Porphyromonas gingivalis, a main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis requires heme as a source of iron and protoporphyrin IX for its survival and the ability to establish an infection. Porphyromonas gingivalis is able to accumulate a defensive cell-surface heme-containing pigment in the form of μ-oxo bisheme. The main sources of heme for P. gingivalis in vivo are hemoproteins present in saliva, gingival crevicular fluid, and erythrocytes. To acquire heme, P. gingivalis uses several mechanisms. Among them, the best characterized are those employing hemagglutinins, hemolysins, and gingipains (Kgp, RgpA, RgpB), TonB-dependent outer-membrane receptors (HmuR, HusB, IhtA), and hemophore-like proteins (HmuY, HusA). Proteins involved in intracellular heme transport, storage, and processing are less well characterized (e.g. PgDps). Importantly, P. gingivalis may also use the heme acquisition systems of other bacteria to fulfill its own heme requirements. Porphyromonas gingivalis displays a novel paradigm for heme acquisition from hemoglobin, whereby the Fe(II)-containing oxyhemoglobin molecule must first be oxidized to methemoglobin to facilitate heme release. This process not only involves P. gingivalis arginine- and lysine-specific gingipains, but other proteases (e.g. interpain A from Prevotella intermedia) or pyocyanin produced by Pseudomonas aeruginosa. Porphyromonas gingivalis is then able to fully proteolyze the more susceptible methemoglobin substrate to release free heme or to wrest heme from it directly through the use of the HmuY hemophore.

Heme acquisition mechanisms of Porphyromonas gingivalis – strategies used in a polymicrobial community in a heme-limited host environment

Objective In recent years metagenomic analysis has become more accessible for the characterization of biological specimens. There has been an important increase of studies using this technique for subgingival human samples. To date, there are no updated systematic reviews on the relationship between oral microbiota and periodontal disease. The aim of the present systematic review was to update data about studies concerning the influences of changes in oral microbiota composition on the periodontal status in human subjects. Materials and methods An electronic search was conducted in four databases (MEDLINE, Scopus, CENTRAL and Web of Science) for articles published in English from January 2014 to April 2018. In vitro or animal studies, case reports, case series, retrospective studies, review articles, abstracts and discussions were excluded. Also, studies that evaluated less than 5 microbial species, only viruses or already known periodontal pathogens were excluded. Two independent researches selected the studies and extracted the data. The quality of evidence was assessed as high, moderate or low for each microorganism. Results Eight studies and three additional publications recovered from the bibliography search of the selected articles were included in the review. The Bacteria domain was the main detected among the others and it included 53 species. The review confirmed the presence of recognized periodontal pathogens such as the members of the red complex but also identified, with high weight of evidence, the presence of new pathogens. Conclusions The results of this systematic review support high evidence for the association of 3 new species/genera with the etiology of periodontitis. Future investigations on the actual role of these new pathogens in the onset and progression of the disease are needed.

Relationship between oral microbiota and periodontal disease: a systematic review.

大豆根瘤菌（Bradyrhizobium japonicum）苹...

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.

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Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments

Porphyromonas gingivalis is considered the principal etiologic agent and keystone pathogen of chronic periodontitis. As an auxotrophic bacterium, it must acquire heme to survive and multiply at the infection site. P. gingivalis HmuY is the first member of a novel family of hemophore-like proteins. Bacterial heme-binding proteins usually use histidine-methionine or histidine-tyrosine residues to ligate heme-iron, whereas P. gingivalis HmuY uses two histidine residues. We hypothesized that other 'red complex' members, i.e. Tannerella forsythia and Treponema denticola might utilize similar heme uptake mechanisms to the P. gingivalis HmuY. Comparative and phylogenetic analyses suggested differentiation of HmuY homologs and low conservation of heme-coordinating histidine residues present in HmuY. The homologs were subjected to duplication before divergence of Bacteroidetes lineages, which could facilitate evolution of functional diversification. We found that T. denticola does not code an HmuY homolog. T. forsythia protein, termed as Tfo, binds heme, but preferentially in the ferrous form, and sequesters heme from the albumin-heme complex under reducing conditions. In agreement with that, the 3D structure of Tfo differs from that of HmuY in the folding of heme-binding pocket, containing two methionine residues instead of two histidine residues coordinating heme in HmuY. Heme binding to apo-HmuY is accompanied by movement of the loop carrying the His166 residue, closing the heme-binding pocket. Molecular dynamics simulations (MD) demonstrated that this conformational change also occurs in Tfo. In conclusion, our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme-binding properties.

/pdf/tannerella-forsythiatfo-belongs-toporphyromonas-51zf31jwyh.pdf

Tannerella forsythiaTfo belongs toPorphyromonas gingivalisHmuY-like family of proteins but differs in heme-binding properties.

