similarly to classical bacterial hemophores that bind heme with high affinity and deliver it to the TonB-dependent outer membrane receptors [ 30 ]. However, in contrast to secreted hemophores, HmuY family proteins are mainly composed of β-structures, are associated with the outer membrane of the bacterial cell and outer membrane vesicles through the lipid anchor, and could subsequently be shed from their surface by limited proteolytic processing [ 26 , 27 , 28 , 29 , 31 ]. Proteins belonging to the HmuY family also use different amino acid residues to bind heme.…”

“…Proteins belonging to the HmuY family also use different amino acid residues to bind heme. Histidine residues (H134 and H166; numbering of amino acid residues according to the full-length, unprocessed proteins is used) coordinating the heme iron in P. gingivalis HmuY [ 29 , 32 ] are unique and generally poorly conserved in the majority of sequences of HmuY homologs identified so far, which mainly utilize methionine residues [ 26 , 27 , 28 ].…”