An Investigation of Porphyromonas gingivalis

Peptidylarginine Deiminase: A Putative Virulence Factor in an

Animal Model of Inflammation

SYATIRAH NAJMI ABDULLAH

BSc.Hons(Biomedical Sc.)

Thesis submitted for the degree of Master of Science in Dentistry

School of Dentistry

The University of Adelaide

November 2010

ii

TABLE OF CONTENTS Table of Contents ........................................................................................................................................... ii

List of Figures ................................................................................................................................................ ix

List of Tables ................................................................................................................................................. xi

Abbreviations ................................................................................................................................................ xii

Abstract ........................................................................................................................................................ xvi

Declaration ................................................................................................................................................. xviii

Acknowledgment ......................................................................................................................................... xix

Chapter 1 ....................................................................................................................................................... 1

1 A Review of the Literature ................................................................................................................... 2

1.1 Introduction ..................................................................................................................... 2

1.2 Periodontal Disease ....................................................................................................... 4

1.2.1 Introduction .............................................................................................................. 4

1.2.2 Types of periodontal disease .................................................................................. 7

1.2.3 Microorganisms linked to the aetiology of periodontal disease............................. 8

1.3 Porphyromonas gingivalis and Periodontal Disease ..................................................10

1.3.1 Porphyromonas gingivalis virulence factors .........................................................11

1.3.2 Peptidylarginine deiminases .................................................................................13

1.3.3 Peptide metabolism by Porphyromonas gingivalis ..............................................15

1.4 Citrullination ..................................................................................................................16

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1.4.1 Citrullination and the immune system ..................................................................17

1.4.2 Citrullinated human proteins .................................................................................18

1.5 Rheumatoid Arthritis.....................................................................................................20

1.5.1 Introduction and history .........................................................................................20

1.5.2 Features of rheumatoid arthritis ............................................................................24

1.6 Rheumatoid Arthritis and Citrullination ........................................................................25

1.6.1 Citrullination in RA patients and clinical markers .................................................26

1.7 Research Questions.....................................................................................................27

Chapter 2 ..................................................................................................................................................... 28

2 Investigation of Porphyromonas gingivalis Peptidylarginine Deiminase ......................................... 29

2.1 Citrulline Assay .............................................................................................................30

2.1.1 Colour detection reagent .......................................................................................30

2.1.2 Standard curve to determine citrulline concentration ..........................................31

2.2 Citrullination by Mammalian Peptidylarginine Deiminase ..........................................33

2.2.1 Citrullination of BAEE by mPAD ...........................................................................33

2.2.2 Citrullination of free arginine by mPAD ................................................................36

2.2.3 Discussion .............................................................................................................38

2.3 Cultivation of Porphyromonas gingivalis .....................................................................38

iv

2.4 Cell Harvesting and Preparation .................................................................................40

2.5 Protein Assay ...............................................................................................................40

2.5.1 Method ...................................................................................................................40

2.5.2 Results ...................................................................................................................40

2.6 Citrullination of BAEE by Porphyromonas gingivalis ..................................................43

2.6.1 Method ...................................................................................................................43

2.6.2 Results and discussion .........................................................................................44

2.7 Characterisation of P. gingivalis Peptidylarginine Deiminase ....................................46

2.7.1 Effect of environmental pH on PgPAD activity .....................................................46

2.7.2 Effect of elevated temperature and enzyme localisation .....................................48

2.7.3 Peptidylarginine deiminase specificity for peptidylarginine position ...................51

2.7.4 Arginine analogues as substrates and competitive inhibitors for citrullination ...53

2.8 Citrullination of Arginine-containing Proteins ..............................................................56

2.8.1 Citrullination of yeast enolase by mPAD ..............................................................56

2.8.2 Citrullination of arginine containing proteins by PgPAD ......................................59

2.9 Effect of Gingipains on PgPAD Activity .......................................................................60

2.9.1 Gingipain activity assay.........................................................................................61

2.9.2 Effect of gingipains inhibitors using azoalbumin assay .......................................63

v

2.9.3 Effect of gingipain inhibitors on citrullination of albumin by PgPAD....................65

2.9.4 Effect of gingipain inhibitors on citrullination of BAEE by PgPAD .......................67

Chapter 3 ..................................................................................................................................................... 68

3 A Histological Survey of Infiltrated Cells in Selected Tissues of Adjuvant Arthritis induced rats Pre-treated with Heat Killed Porphyromonas gingivalis. .................................................................... 69

3.1 Introduction ...................................................................................................................69

3.2 Hypotheses ...................................................................................................................71

