Role of Porphyromonas gingivalis HmuY in Immunopathogenesis of Chronic Periodontitis.

Periodontitis is a multifactorial disease, with participation of bacterial, environmental, and host factors. It results from synergistic and dysbiotic multispecies microorganisms, critical "keystone pathogens," affecting the whole bacterial community. The purpose of this study was to review the role of Porphyromonas gingivalis in the immunopathogenesis of chronic periodontitis, with special attention paid to HmuY. The host response during periodontitis involves the innate and adaptive immune system, leading to chronic inflammation and progressive destruction of tooth-supporting tissues. In this proinflammatory process, the ability of P. gingivalis to evade the host immune response and access nutrients in the microenvironment is directly related to its survival, proliferation, and infection. Furthermore, heme is an essential nutrient for development of these bacteria, and HmuY is responsible for its capture from host heme-binding proteins. The inflammatory potential of P. gingivalis HmuY has been shown, including induction of high levels of proinflammatory cytokines and CCL2, decreased levels of IL-8, and increased levels of anti-HmuY IgG and IgG1 antibodies in individuals with chronic periodontitis. Therefore, the HmuY protein might be a promising target for therapeutic strategies and for development of diagnostic methods in chronic periodontitis, especially in the case of patients with chronic periodontitis not responding to treatment, monitoring, and maintenance therapy.

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.

Potential role for Streptococcus gordonii-derived hydrogen peroxide in heme acquisition by Porphyromonas gingivalis.

Streptococcus gordonii, an accessory pathogen and early colonizer of plaque, co-aggregates with many oral species including Porphyromonas gingivalis. It causes α-hemolysis on blood agar, a process mediated by H2 O2 and thought to involve concomitant oxidation of hemoglobin (Hb). Porphyromonas gingivalis has a growth requirement for heme, which is acquired mainly from Hb. The paradigm for Hb heme acquisition involves the initial oxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb), followed by heme release and extraction through the actions of K-gingipain protease and/or the HmuY hemophore-like protein. The ability of S. gordonii to mediate Hb oxidation may potentially aid heme capture during co-aggregation with P. gingivalis. Hemoglobin derived from zones of S. gordonii α-hemolysis was found to be metHb. Generation of metHb from oxyHb by S. gordonii cells was inhibited by catalase, and correlated with levels of cellular H2 O2 production. Generation of metHb by S. gordonii occurred through the higher Hb oxidation state of ferrylhemoglobin. Heme complexation by the P. gingivalis HmuY was employed as a measure of the ease of heme capture from metHb. HmuY was able to extract iron(III)protoporphyrin IX from metHb derived from zones of S. gordonii α-hemolysis and from metHb generated by the action of S. gordonii cells on isolated oxyHb. The rate of HmuY-Fe(III)heme complex formation from S. gordonii-mediated metHb was greater than from an equivalent concentration of auto-oxidized metHb. It is concluded that S. gordonii may potentially aid heme acquisition by P. gingivalis by facilitating metHb formation in the presence of oxyHb.

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

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.

Oligosaccharides released by hydrazinolysis from Tamm-Horsfall protein of various human donors contain similar high-mannose glycans.

As pathophysiological functions claimed for Tamm-Horsfall protein (THP) are related to its sugar moiety, we examined influence of pregnancy and various diseases on high-mannose chains. Hydrazinolysis was used to liberate oligosaccharides from THP polypeptide backbone. After HPLC separation of fluorescently labelled glycans similar profiles of neutral oligosaccharides were observed in THP of healthy subjects, pregnant women, patients with Bartter's syndrome, patients with acute lymphoblastic leukemia and a patient with carbohydrate deficient glycoprotein syndrome. THP contains Man-5, Man-6 and Man-7 glycans, with the preponderant amount of Man-6 glycan (about 7% of total THP oligosaccharides). No statistically significant differences were found in THP high-mannose glycans profiles between control subjects and pregnant women or patients. It is likely that neither pregnancy nor the pathological conditions examined affect high-mannose chains. In our opinion hydrazinolysis as a method of oligosaccharides liberation, in contrast to enzymatic deglycosylation, is more appropriate for analysis of the sugar moiety of THP.

Screening of cytoplasmic DNA diversity between and within Lupinus mutabilis Sweet and Lupinus albus sensu lato by restriction fragment length polymorphism (RFLP).

