Lysine-specific gingipain K and heme/hemoglobin receptor HmuR are involved in heme utilization in Porphyromonas gingivalis

We have previously reported on the identification and characterization of the Porphyromonas gingivalis A7436 strain outer membrane receptor HmuR, which is involved in the acquisition of hemin and hemoglobin. We demonstrated that HmuR interacts with the lysine- (Kgp) and arginine- (HRgpA) specific proteases (gingipains) and that Kgp and HRgpA can bind and degrade hemoglobin. Here, we report on the physiological significance of the HmuR-Kgp complex in heme utilization in P. gingivalis through the construction and characterization of a defined kgp mutant and a hmuR kgp double mutant in P. gingivalis A7436. The P. gingivalis kgp mutant exhibited a decreased ability to bind both hemin and hemoglobin. Growth of this strain with hemoglobin was delayed and its ability to utilize hemin as a sole iron source was diminished as compared to the wild type strain. Inactivation of both the hmuR and kgp genes resulted in further decreased ability of P. gingivalis to bind hemoglobin and hemin, as well as diminished ability to utilize either hemin or hemoglobin as a sole iron source. Collectively, these in vivo results further confirmed that both HmuR and Kgp are involved in the utilization of hemin and hemoglobin in P. gingivalis A7436.     

Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes

Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis , an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA , kgp and hemagglutinin hagA genes are responsible for coaggregation of P. gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA -deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus , whereas the kgp -null and rgpA rgpB -deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA , kgp , and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.     

Hemagglutinin/Adhesin domains of Porphyromonas gingivalis play key roles in coaggregation with Treponema denticola

Porphyromonas gingivalis and Treponema denticola are major pathogens of periodontal disease. Coaggregation between microorganisms plays a key role in the colonization of the gingival crevice and the organization of periodontopathic biofilms. We investigated the involvement of surface ligands of P. gingivalis in coaggregation. Two triple mutants of P. gingivalis lacking Arg-gingipain A (RgpA), Lys-gingipain (Kgp) and Hemagglutinin A (HagA) or RgpA, Arg-gingipain B (RgpB) and Kgp showed significantly decreased coaggregation with T. denticola, whereas coaggregation with a major fimbriae (FimA)-deficient mutant was the same as that with the P. gingivalis wild-type parent strain. rgpA, kgp and hagA code for proteins that contain 44 kDa Hgp44 adhesin domains. The coaggregation activity of an rgpA kgp mutant was significantly higher than that of the rgpA kgp hagA mutant. Furthermore, anti-Hgp44 immunoglobulin G reduced coaggregation between P. gingivalis wild type and T. denticola. Treponema denticola sonicates adhered to recombinant Rgp domains. Coaggregation following co-culture of the rgpA kgp hagA mutant expressing the RgpB protease with the rgpA rgpB kgp mutant expressing the unprocessed HagA protein was enhanced compared with that of each triple mutant with T. denticola. These results indicate that the processed P. gingivalis surface Hgp44 domains are key adhesion factors for coaggregation with T. denticola.     

Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis. Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA.

Porphyromonas gingivalis produces arginine-specific cysteine proteinase (Arg-gingipain, RGP) and lysine-specific cysteine proteinase (Lys-gingipain, KGP) in the extracellular and cell-associated forms. Two separate genes (rgpA and rgpB) and a single gene (kgp) have been found to encode RGP and KGP, respectively. We constructed rgpA rgpB kgp triple mutants by homologous recombination with cloned rgp and kgp DNA interrupted by drug resistance gene markers. The triple mutants showed no RGP or KGP activity in either cell extracts or culture supernatants. The culture supernatants of the triple mutants grown in a rich medium had no proteolytic activity toward bovine serum albumin or gelatin derived from human type I collagen. Moreover, the mutants did not grow in a defined medium containing bovine serum albumin as the sole carbon/energy source. These results indicate that the proteolytic activity of P. gingivalis toward bovine serum albumin and gelatin derived from human type I collagen appears to be attributable to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for hemagglutination and hemoglobin binding that are also located in the C-terminal regions of rgpA and kgp. A rgpA kgp hagA triple mutant constructed in this study exhibited no hemagglutination using sheep erythrocytes or hemoglobin binding activity, as determined by a solid-phase binding assay with horseradish peroxidase-conjugated human hemoglobin, indicating that the adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin, leading to heme acquisition.     

