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.

Biological Stimuli-Induced Phase Transition of a Synthesized Block Copolymer: Preferential Interactions between PNIPAM-b-PNVCL and Heme Proteins.

The beguiling world of functional polymers is dominated by thermoresponsive polymers with unique structural and molecular attributes. Limited work has been reported on the protein-induced conformational transition of block copolymers; furthermore, the literature lacks a clear understanding of the influence of proteins on the phase behavior of thermoresponsive copolymers. Herein, we have synthesized poly(N-isopropylacrylamide)-b-poly(N-vinylcaprolactam) (PNIPAM-b-PNVCL) by RAFT polymerization using N-isopropylacrylamide and N-vinylcaprolactam. Furthermore, using various biophysical techniques, we have explored the effect of cytochrome c (Cyt c), myoglobin (Mb), and hemoglobin (Hb) with varying concentrations on the aggregation behavior of PNIPAM-b-PNVCL. Absorption and steady-state fluorescence spectroscopy measurements were performed at room temperature to examine the copolymerization effect on fluorescent probe binding and biomolecular interactions between PNIPAM-b-PNVCL and proteins. Furthermore, temperature-dependent fluorescence spectroscopy and dynamic light scattering studies were performed to get deeper insights into the lower critical solution temperature (LCST) of PNIPAM-b-PNVCL. Small-angle neutron scattering (SANS) was also employed to understand the copolymer behavior in the presence of heme proteins. With the incorporation of proteins to PNIPAM-b-PNVCL aqueous solution, LCST has been varied to different extents owing to the preferential, molecular, and noncovalent interactions between PNIPAM-b-PNVCL and proteins. The present study can pave new insights between heme proteins and block copolymer interactions, which will help design biomimetic surfaces and aid in the strategic fabrication of copolymer-protein bioconjugates.

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.

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.

Nitrate and Phosphate Transporters Rescue Fluoride Toxicity in Yeast.

Organisms are exposed to fluoride in the air, water, and soil. Yeast and other microbes utilize fluoride channels as a method to prevent intracellular fluoride accumulation and mediate fluoride toxicity. Consequently, deletion of fluoride exporter genes (FEX) in S. cerevisiae resulted in over 1000-fold increased fluoride sensitivity. We used this FEX knockout strain to identify genes, that when overexpressed, are able to partially relieve the toxicity of fluoride exposure. Overexpression of five genes, SSU1, YHB1, IPP1, PHO87, and PHO90, increase fluoride tolerance by 2- to 10-fold. Overexpression of these genes did not provide improved fluoride resistance in wild-type yeast, suggesting that the mechanism is specific to low fluoride toxicity in yeast. Ssu1p and Yhb1p both function in nitrosative stress response, which is induced upon fluoride exposure along with metal influx. Ipp1p, Pho87p, and Pho90p increase intracellular orthophosphate. Consistent with this observation, fluoride toxicity is also partially mitigated by the addition of high levels of phosphate to the growth media. Fluoride inhibits phosphate import upon stress induction and causes nutrient starvation and organelle disruption, as supported by gene induction monitored through RNA-Seq. The combination of observations suggests that transmembrane nutrient transporters are among the most sensitized proteins during fluoride-instigated stress.

Enhanced bacterial cellulose production by Gluconacetobacter xylinus via expression of Vitreoscilla hemoglobin and oxygen tension regulation.

