Bacteroides fragilis expresses three proteins similar to Porphyromonas gingivalis HmuY: Hemophore-like proteins differentially evolved to participate in heme acquisition in oral and gut microbiomes.

Oral and gut microbiomes are important for the maintenance of homeostasis in the human body. Altered or disturbed mutualism between their members results in dysbiosis with local injury and subsequent systemic diseases. The high bacterial density causes intense competition among microbiome residents to acquire nutrients, including iron and heme, the latter of high importance for heme auxotrophic members of the Bacteroidetes phylum. Our main hypothesis is that the heme acquisition mechanism, with the leading role played by a novel HmuY family of hemophore-like proteins, can be used to fulfill nutritional requirements and increase virulence. We characterized HmuY homologs expressed by Bacteroides fragilis and compared their properties with the first representative of this family, the HmuY protein of Porphyromonas gingivalis. In contrast to other Bacteroidetes members, B. fragilis produces three HmuY homologs (Bfr proteins). All bfr transcripts were produced at higher levels in bacteria starved of iron and heme (fold change increase ~60, ~90, and ~70 for bfrA, bfrB, and bfrC, respectively). X-ray protein crystallography showed that B. fragilis Bfr proteins are structurally similar to P. gingivalis HmuY and to other homologs, except for differences in the potential heme-binding pockets. BfrA binds heme, mesoheme, and deuteroheme, but preferentially under reducing conditions, using Met175 and Met146 to coordinate heme iron. BfrB binds iron-free protoporphyrin IX and coproporphyrin III, whereas BfrC does not bind porphyrins. HmuY is capable of heme sequestration from BfrA, which might increase the ability of P. gingivalis 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.

To acquire heme, Porphyromonas gingivalis uses a hemophore-like protein (HmuY). HmuY sequesters heme from host hemoproteins or heme-binding proteins produced by cohabiting bacteria, and delivers it to the TonB-dependent outer-membrane receptor (HmuR). Although three-dimensional protein structures of members of the novel HmuY family are overall similar, significant differences exist in their heme-binding pockets. Histidines (H134 and H166) coordinating the heme iron in P. gingivalis HmuY are unique and poorly conserved in the majority of its homologs, which utilize methionines. To examine whether changes observed in the evolution of these proteins in the Bacteroidetes phylum might result in improved heme binding ability of HmuY over its homologs, we substituted histidine residues with methionine residues. Compared to the native HmuY, site-directed mutagenesis variants bound Fe(III)heme with lower ability in a similar manner to Bacteroides vulgatus Bvu and Tannerella forsythia Tfo. However, a mixed histidine-methionine couple in the HmuY was sufficient to bind Fe(II)heme, similarly to T. forsythia Tfo, Prevotella intermedia PinO and PinA. Double substitution resulted in abolished heme binding. The structure of HmuY heme-binding pocket may have been subjected to evolution, allowing for P. gingivalis to gain an advantage in heme acquisition regardless of environmental redox conditions.

Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3.

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.

Prevotella intermedia produces two proteins homologous to Porphyromonas gingivalis HmuY but with different heme coordination mode.

As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different from that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, PinO and PinA could use one methionine residue to fulfill this function, with potential support of additional methionine residue/s. The P. intermedia proteins sequestered heme only from the host albumin-heme complex under reducing conditions. Our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme coordination modes. The newer data presented in this manuscript on HmuY homologs produced by P. intermedia sheds more light on the novel mechanism of heme uptake, could be helpful in discovering their biological function, and in developing novel therapeutic approaches.

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

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.

Glycation of Host Proteins Increases Pathogenic Potential of Porphyromonas gingivalis.

