Inactivation of epiderm al growth factor by Porphyromonas...

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

on May 23, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

2

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

3

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

4

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

5

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

6

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

7

(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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

8

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

9

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

10

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

11

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

274


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

12

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

13

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

14

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

15

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

352


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

16

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

17

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

18

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

19

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

20

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

21

Reference List 468 469

1. Albandar, J. M., and T. E. Rams. 2002. Global epidemiology of periodontal 470 diseases: an overview. Periodontol 2000 29:7-10. 471

2. Andersen, P., M. W. Pedersen, A. Woetmann, M. Villingshoj, M. T. Stockhausen, 472 N. Odum, and H. S. Poulsen. 2008. EGFR induces expression of IRF-1 via STAT1 473 and STAT3 activation leading to growth arrest of human cancer cells. Int J Cancer 474 122:342-349. 475

3. Anitua, E., M. Troya, and G. Orive. 2011. Plasma Rich in Growth Factors Promotes 476 Gingival Tissue Regeneration by Stimulating Fibroblast Proliferation and Migration 477 and by Blocking TGF-beta1-Induced Myodifferentiation. J Periodontol. 478

4. Aruni, W., E. Vanterpool, D. Osbourne, F. Roy, A. Muthiah, Y. Dou, and H. M. 479 Fletcher. 2011. Sialidase and sialoglycoproteases can modulate virulence in 480 Porphyromonas gingivalis. Infect. Immun. 79:2779-2791. 481

5. Ashimoto, A., C. Chen, I. Bakker, and J. Slots. 1996. Polymerase chain reaction 482 detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and 483 advanced periodontitis lesions. Oral microbiology and immunology 11:266-273. 484

6. Chang, K. M., N. Lehrhaupt, L. M. Lin, J. Feng, C. Y. Wu-Wang, and S. L. 485 Wang. 1996. Epidermal growth factor in gingival crevicular fluid and its binding 486 capacity in inflamed and non-inflamed human gingiva. Archives of oral biology 487 41:719-724. 488

7. Citri, A., and Y. Yarden. 2006. EGF-ERBB signalling: towards the systems level. 489 Nature reviews. Molecular cell biology 7:505-516. 490

8. Cobb, C. M., K. B. Williams, and M. M. Gerkovitch. 2009. Is the prevalence of 491 periodontitis in the USA in decline? Periodontol 2000 50:13-24. 492

9. Cochran, D. L., and J. M. Wozney. 1999. Biological mediators for periodontal 493 regeneration. Periodontol 2000 19:40-58. 494

10. Colombo, A. P., S. Bennet, S. L. Cotton, J. M. Goodson, R. Kent, A. D. Haffajee, 495 S. S. Socransky, H. Hasturk, T. E. Van Dyke, F. E. Dewhirst, and B. J. Paster. 496 2012. Impact of Periodontal Therapy on the Subgingival Microbiota of Severe 497 Periodontitis: Comparison between Good Responders and "Refractory" Subjects by 498 the Human Oral Microbe Identification Microarray (HOMIM). J Periodontol. 499

11. Colombo, A. P., S. K. Boches, S. L. Cotton, J. M. Goodson, R. Kent, A. D. 500 Haffajee, S. S. Socransky, H. Hasturk, T. E. Van Dyke, F. Dewhirst, and B. J. 501 Paster. 2009. Comparisons of subgingival microbial profiles of refractory 502 periodontitis, severe periodontitis, and periodontal health using the human oral 503 microbe identification microarray. J Periodontol 80:1421-1432. 504

12. Curtis, M. A., H. K. Kuramitsu, M. Lantz, F. L. Macrina, K. Nakayama, J. 505 Potempa, E. C. Reynolds, and J. Aduse-Opoku. 1999. Molecular genetics and 506 nomenclature of proteases of Porphyromonas gingivalis. Journal of periodontal 507 research 34:464-472. 508

13. Cuthbert, G. L., S. Daujat, A. W. Snowden, H. Erdjument-Bromage, T. 509 Hagiwara, M. Yamada, R. Schneider, P. D. Gregory, P. Tempst, A. J. Bannister, 510 and T. Kouzarides. 2004. Histone deimination antagonizes arginine methylation. Cell 511 118:545-553. 512

14. Darveau, R. P. 2009. The oral microbial consortium's interaction with the periodontal 513 innate defense system. DNA and cell biology 28:389-395. 514

15. de Pablo, P., I. L. C. Chapple, C. D. Buckley, and T. Dietrich. 2009. Periodontitis 515 in systemic rheumatic diseases. Nature Reviews Rheumatology 5:218-224. 516


