Species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of Porphyromonas gingivalis HmuY
© Olczak et al; licensee BioMed Central Ltd. 2010
Received: 6 October 2009
Accepted: 4 May 2010
Published: 4 May 2010
Porphyromonas gingivalis is a major etiological agent of chronic periodontitis. The aim of this study was to examine the species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of the P. gingivalis heme-binding protein HmuY.
HmuY is a unique protein of P. gingivalis since only low amino-acid sequence homology has been found to proteins encoded in other species. It is exposed on the cell surface and highly abundant in the outer membrane of the cell, in outer-membrane vesicles, and is released into culture medium in a soluble form. The protein is produced constitutively at low levels in bacteria grown under high-iron/heme conditions and at higher levels in bacteria growing under the low-iron/heme conditions typical of dental plaque. HmuY is immunogenic and elicits high IgG antibody titers in rabbits. It is also engaged in homotypic biofilm formation by P. gingivalis. Anti-HmuY antibodies exhibit inhibitory activity against P. gingivalis growth and biofilm formation.
Here it is demonstrated that HmuY may play a significant role not only in heme acquisition, but also in biofilm accumulation on abiotic surfaces. The data also suggest that HmuY, as a surface-exposed protein, would be available for recognition by the immune response during chronic periodontitis and the production of anti-HmuY antibodies may inhibit biofilm formation.
Periodontitis is a complex process affecting tooth-supporting tissues . The pathogenesis of periodontal diseases is largely attributed to localized inflammation, which results from interaction between host and microbial factors . The most common etiological agent of chronic periodontitis is Porphyromonas gingivalis, a Gram-negative anaerobic black-pigmented bacterium . On tooth surfaces, P. gingivalis is a constituent of the complex multispecies biofilm known as dental plaque, which has properties of other biofilms found in the human body and in the environment. P. gingivalis can also colonize the tissues and cells of the gingival epithelium . The bacterium can not only invade, but also accumulate inside gingival epithelial cells [5, 6]. Recent evidence demonstrates that the effect of periodontitis might have systemic consequences since the bacterium can spread systemically and locate to other tissues [7–10].
Bacteria living in a biofilm have a physiology different from that of planktonic cells and they generally live under nutrient limitation, including that of iron and heme. The uptake of heme as iron and protoporphyrin IX is an important mechanism by which P. gingivalis and other pathogenic bacteria obtain these compounds for their survival and their ability to establish an infection [11, 12]. Gram-negative bacteria utilize outer-membrane receptors to acquire heme from host hemoproteins directly or through a hemophore or lipoprotein and then transport the captured heme into the cell. In the case of P. gingivalis, one of the systems of heme acquisition consists of HmuR and HmuY proteins . HmuR is an outer-membrane TonB-dependent receptor involved in heme transport through the outer membrane [13–16], whereas HmuY is a heme-binding lipoprotein associated with the outer membrane of the bacterial cell [17–21]. A detailed characterization of the HmuY-heme complex demonstrated that heme, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment . In that report we also identified histidines 134 and 166 as potential heme ligands. Recent crystallographic analysis of the HmuY-heme complex confirmed these data and showed that the protein exhibits a unique structure composed of an all-β fold . Our studies also showed that HmuY may be functional in the form of dimers/tetramers [19, 21]. It seems that dimeric HmuY takes up heme and this leads to tetramerization under occlusion of the heme binding sites. Tetrameric HmuY would protect heme from host scavengers and delivered it to HmuR. On the basis of our mutational analysis of HmuY heme ligands , an initial step in heme transfer could involve disruption of only one of the two axial histidine ligands, as found for Serratia marcescens hemophore HasA . Once bound by HmuR, heme is translocated across the outer membrane into the periplasm with the assistance of TonB and further heme transport requires the presence of binding proteins to escort it across the periplasm to the cytoplasm. This step might be performed by other hmu operon proteins, so far not characterized [17, 19]. HmuY, especially in the form associated with the outer membrane, may also store heme and protect the bacterial cell from damage induced by free hemin.
It is likely that HmuY lipoprotein may play a role not only in heme acquisition, but also in the host pathogen response. Therefore the aim of this study was to analyze the surface exposure and expression of HmuY protein in P. gingivalis. In addition, in this report we examined the participation of HmuY protein in biofilm formation.
Results and Discussion
HmuY is a unique P. gingivalis protein
HmuY is exposed on the surface of P. gingivalis cells
HmuY is one of the dominant proteins produced under low-iron/heme conditions by P. gingivalis
In contrast, others have shown that P. gingivalis enhanced hmuY mRNA expression in response to low cell density rather than to low iron concentration . The authors found that the expressions of the hmuY and hmuR genes were highest in P. gingivalis grown in the early log phase, when the cell density is low, but expression levels were significantly decreased in the late log phase, when cell density is much higher. They also suggested that the expression of hmuY mRNA in P. gingivalis cells grown in the same cell densities was similar regardless of the presence of heme. These results are different from those demonstrating higher hmuY mRNA expression levels in P. gingivalis cells grown under low-heme conditions and in biofilm, the latter resembling high-cell-density conditions [35–37]. Our results presented in this study corroborate the latter findings and demonstrate that HmuY protein is constitutively produced in the cell at low levels when bacteria are grown under high-iron/heme conditions; however, significantly higher protein levels are found in cells grown under low-iron/heme conditions, maintained in vitro by the addition of an iron chelator or human serum to the heme-free medium (figure 3). These experiments were performed using P. gingivalis cultures grown in the first passage of starvation, thus allowing achieving similar cell densities, especially in the early growth phase (data not shown).
HmuY participates in homotypic biofilm accumulation
To facilitate adaptation to life within the oral cavity, P. gingivalis must be capable of sensing and responding to the prevailing environmental conditions, including nutrient availability, cell density, and the presence of other bacteria. It has been recently shown that P. gingivalis possesses the luxS gene and produces a functional AI-2 autoinducer . In P. gingivalis, among the many different bacterial features that are regulated by quorum sensing using LuxS protein is the expression of genes involved in iron and heme acquisition, including the heme receptor HmuR [41, 42]. Although the authors analyzed hmuR gene expression only, it is highly possible that the expressions of other components of hmu operon, such as hmuY, may also be regulated by LuxS signaling. It has been shown that LuxS is also required in P. gingivalis for the development of biofilm under low-heme conditions , which additionally supports an involvement of HmuY in both heme uptake and biofilm accumulation.
Anti-HmuY antibodies inhibit P. gingivalis growth and biofilm accumulation
As the prevalence of antibiotic-resistant strains of bacteria increases, novel ways of treating infections need to be developed. This is particularly important with respect to periodontal diseases, which are the most common chronic bacterial infections of man. First of all, HmuY may be important for a better understanding of the pathology caused by P. gingivalis. The surface exposure, high abundance, and immunogenicity of P. gingivalis HmuY protein suggest that its detailed examination may yield novel diagnostic methods. Knowledge of the molecular bases of the host immune response against P. gingivalis HmuY may be further essential for developing approaches to control and treat chronic periodontitis. To confirm these hypotheses, studies of anti-HmuY antibodies produced in patients with various forms of periodontal diseases and the influence of HmuY and anti-HmuY antibodies on the experimental periodontitis in a mouse model are now underway.
Amino-acid sequence analyses
HmuY homologues were identified using the Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi) . Prediction of signal peptides was performed with the LipoP 1.0 Server http://www.cbs.dtu.dk/services/LipoP/ . Mature protein sequences were aligned using the CLUSTALW2 program  with the default alignment parameters: GONNET 250 protein weight matrix, gap opening penalty 10.00, gap extension penalty 0.2, penalty for closing a gap-1, and penalty for gap separation 4. The phylogenetic tree was constructed with the neighbor-joining method . Bootstrap analysis was performed using 1000 replicates with the CLUSTALW2 program. The tree was drawn with the NJplot program .
Strains and growth conditions
The P. gingivalis wild-type strains (A7436, W83, and ATCC 33277), the hmuY deletion mutant constructed in the A7436 strain (TO4), and the Bacteroides fragilis strain were grown anaerobically on blood agar plates (ABA; Biocorp), in Schaedler broth (Biocorp) and then cultured in basal medium alone (BM), BM supplemented with 1 mg/ml hemin (BM+Hm), 5% human serum (BM+serum), or 160 μM dipyridyl (BM+DIP) as described previously . To avoid autolysis, the bacteria were grown for a time not exceeding 48 h . E. coli cells were cultured as indicated in previous reports [18, 19].
HmuY expression and purification
P. gingivalis apo-HmuY lacking the first 25 residues (NCBI accession no. CAM 31898) was expressed using pHmuY11 plasmid and E. coli ER2566 cells (New England Biolabs) and purified from a soluble fraction of E. coli lysate as previously described . The protein concentration was determined as previously reported .
Immunization of rabbits
A non-lipidated form of HmuY (the protein lacking the first 25 amino-acid residues comprising the signal peptide sequence, the following cysteine, and four additional amino acids, GKKK) was used to immunize rabbits (Lampire) with Freund's complete adjuvant. Purified HmuY (0.2 mg per injection) was injected subcutaneously. The animals were boosted on days 7,14, 28, 56, and 84 of the immunization schedule and bled on days 1 (pre-immune serum), 42 (test I serum), 70 (test II serum), and 98 (final-bleed immune serum). The IgG fraction was purified from serum using a HiTrap protein A column according to the manufacturer's instructions (Amersham Pharmacia).
Protease accessibility assay
To detect HmuY on the surface of the cell, wild-type (A7436, W83), hmuY-mutant (TO4), and E. coli cells over-expressing membrane-associated HmuY  were washed with 20 mM sodium phosphate buffer, pH 7.6, containing 140 mM NaCl (PBS) and re-suspended in 50 mM Tris/HCl, pH 7.6, containing 140 mM NaCl and 10 mM MgCl2 to an optical density (OD) of 0.1. The cell suspension was incubated with proteinase K (0.25 mg/ml) for 30 min at 37°C. After incubation, protease inhibitor cocktail (Complete; Roche) was added to stop the reaction, the cells were pelleted, suspended in PBS, and finally the samples were boiled in SDS-PAGE sample buffer. Then the proteins were separated by 15% SDS-PAGE and detected by Western blotting as described below.
Preparation of cells and proteins for SDS-PAGE and Western blotting
P. gingivalis cultures were centrifuged for 30 min at 20,000 × g at 4°C and the supernatants were filtered through a 0.22-μm pore-size filter (Roth). Bacterial pellets were washed with PBS and suspended in PBS to OD660 = 0.1. To separate outer-membrane vesicles, the filtered culture medium was centrifuged for 2 h at 100,000 × g. For HmuY expression analysis, samples corresponding to 5 μl of the bacterial culture at OD660 = 0.1 or 20 μl of the culture medium were separated by 15% SDS-PAGE and transferred onto nitrocellulose membranes (Schleicher & Schuell). Nonspecific binding sites were blocked with 5% skim milk in PBS. HmuY was visualized with polyclonal anti-HmuY rabbit serum (Lampire) and secondary goat anti-rabbit IgG antibodies conjugated with horseradish peroxidase (HRP; Sigma), both used at 1:10,000 dilutions. The reaction was developed using chemiluminescence reagents (Western Lightning Plus-ECL; Perkin Elmer). To determine P. gingivalis autolysis, the presence of Fur was examined in both cells and culture medium using Western blotting with rabbit polyclonal antibodies raised against the synthetic peptide derived from the amino-acid sequence of Fur (CILADKDLRPPRFSY; GeneScript).
Enzyme-Linked Immunosorbent Assay (ELISA)
Levels of anti-HmuY antibodies in rabbit sera were determined by ELISA. For this purpose, 96-well polystyrene plates (Polysorp; Nunc) were coated for 1 h at 37°C with 100 μl/well HmuY in PBS. The plates were washed three times with 200 μl of PBS prior to blocking for 1 h at 37°C with 200 μl of 2% bovine serum albumin (BSA) dissolved in PBS and then washed three times with 200 μl of PBS. Two-fold serum dilutions or 1:10,000 serum dilutions (100 μl of pre-immune, test I, test II, and immune serum) were prepared in PBS and incubated for 1 h at 37°C. After washing, antibody binding was detected using goat anti-rabbit IgG conjugated with HRP. After three final washes, a substrate solution (100 μl) containing 0.05% o-phenylenediamine (Sigma) with 0.01% H2O2 was added for color development at room temperature. The reaction was stopped after 15 min by adding 25 μl of 12.5% H2SO4 and the absorbance was measured at 450 nm using a Multiskan Ascent microplate reader (Thermo Electron Corporation).