The non-enzymatic addition of glucose (glycation) to circulatory and tissue proteins is a ubiquitous pathophysiological consequence of hyperglycemia in diabetes. Given the high incidence of periodontitis and diabetes and the emerging link between these conditions, it is of crucial importance to define the basic virulence mechanisms employed by periodontopathogens such as Porphyromonas gingivalis in mediating the disease process. The aim of this study was to determine whether glycated proteins are more easily utilized by P. gingivalis to stimulate growth and promote the pathogenic potential of this bacterium. We analyzed the properties of three commonly encountered proteins in the periodontal environment that are known to become glycated and that may serve as either protein substrates or easily accessible heme sources. In vitro glycated proteins were characterized using colorimetric assays, mass spectrometry, far- and near-UV circular dichroism and UV-visible spectroscopic analyses and SDS-PAGE. The interaction of glycated hemoglobin, serum albumin and type one collagen with P. gingivalis cells or HmuY protein was examined using spectroscopic methods, SDS-PAGE and co-culturing P. gingivalis with human keratinocytes. We found that glycation increases the ability of P. gingivalis to acquire heme from hemoglobin, mostly due to heme sequestration by the HmuY hemophore-like protein. We also found an increase in biofilm formation on glycated collagen-coated abiotic surfaces. We conclude that glycation might promote the virulence of P. gingivalis by making heme more available from hemoglobin and facilitating bacterial biofilm formation, thus increasing P. gingivalis pathogenic potential in vivo.

Novel synthetic peptide derived from Porphyromonas gingivalis Lys-gingipain detects IgG-mediated host response in periodontitis.

Porphyromonas gingivalis is a keystone pathogen in periodontitis. Analysis of the immunogenicity of its virulence factors may provide insight into the host response to this infection. The Kgp12 (IEDB Epitope ID 763561), an epitope of Lys-gingipain (Kgp) virulence factor from P. gingivalis ATCC 33277, elicits an immunoglobulin G (IgG) immunoreactivity with low cross-reactivity and, therefore, more specificity. The aim of the present study was to determine in silico the localization of Kgp12 within the protein and to evaluate the IgG host response to this novel Kgp peptide through its capacity to differentiate individuals with different periodontal status. Sera of 71 volunteers were tested by indirect ELISA to detect the IgG immunoreactivity specific to Kgp12, as well as to the protein HmuY and to the sonicated total extract of P. gingivalis ATCC33277, both used as gold standard. The participants had no systemic disease and were classified according to periodontal clinical parameters to comparison, firstly, into periodontitis (P) and without periodontitis (WP) groups and, secondly, into periodontitis (P), gingivitis (G) and clinically health (CH) ones. All the antigens tested, Kgp12 (p = 0.02), HmuY (p = 0.00) and P. gingivalis extract (p = 0.03), could differentiate P from WP groups considering IgG serum levels. P group also had higher IgG levels specific to Kgp12 (p = 0.03), HmuY (p < 0.01) and P. gingivalis extract (p = 0.01) when compared to G group. We conclude that the Kgp12 synthetic peptide was useful to detect the IgG-mediated host response signaling that it is a promising epitope to analyze the immunogenicity of P. gingivalis.

Porphyromonas gingivalis HmuY and Streptococcus gordonii GAPDH-Novel Heme Acquisition Strategy in the Oral Microbiome.

The oral cavity of healthy individuals is inhabited by commensals, with species of Streptococcus being the most abundant and prevalent in sites not affected by periodontal diseases. The development of chronic periodontitis is linked with the environmental shift in the oral microbiome, leading to the domination of periodontopathogens. Structure-function studies showed that Streptococcus gordonii employs a "moonlighting" protein glyceraldehyde-3-phosphate dehydrogenase (SgGAPDH) to bind heme, thus forming a heme reservoir for exchange with other proteins. Secreted or surface-associated SgGAPDH coordinates Fe(III)heme using His43. Hemophore-like heme-binding proteins of Porphyromonas gingivalis (HmuY), Prevotella intermedia (PinO) and Tannerella forsythia (Tfo) sequester heme complexed to SgGAPDH. Co-culturing of P. gingivalis with S. gordonii results in increased hmuY gene expression, indicating that HmuY might be required for efficient inter-bacterial interactions. In contrast to the DhmuY mutant strain, the wild type strain acquires heme and forms deeper biofilm structures on blood agar plates pre-grown with S. gordonii. Therefore, our novel paradigm of heme acquisition used by P. gingivalis appears to extend to co-infections with other oral bacteria and offers a mechanism for the ability of periodontopathogens to obtain sufficient heme in the host environment. Importantly, P. gingivalis is advantaged in terms of acquiring heme, which is vital for its growth survival and virulence.

