John W. Smalley

The degradation of type I collagen and human plasma fibronectin by the trypsin-like enzyme and extracellular membrane vesicles of Bacteroides gingivalis W50

A soluble trypsin-like enzyme (STE) was purified from a cell- and particle-free culture supernatant of this bacterium by a combination of ultra-centrifugation, ammonium-sulphate precipitation and gel-filtration chromatography on Sephacryl S-200. Trypsin-like activity in the culture supernatant was associated with a 58 kDa peptide and also with a higher molecular-weight complex. The STE and extracellular vesicle (ECV) fraction of B. gingivalis W50 rapidly degraded human plasma fibronectin in the presence and the absence of 10 mM dithiothreitol (DTT). The STE yielded a range of lower molecular-weight fibronectin digestion products. Under conditions where little activity was expressed by mammalian trypsin, both STE and ECV depolymerized a denatured and a native type I collagen substrate. Quantitative and qualitative differences were observed in the patterns of digestion products generated by both STE and ECV fraction following incubation with and without 10mM DTT. Inclusion of DTT appeared to reduce the degradative effect of both ECV and STE towards the type I collagen and plasma fibronectin substrates.

Trypsin-like Enzyme Activity of the Extracellular Membrane Vesicles of Bacteroides gingivalis W50

Trypsin-like enzyme activity in spent culture media from 3-d-old batch cultures of Bacteroides gingivalis W50 was measured by using the hydrolysis of N alpha-benzoyl-L-arginine-p-nitroanilide. The cell-free culture medium was fractionated by differential centrifugation at 10,000 g and 75,000 g, yielding two particulate fractions and a soluble supernatant fraction. About 80% of the total recoverable activity was associated with the particulate fractions, the remainder being in the supernatant. Electron microscopy of ruthenium-red/osmium stained ultrathin sections of the pellet fractions showed them to be composed of vesicular particles (extracellular vesicles), between 50 and 250 nm in diameter. Enzyme activity in all three fractions was enhanced by dithiothreitol. Gel-permeation chromatography of the soluble fraction yielded one peak of activity which contained 64 kDa and 58 kDa polypeptides. Enzyme activity from the vesicular fractions could be solubilized by sonication, giving a similar chromatographic profile to the supernatant fraction. The main peak of activity was composed of 64 kDa and 58 kDa polypeptides. In addition, there was a higher molecular mass enzyme activity peak composed of the 64 kDa and 58 kDa components along with 111 kDa, 93 kDa and 70 kDa polypeptides. We conclude that the trypsin-like enzyme of B. gingivalis is released as a soluble protein and is also associated with extracellular vesicles, in which it may exist as a soluble component and also as a protein complex.

HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis.

Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

Haemin-binding proteins of Porphyromonas gingivalis W50 grown in a chemostat under haemin-limitation

Porphyromonas gingivalis W50 was grown in a chemostat at pH 7.3 under haemin-limitation and haemin-excess at a constant mean doubling time of 6.9 h. Outer membranes (OM) were extracted from whole cells using EDTA and compared by SDS-PAGE. Haemin-limited cells expressed novel outer membrane proteins (OMPs) of mol. mass 115, 113 and 19 kDa when samples were solubilized at 100 degrees C. A 46 kDa OMP was observed in haemin-excess cells but not in those from haemin-limited conditions. Tetramethylbenzidine (TMBZ) staining of gels, after OM solubilization at 20 degrees C, was used to detect haemin-binding proteins (HBPs). HBPs were observed only in OM from haemin-limited cells. The major HBP (mol. mass 32.4 kDa) corresponded to a similar sized Kenacid-blue-stained protein which was not observed in haemin-excess-derived OM. Haemin-limited cells and OM displayed a ladder-like series of Kenacid-blue-stained proteins. Lighter TMBZ-stained proteins of mol. mass 51, 53, 56 and 60 kDa, with mobilities corresponding to those of silver-stained LPS components, were observed in haemin-limited OM. No soluble HBPs were detected extracellularly. The greater number of HBPs expressed by cells grown under haemin-limitation may reflect an additional cell surface receptor system for haemin acquisition under low environmental levels of this essential cofactor.