3.3 Experimental Animal Model for Chronic Inflammation and Adjuvant Arthritis ...........71

3.3.1 Animal model .........................................................................................................71

3.3.2 Preparation of periodontogenic stimulus ..............................................................72

3.3.3 Preparation of arthritogenic stimulus ....................................................................73

3.3.4 Assessment of clinical polyarthritis .......................................................................73

3.4 Collection and Processing of Tissue Samples and Sponges ....................................73

3.4.1 Decalcification of the heads ..................................................................................73

3.4.2 Collection and processing of sponges and spleen for routine histology .............74

3.4.3 Dissection of decalcified heads and sponges for routine histology ....................74

3.4.4 Tissue processing .................................................................................................74

3.5 Staining .........................................................................................................................75

3.5.1 Routine haematoxylin and eosin staining.............................................................75

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3.5.2 Histochemical staining for identification of osteoclasts .......................................75

3.5.3 TRAP control tissue...............................................................................................76

3.5.4 Cell counting ..........................................................................................................79

3.5.5 Immunohistochemical detection of citrullinated protein in sponge inplants ........79

3.6 Results ..........................................................................................................................81

3.6.1 Histological Survey ................................................................................................81

3.6.2 Polymorphonuclear cells in bone marrow spaces as a measure of proliferation ...

89

3.6.3 Cellular infiltrate in implanted sponges ................................................................92

3.6.4 In situ detection of citrullinated protein in the sponge infiltrate ...........................96

3.6.5 Identification of osteoclasts .................................................................................100

Chapter 4 ................................................................................................................................................... 104

4 Discussion ........................................................................................................................................ 105

4.1 Introduction .................................................................................................................105

4.2 Characterisation of P. gingivalis PAD .......................................................................106

4.2.1 PgPAD Specificity ...............................................................................................108

4.2.2 Citrullination of proteins by PgPAD ....................................................................109

4.2.3 Influence of gingipains on citrullination ..............................................................111

4.2.4 Role of PgPAD in arginine metabolism ..............................................................112

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4.2.5 Exacerbation of the inflammatory response in AA by prior exposure to P.

gingivalis 114

4.3 Conclusion ..................................................................................................................115

Bibliography ............................................................................................................................................... 117

Appendices ................................................................................................................................................ 130

Appendix 1.1 Protein Assay ..............................................................................................131

Appendix 1.2: Citrullination of BAEE by rabbit muscle PAD (Sigma no.: p1584) ............133

Appendix 1.3: Tissue processing .......................................................................................134

Appendix 1.4 Hematoxylin and eosin staining..................................................................136

Appendix 1.5 Preparation of stock solution for TRAP staining ........................................137

Appendix 1.6 Immunohistochemistry – Citrullinated protein............................................138

Appendix 2 Results .............................................................................................................140

Appendix 2.1 Citrullination activity of rabbit muscle PAD at absorbance of 530nm .......140

Appendix 2.2 Citrullination activity of Porphyromonas gingivalis PAD at absorbance of

530nm 140

Appendix 2.3 Effect of environmental pH .........................................................................141

Appendix 2.4 Peptidylarginine deiminase specificity for arginine position......................141

Appendix 2.5 Arginine analogues as potential substrates and potential competitive

inhibitors for citrullination .........................................................................................................142

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Appendix 2.6 Gingipain assay absorbance reading at 440nm ........................................142

Appendix 2.7 Citrullination of yeast enolase by mPAD at absorbance of 530nm ..........143

Appendix 2.8 Effect of gingipains inhibitors using azoalbumin assay absorbance reading

at 440nm 143

Appendix 2.9 Effect of gingipain on citrullination of BAEE by PgPAD at absorbance of

530nm 144

Appendix 2.10 Effect of gingipain on citrullination of albumin by PgPAD at absorbance of

530nm 144

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LIST OF FIGURES Figure 1.1: Surgical exposure of bone loss (arrow) resulting from periodontitis as adapted from

(Pihlstrom et al. 2005). ..................................................................................................................... 5

Figure 1.2: Electron microscopic image of Porphyromonas gingivalis taken from

http://www.microbiologybytes.com. ...............................................................................................11

Figure 1.3: Citrullination activity by P. gingivalis PAD. .................................................................14

Figure 1.4: The process of citrullination of arginine to citrulline catalysed by mammalian PAD.