PgFur participates differentially in expression of virulence factors in more virulent A7436 and less virulent ATCC 33277 Porphyromonas gingivalis strains.

BACKGROUND: Porphyromonas gingivalis is considered a keystone pathogen responsible for chronic periodontitis. Although several virulence factors produced by this bacterium are quite well characterized, very little is known about regulatory mechanisms that allow different strains of P. gingivalis to efficiently survive in the hostile environment of the oral cavity, a typical habitat characterized by low iron and heme concentrations. The aim of this study was to characterize P. gingivalis Fur homolog (PgFur) in terms of its role in production of virulence factors in more (A7436) and less (ATCC 33277) virulent strains. RESULTS: Expression of a pgfur depends on the growth phase and iron/heme concentration. To better understand the role played by the PgFur protein in P. gingivalis virulence under low- and high-iron/heme conditions, a pgfur-deficient ATCC 33277 strain (TO16) was constructed and its phenotype compared with that of a pgfur A7436-derived mutant strain (TO6). In contrast to the TO6 strain, the TO16 strain did not differ in the growth rate and hemolytic activity compared with the ATCC 33277 strain. However, both mutant strains were more sensitive to oxidative stress and they demonstrated changes in the production of lysine- (Kgp) and arginine-specific (Rgp) gingipains. In contrast to the wild-type strains, TO6 and TO16 mutant strains produced larger amounts of HmuY protein under high iron/heme conditions. We also demonstrated differences in production of glycoconjugates between the A7436 and ATCC 33277 strains and we found evidence that PgFur protein might regulate glycosylation process. Moreover, we revealed that PgFur protein plays a role in interactions with other periodontopathogens and is important for P. gingivalis infection of THP-1-derived macrophages and survival inside the cells. Deletion of the pgfur gene influences expression of many transcription factors, including two not yet characterized transcription factors from the Crp/Fnr family. We also observed lower expression of the CRISPR/Cas genes. CONCLUSIONS: We show here for the first time that inactivation of the pgfur gene exerts a different influence on the phenotype of the A7436 and ATCC 33277 strains. Our findings further support the hypothesis that PgFur regulates expression of genes encoding surface virulence factors and/or genes involved in their maturation.

Apoptosis Transcriptional Profile Induced by Porphyromonas gingivalis HmuY.

This study aimed at evaluating the transcriptional profile of apoptosis-related genes after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) derived from individuals with periodontitis (P) and healthy nonperiodontitis (NP) control subjects with P. gingivalis HmuY protein. PBMCs from the P and NP groups were stimulated with HmuY P. gingivalis protein, and the expression of genes related to apoptosis was assessed by custom real-time polymerase chain reaction array (Custom RT2 PCR Array). Compared with the NP group, the P group showed low relative levels of apoptosis-related gene expression, downregulated for FAS, FAS ligand, TNFSF10 (TRAIL), BAK1, CASP9, and APAF1 after P. gingivalis HmuY protein stimulation. Furthermore, the P group exhibited low levels of relative gene expression, downregulated for CASP7 when the cells were not stimulated. Our data suggest that P. gingivalis HmuY protein might participate differently in the modulation of the intrinsic and extrinsic apoptosis pathways.

SLC35A5 Protein-A Golgi Complex Member with Putative Nucleotide Sugar Transport Activity.

Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising nucleotide sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N- nor O-glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in nucleotide sugar transport.

Antimicrobial activity of stable hemiaminals against Porphyromonas gingivalis.