A combination of both arginine- and lysine-specific gingipain activity of Porphyromonas gingivalis is necessary for the generation of the micro-oxo bishaem-containing pigment from haemoglobin.

The black pigment of Porphyromonas gingivalis is composed of the mu-oxo bishaem complex of Fe(III) protoporphyrin IX (mu-oxo oligomer, dimeric haem), namely [Fe(III)PPIX]2O. P. gingivalis W50 and Rgp (Arg-gingipain)- and Kgp (Lys-gingipain)-deficient mutants K1A, D7, E8 and W501 [Aduse-Opoku, Davies, Gallagher, Hashim, Evans, Rangarajan, Slaney and Curtis (2000) Microbiology 146, 1933-1940] were grown on horse blood/agar for 14 days and examined for the production of mu-oxo bishaem. Mu-oxo Bishaem was detected by UV-visible, Mössbauer and Raman spectroscopies in wild-type W50 and in the black-pigmented RgpA- and RgpB-deficient mutants (W501 and D7 respectively), whereas no haem species were detected in the straw-coloured colonies of Kgp-deficient strain K1A. The dark brown pigment of the double RgpA/RgpB knockout mutant (E8) was not composed of mu-oxo bishaem, but of a high-spin monomeric Fe(III) protoporphyrin IX species (possibly a haem-albumin complex). In vitro incubation of oxyhaemoglobin with cells of the W50 strain and the RgpA- and RgpB-deficient mutants (W501 and D7) resulted in the formation of mu-oxo bishaem via methaemoglobin as an intermediate. Although the Kgp-deficient strain K1A converted oxyhaemoglobin into methaemoglobin, this was not further degraded into mu-oxo bishaem. The double RgpA/RgpB knockout was also not capable of producing mu-oxo bishaem from oxyhaemoglobin, but instead generated a haemoglobin haemichrome. Inhibition of Arg-X protease activity of W50, W501, D7 and K1A with leupeptin, under conditions where Lys-X protease activity was unaffected, prevented the production of mu-oxo bishaem from oxyhaemoglobin, but resulted in the formation of a haemoglobin haemichrome. These results show that one or both of RgpA and RgpB gingipains, in addition to the lysine-specific gingipain, is necessary for the production of mu-oxo bishaem from haemoglobin by whole cells of P. gingivalis.

Interactions of Porphyromonas gingivalis with oxyhaemoglobin and deoxyhaemoglobin.

When grown on blood-containing solid media, the anaerobic periodontal pathogen Porphyromonas gingivalis produces a haem pigment, the major component of which is the mu-oxo bishaem of iron protoporphyrin IX [Smalley, Silver, Marsh and Birss (1998) Biochem. J. 331, 681-685]. In this study, mu-oxo bishaem generation by P. gingivalis from oxy- and deoxyhaemoglobin was examined. Bacterial cells were shown to convert oxyhaemoglobin into methaemoglobin, which was degraded progressively, generating a mixture of both monomeric and mu-oxo dimeric iron protoporphyrin IX. The rate of methaemoglobin formation was accelerated in the presence of bacterial cells, but was inhibited by N-ethylmaleimide and tosyl-lysylchloromethylketone. Interaction of cells with deoxyhaemoglobin resulted in formation of an iron(III) haem species (Soret gamma(max), 393 nm), identified as pure mu-oxo bishaem.

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.

