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.

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.

Mechanism of methaemoglobin breakdown by the lysine-specific gingipain of the periodontal pathogen Porphyromonas gingivalis.

Abstract The R- and K-gingipain proteases of Porphyromonas gingivalis are involved in proteolysis of haemoglobin from which the defensive dimeric haem pigment is formed. Whilst oxyhaemoglobin is refractory towards K-gingipain, methaemoglobin is rapidly degraded. Ligation of methaemoglobin with N3-, which effectively blocks haem dissociation from the protein, prevented haemoglobin breakdown. Haem-free globin was rapidly degraded by K-gingipain. These data emphasise the need for haemoglobin oxidation which encourages haem dissociation and makes the haem-free globin susceptible to proteolytic attack.

Sequential action of R- and K-specific gingipains of Porphyromonas gingivalis in the generation of the haem-containing pigment from oxyhaemoglobin.

The arginine- and lysine-specific gingipains of Porphyromonas gingivalis have been implicated in the degradation of haemoglobin from which the black mu-oxo haem dimer-containing pigment is generated. Here, we examined interactions of oxyhaemoglobin (oxyHb) with the Arg-(R)-specific (HRgpA) and Lys-(K)-specific (Kgp) gingipains. Incubation of oxyHb with HRgpA resulted in formation of methaemoglobin (metHb), which could be prevented by the R-gingipain specific inhibitor leupeptin. oxyHb-Kgp interactions resulted in formation of a haemoglobin haemichrome. This was inhibited by the lysine-specific protease inhibitor Z-Phe-Lys-acyloxymethylketone (Z-FKck). metHb, formed by treatment of oxyHb with either NaNO(2) or by pre-incubation with HRgpA, was rapidly degraded by Kgp compared to oxyHb. metHb degradation by Kgp was also inhibited Z-FKck. Together these data show that R-gingipain activity is crucial for converting oxyHb into the metHb form which is rendered more susceptible to Kgp degradation for the eventual release of iron(III) protoporphyrin IX and production of the mu-oxo haem dimer. This explains previous observations [J.W. Smalley, M.F. Thomas, A.J. Birss, R. Withnall, J. Silver, Biochem. J. 379 (2004) 833-840.] of the requirement for a combination of both R- and K-gingipains for pigment production from oxyhaemoglobin by P. gingivalis.

Characterization of a bifunctional catalase-peroxidase of Burkholderia cenocepacia.

Isolates of Burkholderia cenocepacia express a putative haem-binding protein (molecular mass 97 kDa) that displays intrinsic peroxidase activity. Its role has been re-evaluated, and we now show that it is a bifunctional catalase-peroxidase, with activity against tetramethylbenzidine (TMB), o-dianisidine, pyrogallol, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic) acid (ABTS). Both peroxidase and catalase activities are optimal at pH 5.5-6.0. The gene encoding this enzyme was cloned and expressed in Escherichia coli. We have named it katG because of its similarity to other katGs, including that from Burkholderia pseudomallei. It is substantially similar to a previously described catalase-peroxidase of B. cenocepacia (katA). MS analysis indicated that the initial katG translation product may be post-translationally modified in B. cenocepacia to give rise to the mature 97-kDa catalase-peroxidase.

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.

The haem pigment of the oral anaerobes Prevotella nigrescens and Prevotella intermedia is composed of iron(III) protoporphyrin IX in the monomeric form.

The haem pigment of Porphyromonas gingivalis is composed of micro -oxo bishaem, [Fe(III)PPIX](2)O, but the nature of that generated by Prevotella species has not been established. Mössbauer, Raman and UV-visible spectrophotometry were used to characterize the haem pigment of Prevotella intermedia and Prevotella nigrescens. Mössbauer and Raman spectroscopy revealed the major haem species to be monomeric iron protoporphyrin IX, Fe(III)PPIX.OH (haematin). The terminal growth pH of both species on blood agar was between 5.8 and 6.0, which favours the formation and maintenance of monomeric Fe(III)PPIX.OH. Incubation of Pr. nigrescens and Pr. intermedia with oxyhaemoglobin at pH 6.5 resulted in formation of aquomethaemoglobin which was degraded to generate Fe(III)PPIX.OH which in turn became cell-associated, whilst incubation at pH 7.5 resulted in formation of [Fe(III)PPIX](2)O. It is concluded that both Prevotella species degrade oxyhaemoglobin to form [Fe(III)PPIX](2)O as an intermediate, which is converted to Fe(III)PPIX.OH through a depression in pH. The low pH encourages cell-surface deposition of insoluble Fe(III)PPIX.OH which would act as a barrier against oxygen and reactive oxygen species, and also protect against H(2)O(2) through its inherent catalase activity.

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.