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

22

16. Fitsialos, G., A. A. Chassot, L. Turchi, M. A. Dayem, K. LeBrigand, C. 517 Moreilhon, G. Meneguzzi, R. Busca, B. Mari, P. Barbry, and G. Ponzio. 2007. 518 Transcriptional signature of epidermal keratinocytes subjected to in vitro scratch 519 wounding reveals selective roles for ERK1/2, p38, and phosphatidylinositol 3-kinase 520 signaling pathways. The Journal of biological chemistry 282:15090-15102. 521

17. Friedewald, V. E., K. S. Kornman, J. D. Beck, R. Genco, A. Goldfine, P. Libby, S. 522 Offenbacher, P. M. Ridker, T. E. Van Dyke, and W. C. Roberts. 2009. The 523 American Journal of Cardiology and Journal of Periodontology Editors' Consensus: 524 Periodontitis and Atherosclerotic Cardiovascular Disease. J Periodontol 80:1021-525 1032. 526

18. Green, H., and O. Kehinde. 1979. Formation of normally differentiated 527 subcutaneous fat pads by an established preadipose cell line. J Cell Physiol 101:169-528 171. 529

19. Green, H., and O. Kehinde. 1974. Sublines of Mouse 3t3 Cells That Accumulate 530 Lipid. Cell 1:113-116. 531

20. Guo, Q., and W. Fast. 2011. Citrullination of inhibitor of growth 4 (ING4) by 532 peptidylarginine deminase 4 (PAD4) disrupts the interaction between ING4 and p53. 533 The Journal of biological chemistry 286:17069-17078. 534

21. Guo, Y., K. A. Nguyen, and J. Potempa. 2010. Dichotomy of gingipains action as 535 virulence factors: from cleaving substrates with the precision of a surgeon's knife to a 536 meat chopper-like brutal degradation of proteins. Periodontol 2000 54:15-44. 537

22. Hajishengallis, G. 2009. Porphyromonas gingivalis-host interactions: open war or 538 intelligent guerilla tactics? Microbes and infection / Institut Pasteur 11:637-645. 539

23. Holt, S. C., and J. L. Ebersole. 2005. Porphyromonas gingivalis, Treponema 540 denticola, and Tannerella forsythia: the "red complex", a prototype polybacterial 541 pathogenic consortium in periodontitis. Periodontol 2000 38:72-122. 542

24. Howell, T. H., G. Martuscelli, and J. Oringer. 1996. Polypeptide growth factors for 543 periodontal regeneration. Current opinion in periodontology 3:149-156. 544

25. Huang, H. W., S. K. Mohan, and C. Yu. 2010. The NMR solution structure of 545 human epidermal growth factor (hEGF) at physiological pH and its interactions with 546 suramin. Biochemical and biophysical research communications 402:705-710. 547

26. Irwin, C. R., S. L. Schor, and M. W. Ferguson. 1991. Expression of EGF-receptors 548 on epithelial and stromal cells of normal and inflamed gingiva. Journal of periodontal 549 research 26:388-394. 550

27. Jorissen, R. N., F. Walker, N. Pouliot, T. P. Garrett, C. W. Ward, and A. W. 551 Burgess. 2003. Epidermal growth factor receptor: mechanisms of activation and 552 signalling. Experimental cell research 284:31-53. 553

28. Kadowaki, T., R. Takii, K. Yamatake, T. Kawakubo, T. Tsukuba, and K. 554 Yamamoto. 2007. A role for gingipains in cellular responses and bacterial survival in 555 Porphyromonas gingivalis-infected cells. Frontiers in bioscience : a journal and virtual 556 library 12:4800-4809. 557

29. Kearney, P. L., M. Bhatia, N. G. Jones, L. Yuan, M. C. Glascock, K. L. 558 Catchings, M. Yamada, and P. R. Thompson. 2005. Kinetic characterization of 559 protein arginine deiminase 4: a transcriptional corepressor implicated in the onset and 560 progression of rheumatoid arthritis. Biochemistry 44:10570-10582. 561

30. Kilsgard, O., P. Andersson, M. Malmsten, S. L. Nordin, H. M. Linge, M. 562 Eliasson, E. Sorenson, J. S. Erjefalt, J. Bylund, A. I. Olin, O. E. Sorensen, and A. 563 Egesten. 2012. Peptidylarginine deiminases present in the airways during tobacco 564 smoking and inflammation can citrullinate the host defense peptide LL-37, resulting in 565


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

23

altered activities. American journal of respiratory cell and molecular biology 46:240-566 248. 567

