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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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Native PAGE showing the HmuY-haem complex formed through co-incubation of oxyhaemoglobin with HRgpA plus HmuY.Oxyhaemoglobin was co-incubated with HmuY plus HRgpA (CBB stained gel A and TMB/H2O2 stained gel B). Note that a small amount of the HmuY-haem complex was formed after 24 h incubation of oxyhaemoglobin plus HmuY only (CBB stained gel track C and TMB/H2O2 stained gel track D), attributed to pickup of haem lost from oxyhaemoglobin as a result of auto-oxidation. The oxyhaemoglobin concentration was 16 µM (on a haemoglobin subunit basis), as was HmuY, whilst HRgpA was present at 0.4 µM.
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pone-0017182-g008: Native PAGE showing the HmuY-haem complex formed through co-incubation of oxyhaemoglobin with HRgpA plus HmuY.Oxyhaemoglobin was co-incubated with HmuY plus HRgpA (CBB stained gel A and TMB/H2O2 stained gel B). Note that a small amount of the HmuY-haem complex was formed after 24 h incubation of oxyhaemoglobin plus HmuY only (CBB stained gel track C and TMB/H2O2 stained gel track D), attributed to pickup of haem lost from oxyhaemoglobin as a result of auto-oxidation. The oxyhaemoglobin concentration was 16 µM (on a haemoglobin subunit basis), as was HmuY, whilst HRgpA was present at 0.4 µM.

Mentions: Chemostat studies [3] have shown the presence of arginine-specific protease activity in haem-limited cultures expressing haem-binding proteins [4], [18]. Since P. gingivalis may concomitantly deploy the HmuY haemophore and gingipain proteases, we investigated the effect of co-incubation of oxyhaemoglobin with HmuY in the presence of HRgpA to mimic the in vivo situation more closely. It is noteworthy in this respect, that HmuY is absolutely resistant to degradation by either R- or K-gingipains [9]. For this, HmuY (16 µM) was incubated with 16 µM oxyhaemoglobin (with respect to haemoglobin subunit) in the presence of HRgpA (400 nM) and the haem transfer from oxyhaemoglobin to HmuY monitored spectroscopically and by native PAGE (Figs. 7 and 8, respectively). In the presence of both HRgpA and HmuY, the oxyhaemoglobin spectrum was transformed after 24 h into one typical of the HmuY-ferrihaem complex with a 411 nm Soret band and 528 nm and weak 559 nm visible bands (Fig. 7).


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)

Native PAGE showing the HmuY-haem complex formed through co-incubation of oxyhaemoglobin with HRgpA plus HmuY.Oxyhaemoglobin was co-incubated with HmuY plus HRgpA (CBB stained gel A and TMB/H2O2 stained gel B). Note that a small amount of the HmuY-haem complex was formed after 24 h incubation of oxyhaemoglobin plus HmuY only (CBB stained gel track C and TMB/H2O2 stained gel track D), attributed to pickup of haem lost from oxyhaemoglobin as a result of auto-oxidation. The oxyhaemoglobin concentration was 16 µM (on a haemoglobin subunit basis), as was HmuY, whilst HRgpA was present at 0.4 µM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017182-g008: Native PAGE showing the HmuY-haem complex formed through co-incubation of oxyhaemoglobin with HRgpA plus HmuY.Oxyhaemoglobin was co-incubated with HmuY plus HRgpA (CBB stained gel A and TMB/H2O2 stained gel B). Note that a small amount of the HmuY-haem complex was formed after 24 h incubation of oxyhaemoglobin plus HmuY only (CBB stained gel track C and TMB/H2O2 stained gel track D), attributed to pickup of haem lost from oxyhaemoglobin as a result of auto-oxidation. The oxyhaemoglobin concentration was 16 µM (on a haemoglobin subunit basis), as was HmuY, whilst HRgpA was present at 0.4 µM.
Mentions: Chemostat studies [3] have shown the presence of arginine-specific protease activity in haem-limited cultures expressing haem-binding proteins [4], [18]. Since P. gingivalis may concomitantly deploy the HmuY haemophore and gingipain proteases, we investigated the effect of co-incubation of oxyhaemoglobin with HmuY in the presence of HRgpA to mimic the in vivo situation more closely. It is noteworthy in this respect, that HmuY is absolutely resistant to degradation by either R- or K-gingipains [9]. For this, HmuY (16 µM) was incubated with 16 µM oxyhaemoglobin (with respect to haemoglobin subunit) in the presence of HRgpA (400 nM) and the haem transfer from oxyhaemoglobin to HmuY monitored spectroscopically and by native PAGE (Figs. 7 and 8, respectively). In the presence of both HRgpA and HmuY, the oxyhaemoglobin spectrum was transformed after 24 h into one typical of the HmuY-ferrihaem complex with a 411 nm Soret band and 528 nm and weak 559 nm visible bands (Fig. 7).

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