Seven populations and five mutant lines of the Andean lupin and four species from the section Albus were screened for their mitochondrial and chloroplast polymorphisms. For this purpose the RFLP method with EcoRI as a restriction enzyme was used. Lupinus luteus, Lupinus albus and Phaseolus vulgaris organellar clones as well as amplified fragments were used as probes. We found that mitochondrial probes were more suitable than chloroplast probes for identification of inter- and intra-specific variations within the examined material. Most mitochondrial probes differentiate the two species investigated. A high level of mitochondrial polymorphism was observed among the populations of L. mutabilis in contrast to monomorphism among the species in the section Albus. A limited polymorphism was detected between the mutant lines of L. mutabilis. We conclude from this study that the mitochondrial RFLP analysis is a valuable tool for identification of variability among Andean lupin populations.

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

Porphyromonas gingivalis is a key pathogen responsible for initiation and progression of chronic periodontitis. Little is known about the regulatory mechanisms of iron and heme uptake that allow P. gingivalis to express virulence factors and survive in the hostile environment of the oral cavity, so we initiated characterization of a P. gingivalis Fur homolog (PgFur). Many Fur paralogs found in microbial genomes, including Bacteroidetes, confirm that Fur proteins have a tendency to be subjected to a sub- or even neofunctionalization process. PgFur revealed extremely high sequence divergence, which could be associated with its functional dissimilarity in comparison with other Fur homologs. A fur mutant strain constructed by insertional inactivation exhibited retarded growth during the early growth phase and a significantly lower tendency to form a homotypic biofilm on abiotic surfaces. The mutant also showed significantly weaker adherence and invasion to epithelial cells and macrophages. Transcripts of many differentially regulated genes identified in the fur mutant strain were annotated as hypothetical proteins, suggesting that PgFur can play a novel role in the regulation of gene expression. Inactivation of the fur gene resulted in decreased hmuY gene expression, increased expression of other hmu components and changes in the expression of genes encoding hemagglutinins and proteases (mainly gingipains), HtrA, some extracytoplasmic sigma factors and two-component systems. Our data suggest that PgFur can influence in vivo growth and virulence, at least in part by affecting iron/heme acquisition, allowing efficient infection through a complex regulatory network.

Synthesis of microspheres of triuranium octaoxide by simultaneous water and nitrate extraction from ascorbate-uranyl sols.

A new method for synthesis of uranium oxide microspheres (diameter <100 µm) has been developed. It is a variant of our patented Complex Sol-Gel Process, which has been used to synthesize high-quality powders of a wide variety of complex oxides. Starting uranyl-nitrate-ascorbate sols were prepared by addition of ascorbic acid to uranyl nitrate hexahydrate solution and alkalizing by aqueous ammonium hydroxide and then emulsified in 2-ethylhexanol-1 containing 1v/o SPAN-80. Drops of emulsion were firstly gelled by extraction of water by the solvent. Destruction of the microspheres during thermal treatment, owing to highly reactive components in the gels, requires modification of the gelation step by Double Extraction Process-simultaneously extraction of water and nitrates using Primene JMT, which completely eliminates these problem. Final step was calcination in air of obtained microspheres of gels to triuranium octaoxide.

Evidence of mutualism between two periodontal pathogens: co-operative haem acquisition by the HmuY haemophore of Porphyromonas gingivalis and the cysteine protease interpain A (InpA) of Prevotella intermedia.

Haem (iron protoporphyrin IX) is both an essential growth factor and a virulence regulator of the periodontal pathogens Porphyromonas gingivalis and Prevotella intermedia, which acquire it through the proteolytic degradation of haemoglobin and other haem-carrying plasma proteins. The haem-binding lipoprotein HmuY haemophore and the gingipain proteases of P. gingivalis form a unique synthrophic system responsible for capture of haem from haemoglobin and methaemalbumin. In this system, methaemoglobin is formed from oxyhaemoglobin by the activities of gingipain proteases and serves as a facile substrate from which HmuY can capture haem. This study examined the possibility of cooperation between HmuY and the cysteine protease interpain A (InpA) of Pr. intermedia in the haem acquisition process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to be resistant to proteolysis and so able to cooperate with InpA to extract haem from haemoglobin, which was proteolytically converted to methaemoglobin by the protease. Spectroscopic pH titrations showed that both the iron(II) and iron(III) protoporphyrin IX-HmuY complexes were stable over the pH range 4-10, demonstrating that the haemophore could function over a range of pH that may be encountered in the dental plaque biofilm. This is the first demonstration of a bacterial haemophore working in conjunction with a protease from another bacterial species to acquire haem from haemoglobin and may represent mutualism between P. gingivalis and Pr. intermedia co-inhabiting the periodontal pocket.