Porphyromonas gingivalis is a major etiologic agent and a key pathogen responsible for the development and progression of chronic periodontitis. Controlling the number of periodontal pathogens is one of the primary actions for maintaining oral health; therefore, active compounds with a capacity to exert antimicrobial activity have received considerable attention as they may represent potential new therapeutic agents for the treatment of chronic periodontitis. Heterocyclic compounds possessing 1,2,4- or 1,2,3-triazoles are known for several biological activities, including antibacterial properties. Among them are stable hemiaminals which can be obtained in reaction between nitrobenzaldehyde derivatives and 4-amino-1,2,4-triazole or 4-amino-3,5-dimethyl-1,2,4-triazole. In this study, we selected two relatively stable hemiaminals: (2,4-dinitrophenyl)(4H-1,2,4-triazole-4-ylamino)methanol (24DNTAM) and (2,4-dinitrophenyl)(4H-3,5-dimethyl-1,2,4-triazole-4-ylamino)methanol (24DNDMTAM). Both compounds showed promising anti-P. gingivalis activity, higher against ATCC 33277 strain as compared to A7436 strain. The lowest hemiaminal concentration inhibiting visible planktonic bacterial growth under high-iron/heme conditions was ∼0.06 mg/ml, and the lowest hemiaminal concentration showing killing of bacteria was ∼0.25 mg/ml. Antimicrobial activity was also observed against P. gingivalis grown on blood agar plates. Slightly higher antimicrobial activity of both compounds was observed when P. gingivalis was grown in co-cultures with epithelial HeLa cells under low-iron/heme conditions, which mimic those occurring in vivo. 24DNTAM was more effective against P. gingivalis, but exhibited higher cytotoxic activity against epithelial and red blood cells, as compared with 24DNDMTAM. We conclude that both hemiaminals might originate a novel group of biologically important molecules.

UDP-galactose (SLC35A2) and UDP-N-acetylglucosamine (SLC35A3) Transporters Form Glycosylation-related Complexes with Mannoside Acetylglucosaminyltransferases (Mgats).

UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; SLC35A3) form heterologous complexes in the Golgi membrane. NGT occurs in close proximity to mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetylglucosaminyltransferase (Mgat5). In this study we analyzed whether NGT and both splice variants of UGT (UGT1 and UGT2) are able to interact with four different mannoside acetylglucosaminyltransferases (Mgat1, Mgat2, Mgat4B, and Mgat5). Using an in situ proximity ligation assay, we found that all examined glycosyltransferases are in the vicinity of these UDP-sugar transporters both at the endogenous level and upon overexpression. This observation was confirmed via the FLIM-FRET approach for both NGT and UGT1 complexes with Mgats. This study reports for the first time close proximity between endogenous nucleotide sugar transporters and glycosyltransferases. We also observed that among all analyzed Mgats, only Mgat4B occurs in close proximity to UGT2, whereas the other three Mgats are more distant from UGT2, and it was only possible to visualize their vicinity using proximity ligation assay. This strongly suggests that the distance between these protein pairs is longer than 10 nm but at the same time shorter than 40 nm. This study adds to the understanding of glycosylation, one of the most important post-translational modifications, which affects the majority of macromolecules. Our research shows that complex formation between nucleotide sugar transporters and glycosyltransferases might be a more common phenomenon than previously thought.

HmuY is an important virulence factor for Porphyromonas gingivalis growth in the heme-limited host environment and infection of macrophages.

Porphyromonas gingivalis, the main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis, is a haem auxotroph, and the uptake of this compound is essential for its survival and the ability to establish an infection. The aim of this study was to examine the role of a hemophore-like HmuY protein in P. gingivalis growth and infection of macrophages. Inactivation of the hmuY gene caused reduced P. gingivalis growth in vitro in the presence of serum as a heme sole source, as well as in vivo co-cultures with THP-1-derived macrophages. This resulted in diminished invasion efficiency of macrophages by live bacteria lacking functional hmuY gene. Both features were partially restored after addition of the purified HmuY protein, which was internalized when added either together with the hmuY mutant strain or alone to macrophage cultures. We conclude that HmuY is an important virulence factor of P. gingivalis for infection of macrophages in a heme-limited host environment.

UDP-N-acetylglucosamine transporter (SLC35A3) regulates biosynthesis of highly branched N-glycans and keratan sulfate.

SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply ß-1,3-N-acetylglucosaminyl-transferase 7 (ß3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein ß-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.

Short N-terminal region of UDP-galactose transporter (SLC35A2) is crucial for galactosylation of N-glycans.

UDP-galactose transporter (UGT) and UDP-N-acetylglucosamine transporter (NGT) form heterologous complexes in the Golgi apparatus (GA) membrane. We aimed to identify UGT region responsible for galactosylation of N-glycans. Chimeric proteins composed of human UGT and either NGT or CMP-sialic acid transporter (CST) localized to the GA, and all but UGT/CST chimera corrected galactosylation defect in UGT-deficient cell lines, although at different efficiency. Importantly, short N-terminal region composed of 35 N-terminal amino-acid residues of UGT was crucial for galactosylation of N-glycans. The remaining molecule must be derived from NGT not CST, confirming that the role played by UGT and NGT is coupled.

Differential roles of tryptophan residues in conformational stability of Porphyromonas gingivalis HmuY hemophore.

BACKGROUND: We have previously shown that the P. gingivalis HmuY hemophore-like protein binds heme and scavenges heme from host hemoproteins to further deliver it to the cognate heme receptor HmuR. The aim of this study was to characterize structural features of HmuY variants in the presence and absence of heme with respect to roles of tryptophan residues in conformational stability. RESULTS: HmuY possesses tryptophan residues at positions 51 and 73, which are conserved in HmuY homologs present in a variety of bacteria, and a tryptophan residue at position 161, which has been found only in HmuY identified in P. gingivalis strains. We expressed and purified the wildtype HmuY and its protein variants with single tryptophan residues replaced by alanine or tyrosine residues. All HmuY variants were subjected to thermal denaturation and fluorescence spectroscopy analyses. Replacement of the most buried W161 only moderately affects protein stability. The most profound effect of the lack of a large hydrophobic side chain in respect to thermal stability is observed for W73. Also replacement of the W51 exposed on the surface results in the greatest loss of protein stability and even the large aromatic side chain of a tyrosine residue has little potential to substitute this tryptophan residue. Heme binding leads to different exposure of the tryptophan residue at position 51 to the surface of the protein. Differences in structural stability of HmuY variants suggest the change of the tertiary structure of the protein upon heme binding. CONCLUSIONS: Here we demonstrate differential roles of tryptophan residues in the protein conformational stability. We also propose different conformations of apo- and holoHmuY caused by tertiary changes which allow heme binding to the protein.

Porphyromonas endodontalis HmuY differentially participates in heme acquisition compared to the Porphyromonas gingivalis and Tannerella forsythia hemophore-like proteins.

Introduction: Porphyromonas gingivalis and Porphyromonas endodontalis belong to the Bacteroidota phylum. Both species inhabit the oral cavity and can be associated with periodontal diseases. To survive, they must uptake heme from the host as an iron and protoporphyrin IX source. Among the best-characterized heme acquisition systems identified in members of the Bacteroidota phylum is the P. gingivalis Hmu system, with a leading role played by the hemophore-like HmuY (HmuYPg) protein. Methods: Theoretical analysis of selected HmuY proteins and spectrophotometric methods were employed to determine the heme-binding mode of the P. endodontalis HmuY homolog (HmuYPe) and its ability to sequester heme. Growth phenotype and gene expression analysis of P. endodontalis were employed to reveal the importance of the HmuYPe and Hmu system for this bacterium. Results: Unlike in P. gingivalis, where HmuYPg uses two histidines for heme-iron coordination, other known HmuY homologs use two methionines in this process. P. endodontalis HmuYPe is the first characterized representative of the HmuY family that binds heme using a histidine-methionine pair. It allows HmuYPe to sequester heme directly from serum albumin and Tannerella forsythia HmuYTf, the HmuY homolog which uses two methionines for heme-iron coordination. In contrast to HmuYPg, which sequesters heme directly from methemoglobin, HmuYPe may bind heme only after the proteolytic digestion of hemoglobin. Conclusions: We hypothesize that differences in components of the Hmu system and structure-based properties of HmuY proteins may evolved allowing different adaptations of Porphyromonas species to the changing host environment. This may add to the superior virulence potential of P. gingivalis over other members of the Bacteroidota phylum.

Comparative analysis of Porphyromonas gingivalis A7436 and ATCC 33277 strains reveals differences in the expression of heme acquisition systems.

Porphyromonas gingivalis strains exhibit different phenotypes in vitro, different virulence potential in animal models, and different associations with human diseases, with strains classified as virulent/more virulent (e.g., A7436 and W83) or as less virulent/avirulent (e.g., ATCC 33277). In this study, we comparatively analyzed the A7436 and ATCC 33277 strains to better understand their variability. Global gene expression analysis in response to heme and iron limitation revealed more pronounced differences in the A7436 than in the ATCC 33277 strain; however, in both strains, the largest changes were observed in genes encoding hypothetical proteins, genes whose products participate in energy metabolism, and in genes encoding proteins engaged in transport and binding proteins. Our results confirmed that variability between P. gingivalis strains is due to differences in the arrangement of their genomes. Analysis of gene expression of heme acquisition systems demonstrated that not only the availability of iron and heme in the external environment but also the ability to store iron intracellularly can influence the P. gingivalis phenotype. Therefore, we assume that differences in virulence potential may also be due to differences in the production of systems involved in iron and heme acquisition, mainly the Hmu system. In addition, our study showed that hemoglobin, in a concentration-dependent manner, differentially influences the virulence potential of P. gingivalis strains. We conclude that iron and heme homeostasis may add to the variability observed between P. gingivalis strains. IMPORTANCE: Periodontitis belongs to a group of multifactorial diseases, characterized by inflammation and destruction of tooth-supporting tissues. P. gingivalis is one of the most important microbial factors involved in the initiation and progression of periodontitis. To survive in the host, the bacterium must acquire heme as a source of iron and protoporphyrin IX. P. gingivalis strains respond differently to changing iron and heme concentrations, which may be due to differences in the expression of systems involved in iron and heme acquisition. The ability to accumulate iron intracellularly, being different in more and less virulent P. gingivalis strains, may influence their phenotypes, production of virulence factors (including proteins engaged in heme acquisition), and virulence potential of this bacterium.

Hemophore-like proteins of the HmuY family in the oral and gut microbiome: unraveling the mystery of their evolution.

SUMMARY: Heme (iron protoporphyrin IX, FePPIX) is the main source of iron and PPIX for host-associated pathogenic bacteria, including members of the Bacteroidota (formerly Bacteroidetes) phylum. Porphyromonas gingivalis, a keystone oral pathogen, uses a unique heme uptake (Hmu) system, comprising a hemophore-like protein, designated as the first member of the novel HmuY family. Compared to classical, secreted hemophores utilized by Gram-negative bacteria or near-iron transporter domain-based hemophores utilized by Gram-positive bacteria, the HmuY family comprises structurally similar proteins that have undergone diversification during evolution. The best characterized are P. gingivalis HmuY and its homologs from Tannerella forsythia (Tfo), Prevotella intermedia (PinO and PinA), Bacteroides vulgatus (Bvu), and Bacteroides fragilis (BfrA, BfrB, and BfrC). In contrast to the two histidine residues coordinating heme iron in P. gingivalis HmuY, Tfo, PinO, PinA, Bvu, and BfrA preferentially use two methionine residues. Interestingly, BfrB, despite conserved methionine residue, binds the PPIX ring without iron coordination. BfrC binds neither heme nor PPIX in keeping with the lack of conserved histidine or methionine residues used by other members of the HmuY family. HmuY competes for heme binding and heme sequestration from host hemoproteins with other members of the HmuY family to increase P. gingivalis competitiveness. The participation of HmuY in the host immune response confirms its relevance in relation to the survival of P. gingivalis and its ability to induce dysbiosis not only in the oral microbiome but also in the gut microbiome or other host niches, leading to local injuries and involvement in comorbidities.