........................................................................................................................................................17

Figure 1.5: La Familia de Jordaens en un Jardín Jacob Jordaens ..............................................21

Figure 1.6 Changes that occur in the synovial joint as a result of RA. ........................................24

Figure 2.1: A standard curve for the coulorimetric determination of citrulline concentration. .....32

Figure 2.2: The citrullination of BAEE by rabbit muscle PAD. .....................................................35

Figure 2.3: The citrullination activity of mPAD against free arginine. ..........................................37

Figure 2.4: A Gram stain of P. gingivalis W50 under light microscopy oil immersion at 1,000 x

magnification. .................................................................................................................................39

Figure 2.5: The standard curve of protein concentration at a wavelength of 595 nm. ................42

Figure 2.6: The microtitre plate colourimetric method used to detect citrulline. ..........................44

Figure 2.7: Rate of citrullination of BAEE by P. gingivalis W50 cells...........................................45

Figure 2.8: The effect of environmental pH on citrullination of BAEE by PgPAD. ......................47

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Figure 2.9: The effect of heat and sonication on PgPAD activity.................................................50

Figure 2.10: Citrullination of yeast enolase by mPAD. .................................................................58

Figure 2.11: The proteolytic activity of P. gingivalis washed cells using the azoalbumin assay.

........................................................................................................................................................62

Figure 3.1: Tartrate-resistant acid phosphatase (TRAP) staining................................................77

Figure 3.2: Histology of the normal tissue sections of rats...........................................................82

Figure 3.3: Lateral sagittal section from normal rat's head stained by routine H&E (Low power)

........................................................................................................................................................84

Figure 3.4: Lateral sagittal section of normal rat head showing maxillary region (Low power). .86

Figure 3.5: Lateral sagittal section of lower jaw from a normal rat. .............................................88

Figure 3.6: Cross section of bone marrow taken from area of interest of the head (High power).

........................................................................................................................................................90

Figure 3.7: Sections taken from sponges and tissue surroundings stained by routine H&E ......93

Figure 3.8: Polyclonal anti-citrulline antibody staining of sponges and tissues taken from the rat

flank.................................................................................................................................................97

Figure 3.9: The TRAP staining of rats from three different groups. ...........................................101

Figure 4.1: Role of peptidylarginine deiminase in energy production of P. gingivalis. ..............113

Figure 4.2: Shandon Citadel 2000 automatic tissue processor .................................................134

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LIST OF TABLES Table 1.1: The 2010 American College of Rheumatology/ European League against

rheumatism classification criteria for rheumatoid arthritis. ...........................................................23

Table 2.1: Comparison of PAD activity in intact heat killed and sonicated cells. ........................49

Table 2.2: Peptides used as substrates for PgPAD. ....................................................................52

Table 2.3: PgPAD activity against arginine-containing peptides and free arginine. ...................53

Table 2.4: The citrullination of various arginine analogues and their ability to act as competitive

inhibitors of the reaction. ................................................................................................................55

Table 2.5: Arginine-containing proteins and their rates of citrullination by PgPAD .....................60

Table 2.6: The inhibition of cellular proteolytic activity. ................................................................64

Table 2.7: The effect of gingipain inhibitors on PgPAD activity ...................................................66

Table 2.8: The direct effect of gingipain inhibitors on PgPAD .....................................................67

Table 3.1: PMN proliferation in bone marrow (section stained by H&E). ....................................92

Table 3.2: PMN cells observed within the infiltrate of implanted sponges with and without

HKPg. ..............................................................................................................................................96

Table 4.1 Reagents used for the experiment and their concentration and volume ..................133

Table 4.2: The step for Shandon Citadel 2000 automatic tissue processor for impregnation of

tissue .............................................................................................................................................135

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ABBREVIATIONS ACPA Anti-citrullinated protein antibodies