Gallium(III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells

Porphyromonas gingivalis acquires heme for growth, and initiation and progression of periodontal diseases. One of its heme acquisition systems consists of the HmuR and HmuY proteins. This study analyzed the antimicrobial activity of non-iron metalloporphyrins against P. gingivalis during planktonic growth, biofilm formation, epithelial cell adhesion and invasion, and employed hmuY, hmuR and hmuY-hmuR mutants to assess the involvement of HmuY and HmuR proteins in the acquisition of metalloporphyrins. Iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) supported planktonic growth of P. gingivalis cells, biofilm accumulation, as well as survival, adhesion and invasion of HeLa cells in a way analogous to protoheme. In contrast, cobalt(III), gallium(III) and copper(II) protoporphyrin IX exhibited antimicrobial activity against P. gingivalis, and thus represent potentially useful antibacterial compounds with which to target P. gingivalis. P. gingivalishmuY, hmuR and hmuY-hmuR mutants showed decreased growth and infection of epithelial cells in the presence of all metalloporphyrins examined. In conclusion, the HmuY protein may not be directly involved in transport of free metalloporphyrins into the bacterial cell, but it may also play a protective role against metalloporphyrin toxicity by binding an excess of these compounds.

Role of the cysteine protease interpain A of Prevotella intermedia in breakdown and release of haem from haemoglobin.

The gram-negative oral anaerobe Prevotella intermedia forms an iron(III) protoporphyrin IX pigment from haemoglobin. The bacterium expresses a 90 kDa cysteine protease, InpA (interpain A), a homologue of Streptococcus pyogenes streptopain (SpeB). The role of InpA in haemoglobin breakdown and haem release was investigated. At pH 7.5, InpA mediated oxidation of oxyhaemoglobin to hydroxymethaemoglobin [in which the haem iron is oxidized to the Fe(III) state and which carries OH- as the sixth co-ordinate ligand] by limited proteolysis of globin chains as indicated by SDS/PAGE and MALDI (matrix-assisted laser-desorption ionization)-TOF (time-of-flight) analysis. Prolonged incubation at pH 7.5 did not result in further haemoglobin protein breakdown, but in the formation of a haemoglobin haemichrome (where the haem Fe atom is co-ordinated by another amino acid ligand in addition to the proximal histidine residue) resistant to degradation by InpA. InpA-mediated haem release from hydroxymethaemoglobin-agarose was minimal compared with trypsin at pH 7.5. At pH 6.0, InpA increased oxidation at a rate greater than auto-oxidation, producing aquomethaemoglobin (with water as sixth co-ordinate ligand), and resulted in its complete breakdown and haem loss. Aquomethaemoglobin proteolysis and haem release was prevented by blocking haem dissociation by ligation with azide, whereas InpA proteolysis of haem-free globin was rapid, even at pH 7.5. Both oxidation of oxyhaemoglobin and breakdown of methaemoglobin by InpA were inhibited by the cysteine protease inhibitor E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane]. In summary, we conclude that InpA may play a central role in haem acquisition by mediating oxyhaemoglobin oxidation, and by degrading aquomethaemoglobin in which haem-globin affinity is weakened under acidic conditions.

The influence of fluoride on the uptake of radiosulphate by rat incisor odontoblasts in vitro

A tissue culture system was used consisting of odontoblasts still attached to the mineralized elements of rat incisor teeth, to study the incorporation of [35S]-sulphate into sulphated glycosaminoglycans. Chondroitin-4-sulphate was identified as the sole sulphated glycosaminoglycan present; temporal incorporation of [35S]-sulphate showed that the label was distributed between cetylpyridinium chloride (CPC)-precipitable material and non-CPC precipitable fraction. The non-CPC material possessed a 4-fold greater specific activity labelled-sulphate per unit uronic acid than the CPC-fraction. Fluoride ion at final concentrations of 2.95 mM and 5.90 mM elicited a dose-dependent graded response on the ability of the CPC and non-CPC fractions to incorporate [35S]-sulphate. Fluoride may thus interfere with the ability of the odontoblast to synthesise or transfer active sulphate into the sulphated glycosaminoglycans furnished by this cell.