Hemophore-like proteins of the HmuY family in the oral and gut microbiome: unraveling the mystery of their evolution.

SUMMARY: Heme (iron protoporphyrin IX, FePPIX) is the main source of iron and PPIX for host-associated pathogenic bacteria, including members of the Bacteroidota (formerly Bacteroidetes) phylum. Porphyromonas gingivalis, a keystone oral pathogen, uses a unique heme uptake (Hmu) system, comprising a hemophore-like protein, designated as the first member of the novel HmuY family. Compared to classical, secreted hemophores utilized by Gram-negative bacteria or near-iron transporter domain-based hemophores utilized by Gram-positive bacteria, the HmuY family comprises structurally similar proteins that have undergone diversification during evolution. The best characterized are P. gingivalis HmuY and its homologs from Tannerella forsythia (Tfo), Prevotella intermedia (PinO and PinA), Bacteroides vulgatus (Bvu), and Bacteroides fragilis (BfrA, BfrB, and BfrC). In contrast to the two histidine residues coordinating heme iron in P. gingivalis HmuY, Tfo, PinO, PinA, Bvu, and BfrA preferentially use two methionine residues. Interestingly, BfrB, despite conserved methionine residue, binds the PPIX ring without iron coordination. BfrC binds neither heme nor PPIX in keeping with the lack of conserved histidine or methionine residues used by other members of the HmuY family. HmuY competes for heme binding and heme sequestration from host hemoproteins with other members of the HmuY family to increase P. gingivalis competitiveness. The participation of HmuY in the host immune response confirms its relevance in relation to the survival of P. gingivalis and its ability to induce dysbiosis not only in the oral microbiome but also in the gut microbiome or other host niches, leading to local injuries and involvement in comorbidities.

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.

Porphyromonas gingivalis HmuY and Streptococcus gordonii GAPDH-Novel Heme Acquisition Strategy in the Oral Microbiome.

The oral cavity of healthy individuals is inhabited by commensals, with species of Streptococcus being the most abundant and prevalent in sites not affected by periodontal diseases. The development of chronic periodontitis is linked with the environmental shift in the oral microbiome, leading to the domination of periodontopathogens. Structure-function studies showed that Streptococcus gordonii employs a "moonlighting" protein glyceraldehyde-3-phosphate dehydrogenase (SgGAPDH) to bind heme, thus forming a heme reservoir for exchange with other proteins. Secreted or surface-associated SgGAPDH coordinates Fe(III)heme using His43. Hemophore-like heme-binding proteins of Porphyromonas gingivalis (HmuY), Prevotella intermedia (PinO) and Tannerella forsythia (Tfo) sequester heme complexed to SgGAPDH. Co-culturing of P. gingivalis with S. gordonii results in increased hmuY gene expression, indicating that HmuY might be required for efficient inter-bacterial interactions. In contrast to the DhmuY mutant strain, the wild type strain acquires heme and forms deeper biofilm structures on blood agar plates pre-grown with S. gordonii. Therefore, our novel paradigm of heme acquisition used by P. gingivalis appears to extend to co-infections with other oral bacteria and offers a mechanism for the ability of periodontopathogens to obtain sufficient heme in the host environment. Importantly, P. gingivalis is advantaged in terms of acquiring heme, which is vital for its growth survival and virulence.

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.

Tannerella forsythia Tfo belongs to Porphyromonas gingivalis HmuY-like family of proteins but differs in heme-binding properties.

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.

Breakdown of albumin and haemalbumin by the cysteine protease interpain A, an albuminase of Prevotella intermedia.

BACKGROUND: Prevotella intermedia is a Gram-negative black-pigmenting oral anaerobe associated with periodontitis in humans, and has a haem requirement for growth, survival and virulence. It produces an iron porphyrin-containing pigment comprising monomeric iron (III) protoporphyrin IX (Fe(III)PPIX.OH; haematin). The bacterium expresses a 90-kDa cysteine protease termed interpain A (InpA) which both oxidizes and subsequently degrades haemoglobin, releasing haem. However, it is not known whether the enzyme may play a role in degrading other haem-carrying plasma proteins present in the gingival sulcus or periodontal pocket from which to derive haem. This study evaluated the ability of InpA to degrade apo- and haem-complexed albumin. RESULTS: Albumin breakdown was examined over a range of pH and in the presence of reducing agent; conditions which prevail in sub- and supra-gingival plaque. InpA digested haemalbumin more efficiently than apoalbumin, especially under reducing conditions at pH 7.5. Under these conditions InpA was able to substantially degrade the albumin component of whole human plasma. CONCLUSIONS: The data point to InpA as an efficient "albuminase" with the ability to degrade the minor fraction of haem-bound albumin in plasma. InpA may thus contribute significantly to haem acquisition by P. intermedia under conditions of low redox potential and higher pH in the inflamed gingival crevice and diseased periodontal pocket where haem availability is tightly controlled by the host.

Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

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.

Pyocyanina contributory factor in haem acquisition and virulence enhancement of Porphyromonas gingivalis in the lung [corrected].

Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain production, may contribute to virulence of P. gingivalis and disease severity when co-infecting with P. aeruginosa in the lung.