31. King, G. N., and D. L. Cochran. 2002. Factors that modulate the effects of bone 568 morphogenetic protein-induced periodontal regeneration: a critical review. J 569 Periodontol 73:925-936. 570

32. Lamont, R. J., and H. F. Jenkinson. 1998. Life below the gum line: pathogenic 571 mechanisms of Porphyromonas gingivalis. Microbiology and molecular biology 572 reviews : MMBR 62:1244-1263. 573

33. Lemmon, M. A., and J. Schlessinger. 2010. Cell signaling by receptor tyrosine 574 kinases. Cell 141:1117-1134. 575

34. Li, P., D. Wang, H. Yao, P. Doret, G. Hao, Q. Shen, H. Qiu, X. Zhang, Y. Wang, 576 and G. Chen. 2010. Coordination of PAD4 and HDAC2 in the regulation of p53-577 target gene expression. Oncogene 29:3153-3162. 578

35. Lo Bue, A. M., G. Nicoletti, M. A. Toscano, B. Rossetti, G. Cali, and F. 579 Condorelli. 1999. Porphyromonas gingivalis prevalence related to other micro-580 organisms in adult refractory periodontitis. The new microbiologica 22:209-218. 581

36. Loe, H. 1993. Periodontal diseases: a brief historical perspective. Periodontol 2000 582 2:7-12. 583

37. Loos, T., A. Mortier, M. Gouwy, I. Ronsse, W. Put, J. P. Lenaerts, J. Van 584 Damme, and P. Proost. 2008. Citrullination of CXCL10 and CXCL11 by 585 peptidylarginine deiminase: a naturally occurring posttranslational modification of 586 chemokines and new dimension of immunoregulation. Blood 112:2648-2656. 587

38. Lu, L., P. S. Reinach, and W. W. Kao. 2001. Corneal epithelial wound healing. 588 Experimental biology and medicine 226:653-664. 589

39. Makrygiannakis, D., M. Hermansson, A. K. Ulfgren, A. P. Nicholas, A. J. 590 Zendman, A. Eklund, J. Grunewald, C. M. Skold, L. Klareskog, and A. I. 591 Catrina. 2008. Smoking increases peptidylarginine deiminase 2 enzyme expression in 592 human lungs and increases citrullination in BAL cells. Ann Rheum Dis 67:1488-1492. 593

40. Mangat, P., N. Wegner, P. J. Venables, and J. Potempa. 2010. Bacterial and human 594 peptidylarginine deiminases: targets for inhibiting the autoimmune response in 595 rheumatoid arthritis? Arthritis research & therapy 12:209. 596

41. Martin, P. 1997. Wound healing--aiming for perfect skin regeneration. Science 597 276:75-81. 598

42. Matsuda, N., W. L. Lin, N. M. Kumar, M. I. Cho, and R. J. Genco. 1992. 599 Mitogenic, chemotactic, and synthetic responses of rat periodontal ligament 600 fibroblastic cells to polypeptide growth factors in vitro. J Periodontol 63:515-525. 601

43. McAllister, B. S., F. Leeb-Lundberg, J. T. Mellonig, and M. S. Olson. 1995. The 602 functional interaction of EGF and PDGF with bradykinin in the proliferation of human 603 gingival fibroblasts. J Periodontol 66:429-437. 604

44. McGraw, W. T., J. Potempa, D. Farley, and J. Travis. 1999. Purification, 605 characterization, and sequence analysis of a potential virulence factor from 606 Porphyromonas gingivalis, peptidylarginine deiminase. Infect. Immun. 67:3248-3256. 607

45. McGwire, G. B., and R. A. Skidgel. 1995. Extracellular conversion of epidermal 608 growth factor (EGF) to des-Arg53-EGF by carboxypeptidase M. The Journal of 609 biological chemistry 270:17154-17158. 610

46. Michalik, M., M. Pierzchalska, A. Pabianczyk-Kulka, and W. Korohoda. 2003. 611 Procaine-induced enhancement of fluid-phase endocytosis and inhibition of exocytosis 612 in human skin fibroblasts. European journal of pharmacology 475:1-10. 613

47. Mogi, M., J. Otogoto, N. Ota, H. Inagaki, M. Minami, and K. Kojima. 1999. 614 Interleukin 1 beta, interleukin 6, beta 2-microglobulin, and transforming growth 615


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

24

factor-alpha in gingival crevicular fluid from human periodontal disease. Archives of 616 oral biology 44:535-539. 617

48. Mortier, A., T. Loos, M. Gouwy, I. Ronsse, J. Van Damme, and P. Proost. 2010. 618 Posttranslational modification of the NH2-terminal region of CXCL5 by proteases or 619 peptidylarginine Deiminases (PAD) differently affects its biological activity. The 620 Journal of biological chemistry 285:29750-29759. 621