Degradation of Host Heme Proteins by Lysine- and Arginine-Specific Cysteine Proteinases (Gingipains) of Porphyromonas gingivalis

Porphyromonas gingivalis can use hemoglobin bound to haptoglobin and heme complexed to hemopexin as heme sources; however, the mechanism by which hemin is released from these proteins has not been defined. In the present study, using a variety of analytical methods, we demonstrate that lysine-specific cysteine proteinase of P. gingivalis (gingipain K, Kgp) can efficiently cleave hemoglobin, hemopexin, haptoglobin, and transferrin. Degradation of hemopexin and transferrin in human serum by Kgp was also detected; however, we did not observe extensive degradation of hemoglobin in serum by Kgp. Likewise the beta-chain of haptoglobin was partially protected from degradation by Kgp in a haptoglobin-hemoglobin complex. Arginine-specific gingipains (gingipains R) were also found to degrade hemopexin and transferrin in serum; however, this was observed only at relatively high concentrations of these enzymes. Growth of P. gingivalis strain A7436 in a minimal media with normal human serum as a source of heme correlated not only with the ability of the organism to degrade hemoglobin, haptoglobin, hemopexin, and transferrin but also with an increase in gingipain K and gingipain R activity. The ability of gingipain K to cleave hemoglobin, haptoglobin, and hemopexin may provide P. gingivalis with a usable source of heme for growth and may contribute to the proliferation of P. gingivalis within periodontal pockets in which erythrocytes are abundant.     

Coaggregation of Porphyromonas gingivalis and Prevotella intermedia.

Porphyromonas gingivalis cells coaggregated with Prevotella intermedia cells. The coaggregation was inhibited with L-arginine, L-lysine, Nalpha-p-tosyl-L-lysine chloromethyl ketone, trypsin inhibitor, and leupeptin. Heat- and proteinase K-treated P. gingivalis cells showed no coaggregation with P. intermedia cells, whereas heat and proteinase K treatments of P. intermedia cells did not affect the coaggregation. The vesicles from P. gingivalis culture supernatant aggregated with P. intermedia cells, and this aggregation was also inhibited by addition of L-arginine or L-lysine and by heat treatment of the vesicles. The rgpA rgpB, rgpA kgp, rgpA rgpB kgp, and rgpA kgp hagA mutants of P. gingivalis did not coaggregate with P. intermedia. On the other hand, the fimA mutant lacking the FimA fimbriae showed coaggregation with P. intermedia as well as the wild type parent. These results strongly imply that a heat-labile and proteinous factor on the cell surface of P gingivalis, most likely the gingipain-adhesin complex, is involved in coaggregation of P. gingivalis and P. intermedia.     

Human lactoferrin binds and removes the hemoglobin receptor protein of the periodontopathogen Porphyromonas gingivalis

Porphyromonas gingivalis possesses a hemoglobin receptor (HbR) protein on the cell surface as one of the major components of the hemoglobin utilization system in this periodontopathogenic bacterium. HbR is intragenically encoded by the genes of an arginine-specific cysteine proteinase (rgpA), lysine-specific cysteine proteinase (kgp), and a hemagglutinin (hagA). Here, we have demonstrated that human lactoferrin as well as hemoglobin have the abilities to bind purified HbR and the cell surface of P. gingivalis through HbR. The interaction of lactoferrin with HbR led to the release of HbR from the cell surface of P. gingivalis. This lactoferrin-mediated HbR release was inhibited by the cysteine proteinase inhibitors effective to the cysteine proteinases of P. gingivalis. P. gingivalis could not utilize lactoferrin for its growth as an iron source and, in contrast, lactoferrin inhibited the growth of the bacterium in a rich medium containing hemoglobin as the sole iron source. Lactoferricin B, a 25-amino acid-long peptide located at the N-lobe of bovine lactoferrin, caused the same effects on P. gingivalis cells as human lactoferrin, indicating that the effects of lactoferrin might be attributable to the lactoferricin region. These results suggest that lactoferrin has a bacteriostatic action on P. gingivalis by binding HbR, removing it from the cell surface, and consequently disrupting the iron uptake system from hemoglobin.     