AKA Anti-keratin antibodies

anti-CCP Anti-citrullinated cyclic peptide antibodies

APF Anti-perinuclear factor

Arg-Xaa Arginine carboxy terminal peptide bond

ATP Adenosine triphosphatase

AU Absorbance unit

BAEE Benzoyl-arginine ethyl ester

BHI Brain-heart infusion

BSA Bovine serum albumin

Ca2+ Calcium ion

CaCl2 Calcium chloride

CO2 Carbon dioxide

C-terminal Carboxy terminal

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

xiii

EGTA ethylene glycol tetraacetic acid

et al. et alia

FeCl3 Iron (III) Chloride

FMN Flavin mononucleotide

g Gravitational force

GCF Gingival crevicular fluid

H2 Hydrogen

HCl Acid hydrochloric

IgA Immunoglobulin A

IgG Immunoglobulin G

kDa kilo Dalton

Kgp Lysine gingipain

Lys-Xaa Lysine carboxy terminal peptide bond

M Molar

mg cell protein-1.min-1 Milligram per cell protein per minutes

mM milliMolar

mPAD Rabbit muscle/ mammalian PAD

xiv

N2 Nitrogen

nm nanometre

nmoles nanomoles

nmoles citrulline.unit-1.min-1 nanomoles citrulline per unit per minute

N-terminal Amino terminal

oC Degree Celcius

OD560 Optical Density at 560nm

PAD Peptidylarginine deiminase

PD Periodontal disease

Pg Porphyromonas gingivalis

PgPAD Porphyromonas gingivalis peptidylarginine deiminase

PMN Polymorphonuclear

RA Rheumatoid arthritis

RF Rheumatoid factor

Rgp Arginine gingipain

R-group Amino acid functional group

Sp. Species

xv

TNF- Tumor necrosis factor alpha

TPCK Tosyl phenylalanyl chloromethyl ketone

Tris-HCl Tri sulphate – hydrochloric acid

v/v Volume per volume

w/v Weight per volume

μL microlitre

μm micrometre

met-arg-phe Methionine-arginine-phenylalanine

H2O2 Hydrogen peroxide

xvi

ABSTRACT Porphyromonas gingivalis, an oral periodontopathogen linked to chronic periodontitis

expresses peptidylarginine deiminase (PAD), an enzyme that converts peptide-bound arginine

to citrulline. A relationship between human PADs and chronic inflammatory diseases has been

proposed. Citrullinated -enolase is a candidate auto-antigen in rheumatoid arthritis. Vimentin

and fibrin are also likely target proteins in disease development. This study partially

characterised the enzyme and the ability of P. gingivalis cells to citrullinate peptides and these

rheumatoid arthritis relevant proteins. In addition, the influence of gingipains, key P. gingivalis

virulence factors, on PgPAD activity was investigated. A limited histological survey was

performed on selected tissues to investigate the effect of P. gingivalis in an animal model of

adjuvant arthritis.

A colourimetric assay to quantify citrulline was developed and used to determine the effect of

environmental pH and temperature on enzyme activity. Enzyme localization was investigated

by comparing reaction rates of whole cells to cell sonicates. Enzyme specificity was determined

by incubation of cells with a range of arginine analogues and arginine-containing peptides. The

rates of citrullination of enolase, vimentin and fibrin by P. gingivalis cells were calculated. The

influence of the gingipains on citrullination was measured by comparing the rate of citrullination

of albumin in the presence and absence of the proteolytic inhibitors tosyl phenylalanyl

chloromethyl ketone and leupeptin. Tissue sections from three regions of the animal heads

were stained for polymorphonuclear cells and osteoclasts. In addition sponge samples were

surveyed for polymorphonuclear cells and citrullinated proteins detected using

immunohistochemical technique.

xvii

PgPAD activity was heat stable, predominantly cell-surface expressed and exhibited optimal

activity between pH 7.5 and 8. The enzyme was highly specific for arginine and citrullinated

arginine residues in all positions in the peptides tested. PgPAD was able to citrullinate all

rheumatoid arthritis relevant proteins, at rates slower than peptides. Inhibition of the gingipains

failed to influence the rate of citrullination of albumin. In the adjuvant arthritis animal study, pre-

treatment with P. gingivalis produced increased inflammatory cellular infiltrate at the site of

exposure but no similar affect in the head tissue. There was a significant increase numbers of

polymorphonuclear cells in the bone marrow from the head region and in the implanted sponge

infiltrate from rats with prior exposure to P. gingivalis. Although citrullinated proteins were

detected in sponge sections from both adjuvant arthritis-induced rat groups, no difference

between them was observed. A similar result was seen with osteoclasts, as both groups

exhibited increased numbers over the control group.

This study has shown that P. gingivalis peptidylarginine deiminase has potential to influence

the inflammatory process by citrullinating arginine containing peptides and rheumatoid arthritis

relevant proteins. An examination of rats exposed to the bacterium in an animal model of

rheumatoid arthritis did not appear to exacerbate inflammation in selected tissues.

xviii

DECLARATION

This work contains no material which has been accepted for the award of any other degree or

diploma in any university or other tertiary institution to Syatirah Najmi Abdullah and, to the best

of my knowledge and belief, contains no material previously published or written by another

person, except where due reference has been made in the text.

I give consent to this copy of my thesis, when deposited in the University Library, being made

available for loan and photocopying, subject to the provisions of the Copyright Act 1968. I also

give permission for the digital version of my thesis to be made available on the web, via the

University’s digital research repository, the Library catalogue, the Australasian Digital Theses

Program (ADTP) and also through web search engines, unless permission has been granted

by the University to restrict access for a period of time.