49. Moulin, V. 1995. Growth factors in skin wound healing. European journal of cell 622 biology 68:1-7. 623

50. Naito, M., H. Hirakawa, A. Yamashita, N. Ohara, M. Shoji, H. Yukitake, K. 624 Nakayama, H. Toh, F. Yoshimura, S. Kuhara, M. Hattori, T. Hayashi, and K. 625 Nakayama. 2008. Determination of the genome sequence of Porphyromonas 626 gingivalis strain ATCC 33277 and genomic comparison with strain W83 revealed 627 extensive genome rearrangements in P. gingivalis. DNA research : an international 628 journal for rapid publication of reports on genes and genomes 15:215-225. 629

51. Nelson, K. E., R. D. Fleischmann, R. T. DeBoy, I. T. Paulsen, D. E. Fouts, J. A. 630 Eisen, S. C. Daugherty, R. J. Dodson, A. S. Durkin, M. Gwinn, D. H. Haft, J. F. 631 Kolonay, W. C. Nelson, T. Mason, L. Tallon, J. Gray, D. Granger, H. Tettelin, H. 632 Dong, J. L. Galvin, M. J. Duncan, F. E. Dewhirst, and C. M. Fraser. 2003. 633 Complete genome sequence of the oral pathogenic Bacterium porphyromonas 634 gingivalis strain W83. J Bacteriol 185:5591-5601. 635

52. Niederman, R., B. Brunkhorst, S. Smith, R. N. Weinreb, and M. I. Ryder. 1990. 636 Ammonia as a potential mediator of adult human periodontal infection: inhibition of 637 neutrophil function. Archives of oral biology 35 Suppl:205S-209S. 638

53. Nordlund, L., M. Hormia, L. Saxen, and I. Thesleff. 1991. Immunohistochemical 639 localization of epidermal growth factor receptors in human gingival epithelia. Journal 640 of periodontal research 26:333-338. 641

54. O'Brien-Simpson, N. M., R. D. Pathirana, G. D. Walker, and E. C. Reynolds. 642 2009. Porphyromonas gingivalis RgpA-Kgp proteinase-adhesin complexes penetrate 643 gingival tissue and induce proinflammatory cytokines or apoptosis in a concentration-644 dependent manner. Infect. Immun. 77:1246-1261. 645

55. O-Brien-Simpson, N. M., P. D. Veith, S. G. Dashper, and E. C. Reynolds. 2003. 646 Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire. 647 Current protein & peptide science 4:409-426. 648

56. Ogiso, H., R. Ishitani, O. Nureki, S. Fukai, M. Yamanaka, J. H. Kim, K. Saito, A. 649 Sakamoto, M. Inoue, M. Shirouzu, and S. Yokoyama. 2002. Crystal structure of the 650 complex of human epidermal growth factor and receptor extracellular domains. Cell 651 110:775-787. 652

57. Okada, H., and S. Murakami. 1998. Cytokine expression in periodontal health and 653 disease. Critical reviews in oral biology and medicine : an official publication of the 654 American Association of Oral Biologists 9:248-266. 655

58. Olayioye, M. A., R. M. Neve, H. A. Lane, and N. E. Hynes. 2000. The ErbB 656 signaling network: receptor heterodimerization in development and cancer. The 657 EMBO journal 19:3159-3167. 658

59. Parkar, M. H., L. Kuru, M. Giouzeli, and I. Olsen. 2001. Expression of growth-659 factor receptors in normal and regenerating human periodontal cells. Archives of oral 660 biology 46:275-284. 661

60. Proost, P., T. Loos, A. Mortier, E. Schutyser, M. Gouwy, S. Noppen, C. Dillen, I. 662 Ronsse, R. Conings, S. Struyf, G. Opdenakker, P. C. Maudgal, and J. Van 663 Damme. 2008. Citrullination of CXCL8 by peptidylarginine deiminase alters receptor 664


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

25

usage, prevents proteolysis, and dampens tissue inflammation. The Journal of 665 experimental medicine 205:2085-2097. 666

61. Pullar, C. E., B. S. Baier, Y. Kariya, A. J. Russell, B. A. Horst, M. P. 667 Marinkovich, and R. R. Isseroff. 2006. beta4 integrin and epidermal growth factor 668 coordinately regulate electric field-mediated directional migration via Rac1. Molecular 669 biology of the cell 17:4925-4935. 670