A novel type of two-component regulatory system affecting gingipains in Porphyromonas gingivalis

We surveyed the Porphyromonas gingivalis W83 genome database for homologues of FimS, the first two-component sensor histidine kinase, which could possibly control virulence factors. Including fimS, we found six putative sensor kinase genes in the genome. The gene encoding one of the homologues was cloned from a P. gingivalis plasmid library, sequenced, and analyzed using its mutants. Two gene-disruption mutants were created in strain ATCC 33277 by introducing a drug cassette into the gene. The mutants formed nonpigmented colonies, indicating that they might be defective in proteinase production, a characteristic of this organism. Proteinase activities, measured as arginine- and lysine-specific (Rgp and Kgp gingipains, respectively) activities, of the mutants were almost half those of the parent strain. Unlike the parent and wildtype strains, most of the gingipain activities were detected in the culture supernatant, not in cells, of the mutants. Abnormal production of gingipains was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses. These results strongly suggest that this newly-discovered two-component sensor kinase is involved in maturation and proper localization of gingipains to the outer membrane through an unknown mechanism. The gene encoding the sensor histidine kinase was designated gppX, which represents regulation (X) of gingipains and black pigmentation in P. gingivalis.     

Porphyromonas gingivalis proteinases as virulence determinants in progression of periodontal diseases

Porphyromonas gingivalis, one of the major causative agents of periodontal diseases, produces large amounts of arginine- and lysine-specific cysteine proteinases in cell-associated and secretory forms, which are now referred to as Arg-gingipain (Rgp) and Lys-gingipain (Kgp), respectively. A number of studies have revealed that these proteinases are closely associated with the periodontopathogenesis of this bacterium: destruction of periodontal connective tissues, disruption of host defense mechanisms, and development and maintenance of inflammation in periodontal pockets. With respect to the physiology of the bacterium, Rgp and Kgp are indispensable for it to obtain nutrients from the environment, since it cannot utilize saccharides as carbon/energy sources for growth and totally depends on peptides and amino acids that are provided from environmental proteins by Rgp and Kgp. Furthermore, proteolytic activities of Rgp and Kgp contribute to processing/maturation of various cell-surface proteins of P. gingivalis, such as fimA fimbrilin (a subunit of major fimbriae), 75-kDa protein (a subunit of minor fimbriae), hemagglutinins, and the hemoglobin receptor protein, which are important for the bacterium to colonize and proliferate in the gingival crevice and to invade the periodontium. These findings strongly indicate critical roles of Rgp and Kgp in the virulence of P. gingivalis.     

Porphyromonas gingivalis selectively up-regulates the HIV-1 coreceptor CCR5 in oral keratinocytes

Primary infection of oral epithelial cells by HIV-1, if it occurs, could promote systemic infection. Most primary systemic infections are associated with R5-type HIV-1 targeting the R5-specific coreceptor CCR5, which is not usually expressed on oral keratinocytes. Because coinfection with other microbes has been suggested to modulate cellular infection by HIV-1, we hypothesized that oral keratinocytes may up-regulate CCR5 in response to the oral endogenous pathogen Porphyromonas gingivalis by cysteine-protease (gingipains) activation of the protease-activated receptors (PARs) or LPS signaling through the TLRs. The OKF6/TERT-2-immortalized normal human oral keratinocyte line expressed CXCR4, whereas CCR5 was not detectable. When exposed to P. gingivalis ATCC 33277, TERT-2 cells induced greater time-dependent expression of CCR5-specific mRNA and surface coreceptors than CXCR4. By comparing arg- (Rgp) and lys-gingipain (Kgp) mutants, a mutant deficient in both proteases, and the action of trypsin, P. gingivalis Rgp was strongly suggested to cleave PAR-1 and PAR-2 to up-regulate CCR5. CCR5 was also slightly up-regulated by an isogenic gingipain-deficient mutant, suggesting the presence of a nongingipain-mediated mechanism. Purified P. gingivalis LPS also up-regulated CCR5. Blocking TLR2 and TLR4 receptors with Abs attenuated induction of CCR5, suggesting LPS signaling through TLRs. P. gingivalis, therefore, selectively up-regulated CCR5 by two independent signaling pathways, Rgp acting on PAR-1 and PAR-2, and LPS on TLR2 and TLR4. By inducing CCR5 expression, P. gingivalis coinfection could promote selective R5-type HIV-1 infection of oral keratinocytes.     

Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis

The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.     

Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis.

Arginine-specific cysteine proteinase (Arg-gingipain [RGP], a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB+ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins.     