Oxygen plays a key role during bacterial cellulose (BC) biosynthesis by Gluconacetobacter xylinus. In this study, the Vitreoscilla hemoglobin (VHb)-encoding gene vgb, which has been widely applied to improve cell survival during hypoxia, was heterologously expressed in G. xylinus via the pBla-VHb-122 plasmid. G. xylinus and G. xylinus-vgb + were statically cultured under hypoxic (10 and 15% oxygen tension in the gaseous phase), atmospheric (21%), and oxygen-enriched conditions (40 and 80%) to investigate the effect of oxygen on cell growth and BC production. Irrespective of vgb expression, we found that cell density increased with oxygen tension (10-80%) during the exponential growth phase but plateaued to the same value in the stationary phase. In contrast, BC production was found to significantly increase at lower oxygen tensions. In addition, we found that BC production at oxygen tensions of 10 and 15% was 26.5 and 58.6% higher, respectively, in G. xylinus-vgb + than that in G. xylinus. The maximum BC yield and glucose conversion rate, of 4.3 g/L and 184.7 mg/g, respectively, were observed in G. xylinus-vgb + at an oxygen tension of 15%. Finally, BC characterization suggested that hypoxic conditions enhance BC's mass density, Young's modulus, and thermostability, with G. xylinus-vgb + synthesizing softer BC than G. xylinus under hypoxia as a result of a decreased Young's modulus. These results will facilitate the use of static culture for the production of BC.

Molecularly Imprinted Electropolymer for a Hexameric Heme Protein with Direct Electron Transfer and Peroxide Electrocatalysis.

For the first time a molecularly imprinted polymer (MIP) with direct electron transfer (DET) and bioelectrocatalytic activity of the target protein is presented. Thin films of MIPs for the recognition of a hexameric tyrosine-coordinated heme protein (HTHP) have been prepared by electropolymerization of scopoletin after oriented assembly of HTHP on a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold electrodes. Cavities which should resemble the shape and size of HTHP were formed by template removal. Rebinding of the target protein sums up the recognition by non-covalent interactions between the protein and the MIP with the electrostatic attraction of the protein by the SAM. HTHP bound to the MIP exhibits quasi-reversible DET which is reflected by a pair of well pronounced redox peaks in the cyclic voltammograms (CVs) with a formal potential of -184.4 ± 13.7 mV vs. Ag/AgCl (1 M KCl) at pH 8.0 and it was able to catalyze the cathodic reduction of peroxide. At saturation the MIP films show a 12-fold higher electroactive surface concentration of HTHP than the non-imprinted polymer (NIP).

Porphyromonas gingivalis and related bacteria: from colonial pigmentation to the type IX secretion system and gliding motility.

Porphyromonas gingivalis is a gram-negative, non-motile, anaerobic bacterium implicated as a major pathogen in periodontal disease. P. gingivalis grows as black-pigmented colonies on blood agar, and many bacteriologists have shown interest in this property. Studies of colonial pigmentation have revealed a number of important findings, including an association with the highly active extracellular and surface proteinases called gingipains that are found in P. gingivalis. The Por secretion system, a novel type IX secretion system (T9SS), has been implicated in gingipain secretion in studies using non-pigmented mutants. In addition, many potent virulence proteins, including the metallocarboxypeptidase CPG70, 35 kDa hemin-binding protein HBP35, peptidylarginine deiminase PAD and Lys-specific serine endopeptidase PepK, are secreted through the T9SS. These findings have not been limited to P. gingivalis but have been extended to other bacteria belonging to the phylum Bacteroidetes. Many Bacteroidetes species possess the T9SS, which is associated with gliding motility for some of these bacteria.

Effects of cascaded vgb promoters on poly(hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli grown micro-aerobically.

