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

Effect of ferrihaem binding on the electrophoretic mobility of HmuY.

<p>The presence of haem in the complex with HmuY is confirmed by haem-peroxidise activity (bottom panel).</p

Spectrum showing the ferrihaem-HmuY complex (red line) formed after 24 h co-incubation of oxyhaemoglobin with HRgpA and HmuY.

<p>The oxyhaemoglobin and HmuY concentrations were both 16 µM, whilst HRgpA was at 0.4 µM.</p

Formation of the ferrohaem-HmuY complex during incubation of HmuY with deoxyhaemoglobin-agarose.

<p>Haemoglobin-agarose was deoxygenated with sodium dithionite and maintained anaerobically during the reaction with HmuY. Concentration of haemoglobin was 16 µM (with respect to haemoglobin subunit) as was HmuY. See text for details. The absorbance below 375 nm is due to the presence of dithionite. A small amount of deoxyhaemoglobin (∼0.1% of the total) was released from the control deoxyhaemoglobin-agarose after 40 min incubation in the absence of HmuY.</p

HmuY-haem complex formation during the co-incubation of oxyhaemoglobin with both HmuY and Kgp.

<p>HmuY and oxyhaemoglobin (both at 16 µM) were incubated at 37°C with Kgp (0.2 µM) and sampled periodically, and subjected to native PAGE. Gel tracks were loaded with ∼12 µg total protein.</p

Difference spectra showing HmuY-haem complex formation from HRgpA-methaemoglobin (A) and NaNO₂-induced methaemoglobin (B).

<p>The difference spectra in the visible region show the appearance of the 525 nm and 558 nm bands attributable to formation of the HmuY-ferrihaem complex. Note that the relative extinctions at 527 nm and 558 nm are reversed compared to those in the ferrihaem-HmuY complex shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017182#pone-0017182-g001" target="_blank">Fig. 1</a>, and occur as a result of subtraction of the spectra. Starting concentrations of haemoglobin were 16 µM (haemoglobin subunit basis) and contained 77 and 87% methaemoglobin for the HRgpA- and NaNO₂-treated oxyhaemoglobin, respectively. HRgpA and NaNO₂ concentrations were 0.4 µM and 64 µM, respectively.</p

Formation of the HmuY-ferrihaemcomplex during incubation of methaemoglobin-agarose in the presence of human serum albumin.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017182#pone-0017182-g004" target="_blank">Fig. 4</a>, 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.</p

Formation of the ferrihaem-HmuY complex during incubation of human methaemalbumin with HmuY.

<p>Methaemalbumin (16 µM) was incubated with an equimolar amount of HmuY at 37°C. Arrows denote changes in the spectra with time at the indicated wavelengths.</p