[Structure and function of Tamm-Horsfall protein in human physiologic and pathologic states]. / Struktura i funkcja bialka Tamma-Horsfalla w stanach fizjologicznych i patologicznych czlowieka.

Tamm-Horsfall protein (THP), the most abundant glycoprotein in normal human urine, is produced by kidney. THP is heavily glycosylated by N-linked glycans, which are responsible for most of its properties. The sugar moiety is also modified in some diseases. THP plays an important role in ion transport and in maintaining water impermeability of the TALH and also protects the urinary system from uropathogens. It is also involved in immunoregulatory processes. THP was reported to influence many pathological conditions, such as formation of kidney stones, development of interstitial nephritis and tubular blockade.

The sugar moiety of Tamm-Horsfall protein is affected by the carbohydrate-deficient glycoprotein type I syndrome. A case study.

As the sugar moiety of Tamm-Horsfall protein (THP) is affected by many pathological conditions, the aim of this study was to examine the influence of carbohydrate-deficient glycoprotein syndrome (CDG) on THP glycans. THP was isolated from urine of one patient with CDG type I and N-glycan profiling, analysis of monosaccharide content, determination of THP reactivity with specific lectins and with anti-THP antibodies were performed. THP of the CDG patient showed markedly lower amounts of all monosaccharides. Diminished amounts of lactosamine-type chains, galactose and alpha2,3 linked sialic acid were expressed in lower reactivity with PHA-L, DSA and MAA, respectively. These modifications were reflected in altered proportions of tetrasialylated and disialylated oligosaccharide chains. THP of the CDG patient reacted slightly more with anti-THP antibodies. Our results indicate that the CDG type I affects the THP sugar moiety and slightly enhances the THP immunoreactivity.

Characterization and expression of HmuR, a TonB-dependent hemoglobin receptor of Porphyromonas gingivalis.

The gram-negative pathogen Porphyromonas gingivalis requires hemin for growth. Hemoglobin bound to haptoglobin and hemin complexed to hemopexin can be used as heme sources, indicating that P. gingivalis must have a means to remove the hemin from these host iron-binding proteins. However, the specific mechanisms utilized by P. gingivalis for the extraction of heme from heme-binding proteins and for iron transport are poorly understood. In this study we have determined that a newly identified TonB-dependent hemoglobin-hemin receptor (HmuR) is involved in hemoglobin binding and utilization in P. gingivalis A7436. HmuR shares amino acid homology with TonB-dependent outer membrane receptors of gram-negative bacteria involved in the acquisition of iron from hemin and hemoglobin, including HemR of Yersinia enterocolitica, ShuA of Shigella dysenteriae, HpuB of Neisseria gonorrhoeae and N. meningitidis, HmbR of N. meningitidis, HgbA of Haemophilus ducreyi, and HgpB of H. influenzae. Southern blot analysis confirmed the presence of the hmuR gene and revealed genetic variability in the carboxy terminus of hmuR in P. gingivalis strains 33277, 381, W50, and 53977. We also identified directly upstream of the hmuR gene a gene which we designated hmuY. Upstream of the hmuY start codon, a region with homology to the Fur binding consensus sequence was identified. Reverse transcription-PCR analysis revealed that hmuR and hmuY were cotranscribed and that transcription was negatively regulated by iron. Inactivation of hmuR resulted in a decreased ability of P. gingivalis to bind hemoglobin and to grow with hemoglobin or hemin as sole iron sources. Escherichia coli cells expressing recombinant HmuR were shown to bind hemoglobin and hemin. Furthermore, purified recombinant HmuR was demonstrated to bind hemoglobin. Taken together, these results indicate that HmuR serves as the major TonB-dependent outer membrane receptor involved in the utilization of both hemin and hemoglobin in P. gingivalis.