Micro-aeration is a situation that will be encountered in bacterial cell growth especially when the saturated dissolved oxygen level cannot match the demand from cells grown to a high density. Therefore, it is desirable to separate aerobic growth and micro-aerobic product formation into two stages using methods including anaerobic or micro-aerobic promoters that are inducible under low aeration intensity. Eleven potential low aeration-inducible promoters were cloned and studied for their induction strengths under micro-aerobic conditions. Of them, Vitreoscilla hemoglobin promoter (P vgb ) was found to be the strongest among all 11 promoters. At the same time, six E. coli hosts harboring poly(R-3-hydroxybutyrate) (PHB) synthesis operon phaCAB were compared for their ability to accumulate poly(hydroxyalkanoates) (PHA). E. coli S17-1 was demonstrated to be the best host achieving a 70 % (mass fraction) PHB in the cell dry weigh (CDW) after 48 h under micro-aerobic growth. Cascaded P vgb repeats (P nvgb ) were investigated for enhanced expression level under micro-aerobic growth. The highest PHA production was obtained when a promoter containing eight cascaded P vgb repeats (P 8vgb ) was used, 5.37 g/l CDW containing 90 % PHB was obtained from recombinant in E. coli S17-1. Cells grew further to 6.30 g/l CDW containing 91 % PHB when oxygen-responsive transcription factor ArcA (arcA) was deleted in the same recombinant E. coli S17-1. This study revealed that vgb promoter containing cascaded P vgb repeats (P 8vgb ) is useful for product formation under low aeration intensity.

Electrocatalytic features of a heme protein attached to polymer-functionalized magnetic nanoparticles.

Direct electron-transfer and electrocatalytic kinetics of covalently attached myoglobin (MB) films on magnetic nanoparticles (MB-MNP(covalent)), in comparison to the corresponding physisorbed films and individual components, are reported for the first time. MB-MNP(covalent) ("-" denotes a covalent linkage) was adsorbed onto a cationic poly(ethyleneimine) layer (PEI) coated high-purity graphite (HPG) electrode. Similarly, films of myoglobin physisorbed on magnetic nanoparticles (MB/MNP(adsorbed), "/" denotes a noncovalent nature), only MB, or only MNP were constructed on HPG/PEI electrodes for comparison. The observed electron-transfer rate constants (k(s), s(-1)) were in the following order: MB-MNP(covalent) (69 ± 6 s(-1)), MB/MNP(adsorbed) (37 ± 2 s(-1)), only MB (27 ± 2 s(-1)), and only MNP (16 ± 3 s(-1)). The electrocatalytic properties of these films were investigated with the aid of tert-butylhydroperoxide as a model reactant, and its reduction kinetics were examined. We observed the following order of catalytic current density: MB-MNP(covalent) > MB/MNP(adsorbed) > only MNP > only MB, in agreement with the electron-transfer (ET) rates of MB-MNP(covalent) and MB/MNP(adsorbed) films. The crucial function of MNP in favorably altering the direct ET and electrocatalytic properties of both covalently bound MB and physisorbed MB molecules are discussed. In addition, the occurrence of a highly enhanced electron-hopping mechanism in the designed covalent MB-MNP(covalent) films over the corresponding physisorbed MB/MNP(adsorbed) film is proposed. The enhanced electron-transfer rates and catalytic current density suggest the advantages of using metalloenzymes covalently attached to polymer-functionalized magnetic nanoparticles for the development of modern highly efficient miniature biosensors and bioreactors.

Magnetic separation of malaria-infected red blood cells in various developmental stages.

Malaria is a serious disease that threatens the public health, especially in developing countries. Various methods have been developed to separate malaria-infected red blood cells (i-RBCs) from blood samples for clinical diagnosis and biological and epidemiological research. In this study, we propose a simple and label-free method for separating not only late-stage but also early-stage i-RBCs on the basis of their paramagnetic characteristics due to the malaria byproduct, hemozoin, by using a magnetic field gradient. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with a ferromagnetic wire fixed on a glass slide. To evaluate the performance of the microfluidic device containing the ferromagnetic wire, lateral displacement of NaNO2-treated RBCs, which also have paramagnetic characteristics, was observed at various flow rates. The results showed excellent agreement with theoretically predicted values. The same device was applied to separate i-RBCs. Late-stage i-RBCs (trophozoites and schizonts), which contain optically visible black dots, were separated with a recovery rate of approximately 98.3%. In addition, using an optimal flow rate, early-stage (ring-stage) i-RBCs, which had been difficult to separate because of their low paramagnetic characteristics, were successfully separated with a recovery rate of 73%. The present technique, using permanent magnets and ferromagnetic wire in a microchannel, can effectively separate i-RBCs in various developmental stages so that it could provide a potential tool for studying the invasion mechanism of the malarial parasite, as well as performing antimalarial drug assays.

