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

Smalley JW, Byrne DP, Birss AJ, Wojtowicz H, Sroka A, Potempa J, Olczak T - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, [corrected] University of Liverpool, Liverpool, United Kingdom. josmall@liv.ac.uk

ABSTRACT
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.

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Related in: MedlinePlus

Demonstration HmuY-ferrihaem complex formation after exposure of HRgpA-induced methaemoglobin to HmuY.Before exposure, black line; 24 h after exposure, red line. For clarity, only the initial and final spectra are shown. HRgpA and HmuY were used at 0.4 and 16 µM, respectively. Starting concentration of haemoglobin was 16 µM (with respect to haemoglobin subunit) and comprised 77% methaemoglobin.
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pone-0017182-g005: Demonstration HmuY-ferrihaem complex formation after exposure of HRgpA-induced methaemoglobin to HmuY.Before exposure, black line; 24 h after exposure, red line. For clarity, only the initial and final spectra are shown. HRgpA and HmuY were used at 0.4 and 16 µM, respectively. Starting concentration of haemoglobin was 16 µM (with respect to haemoglobin subunit) and comprised 77% methaemoglobin.

Mentions: In vivo, at periodontitis sites where bleeding may occur, any oxyhaemoglobin released from erythrocytes must firstly be oxidized to the methaemoglobin form before haem can be sequestered by HmuY. Therefore the process of haem acquisition and uptake by the HmuY/HmuR system should be dependent upon gingipain protease-mediated haemoglobin oxidation [12], [13]. To experimentally verify this hypothesis, an HRgpA-induced methaemoglobin preparation (total haemoglobin concentration 16 µM and comprising 77% in the oxidised form) was incubated with HmuY (16 µM). A control NaNO2-induced methaemoglobin preparation (containing 83% in the oxidised form) was also incubated with HmuY as above for comparison. With time, spectral features were observed indicative of conversion of R-gingipain-induced methaemoglobin into the ferrihaem-HmuY complex, i.e., red shift of the Soret band from 406 to 411 nm, loss of A500 nm and A630 nm, and increases in A527 nm and A558 nm, as shown in Fig. 5. These spectral features were also observed for the incubation of HmuY with the NaNO2-induced methaemoglobin preparation (data not presented).


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

Smalley JW, Byrne DP, Birss AJ, Wojtowicz H, Sroka A, Potempa J, Olczak T - PLoS ONE (2011)

Demonstration HmuY-ferrihaem complex formation after exposure of HRgpA-induced methaemoglobin to HmuY.Before exposure, black line; 24 h after exposure, red line. For clarity, only the initial and final spectra are shown. HRgpA and HmuY were used at 0.4 and 16 µM, respectively. Starting concentration of haemoglobin was 16 µM (with respect to haemoglobin subunit) and comprised 77% methaemoglobin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040768&req=5

pone-0017182-g005: Demonstration HmuY-ferrihaem complex formation after exposure of HRgpA-induced methaemoglobin to HmuY.Before exposure, black line; 24 h after exposure, red line. For clarity, only the initial and final spectra are shown. HRgpA and HmuY were used at 0.4 and 16 µM, respectively. Starting concentration of haemoglobin was 16 µM (with respect to haemoglobin subunit) and comprised 77% methaemoglobin.
Mentions: In vivo, at periodontitis sites where bleeding may occur, any oxyhaemoglobin released from erythrocytes must firstly be oxidized to the methaemoglobin form before haem can be sequestered by HmuY. Therefore the process of haem acquisition and uptake by the HmuY/HmuR system should be dependent upon gingipain protease-mediated haemoglobin oxidation [12], [13]. To experimentally verify this hypothesis, an HRgpA-induced methaemoglobin preparation (total haemoglobin concentration 16 µM and comprising 77% in the oxidised form) was incubated with HmuY (16 µM). A control NaNO2-induced methaemoglobin preparation (containing 83% in the oxidised form) was also incubated with HmuY as above for comparison. With time, spectral features were observed indicative of conversion of R-gingipain-induced methaemoglobin into the ferrihaem-HmuY complex, i.e., red shift of the Soret band from 406 to 411 nm, loss of A500 nm and A630 nm, and increases in A527 nm and A558 nm, as shown in Fig. 5. These spectral features were also observed for the incubation of HmuY with the NaNO2-induced methaemoglobin preparation (data not presented).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, [corrected] University of Liverpool, Liverpool, United Kingdom. josmall@liv.ac.uk

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus