1
Inactivation of epidermal growth factor by Porphyromonas 1
gingivalis as a potential mechanism for periodontal tissue damage 2
3
Krzysztof Pyrca,b*, Aleksandra Milewskaa, Tomasz Kantykaa, Aneta Srokaa, Katarzyna Maresza, 4
Joanna Kozieła, Ky-Anh Nguyenc,d, Jan J. Enghilde, Anders Dahl Knudsene, Jan Potempaa,f 5 6 7 a Microbiology Department, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian 8
University, Gronostajowa 7, 30-387 Krakow, Poland. 9
b Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7, 30–387 Krakow, 10
Poland 11
c Institute of Dental Research, Westmead Centre for Oral Health and Westmead Millennium Institute, 12
Sydney, Australia; 13
d Department of Oral Biology, Faculty of Dentistry, University of Sydney, Sydney, Australia 14
e Department of Molecular Biology and Genetics, Aarhus University, Incuba Science Park, Gustav 15
Wieds vej 10C, 8000 Aarhus C, Denmark 16
f Oral Health and Systemic Disease Research Group, School of Dentistry, University of Louisville, 17
Louisville, KY, USA 18
19 20
21
22
23
24
25
26
27
28 29 * Corresponding author: Krzysztof Pyrc, Microbiology Department, Faculty of Biochemistry 30
Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland; 31
Phone number: +48 12 664 61 21; Fax: +48 12 664 69 02; www: http://virogenetics.info 32
E-mail: [email protected] 33
34
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00830-12 IAI Accepts, published online ahead of print on 22 October 2012
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ABSTRACT 35
Porphyromonas gingivalis is a Gram-negative bacterium associated with the development of 36
periodontitis. The evolutionary success of this pathogen results directly from the presence of 37
numerous virulence factors, including a peptidylarginine deiminase (PPAD), an enzyme, 38
which converts arginine to citrulline in proteins and peptides. Such posttranslational 39
modification is thought to affect the function of many different signaling molecules. Taking 40
into account the importance of tissue remodeling and repair mechanisms for periodontal 41
homeostasis, which are orchestrated by ligands of the epidermal growth factor receptor 42
(EGFR), we investigated the ability of PPAD to distort cross-talk between the epithelium and 43
the EGF signaling pathway. We found that EGF preincubation with purified recombinant 44
PPAD, or a wild-type strain of P. gingivalis, but not with a PPAD-deficient isogenic-mutant, 45
efficiently hindered the ability of the growth factor to stimulate epidermal cell proliferation 46
and migration. In addition, PPAD abrogated EGFR-EGF interaction-dependent stimulation of 47
expression of Suppressor of Cytokine Signaling 3 (SOCS3) and Interferon Regulatory Factor 48
1 (IRF-1). Biochemical analysis clearly showed that the PPAD-exerted effects on EGF 49
activities were solely due to deimination of the C-terminal arginine. Interestingly, 50
citrullination of two internal Arg residues with human endogenous peptidylarginine 51
deiminases did not alter EFG function, arguing that the C-terminal arginine is essential for 52
EGF biological activity. Cumulatively, these data suggest that PPAD-activity-abrogating 53
EGF function in gingival pockets may at least partially contribute to tissue damage and 54
delayed healing within P. gingivalis-infected periodontia. 55
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INTRODUCTION 56
Tissue remodeling and wound healing are extremely complex processes, which include tightly 57
synchronized cell proliferation, migration, and repopulation (38, 41). These responses are of 58
paramount importance for maintenance of homeostasis in the periodontium permanently 59
exposed to mechanical stress, potentially damaging environmental factors and importantly, 60
colonization with pathogenic bacteria. During periods of active inflammation, tissues are 61
exposed to a wide range of cytokines and growth factors released by resident tissue cells or 62
immune cells to modulate healing processes in a coordinated manner (9, 24, 31, 57, 59). 63
These signaling molecules play a major role in the normal periodontal tissue turnover as well 64
as in periodontal repair and regeneration during chronic inflammatory periodontal disease (36, 65
64). Most prominent among these cell-derived factors are ligands for the epidermal growth 66
factor receptor (EGFR), members of the epidermal growth factor (EGF) family (16, 18, 19, 67
69). 68
EGF is expressed as a pro-form, which is proteolytically processed into a biologically 69
active peptide encompassing 53 amino acid residues. The binding of the processed peptide to 70
the EGF receptor (EGFR) induces receptor homo- or hetero-dimerization and subsequent 71
activation of a complex by auto-phosphorylation catalyzed by a tyrosine kinase domain of the 72
EGFR molecule (7, 67). Both EGF and EGFR are expressed in periodontal tissues (6, 74). In 73
healthy human gingiva, EGFR expression is limited to the gingival epithelium but during 74
periodontal disease, resultant tissue damage and the subsequent regeneration process induces 75
a drastic increase in EGFR expression in the periodontal ligaments (26, 49, 59). This 76
observation correlates with the finding that EGF-dependent signaling is involved in regulation 77
of numerous biological pathways in the periodontium, including regulation of cell 78
proliferation, migration and differentiation (6, 53, 59). Thus, the importance of EGF in 79
periodontal health is highlighted by the fact that a single nucleotide polymorphism within the 80
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EGF gene has been found to be associated with development of severe chronic 81
periodontitis (73). 82
Periodontitis is a microbial biofilm-driven chronic inflammatory condition associated 83
with the presence of specific bacterial pathogens, including Porphyromonas gingivalis (5, 14, 84
22, 23, 70). P. gingivalis is a Gram-negative, non-motile, anaerobic asaccharolytic black-85
pigmented bacterium furnished with a wide range of virulence factors, including fimbriae, 86
hemagglutinins and numerous proteinases which are indispensable for colonization, growth 87
and deterrence of antibacterial activity of the immune system (32). Except for these well-88
characterized pathogenicity traits, P. gingivalis also produces other enzymes recognized as 89
potential virulence factors, which also may be involved in disease development and 90
progression (4, 44). One of these poorly characterized enzymes is the P. gingivalis 91
peptidylarginine deiminase (PPAD), which is able to modify proteins by deimination of the 92
arginine residues, thereby converting them to citrulline (44, 66). Despite the low level of 93
sequence similarity, all catalytic and guanidine-binding residues essential for peptidylarginine 94
deiminase activity of eukaryotic enzymes are conserved in PPAD (40). Contrary to 95
mammalian enzymes, PPAD is able to deiminate both free arginine and peptidylarginine, 96
preferentially targeting an Arg residue at the carboxy-terminus of a peptide/polypeptide 97
chain (44). In contrast to peptidylarginine deiminase activity of PPAD, other known bacterial 98
homologous enzymes can only deiminate free arginine or agmatine residues (arginine 99
derivative) (40). 100
Conversion of positively charged arginine into neutral citrulline may affect biological 101
function of a protein in a number of ways: (i) by impacting on the folding and stability of a 102
polypeptide chain, (ii) by altering susceptibility to proteolysis of the modified protein, or 103
(iii) abrogating the biological activity dependent on an exposed Arg residue(s). Indeed, it has 104
been shown that endogenous PAD citrullination of histones (13, 76), chemokines (37), and 105
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bactericidal peptide LL-37 (30), affected genes expression (34, 76), inflammatory 106
reaction (60, 77), and antimicrobial activity in the lungs (30, 39), respectively. 107
The presence of bacterial products, including P. gingivalis outer membrane vesicles 108
(32) and gingipains (54) in gingival tissues distant to the bacterial plaque strongly argues that 109
PPAD can also penetrate deeply into the connective tissue. Outer membrane-associated 110
enzymes such as PPAD could disseminate into the tissues via outer membrane vesicles or 111
diffusion of the soluble form and modify EGF within the inflamed periodontium. Therefore, 112
we hypothesized that PPAD can inactivate EGF and negatively impacting on the course of 113
periodontal tissue regeneration during the quiescence phase of periodontitis or after tooth 114
debridement. Such contention is supported by well-documented observation of refractory to 115
periodontal treatment disease due to the persistent presence of P. gingivalis (10, 11, 35). 116
The current study shows that EGF can be inactivated by PPAD and it is the first report 117
to describe modulation of a eukaryotic signaling molecule by a bacterial PAD enzyme. 118
Biochemical analysis clearly shows that PPAD citrullinates C-terminal arginine of EGF with 119
subsequent impairment of EGF biological activity. The functional analysis of the EGF activity 120
included evaluation of EGF-induced cell proliferation and migration. Furthermore, an assay 121
showing induction of EGF-dependent SOCS3 (Suppressor of Cytokine Signaling 3) and 122
IRF-1 (Interferon Regulatory Factor 1) genes expression was conducted. Surprisingly, only 123
citrullination of the C-terminal arginine residue results in impairment of EGF function, as 124
citrullination of internal residues with human PAD2 and PAD4 enzymes does not abrogate the 125
peptide function. Thus, decreased activity of EGF in gingival pockets may at least partially 126
contribute to the observed tissue damage and delayed healing within human periodontium 127
during P. gingivalis infection. 128
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MATERIALS AND METHODS 129
CELL CULTURE 130
Human primary fibroblasts (46) were maintained in D10 media (Dulbecco-modified Eagle’s 131
medium (DMEM; PAA Laboratories, Germany) supplemented with 10% heat-inactivated 132
fetal bovine serum (PAA Laboratories, Germany), penicillin (100 U mL-1), and streptomycin 133
(100 μg mL-1). Cells were cultured on T25 flasks (TPP, Switzerland) at 37ºC with 5% CO2. 134
Cultures were routinely tested for presence of Mycoplasma spp. and proved negative. 135
136
BACTERIAL CULTURE 137
P. gingivalis strain W83 (51) and W83 Δppad (78) were anaerobically grown in Schaedler 138
broth supplemented with L-cysteine (0.05 g/mL), 1% DTT, menadione (0.5 mg mL-1) and 139
hemin (1 mg mL-1). P. gingivalis strain ATCC33277 and ATCC33277 Δppad (78) were 140
anaerobically grown in BHI medium supplemented with hemin (5 μg mL-1) and vitamin K 141
(0.5 μg mL-1). All cultures were carried on in an anaerobic chamber MACS500 (Don Whitley 142
Scientific Limited, Frederick, MD, USA) in an atmosphere of 80% N2, 10% CO2, 10% H2. 143
Bacteria from stocks stored at -80°C in storage media (BHI supplemented with glycerol) were 144
plated on blood agar and a single colony was used to inoculate broth. The culture was grown 145
anaerobically at 37 °C until OD600 of 1.0 was reached. 146
147
EXPRESSION AND PURIFICATION OF P. GINGIVALIS PAD 148
Expression plasmid pET48b containing gene encoding P. gingivalis PPAD with a 6 × His tag 149
was a kind gift from Natalia Wegner (Kennedy Institute of Rheumatology, London, UK). 150
Briefly, PPAD was expressed in E. coli BL21(DE3)pLysS (Life technologies, Poland) with 151
3 h induction time in presence of 1 mM IPTG at 37 °C. Protein was purified with Ni-152
Sepharose 6 Fast Flow source (GE Healthcare Life Sciences, Germany) and Superdex 75 153
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(GE Healthcare Life Sciences, Germany). Protein purity was evaluated with SDS-PAGE 154
electrophoresis and N-terminal amino acid sequence analysis. PPAD activity was assessed 155
using Nα-Benzyloarginine ethyl ester assay (BAEE) as a substrate and activity was expressed 156
in U/mL (1 U = 1 μmol of citrulline produced within the 1 h of the reaction) (29). 157
158
INFLUENCE OF P. GINGIVALIS ON EGF ACTIVITY 159
Cell proliferation assay was used to investigate P. gingivalis (strains W83 WT, W83Δppad, 160
ATCC33277 WT and ATCC33277Δppad) effect on EFG biological activity. Briefly, 400 ng 161
of EGF was added to P. gingivalis cell culture adjusted to OD600 = 1.0 (total volume of 30 μL) 162
and incubated for 3 h at 37 °C in anaerobic conditions with shaking. Subsequently, bacteria 163
were removed by centrifugation (2,300 × g, 10 min) and the supernatant was used for further 164
analyses. 165
166
INFLUENCE OF PURIFIED HUMAN PAD2 AND PAD4 AND P. GINGIVALIS PAD ON EGF 167
ACTIVITY 168
In order to test, whether incubation with purified PPAD affects EGF activity, 1 μg of EGF 169
was incubated with 0.0126 U of PPAD, PAD2 or PAD4 (Modiquest research) in dilution 170
buffer (100 mM TRIS; 10 mM CaCl2; 2 mM L-cysteine; pH 7.5) or with negative control 171
samples (dilution buffer or 1 × PBS) for 3 h at 37 °C. Following the incubation samples were 172
diluted with D0 medium (Dulbecco-modified Eagle’s medium (DMEM; PAA Laboratories, 173
Germany) supplemented with penicillin (100 U mL-1), and streptomycin (100 μg mL-1) to 174
reach the desired EGF concentration. Samples were used directly following the incubation. 175
176
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CELL PROLIFERATION ASSAY 177
Fibroblasts cultured as described above were seeded on 96-well plates (40 000 cells per well) 178
in D10 medium. Cells were incubated for 24 h at 37 °C with 5% CO2. Subsequently, cells 179
were washed with 1 × PBS and overlaid with 100 μL of fresh DMEM deprived of serum, 180
supplemented with native EGF, bacteria, PAD2, PAD4 or PPAD-treated EGF or dilution 181
buffer. Cells were subsequently cultured for 72 h as described above and media was removed. 182
Each well was washed with 1 × PBS and cells were detached with 1 × trypsin (0.5 mg/ml) 183
supplemented with EDTA (0.22mg ml) (PAA Laboratories, Germany). Cell suspension was 184
mixed with trypan blue (1:1 v/v) and cells were counted in Fuchs-Rosenthal hemocytometer. 185
In this way, absolute number of cells was directly and precisely counted rather than using 186
more indirect methods such as qPCR and proteomics, which determines cell proliferation as a 187
derivative of nucleic acid or protein concentration. 188
189
CELL MIGRATION ASSAY 190
Human fibroblasts cultured as described above were seeded on 12-well plates 191
(500 000 cells/well) coated with collagen (Purecol; Nutacon, The Netherlands) in D10 192
medium. Cells were incubated for 24 h at 37 °C with 5% CO2, reaching 100% confluence. 193
Subsequently, medium was removed, cells were washed with 1 × PBS and native EGF, 194
bacteria and PPAD-treated EGF or control samples (dilution buffer) diluted in fresh D0 195
medium, were added to each well. Medium was supplemented with mitomycin C 196
(10 μg mL-1) to eliminate cell proliferation, which may mask presentation of cell migration. 197
To analyze cell migration, a single linear scratch wound was made centrally across each cell 198
monolayer using a pipette tip (71). Cell migration in the presence of untreated and treated 199
EGF, and control samples was visualized at 24 h post-inoculation using phase-contrast 200
microscopy (Nikon Eclipse Ti). 201
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NUCLEIC ACID EXTRACTION, REVERSE TRANSCRIPTION AND REAL-TIME PCR REACTIONS 202
Total cellular RNA was extracted using TRIreagent (Life technologies, Poland), according to 203
manufacturer’s protocol. Isolated RNA was reverse transcribed with High Capacity cDNA 204
Reverse Transcription kit (Life technologies, Poland), according to manufacturer’s 205
instructions, using 5 μl of the previously extracted RNA. 206
PCR amplification was carried out in a total volume of 10 μL with DreamTaq PCR 207
Master Mix (Fermentas), in the presence of forward and reverse primers (for amplification of 208
β-actin: Bact5: 5’- CCA CAC TGT GCC CAT CTA CG -3’, Bact3: 5’-AGG ATC TTC ATG 209
AGG TAG TCA GTC AG -3’, 500 nM each; for amplification of SOCS3: 5_SOCS3: 5’- 210
AGA GCC TAT TAC ATC TAC TCC GGG -3', 3_SOCS3 5’- TTC CGA CAG AGA TGC 211
TGA AGA GTG -3’; for amplification of IRF-1: 5_IRF1 5’- AGA GCA AGG CCA AGA 212
GGA AGT CAT -3’, 3_IRF1 5’- ACT GTG TAG CTG CTG TGG TCA TCA -3’, 500 nM 213
each) and template DNA (1 μL). The PCR cycling conditions included initial denaturation for 214
3 minutes at 95 °C followed by 27 cycles of 20 sec at 95 °C, 30 sec at 56 °C, 40 sec at 72 °C 215
then followed and 5 min at 72 °C for the final elongation. 216
Semi quantitative real-time PCR amplification was conducted with 2 × SYBRGreen 217
mix (Sigma Aldrich, Poland). Reaction was carried out in a total volume of 10 μL in presence 218
of forward and reverse primers (Bact5, Bact3 or 5_SOCS3, 3_SOCS3, 5_IRF1, 3_IRF1) and 219
template DNA (1 μL) with Rox as a reference dye. The PCR cycling conditions included 220
initial denaturation for 5 minutes at 95 °C followed by 40 cycles of 30 sec at 95 °C, 30 sec at 221
56 °C, 45 sec at 72 °C. Real-time amplification was followed by melting curve assessment to 222
confirm product identity. 223
224
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HPLC ANALYSIS 225
Ten μg of EGF was diluted in 100 μL of 0.1 M Tris-HCl, pH 7.5, 10 mM CaCl2, freshly 226
supplemented with L-Cys to 2 mM concentration. Next, to maintain the molar ratio of EGF to 227
the enzyme at similar levels during sample preparation for activity assay, 63 mU of PPAD 228
was added to the EGF sample and incubated for 3 h at 37 °C, in final volume of 200 μL. 229
Reaction was stopped by addition of trifluoroacetic acid to 0.5% final concentration. Sample 230
was resolved using high-performance liquid chromatography system AKTA Micro (GE 231
Healthcare, Germany) and µRPC C2-C18 4.6/100 ST reverse-phase column in 10-80% 232
acetonitrile gradient using mobile phases A: 0.1% TFA in water and B: 80% acetonitrile with 233
0.08% TFA in water. Elution was monitored with spectrophotometer (λ = 215 nm) and results 234
were recorded. Eluting fractions were collected on the 96-well microplate using AKTA 235
system Frac-950 fraction collector (GE Healthcare, Germany) and fractions from each single 236
HPLC peak were pooled together and vacuum-dried using SpeedVac system (Eppendorf, 237
Poland). 238
239
LC-TANDEM MASS SPECTROMETRY 240
RP HPLC fractions dried under vacuum using SpeedVac system (Eppendorf, Poland) were 241
resuspended in 0.1M NH4HCO3. Samples were reduced and alkylated in solution using 242
10 mM DTT at 37 °C for 30 min followed by incubation with 55 mM iodoacetamide for 243
30 min at RT in the dark. Samples were subsequently digested overnight at 37 °C with 244
0.02 mass equivalents of Sequencing Grade Modified Trypsin (Promega, Germany). The 245
digested samples were acidified using 100% formic acid and purified using 246
C18 RP STAGEtips (Proxeon, Odense, Denmark). The eluate was vacuum-concentrated and 247
resuspended in 0.1% FA (MS eluent A). Samples were subsequently analyzed with 248
Tandem-MS.All experiments were performed on a AB-Sciex TripleTOF 5600 mass 249
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spectrometer with a Nanospray® III ionsource (ABSciex). The high performance liquid 250
chromatrography setup used in conjunction with the mass spectrometer consisted of a 251
Proxeon Easy-nLC™ HPLC system operated using intelligent flow control. A 10cm fused 252
silica column (75μm I.D., 360μm O.D.) packed with from 3μm C18 reverse phase material 253
(Thermo Scientific). Mobile phases consisted of 0.1% Formic acid in water (A) (Sigma-254
Aldrich) and 90% acetonitrile in 0.1% Formic acid in water (B). 12μl tryptic peptide sample 255
was automatically loaded onto the column and rinsed for 5min at a flow rate of 250nl/min 256
followed by a 30-40min gradient from 5% B to 35% B at a constant flow rate of 250nL/min. 257
MS analysis was performed with 35-50 scans per 2.8-3.8 sec cycle with a MS accumulation 258
time of 250ms, MS/MS accumulation times of 100ms and a peptide fragmentation threshold 259
of 150 arbitrary intensity units. Every fifth sample, a calibration was performed automatically, 260
ensuring mass accuracies below 15ppm. The generated data was manually reviewed by 261
checking for the presence of precursor ions with predicted m/z-values matching putative 262
citrullinated EGF peptides. Subsequently, the associated MS/MS spectra were reviewed for 263
fragment ions proving or disproving presence of citrulline. The resulting data was submitted 264
to Mascot search engine (v. 2.3.02, Matrix Science, London, UK) against the Swissprot 265
database (Human database, October 2011). The search was performed with trypsin specificity 266
(1 allowed missed cleavage), carbamidomethyl as fixed modification, and oxidation of 267
methionine as variable modification. The peptide mass tolerance was set to 20 ppm and the 268
fragment mass tolerance was set to 0.05 Da. The instrument setting was ESI-QUAD-TOF 269
which permits b-, y- b-NH4 and y-H2O fragment ion types. The score threshold for peptides 270
was suggested by Mascot at around 20-40 (p<0.05). 271
272
273
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STATISTICAL ANALYSIS 275
All experiments were repeated at least three times and results are expressed as mean ± SD. To 276
determine significance of obtained results, comparison between groups was made using 277
Student’s t test. P values < 0.05 were considered significant. 278
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RESULTS 279
P. GINGIVALIS PEPTIDYLARGININE DEIMINASE (PPAD) IS RESPONSIBLE FOR ABROGATION 280
OF EPIDERMAL GROWTH FACTOR (EGF) ABILITY TO STIMULATE CELL PROLIFERATION 281
During periodontitis, bacteria growing below the gum line actively modulate inflammatory 282
and healing processes in periodontal and gingival tissues orchestrated by cytokines and 283
growth factors. Among the latter, EGF is a potent modulator of cell cycle progression (27) 284
and its inactivation by P. gingivalis may have a negative impact on regeneration of tissue 285
damage. To investigate P. gingivalis effect on EGF activity, primary human fibroblasts were 286
cultured in the presence of EGF, EGF pre-incubated with two P. gingivalis strains (W83 or 287
ATCC33277 differing genetically and phenotypically (50)) or control samples and 288
subsequently, fibroblast cell numbers were counted. As depicted in Figure 1A, incubation of 289
EGF in the presence of wild-type bacteria (both W83 and ATCC33277 strains), invariably led 290
to abolishment of its biological activity to stimulate cell proliferation. 291
Gingipains are among the most important virulence factors produced by P. gingivalis; 292
these proteolytic enzymes target numerous host proteins and are essential for bacterial 293
pathogenicity in vivo (21, 28, 55). Surprisingly, incubation of EGF with gingipain-null mutant 294
(P. gingivalis W83 ∆k/∆rab) also obliterated EGF’s ability to stimulate proliferation of 295
fibroblasts (Figure 1B). This finding showed that gingipains have no effect on EGF activity 296
and indicated that P. gingivalis produces another factor responsible for inactivation of EGF. 297
The presence of the Arg residue at the C-terminus of EGF, the preferential target for 298
PPAD, suggested that conversion of this Arg residue into citrulline by PPAD may inactivate 299
EGF activity. To experimentally verify this assumption, EGF was preincubated with 300
recombinant PPAD and the growth factor activity was assessed in the cell proliferation assay. 301
As shown in Figure 1D such treatment inhibited EGF ability to stimulate proliferation of 302
fibroblasts confirming our contention that PPAD is at least partially responsible for EGF 303
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inactivation by whole P. gingivalis cells. On the other hand, although incubation of EGF with 304
human PAD2 and PAD4 resulted in citrullination of internal Arg residues within the peptide 305
(data not shown), no decrease in EGF activity was observed (Figure 1D). The unique ability 306
of PPAD to negate EGF function was further studied using isogenic PPAD-null mutants in 307
two different genetic backgrounds of P. gingivalis. In contrast to parental strains W83 and 308
ATCC 33277 (Figure 1A), the PPAD-null mutants had no effect on EGF activity (Figure 1C) 309
demonstrating that regardless of the P. gingivalis genetic background PPAD is a sole factor 310
responsible for EGF inactivation. 311
312
PPAD INHIBITS EGF ABILITY TO STIMULATE CELL MIGRATION 313
Apart from its cell proliferation stimulating activity during tissue regeneration, EGF also 314
enhances cell migration (41, 61, 65, 68). To test the effect of PPAD on this well-known EGF 315
activity, primary human fibroblasts were incubated with native EGF, EGF preincubated with 316
P. gingivalis or purified recombinant PPAD, and pertinent control samples in the presence of 317
a cytostatic agent (mitomycin C). Cell migration was analyzed by light microscopy. Obtained 318
results clearly show that pre-incubation of EGF with wild-type bacteria and PPAD, but not 319
with PPAD-null strains, abolished EGF ability to stimulate cell migration (Figure 2). Again, 320
these data confirms that PPAD is the only P. gingivalis-produced factor affecting the 321
biological activity of EGF. 322
323
PPAD ABROGATES EGF-INDUCED STIMULATION OF EXPRESSION OF SOCS3 AND IRF-1 324
Interaction of EGF with EGFR triggers a cascade of events resulting in expression of 325
numerous genes responsible for EGF mediated effect. Among these genes are SOCS3 and 326
IRF-1, a STAT-regulated cytokine-inducible negative regulator of cytokine signaling and an 327
activator of interferons alpha and beta transcription, respectively (2). To see if PPAD can also 328
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abrogate this direct biological effect of EGF ligation to its receptor, cells were incubated for 329
1 h with native and PPAD-pretreated EGF and the level of SOCS3 and IRF-1 mRNA 330
determined with quantitative RT-PCR. As expected, native EGF increased expression of the 331
two genes up to 7-fold over the background level and this effect was significantly reduced by 332
the growth factor pretreatment with PPAD (Figure 3). 333
334
INCUBATION OF EGF WITH PPAD RESULTS IN CITRULLINATION OF ARGININE. 335
Considering the PPAD preference for C-terminal Arg residues (44), it is logical to assume that 336
EGF C-terminal arginine will be modified (Arg53). To verify this hypothesis, native and 337
PPAD-treated EGF were resolved by reverse-phase HPLC. Incubation of EGF with PPAD 338
resulted in the increased retention time of the modified EGF in concordance with increased 339
hydrophobicity of protein due to conversion of arginine to citruline (Figure 4). To confirm 340
that, indeed, the modification was due to PPAD-catalyzed deimination of the C-terminal 341
arginine, tryptic digests of the purified, deiminated form of EGF was subjected to tandem 342
LC-MS analysis. The identity of the detected peptides was confirmed by Mascot search. Two 343
peptides with the mass shift corresponding to the citrullination of arginine (+1 Da) were 344
clearly recognized as DLKWWL-Cit and CQYRDLKWWL-Cit peptides derived from the 345
C-terminus of the EGF molecule. To further confirm the presence of the citrulline, the CID 346
fragmentation ions for these two peptides were manually inspected and shown to contain 347
citrulline as the C-terminal residue. The identification of the EGF-derived peptides bearing 348
Cit53 in PPAD-incubated samples, when compared with respective controls, clearly indicates 349
that PPAD-catalyzed deimination of the C-terminal Arg53 is a sole modification of the 350
molecule (Figure 5). 351
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DISCUSSION 353
Periodontitis is the most prevalent infectious inflammatory disease of humankind. It is 354
estimated that up to 30% of the adult population suffers from periodontitis and approximately 355
8% will result in tooth loss (1, 8). Furthermore, a causative link between periodontal disease 356
and numerous other conditions has been recognized, including rheumatoid arthritis, 357
cardiovascular disease and aspiration pneumonia (15, 17, 75). 358
The physiological role of PPAD in periodontal disease development and progression 359
remains unclear. Numerous hypotheses have been raised, including production of ammonia 360
during deimination process enhances the survival of P. gingivalis within the periodontal 361
pocket, as reported previously for arginine deiminases and agmatine deiminases (44). 362
Ammonia neutralizes the acidic environment and optimizes pH-dependent function of 363
gingipain and PPAD, inactivates hemagglutinins, promotes ATP production, and has negative 364
effects on neutrophil function (44, 52). Furthermore, it can be speculated that PPAD acts as a 365
virulence factor by generating citrullinated peptides, which may assist the bacterium in 366
spreading and circumventing the humoral immune response (44). 367
It has been previously shown that citrullination mediated by eukaryotic PAD enzymes 368
results in modification or abrogation of protein or peptide function, influencing immune 369
responses and tissue remodeling. For example, eukaryotic PAD-mediated citrullination of 370
various signaling molecules, including CXCL-5, CXCL-8, CXCL-10, CXCL-11, CXCL-12 371
and ING4 results in modulation of their activity and most likely is important for their 372
biological functions in vivo (20, 37, 48, 60, 72). Surprisingly, no literature is available 373
regarding bacterial PPAD modification of specific host functional protein(s). Here, we present 374
one of the potential effects of this enzyme on the host homeostasis. 375
EGF together with its receptor (EGFR) functions in a wide range of cellular processes 376
including cell fate determination, proliferation, migration and apoptosis. In gingival 377
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epithelium, enhanced cell proliferation and migration triggered by EGF is associated with 378
turnover, repair and regeneration of periodontal tissues (6, 53, 59). The ubiquitously 379
expressed EGFR is a pleiotropic signal transducer and its EGF-dependent activation triggers 380
major signaling cascades, for instance, the Ras-mitogen-activated protein kinase or the MAP 381
kinase pathway. Activation of these cascades recruits the SOS guanine nucleotide exchange 382
factor to the plasma membrane. Subsequent exchange of the GTP for GDP on the small 383
protein Ras leads to cell proliferation. Another fundamental process in tissue regeneration – 384
enhanced cell motility – is regulated by EGFR-dependent phosphorylation of phospholipase 385
Cγ (PLCγ). Phosphorylated PLCγ catalyzes the formation of two important signaling 386
molecules: inositol triphosphate (IP3) and diacylglycerol (DAG). These transmitters stimulate 387
the release of calcium ions from the smooth endoplasmic reticulum and activate of protein 388
kinase C (PKC), respectively, thus further contributing to the pleiotropic biological effect of 389
EGF (33, 58). 390
EGF is known to be present in saliva, gingival tissues and gingival crevicular fluid, 391
contributing to the maintenance of tissue homeostasis. Previous studies on the levels of EGF 392
in GCF are contradictory. While some reported no marked modulation of EGF levels (47), 393
others observed significant differences in EGF concentrations in GCF from deep and shallow 394
sites in patients with periodontal disease (6). However, studies have shown that during 395
inflammation associated with the development of periodontitis, expression of EGFR is 396
significantly increased, suggesting enhanced sensitivity of gingival tissues to EGF signaling 397
(6). Further, EGF was shown to stimulate proliferation of human periodontal ligament 398
fibroblasts and human gingival fibroblasts (42, 43), apparently through mechanisms as 399
described above. 400
It has been previously shown that P. gingivalis lipopolysaccharide (LPS) modulates the 401
regenerative effect of EGF via down-regulation of EGFR-dependent signaling (63). It was 402
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suggested that this phenomenon is directly related to the fact that both epidermal growth 403
factor and lipopolysaccharide activate the mitogen-activated protein kinases to modulate cell 404
proliferation, cell survival and the release of inflammatory mediators. The observed 405
alterations in EGF signaling caused by P. gingivalis LPS may be mediated by an array of 406
events, including, among other possibilities, the differential recruitment and altered kinetics of 407
activation of upstream mediators in response to LPS and EGF (63). The effect observed in the 408
current study cannot be associated with LPS activity, as no decrease in EGF activity following 409
incubation with LPS-positive Δppad ATCC33277 or Δppad W83 P. gingivalis mutants was 410
observed. One may therefore assume that the effect observed in this study is complementary 411
to previously observed phenomena and together, may lead to severe tissue damage and 412
remodeling. 413
It is of surprise that apparently gingipains, which are considered important virulence 414
factors of P. gingivalis (12, 62), did not show any prominent effect on EGF in vivo, as 415
incubation with Δppad P. gingivalis equipped with a whole set of gingipains did not lead to 416
decrease in EGF activity and incubation with the ∆k/∆rab P. gingivalis W83 gingipain-null 417
mutant led to complete abolishment of EGF activity. Furthermore, incubation with 418
P. gingivalis Δppad strains expressing the whole set of gingipains did not result in a 419
significant degradation of EGF. In contrast, HPLC analysis of PPAD-treated EGF revealed a 420
significant shift of the retention time, suggesting altered peptide charge and increased 421
hydrophobicity. To further confirm the observation, subsequent analysis with mass 422
spectrometry was performed. The sole detected modification of the peptide was citrullination 423
of the C-terminal Arg53. Lack of detectable citrullination of internal arginines is consistent 424
with previous observations that PPAD is specific mostly toward the C-terminal residues and 425
its capability to modify other amino acids is questionable and most likely minimal (44). 426
Furthermore, citrullination of internal EGF Arg residues (Arg41 and Arg45) with human PADs 427
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did not result in decreased EGF activity, which provides further evidence that modification of 428
C-terminal Arg is sufficient to hamper EGF activity. As mentioned above, such an 429
observation is rather surprising, as it was previously reported that excision of the EGF C-430
terminal Arg53 residue is not associated with loss of function (45). Nonetheless, one may 431
assume that modification of the molecule charge may affect the ability of the peptide to 432
interact with its receptor. Comparison of the EGF structure in complex with its receptor as 433
determined by X-ray diffraction (56) or with NMR (25) reveals the unfolded organization of 434
the C-terminal α-helix allowing the essential Leu47 residue to be exposed and available for 435
interaction with site 3 of the EGFR. This is in agreement with the “hand-glove” model of 436
receptor ligand interaction. Based on the described mechanisms, it is hypothesized that 437
citrullination of C-terminal arginine introduces additional hydrophobic interactions, which 438
stabilize the C-terminal helix and prevent structural changes required for the receptor binding. 439
Such a process would likely result in inactivation of the molecule. The proposed mechanism, 440
termed “subtraction by addition” explains both the observed redundancy of C-terminal Arg53 441
for receptor binding (45) and deamination-mediated inactivation of EGF. Although intriguing, 442
as it would provide the structure-function link for EGF inactivation, the proposed mechanism 443
requires structural verification, which is beyond the scope of the current report. 444
The current study is the first to show the direct effect of PPAD on an eukaryotic 445
signaling molecule, showing that this bacterial enzyme is not only active as a source of 446
ammonia, but may also modulate the local microenvironment. EGF is one of the essential 447
factors in wound healing and tissue regeneration and its inactivation may impair regeneration 448
and/or healing of the periodontal tissues. The PPAD-induced disruption of cross-talk between 449
epithelium and the EGF signaling pathway may have pronounced consequences for disease 450
progression (3). Nonetheless, further clinical studies are required to determine the validity of 451
such a hypothesis and its consequence in the treatment of periodontitis. 452
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ACKNOWLEDGMENTS 453
This work was supported by the grant from the Foundation for Polish Science (TEAM project 454
DPS/424-329/10) (JP), the National Institutes of Health, USA (Grants DE 09761 and 455
DE022597 to JP), National Science Centre, Poland (UMO-2011/01/D/NZ6/00269 and 456
2011/01/B/NZ6/00268 to KP and JP, respectively), Ministry of Science and Higher 457
Education, Poland (Iuventus Plus grants IP 2010 033870, IP2011 044371 and IP 2011 458
022171, to KP and TK) and the European Community (FP7-HEALTH-2010-261460 459
“Gums&Joints” and PIRG03-GA-2008-230850 “PerioPain” to JP, JJE, and KM, 460
respectively). The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian 461
University is a beneficiary of the structural funds from the European Union (grant No: 462
POIG.02.01.00-12-064/08 – “Molecular biotechnology for health”). The funders had no role 463
in study design, data collection and analysis, decision to publish, or preparation of the 464
manuscript. 465
466
The authors declare that they have no competing interests. 467
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77. Wegner, N., K. Lundberg, A. Kinloch, B. Fisher, V. Malmstrom, M. Feldmann, 718 and P. J. Venables. 2010. Autoimmunity to specific citrullinated proteins gives the 719 first clues to the etiology of rheumatoid arthritis. Immunol Rev 233:34-54. 720
78. Wegner, N., R. Wait, A. Sroka, S. Eick, K. A. Nguyen, K. Lundberg, A. Kinloch, 721 S. Culshaw, J. Potempa, and P. J. Venables. 2010. Peptidylarginine deiminase from 722 Porphyromonas gingivalis citrullinates human fibrinogen and alpha-enolase: 723 implications for autoimmunity in rheumatoid arthritis. Arthritis Rheum 62:2662-2672. 724
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FIGURES 728
Figure 1. P. gingivalis interferes with EGF-mediated signaling. A. Incubation of EGF with 729
P. gingivalis (strains W83 and ATCC 33277) results in complete inactivation of EGF 730
signaling pathway. B. Incubation of EGF with gingipain-deficient mutant (∆k/∆rab) results in 731
significant decrease in EGF-mediated fibroblast proliferation; C. Incubation of EGF with 732
PPAD-deficient mutants of P. gingivalis (strains W83 and ATCC 33277), in contrast to wild-733
type bacteria, does not result in decreased EGF-mediated fibroblast proliferation. D. Purified 734
PPAD, but not human PAD2 or PAD4, hampers EGF-mediated proliferation of fibroblasts. 735
All results are presented as percent of non-stimulated sample (NC). Significance of observed 736
differences between samples and positive control samples was analyzed with Student’s t-test: 737
n.s. not significant; *** p<0.001; ** p<0.01, * p<0.05. All experiments were repeated three 738
times and results are expressed as mean ± SD. 739
Figure 2. P. gingivalis hampers EGF-mediated cell migration. A. Incubation of EGF with 740
WT P. gingivalis results in complete abolishment of EGF effect on migration of human 741
fibroblasts, whereas P. gingivalis Δppad has no effect on EGF activity. a) Untreated control, 742
b) Control with P. gingivalis W83, c) Control with P. gingivalis Δppad, d) EGF-treated 743
control, e) EGF-treated cells with P. gingivalis W83, f) EGF-treated cells with P. gingivalis 744
Δppad; B. Incubation of EGF with purified PPAD results in complete abolishment of EGF 745
effect on migration of human fibroblasts. a) Untreated control, b) Control with sample buffer, 746
c) Control with PPAD, d) EGF-treated cells, e) EGF-treated cells with sample buffer, f) EGF-747
treated cells with PPAD. Black arrows show the apparent size of linear scratch wound made 748
centrally across each cell monolayer using a pipette tip. All experiments were repeated three 749
times and representative images are presented. 750
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Figure 3. Modulation of SOCS 3 (A) or IRF-1 (B) in presence of EGF and citrullinated 752
EGF. All results are presented as relative quantity to β-actin as a reference gene. Significance 753
of observed differences between test samples and EGF-treated samples were analyzed with 754
Student’s t-test: n.s. not significant; *** p<0.0005. All experiments were repeated three times 755
and results are expressed as mean ± SD. 756
Figure 4. Citrullination of EGF by P. gingivalis PPAD. 10 μg of EGF (black line), EGF in 757
the reaction buffer (faint gray line) and EGF with PPAD (dark gray line) were resolved using 758
reverse phase HPLC. Eluted fractions were collected and after trypsinisation analyzed by 759
tandem LC-MS mass spectrometer (see Figure 5). 760
Figure 5. PPAD modifies C-terminal Arg 53 in mature EGF (A) Tandem MS CID 761
fragmentation spectra of the precursor ion 573.8 detected for EGF treated with PPAD. 762
Spectrum matches the peptide DLKWWEL[Cit]. The y1 ion mass of 176.1 matches the 763
expected mass of a c-terminal citrulline residue. The series of b- andy-ions are listed above 764
the spectra and the additional evidence ions are listed where appropriate; *denotes ions 765
generated by loss of ammonia, 0 loss of water and ++ denotes double charged ions. (B) 766
Modification sites, summarized in the table were detected using collision induced dissociation 767
(CID) mass spectrometry. Sites were confirmed by review of CID fragmentation spectra 768
verifying if peak patterns are best explained by citrullination. The listed y-ions (C-terminal 769
fragments) and b-ions (N-terminal fragments) show the observed evidence ions/peaks 770
confirming citrullination. Nine m/z variants of the four peptides were reviewed for all 771
samples, but only peptides found to be potentially citrullinated are shown. All identified 772
peptides had mass deviations below 12ppm and the average deviation was 3.7ppm. Peptide 773
matches indicate the number of EGF peptide spectra recorded for the sample and sequence 774
coverage indicate the percentage of the EGF sequence covered by detected peptides 775
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