Assignment of the 1H NMR resonances of protein residues in close proximity to the heme of the nitrophorins: similarities and differences among the four proteins from the saliva of the adult blood-sucking insect Rhodnius prolixus.

The nuclear Overhauser effects (NOEs) observed between heme substituent protons and a small number of nearby protein side chain protons in the water-elimination Fourier transform NOE spectroscopy (WEFT-NOESY) spectra of high- and low-spin wild-type nitrophorin (NP) 2 and its ligand complexes have been analyzed and compared with those observed for the same complexes of wild-type NP3. These assignments were made on naturally abundant isotope samples, with the most useful protein side chains being those of Ile120, Leu122, and Leu132 for NP2 and NP3, and Thr121, Leu123, and Leu133 for NP1 and NP4. It is found that the NOEs observed are identical, with extremely similar protein side chain proton chemical shifts. This is strong evidence that the structure of NP3, for which no X-ray crystal structures are available, is essentially identical to that of NP2, at least near the heme binding pocket. Similarly, the NOEs observed between heme substituents and protein side chains for NP1 and NP4 also indicate that the structures of the protein having both A and B heme orientations are very similar to each other, as well as to the proteins with major B heme orientation of NP2 and NP3. These A and B connectivities can be seen, even though the two heme orientations have similar populations in NP1 and NP4, which complicates the analysis of the NOESY spectra. The histamine complex of wild-type NP2 shows significant shifts of the Leu132 side chain protons relative to all other ligand complexes of NP1-NP4 because of the perturbation of the structure near Leu132 caused by the histamine's side chain ammonium hydrogen bond to the Asp29 side chain carboxylate.

Identification of the native N-terminus of the membrane attaching ferriheme protein nitrophorin 7 from Rhodnius prolixus.

All species of the genus Rhodnius have a characteristic red coloration in their salivary glands due to the presence of heme proteins. Some of these secreted proteins, known as nitrophorins (NPs), are responsible for many of the antihemostatic activities of Rhodnius saliva such as anticoagulant and antihistamine. Several NPs have been described (NP1-4 and NP7), where NP7 is the only one with affinity to phospholipid membranes. Computational prediction suggested that NP7 also has an extended N-terminal tail on signal peptide cleavage; however, the complementary DNA does not allow the determination of the exact site of signal peptidase cleavage. On the other hand, according to previous studies, the exact length of the N-terminus has important consequences for the nitric oxide binding properties of NP7. Here, a method was developed to select phospholipid membrane-attaching proteins from homogenized tissue for analysis by mass spectrometry. The method was used to determine the exact N-terminus of the ferriheme protein NP7 from homogenates of the salivary glands of 5th instar nymphal stages of Rhodnius prolixus.

Induction of interleukin (IL)-1ß, IL-10, IL-8 and immunoglobulin G by Porphyromonas gingivalis HmuY in humans.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis, an anaerobic gram-negative bacterium, is associated with chronic periodontitis. This study was undertaken to evaluate the production of interleukin (IL)-1ß, IL-8 and IL-10 by human peripheral blood mononuclear cells (PBMC) stimulated with P. gingivalis antigens and to assess the levels of serum immunoglobulin (Ig)G, IgA and IgG subclasses raised against P. gingivalis HmuY protein. MATERIAL AND METHODS: PBMC from patients with chronic periodontitis (CP) and from nonperiodontitis (NP) control subjects were stimulated with P. gingivalis antigens, and the cytokine levels in the culture supernatants were determined by ELISA. The specificity of serum antibodies raised against HmuY was analyzed by Western blotting and by ELISA. RESULTS: Compared with the NP controls, the CP patients produced higher levels of total serum IgG and IgG1 specific for P. gingivalis HmuY. No differences were found between CP and NP groups in the production of IL-1ß and IL-8 by PBMC stimulated with total P. gingivalis antigens. Only P. gingivalis lipopolysaccharide (LPS) induced higher levels of IL-10 in the CP group. Higher levels of IL-1ß and IL-10 were induced by HmuY than by other antigens derived from the wild-type P. gingivalis strains. In contrast, total antigens derived from the hmuY-deletion mutant strain induced the production of significantly higher levels of IL-8 and significantly lower levels of IL-1ß. CONCLUSION: Our data suggest that P. gingivalis HmuY may be considered an immunogenic protein associated with host-pathogen interactions.

