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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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Formation of the HmuY-ferrihaemcomplex during incubation of methaemoglobin-agarose in the presence of human serum albumin.HmuY, albumin and methaemoglobin were each present at 16 µM (with the haemoglobin on a subunit basis). Panel A, methaemoglobin-agarose co-incubated with both HmuY and albumin. Panel B, methaemoglobin plus albumin only. The experimental protocol was the same as described for Fig. 4, and the incubations were carried out at 37°C. The spectra are background corrected to take account of the small amount of methaemoglobin released at each time period from the haemoglobin agarose during incubation.
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pone-0017182-g011: Formation of the HmuY-ferrihaemcomplex during incubation of methaemoglobin-agarose in the presence of human serum albumin.HmuY, albumin and methaemoglobin were each present at 16 µM (with the haemoglobin on a subunit basis). Panel A, methaemoglobin-agarose co-incubated with both HmuY and albumin. Panel B, methaemoglobin plus albumin only. The experimental protocol was the same as described for Fig. 4, and the incubations were carried out at 37°C. The spectra are background corrected to take account of the small amount of methaemoglobin released at each time period from the haemoglobin agarose during incubation.

Mentions: We tested whether HmuY was capable of binding haem complexed in the form of methaemoglobin but in the presence of serum albumin. It should be noted that methaemalbumin and methaemoglobin share similar spectral features at physiological pH and that the presence of all three components in solution would yield a very complex spectrum. Therefore, we used immobilised methaemoglobin-agarose as the substrate so that the differential formation of either the methaemalbumin or HmuY-ferrihaem complexes (which are spectrally distinct) could be more easily followed. All the three components each at 16 µM (with methaemoglobin on a subunit basis as above) were incubated together at 37°C, and the supernatant solutions were removed periodically from the agarose beads, and the spectra recorded as for the two component (HmuY/methaemoglobin-agarose) system. As can be seen in Fig. 11 (panel A), in the three component mixture a spectrum was observed with a 411 nm Soret band and visible bands at 527 and 558 nm, which was indicative of the formation of the HmuY-ferrihaem species. The intensities of these bands increased with time of incubation as previously demonstrated in Figure 4 for the reaction of HmuY plus methaemoglobin-agarose only. In contrast however, for the incubation of the methaemoglobin-agarose with albumin generated spectra with a 402 nm Soret band and lower intensity bands at 500 and 620 nm, indicative of the formation of the methaemalbumin complex (Fig. 11, panel B).


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)

Formation of the HmuY-ferrihaemcomplex during incubation of methaemoglobin-agarose in the presence of human serum albumin.HmuY, albumin and methaemoglobin were each present at 16 µM (with the haemoglobin on a subunit basis). Panel A, methaemoglobin-agarose co-incubated with both HmuY and albumin. Panel B, methaemoglobin plus albumin only. The experimental protocol was the same as described for Fig. 4, and the incubations were carried out at 37°C. The spectra are background corrected to take account of the small amount of methaemoglobin released at each time period from the haemoglobin agarose during incubation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017182-g011: Formation of the HmuY-ferrihaemcomplex during incubation of methaemoglobin-agarose in the presence of human serum albumin.HmuY, albumin and methaemoglobin were each present at 16 µM (with the haemoglobin on a subunit basis). Panel A, methaemoglobin-agarose co-incubated with both HmuY and albumin. Panel B, methaemoglobin plus albumin only. The experimental protocol was the same as described for Fig. 4, and the incubations were carried out at 37°C. The spectra are background corrected to take account of the small amount of methaemoglobin released at each time period from the haemoglobin agarose during incubation.
Mentions: We tested whether HmuY was capable of binding haem complexed in the form of methaemoglobin but in the presence of serum albumin. It should be noted that methaemalbumin and methaemoglobin share similar spectral features at physiological pH and that the presence of all three components in solution would yield a very complex spectrum. Therefore, we used immobilised methaemoglobin-agarose as the substrate so that the differential formation of either the methaemalbumin or HmuY-ferrihaem complexes (which are spectrally distinct) could be more easily followed. All the three components each at 16 µM (with methaemoglobin on a subunit basis as above) were incubated together at 37°C, and the supernatant solutions were removed periodically from the agarose beads, and the spectra recorded as for the two component (HmuY/methaemoglobin-agarose) system. As can be seen in Fig. 11 (panel A), in the three component mixture a spectrum was observed with a 411 nm Soret band and visible bands at 527 and 558 nm, which was indicative of the formation of the HmuY-ferrihaem species. The intensities of these bands increased with time of incubation as previously demonstrated in Figure 4 for the reaction of HmuY plus methaemoglobin-agarose only. In contrast however, for the incubation of the methaemoglobin-agarose with albumin generated spectra with a 402 nm Soret band and lower intensity bands at 500 and 620 nm, indicative of the formation of the methaemalbumin complex (Fig. 11, panel B).

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