SUMMARYPorphyromonas gingivalis is a key pathogenresponsible for initiation and progression ofchronic periodontitis. Little is known about theregulatory mechanisms of iron and heme uptakethat allow P. gingivalis to express virulence fac-tors and survive in the hostile environment of theoral cavity, so we initiated characterization of aP. gingivalis Fur homolog (PgFur). Many Fur para-logs found in microbial genomes, includingBacteroidetes, conﬁrm that Fur proteins have atendency to be subjected to a sub- or even neo-functionalization process. PgFur revealed extre-mely high sequence divergence, which could beassociated with its functional dissimilarity incomparison with other Fur homologs. A fur mutantstrain constructed by insertional inactivationexhibited retarded growth during the early growthphase and a signiﬁcantly lower tendency to form ahomotypic bioﬁlm on abiotic surfaces. The mutantalso showed signiﬁcantly weaker adherence andinvasion to epithelial cells and macrophages.Transcripts of many differentially regulated genesidentiﬁed in the fur mutant strain were annotatedas hypothetical proteins, suggesting that PgFurcan play a novel role in the regulation of geneexpression. Inactivation of the fur gene resultedin decreased hmuY gene expression, increasedexpression of other hmu components and changesin the expression of genes encoding hemaggluti-nins and proteases (mainly gingipains), HtrA,some extracytoplasmic sigma factors and two-component systems. Our data suggest that PgFurcan inﬂuence in vivo growth and virulence, at leastin part by affecting iron/heme acquisition, allowingefﬁcient infection through a complex regulatorynetwork.INTRODUCTIONPeriodontal diseases are among the most commonchronic infections in humans (Graves et al., 2000;Craig et al., 2003; Hajishengallis, 2011). From theclinical point of view, periodontitis is characterized bydeep periodontal pockets resulting from the loss ofconnective tissue attachment and alveolar bone,which is caused by destructive activity of bacterialagents, such as proteolytic enzymes (Potempa et al.,2003). The severity of the bleeding on probingdepends on the intensity of the gingival inﬂammation(Craig et al., 2003; Nakagawa et al., 2006). Analysesof bacterial species isolated from subgingival samplesrevealed the presence and relative abundance of peri-odontal pathogens, including the ‘red complex’ bacte-ria (Porphyromonas gingivalis, Tannerella forsythiaand Treponema denticola), associated with the clinical

Fur homolog regulates Porphyromonas gingivalis virulence under low-iron/heme conditions through a complex regulatory network.

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.

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

Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.

Anti-HmuY Antibodies Specifically Recognize Porphyromonas gingivalis HmuY Protein but Not Homologous Proteins in Other Periodontopathogens

Porphyromonas gingivalis, a keystone pathogen of chronic periodontitis, uses ferric uptake regulator homolog (PgFur) to regulate production of virulence factors. This study aimed to characterize PgFur protein in regard to its structure-function relationship. We experimentally identified 5’ mRNA sequence encoding 171-amino-acid long PgFur protein in A7436 strain and examined this PgFur version as a full-length protein. Although purified PgFur protein did not bind to the canonical E. coli Fur box, the wild-type phenotype of the mutant pgfur strain was restored partially when expression of the ecfur gene was induced from the native pgfur promoter. The full-length PgFur protein contained one zinc atom per protein monomer, but did not bind iron, manganese, or heme. Single cysteine substitutions of CXXC motifs resulted in phenotypes similar to the mutant pgfur strain. The modified proteins were produced in E. coli at significantly lower levels, were highly unstable, and did not bind zinc. pgfur gene was expressed at higher levels in bacteria cultured for 24 h in the absence of iron and heme or under lower iron/heme availability for 10 h. No influence of high availability of Fe2+, Zn2+, or Mn2+ on pgfur gene expression was observed. To examine the influence of the unusual C-terminal lysine-rich region on potential PgFur function, two chromosomal mutant strains, allowing production of the protein lacking 4 (pgfur4aa) or 13 (pgfur13aa) C-terminal amino-acid residues of the native PgFur protein were constructed. The pgfur13aa strain showed phenotype typical for the mutant pgfur strain, but both the wild-type PgFur protein and its truncated version bound zinc with similar ability. pgfur mutant strain produced higher amounts of HmuY protein compared with the wild-type strain. Potential PgFur ligands, Fe2+, Mn2+, Zn2+, PPIX, or serum components, did not influence HmuY production in the pgfur mutant strain. The mutant pgfur4aa and pgfur13aa strains produced higher levels of HmuY protein. PgFur bound to the hmu operon promoter, further supporting involvement of this protein in regulation of the hmuY gene expression.

/pdf/porphyromonas-gingivalis-pgfur-is-a-member-of-a-novel-fur-4bjfvv9sha.pdf

Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function.

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.

https://www.mdpi.com/1422-0067/22/5/2237/pdf

Michał Śmiga

Papers

Fur homolog regulates Porphyromonas gingivalis virulence under low-iron/heme conditions through a complex regulatory network.

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

Anti-HmuY Antibodies Specifically Recognize Porphyromonas gingivalis HmuY Protein but Not Homologous Proteins in Other Periodontopathogens

Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function.

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