Comparison of Laccases and Hemeproteins Systems in Bioremediation of Organic Pollutants.

AIM: Laccases and peroxidases have attracted great interest for industrial and environmental applications. These enzymes have a broad substrate range and a robust oxidizing ability. Moreover, using mediators or co-oxidants makes it possible to increase their catalytic activity and extend their substrate scope to more resistant chemical structures. BACKGROUND: Fungal laccases and ligninolytic peroxidases, mainly lignin and manganese peroxidases, are the privileged oxidoreductases for bioremediation processes. Nonetheless, an increasing diversity of laccases and peroxidase-type enzymes has been proposed for environmental technologies. OBJECTIVE: This article aims to provide an overview of these enzymes and compare their applicability in the degradation of organic pollutants. METHODS: Fundamental properties of the proteins are covered and applications towards polycyclic aromatic hydrocarbons (PAHs) and pesticides are specially focused. RESULTS: Laccases are multicopper oxidases initially studied for applications in the pulp and paper industry but able to oxidize a variety of environmentally concerning compounds. Relying on O2, laccases do not require peroxides nor auxiliary agents, like Mn2+, although suitable redox mediators are needed to attack the more recalcitrant pollutants (e.g., PAHs). True and pseudo-peroxidases use a stronger oxidant (H2O2) and the redox chemistry at the heme site generates high potential species that allow the oxidation of dyes and some pesticides. CONCLUSION: Lately, research efforts have been directed to enzyme discovery, testing with micropollutants, and improving biocatalysts' stability by immobilization and protein engineering. Further understanding of the effects of natural media components and solvents on the enzymes might lead to competitive enzymatic treatments of highly toxic media.

Unique structure and stability of HmuY, a novel heme-binding protein of Porphyromonas gingivalis.

Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-beta fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection.

Characterization of human-type monoclonal antibodies against reduced form of hemin binding protein 35 from Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: The gram-negative anaerobe Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We previously identified a 35 kDa surface protein (hemin binding protein 35; HBP35) from P. gingivalis that exhibited coaggregation activity, while additional analysis suggested that this protein possessed an ability to bind heme molecules. For development of passive immunotherapy for periodontal diseases, human-type monoclonal antibodies have been prepared using HBP35 as an antigen in TransChromo mice. In the present study, we focused on a single antibody, TCmAb-h13, which is known to inhibit heme binding to recombinant HBP35. The aim of our investigation was to clarify the redox-related function of HBP35 and consider the benefits of human-type monoclonal antibodies. MATERIAL AND METHODS: To examine the antigen recognition capability of TCmAbs with immunoblotting and Biacore techniques, we used the native form as well as several Cys-to-Ser variants of recombinant HBP35. RESULTS: We found that the redox state of recombinant HBP35 was dependent on two Cys residues, (48) C and (51) C, in the thioredoxin active center (WCGxCx). Furthermore, TCmAb-h13 recognized the reduced forms of recombinant HBP35, indicating its inhibitory effect on P. gingivalis growth. CONCLUSION: Hemin binding protein 35 appears to be an important molecule involved in recognition of the redox state of environmental conditions. In addition, TCmAb-h13 had an inhibitory effect on heme binding to recombinant HBP35, thereby interfering with P. gingivalis growth.