Porphyromonas gingivalis-induced platelet aggregation in plasma depends on Hgp44 adhesin but not Rgp proteinase

Evidence from recent epidemiological studies suggests a link between periodontal infections and increased risk of atherosclerosis and related cardiovascular and cerebrovascular events in human subjects. One of the major pathogens of periodontitis, Porphyromonas gingivalis, has the ability to aggregate human platelets in platelet-rich plasma (PRP). Mechanism of P. gingivalis-induced platelet aggregation in PRP was investigated. Proteinase inhibitors toward Arg-gingipain (Rgp) and Lys-gingipain (Kgp) did not suppress P. gingivalis-induced platelet aggregation in PRP, whereas the Rgp inhibitor markedly inhibited P. gingivalis-induced platelet aggregation using washed platelets. Mutant analysis revealed that P. gingivalis-induced platelet aggregation in PRP depended on Rgp-, Kgp- and haemagglutinin A (HagA)-encoding genes that intragenically coded for adhesins such as Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalis-induced platelet aggregation in PRP. P. gingivalis cell-reactive IgG in plasma, and FcgammaRIIa receptor and to a lesser extent GPIbalpha receptor on platelets were found to be a prerequisite for P. gingivalis-induced platelet aggregation in PRP. These results reveal a novel mechanism of platelet aggregation by P. gingivalis.     

Genotype variation and capsular serotypes of Porphyromonas gingivalis from chronic periodontitis and periodontal abscesses

Porphyromonas gingivalis is considered an important pathogen in periodontal disease. While this organism expresses a number of virulence factors, no study combining different virulence polymorphisms has, so far, been conducted. The occurrence of combined virulence (Cv) genotypes in 62 isolates of P. gingivalis was investigated from subjects displaying either chronic periodontitis or periodontal abscess. The Cv genotypes, based on gene variation of fimbriae (fimA), Lys-specific cystein proteinase (kgp) and Arg-specific cystein proteinase (prpR1/rgpA), were evaluated by PCR. The isolates were also subjected to capsular polysaccharide K-serotyping. A total of 18 Cv genotype variants based on fimA: kgp: rgpA were identified, of which II:I:A and II:II:A Cv genotypes (53.3%) were the two most frequently detected combinations. Moreover, 36% of the isolates were K-typeable, with the K6 serotype being the most prevalent (23%). Two isolates had the same genotype as the virulent strain W83. The results indicate that chronic periodontitis is not associated with a particularly virulent clonal type. A highly virulent genotype (e.g. strain W83) of P. gingivalis can be found in certain periodontitis patients.     

Production of protective antibodies against Porphyromonas gingivalis strains by immunization with recombinant gingipain domains

We studied the effect of antibodies against Porphyromonas gingivalis gingipain domains, preparing them against three recombinant fragments of RgpA (catalytic domain, r-Rgp CAT; hemagglutinin domains, r-Rgp 44 and r-Rgps 15-27) and one fragment of Kgp (catalytic domain, r-Kgp CAT). Enhancement of opsonization and killing by human polymorphonuclear leukocytes were measured in the noninvasive FDC 381 and invasive W50 strains of P. gingivalis. Anti-r-Rgp 44 was the most effective in both strains of P. gingivalis. The present findings lead us to recommend RgpA 44 as a candidate immunogen for vaccines against 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.     

Purification and characterization of a 50-kDa cysteine proteinase (gingipain) from Porphyromonas gingivalis.

Porphyromonas gingivalis, a Gram-negative anaerobic rod, has been closely associated with the initiation and progression of periodontal disease. This organism has been shown to produce a large number of proteolytic enzymes which can degrade a variety of tissue proteins, and these are considered to be major virulence factors. One of the proteinases produced by this organism, referred to as gingipain-1, has been purified to homogeneity from P. gingivalis culture medium by a combination of gel filtration and ion-exchange chromatography. The enzyme was found to have a molecular mass near 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a pH optimum in the neutral to alkaline range, and a requirement for cysteine for activation and Ca2+ for stabilization. Amino-terminal sequence analysis indicated that gingipain belongs to a new, so far unknown, subfamily of cysteine proteinases. Three unusual features of this proteinase are: (a) the stimulation of amidolytic activity by glycine-containing dipeptides; (b) a narrow specificity which is limited to peptide bonds containing arginine residues; and (c) resistance to inhibition by proteinase inhibitors in human plasma.     

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.