The Porphyromonas gingivalis HmuY haemophore binds gallium(iii), zinc(ii), cobalt(iii), manganese(iii), nickel(ii), and copper(ii) protoporphyrin IX but in a manner different to iron(iii) protoporphyrin IX.

Porphyromonas gingivalis, a major etiological agent of chronic periodontitis, acquires haem from host haemoproteins through a haem transporter HmuR and a haemophore HmuY. The aim of this study was to analyse the binding specificity of HmuY towards non-iron metalloporphyrins which may be employed as antimicrobials to treat periodontitis. HmuY binds gallium(iii), zinc(ii), cobalt(iii), manganese(iii), nickel(ii), and copper(ii) protoporphyrin IX but in a manner different to iron(iii) protoporphyrin IX which uses His(134) and His(166) as axial ligands. The metal ions in Ga(iii)PPIX and Zn(ii)PPIX can accept only His(166) as an axial ligand, whereas nickel(ii) and copper(ii) interact exclusively with His(134). Two forms of pentacoordinate manganese(iii) are present in the Mn(iii)PPIX-HmuY complex since the metal accepts either His(134) or His(166) as a single axial ligand. The cobalt ion is hexacoordinate in the Co(iii)PPIX-HmuY complex and binds His(134) and His(166) as axial ligands; however, some differences in their environments exist. Despite different coordination modes of the central metal ion, gallium(iii), zinc(ii), cobalt(iii), and manganese(iii) protoporphyrin IX bound to the HmuY haemophore cannot be displaced by excess haem. All of the metalloporphyrins examined bind to a P. gingivalis wild-type strain with higher ability compared to a mutant strain lacking a functional hmuY gene, thus corroborating binding of non-iron metalloporphyrins to purified HmuY protein. Our results further clarify the basis of metalloporphyrin acquisition by P. gingivalis and add to understanding of the interactions with porphyrin derivatives which exhibit antimicrobial activity against P. gingivalis.

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. gingivalis hmuY, 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.

Iron(III) mesoporphyrin IX and iron(III) deuteroporphyrin IX bind to the Porphyromonas gingivalis HmuY hemophore.

Porphyromonas gingivalis acquires heme through an outer-membrane heme transporter HmuR and heme-binding hemophore-like lipoprotein HmuY. Here, we compare binding of iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) to HmuY with that of iron(III) protoporphyrin IX (protoheme) and protoporphyrin IX (PPIX) using spectroscopic methods. In contrast to PPIX, mesoheme and deuteroheme enter the HmuY heme cavity and are coordinated by His134 and His166 residues in a fully analogous way to protoheme binding. However, in the case of deuteroheme two forms of HmuY-iron porphyrin complex were observed differing by a 180° rotation of porphyrin about the α-γ-meso-carbon axis. Since the use of porphyrins either as active photosensitizers or in combination with antibiotics may have therapeutic value for controlling bacterial growth in vivo, it is important to compare the binding of heme derivatives to HmuY.

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.

Transmissible Burkholderia cepacia genomovar IIIa strains bind and convert monomeric iron(III) protoporphyrin IX into the mu-oxo oligomeric form.

Burkholderia cepacia isolates of genomovar III are highly transmissible amongst patients with cystic fibrosis (CF) and express a 97 kDa putative haem-binding protein (HBP) [Smalley, J. W., Charalabous, P., Birss, A. J. & Hart, C. A. (2001). Clin Diagn Lab Immunol 8, 509-514]. An investigation of the interactions of iron(III) protoporphyrin IX with epidemic and non-epidemic strains of B. cepacia to determine the role of the above protein in haem acquisition and binding is reported herein. Spectrophotometric titrations of cell suspensions of genomovar IIIa strains BC7 and C5424 with iron(III) protoporphyrin IX, at pH 7.0, resulted in the depletion of Fe(III)PPIX.OH monomers and formation of the micro -oxo oligomeric species, [Fe(III)PPIX](2)O. Difference spectroscopy indicated a continuous conversion of the monomeric iron(III) protoporphyrin IX into micro -oxo oligomers. Incubations with Fe(III)PPIX.OH monomers at pH 6.5 also showed that cells could shift the equilibrium to generate the micro -oxo oligomeric form. Genomovar I strains ATCC 25416 and LMG 17997 were unable to mediate this conversion. SDS-PAGE of genomovar IIIa strains exposed to Fe(III)PPIX.OH at pH 6.5 followed by tetramethylbenzidine/H(2)O(2) staining revealed, in addition to the 97 kDa HBP, two proteins of 77 and 149 kDa located in the outer membrane which bound Fe(III)PPIX.OH monomers. These proteins were absent from the genomovar I strains. Genomovar IIIa strains BC7 and C5424 showed increased cellular binding of [Fe(III)PPIX](2)O, and as a consequence, displayed increased catalase activities compared to cells of the genomovar I isolates. It is concluded that, in addition to the putative 97 kDa HBP, B. cepacia genomovar IIIa strains express two outer-membrane proteins which function to bind and convert Fe(III)PPIX.OH monomers into the micro -oxo oligomeric form, [Fe(III)PPIX](2)O. The ability to perform this conversion at both neutral and slightly acidic pHs may enable epidemic strains to withstand attack from neutrophil-derived H(2)O(2) in the inflamed CF lung.