62. Pyrc, K., P. Strzyz, A. Milewska, A. Golda, O. Schildgen, and J. Potempa. 2011. 671 Porphyromonas gingivalis enzymes enhance infection with human metapneumovirus 672 in vitro. The Journal of general virology 92:2324-2332. 673

63. Quinchia-Rios, B. H., M. Guerrero, S. Abozeid, B. Bainbridge, R. Darveau, T. 674 Compton, and P. J. Bertics. 2008. Down-regulation of epidermal growth factor 675 receptor-dependent signaling by Porphyromonas gingivalis lipopolysaccharide in life-676 expanded human gingival fibroblasts. Journal of periodontal research 43:290-304. 677

64. Ranney, R. R. 1993. Classification of periodontal diseases. Periodontol 2000 2:13-25. 678 65. Rheinwald, J. G., and H. Green. 1977. Epidermal growth factor and the 679

multiplication of cultured human epidermal keratinocytes. Nature 265:421-424. 680 66. Rodriguez, S. B., B. L. Stitt, and D. E. Ash. 2009. Expression of peptidylarginine 681

deiminase from Porphyromonas gingivalis in Escherichia coli: enzyme purification 682 and characterization. Archives of biochemistry and biophysics 488:14-22. 683

67. Schlessinger, J. 2002. Ligand-induced, receptor-mediated dimerization and activation 684 of EGF receptor. Cell 110:669-672. 685

68. Schultz, G., D. S. Rotatori, and W. Clark. 1991. EGF and TGF-alpha in wound 686 healing and repair. Journal of cellular biochemistry 45:346-352. 687

69. Sidhu, R. S. 1979. Two-dimensional electrophoretic analyses of proteins synthesized 688 during differentiation of 3T3-L1 preadipocytes. The Journal of biological chemistry 689 254:11111-11118. 690

70. Socransky, S. S., A. D. Haffajee, M. A. Cugini, C. Smith, and R. L. Kent, Jr. 691 1998. Microbial complexes in subgingival plaque. Journal of clinical periodontology 692 25:134-144. 693

71. Stephens, P., P. Grenard, P. Aeschlimann, M. Langley, E. Blain, R. Errington, D. 694 Kipling, D. Thomas, and D. Aeschlimann. 2004. Crosslinking and G-protein 695 functions of transglutaminase 2 contribute differentially to fibroblast wound healing 696 responses. J Cell Sci 117:3389-3403. 697

72. Struyf, S., S. Noppen, T. Loos, A. Mortier, M. Gouwy, H. Verbeke, D. Huskens, 698 S. Luangsay, M. Parmentier, K. Geboes, D. Schols, J. Van Damme, and P. Proost. 699 2009. Citrullination of CXCL12 differentially reduces CXCR4 and CXCR7 binding 700 with loss of inflammatory and anti-HIV-1 activity via CXCR4. Journal of 701 immunology 182:666-674. 702

73. Suzuki, A., G. Ji, Y. Numabe, M. Muramatsu, K. Gomi, M. Kanazashi, Y. Ogata, 703 E. Shimizu, Y. Shibukawa, A. Ito, T. Ito, A. Sugaya, T. Arai, S. Yamada, S. 704 Deguchi, and K. Kamoi. 2004. Single nucleotide polymorphisms associated with 705 aggressive periodontitis and severe chronic periodontitis in Japanese. Biochemical and 706 biophysical research communications 317:887-892. 707

74. Tajima, Y., S. Yokose, M. Kashimata, M. Hiramatsu, N. Minami, and N. Utsumi. 708 1992. Epidermal growth factor expression in junctional epithelium of rat gingiva. 709 Journal of periodontal research 27:299-300. 710

75. Terpenning, M. S., G. W. Taylor, D. E. Lopatin, C. K. Kerr, B. L. Dominguez, 711 and W. J. Loesche. 2001. Aspiration pneumonia: Dental and oral risk factors in an 712 older veteran population. Journal of the American Geriatrics Society 49:557-563. 713


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

26

76. Wang, Y., J. Wysocka, J. Sayegh, Y. H. Lee, J. R. Perlin, L. Leonelli, L. S. 714 Sonbuchner, C. H. McDonald, R. G. Cook, Y. Dou, R. G. Roeder, S. Clarke, M. 715 R. Stallcup, C. D. Allis, and S. A. Coonrod. 2004. Human PAD4 regulates histone 716 arginine methylation levels via demethylimination. Science 306:279-283. 717

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

725 726

727


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

27

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

751


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

28

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


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

Inactivation of epiderm al growth factor by Porphyromonas...

Documents

Transcript of Inactivation of epiderm al growth factor by Porphyromonas...