Characterization of hemin-binding protein 35 (HBP35) in Porphyromonas gingivalis: its cellular distribution, thioredoxin activity and role in heme utilization.

BACKGROUND: The periodontal pathogen Porphyromonas gingivalis is an obligate anaerobe that requires heme for growth. To understand its heme acquisition mechanism, we focused on a hemin-binding protein (HBP35 protein), possessing one thioredoxin-like motif and a conserved C-terminal domain, which are proposed to be involved in redox regulation and cell surface attachment, respectively. RESULTS: We observed that the hbp35 gene was transcribed as a 1.1-kb mRNA with subsequent translation resulting in three proteins with molecular masses of 40, 29 and 27 kDa in the cytoplasm, and one modified form of the 40-kDa protein on the cell surface. A recombinant 40-kDa HBP35 exhibited thioredoxin activity in vitro and mutation of the two putative active site cysteine residues abolished this activity. Both recombinant 40- and 27-kDa proteins had the ability to bind hemin, and growth of an hbp35 deletion mutant was substantially retarded under hemin-depleted conditions compared with growth of the wild type under the same conditions. CONCLUSION: P. gingivalis HBP35 exhibits thioredoxin and hemin-binding activities and is essential for growth in hemin-depleted conditions suggesting that the protein plays a significant role in hemin acquisition.

Species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of Porphyromonas gingivalis HmuY.

BACKGROUND: Porphyromonas gingivalis is a major etiological agent of chronic periodontitis. The aim of this study was to examine the species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of the P. gingivalis heme-binding protein HmuY. RESULTS: HmuY is a unique protein of P. gingivalis since only low amino-acid sequence homology has been found to proteins encoded in other species. It is exposed on the cell surface and highly abundant in the outer membrane of the cell, in outer-membrane vesicles, and is released into culture medium in a soluble form. The protein is produced constitutively at low levels in bacteria grown under high-iron/heme conditions and at higher levels in bacteria growing under the low-iron/heme conditions typical of dental plaque. HmuY is immunogenic and elicits high IgG antibody titers in rabbits. It is also engaged in homotypic biofilm formation by P. gingivalis. Anti-HmuY antibodies exhibit inhibitory activity against P. gingivalis growth and biofilm formation. CONCLUSIONS: Here it is demonstrated that HmuY may play a significant role not only in heme acquisition, but also in biofilm accumulation on abiotic surfaces. The data also suggest that HmuY, as a surface-exposed protein, would be available for recognition by the immune response during chronic periodontitis and the production of anti-HmuY antibodies may inhibit biofilm formation.

Hemin-binding protein 35 (HBP35) plays an important role in bacteria-mammalian cells interactions in Porphyromonas gingivalis.

Hemin-binding protein 35 (HBP35) may be an essential protein for bacterial survival in evasion from environmental stress in Porphyromonas gingivalis. The anti-recombinant HBP35 antibody inhibits P. gingivalis hemagglutination. This study considered the role of this protein for hemagglutination and adherence to host cells using the HBP35-deficient mutant (MD774) derived from P. gingivalis FDC381. FDC381 had strong hemagglutination activity, whereas MD774 had no activity. Anti-130-kDa hemagglutinin antibody, mAb-Pg-vc, reacted mainly with the 43- and 49-kDa molecules in the membrane fraction. However, no proteins reacted in the MD774. The hemolytic activity in MD774 was much lower than that in FDC381. Anti-recombinant HBP35 antibody strongly inhibited the P. gingivalis FDC381 adherence to epithelial cells. In addition, MD774 exhibited a significant decrease in the adherence. The hydrophobicity of MD774 was equal to 19.4% of that of FDC381. SDS-PAGE profiling of the membrane fractions of both strains showed very different profiles. Taken together, these findings suggest that HBP35 plays a role, not only in hemin-binding, but also in multiple P. gingivalis binding to erythrocytes, and host epithelial gingival cells. In addition, this protein may directly and/or indirectly affect the virulence of this organism.