Binding specificity of the Porphyromonas gingivalis heme and hemoglobin receptor HmuR, gingipain K, and gingipain R1 for heme, porphyrins, and metalloporphyrins.

Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant P. gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 x 10(-5) M. Recombinant E. coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.

Tamm-Horsfall protein isolated from urine of pregnant and non-pregnant women has similar oligosaccharides.

BACKGROUND: Differences in the immunosuppressive activity of Tamm-Horsfall protein and uromodulin claimed by many investigators are controversial. They have been ascribed to variations in the carbohydrate moiety. METHODS: We isolated urinary glycoproteins from 18 non-pregnant (THP) and 12 pregnant (UM) women using the same method (adsorption on diatomaceous earth) and performed comparative studies including monocarbohydrate analysis and tests of reactivity with lectins, cytokines and serum proteins. RESULTS: No significant differences in monocarbohydrate content or in the reactivity with specific lectins were found between the native urinary glycoproteins or between their proteolytic products. Also, in Sda+ and Sda- donors monitored throughout pregnancy and after delivery, no time-dependent differences in the sugar moiety were observed. We found that THP/UM from Sda+ donors reacted more strongly with PHA-L than THP/UM from Sda- donors, providing further evidence for carbohydrate variations between Sda+ and Sda- determinants. No differences between THP and UM in terms of their reactivity with recombitant human interleukin (rhIL) 1 alpha, recombinant human tumour necrosis factor (rhTNF) alpha and some serum proteins were found, indicating similar biological activity. CONCLUSION: The results of our carbohydrate analysis together with previous data on amino acid analysis indicate that Tamm-Horsfall protein and uromodulin are identical or very similar glycoproteins.

Composition of the sugar moiety of Tamm-Horsfall protein in patients with urinary diseases.

The sugar moiety of Tamm-Horsfall protein (THP) is altered by pathological conditions. The aim of this study was to investigate the composition of THP glycans in urinary diseases. THP was isolated from the urine of patients with urinary tract infection (group A), glomerulonephritis or interstitial nephritis (group B), and Bartter's syndrome (BS) (group C). Monosaccharides, N-glycan profile, THP reactivity with specific lectins and some other proteins were analyzed. THP of patients from groups A, B, and C showed lower amounts of N-acetylgalactosamine (P<0.05, P<0.005, and P<0.05, respectively) than controls; this was reflected in lower reactivity with Phaseolus vulgaris lectin (P<0.005, P<0.05, and P<0.005). Reduced amounts of N-acetylglucosamine were noticed in groups A (P<0. 05) and B (P<0.05). In group A lower amounts of galactose and alpha2, 6-linked sialic acid, as determined by reactivity with Datura stramonium lectin (P<0.005) and Sambucus nigra lectin (P<0.005), were observed. In patients with BS there was a shift from tetrasialylated glycans towards less-sialylated chains. We found also that THP of all patients binds more strongly to IgG(1) (P<0.005, for all patient groups). Our results indicate that the urinary diseases examined affect the THP sugar moiety and the binding of THP to IgG(1).

Alterations of the sugar moiety of Tamm-Horsfall protein in children with malignancies of lymphoid cells.

The aim of this study was to examine whether the sugar moiety of Tamm-Horsfall protein (THP) is affected by pathological processes caused by acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma (NHL). The carbohydrate part of THP was studied by monosaccharide analysis, N-glycan profiling, and reactivity with specific lectins. Our results have shown that THP of ALL or NHL patients, in comparison with healthy subjects, have modified sugar chains. This is expressed in lower contents of N-acetylgalactosamine, alpha2,6-linked sialic acid and alpha1,6-linked fucose as well as in altered proportions of various N-glycans. We have shown that pathological processes also affect the carbohydrate unit of human immunoglobulin G (IgG) isolated from sera of ALL or NHL patients. As compared with healthy subjects, in IgG of the patient group, lower amounts of sialic acid and fucose were observed. These changes did not influence the biological properties of THP as judged by their unaltered ability to bind with interleukin-1alpha, alpha1-acid glycoprotein, serum albumin, transferrin, IgG1 and the light and heavy chains of IgG. Neither the in vivo alterations of IgG caused by ALL or NHL nor its in vitro modifications influence the interaction between IgG and THP.