Whole-cell ELISA, dot-blotting, and FACS analyses
As an additional method of HmuY detection, cell surface staining with anti-HmuY antibodies was performed using whole-cell ELISA, dot-blotting, and flow cytometry (FACS) analyses. P. gingivalis cells grown to OD660 = 1.0 were used for these experiments. For the ELISA and dot-blotting analyses, washed cells at several dilutions were adsorbed on the surface of microtiter plates or nitrocellulose membranes. Nonspecific binding of antibodies was prevented by incubation with 1% bovine serum albumin and 2% bovine fetal serum (Sigma) before the addition of rabbit pre-immune or anti-HmuY immune serum (1:10,000) or purified IgG fractions (100 ng/ml). After 1-h incubation and washing with PBS, goat HRP-conjugated (Sigma) or bovine phycoerythrin-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) at 1:10,000 or 1:500 dilutions were used, respectively. Finally, the cells, wells, and membranes were washed with PBS. For FACS analysis, the cells were fixed with 2% p-formaldehyde. Then absorbance at 450 nm (ELISA), chemiluminescence (dot-blotting analysis), or fluorescence (FACS; Excalibur, Beckton Dickinson) were detected.
Homotypic biofilm formation by P. gingivalis was performed as described by others . Briefly, P. gingivalis cells were grown on ABA plates, then in BM supplemented with hemin or dipyridyl to OD660 = 1.0 and used to inoculate fresh cultures to OD660 = 0.1. Cells in the appropriate medium were transferred (200 μl) into sterile round-bottom microtiter plates (Sarstedt) and incubated under anaerobic conditions at 37°C for 24 or 48 h. The resulting biofilms were washed with PBS, stained with 1% crystal violet, washed with PBS, and de-stained with 96% ethanol. Absorbance (A) was determined at 570 nm using a Multiskan Ascent microplate reader. The assays were repeated at least three times with each strain grown in eight wells. To confirm that the P. gingivalis cells were viable, the biofilm cells were scrapped into the respective medium and the OD at 660 nm and colony-forming unit (CFU) values were evaluated after 24 and 48 h (see Additional file 3). In parallel, bacteria were grown in planktonic form and the OD at 660 nm and CFU values were measured after 24 and 48 h.
Growth and biofilm inhibition studies
Bacteria were grown overnight on ABA plates and then in BM supplemented with hemin or dipyridyl to OD660 = 1.0. After centrifugation, the bacteria were washed and suspended in PBS to OD660 = 0.1. Then 5 ml of the bacterial suspension was centrifuged and the bacteria were incubated in 200 μl of PBS for 1 h at 37°C with the IgG fraction purified from pre-immune or immune anti-HmuY rabbit serum (200 ng). After addition of 5 ml of the appropriate medium, planktonic bacterial growth was monitored by measuring the OD at 660 nm or biofilm formed as described above. Assays were performed three times in duplicate.
Data are expressed as means values ± standard deviations (mean ± SD). Statistical analysis was performed using unpaired Student's t test (GraphPad Prism 5). Values of p < 0.05 were considered statistically significant.
This work was supported in part by grant nos. N401 029 32/0742, N N303 406136, and N N303 518438 from the Ministry of Science and Higher Education, and by Wroclaw Research Center EIT+ under the project "Biotechnologies and advanced medical technologies - BioMed" (POIG 01.01.02-02-003/08/00) financed from the European Regional Development Fund (Operational Program Innovative Economy, 1.1.2) (TO) and the European Social Fund (Human Capital Program, 8.2.2), state, and province government under the project "Grant - support of research through scientific stipends for PhD students" (HW).
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