Signed:

Date:

xix

ACKNOWLEDGMENT I would like to express my deepest sense of gratitude to my Principal supervisor Dr. Neville

Gully, a friend and an excellent mentor, for his patient guidance, constant encouragement and

invaluable suggestions throughout this study. He has been everything that one could ask for as

an advisor.

My Co-supervisors, Dr Elizabeth-Anne Farmer, A/Prof Dr. Richard Logan and Mr. Llew Spargo

for their countenance, valuable contributions and guidance in making this happen.

I am deeply indebted to Mr Victor Marino and Prof Mark Bartold of Colgate Australian Clinical

Dental Research Centre, Dr David Haynes, Ms. Melissa Cantley and Dr Kencana Dharmapatni

of Discipline of Pathology, School of Medical Sciences, The University of Adelaide for their

collaboration and valuable assistance in the AA induced HKPg model research.

My sincere thanks go to Ms. Sandy Hughes and Ms. Marjorie Quinn for their generous

assistance in histology and for being great friends in that quiet yet wonderful time of tissue

sectioning.

To my friends Atika, Jactty, Yi, Arnida, Zati, Fauziah, Nadiyah, Liyana, Yanti, Rafisah, Awan,

Nurul, Suhaiza, Azlina, Aini, Aida, Saidatul, Fiona, Nikki, Kazu, Anh, Judy and all those people

who made this research possible, for sharing experiences and knowledge and an enjoyable

experience for me.

I would like to express my gratitude to my Scholarship sponsor from the Ministry of Higher

Education Malaysia, Universiti Sains Islam Malaysia, and to the Australia Dental Research

Foundation and School of Dentistry, The University of Adelaide, for their financial support.

xx

Finally, I would like to express my deepest gratitude for the constant support, understanding

and love that I received from my parents; Ayah Abdullah and Mak Wan Nasrah, my incredible

sisters; Ainul and Atiqah, brother Shaharen and family during the past years.

1

CHAPTER 1

2

1 A Review of the Literature

1.1 Introduction

Microbial pathogens often interact in complex ways within their mammalian hosts. In human

disease, there are many well-understood examples of microbes colonising bodily fluids and

tissues to the detriment of their host by producing destructive toxins or enzymes, such as in

infections of environmentally accessible mucosal surfaces or in opportunistic infections. Less

well-understood are periodontal diseases (PD) such as periodontitis, where, in susceptible

individuals, a chronic inflammatory response is associated with persistent microbial

colonisation of specific sites. Other examples of microbe-linked chronic inflammation include

rheumatic fever, gastric ulcers, Whipples disease and reactive arthritis. The reasons that the

microbial pathogens are able to colonise tissues successfully in these cases are not clear but

are thought to include strategies to evade the immune response while exploiting a nutrient

advantage.

PDs affect a large proportion of the world population (15%) and have been linked to a consortia

of oral microbes of endogenous origin, such as Actinobacillus actinomycetemcomitans,

Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, Peptostreptococcus

micros and most commonly, Porphyromonas gingivalis (P.gingivalis) (Petersen 2003; Shiloah

et al. 2000; van Winkelhoff et al. 2002). P. gingivalis is thought to be able to successfully

colonise the gingival tissues because the microorganism possesses numerous virulence

factors that facilitate its growth and survival (McGraw et al. 1999; Travis et al. 1997). P.

gingivalis is able to evade the host immune system, manipulate the local environment in order

to supply nutrition for its own continuity and these activities lead to the destruction of host

tissues. The microorganism is highly proteolytic and utilizes environmental peptides and free

3

amino acids such as serine and threonine as sources of energy (Dashper et al. 2001;

Takahashi et al. 2000). In addition, P. gingivalis expresses an enzyme that modifies

peptidylarginine, a unique property among prokaryotes (McGraw et al. 1999).

Periodontal diseases are relatively widespread and in recent years have also been associated

with the development of several other chronic inflammatory diseases, such as, coronary heart

disease, diabetes mellitus and rheumatoid arthritis (RA) (Southerland et al. 2006). The basis of

these associations is unknown, but may be due to non-specific positive feedback loops

involving inflammatory mediators, or to more specific effects, from a common aetiological

agent. The ability of P. gingivalis to citrullinate arginine containing peptides is of special interest

when investigating the potential link between PD and RA, because auto-antibodies against

citrullinated peptides have recently been shown to be both highly specific and sensitive for the

diagnosis of RA (Schellekens et al. 1998). Citrullinated peptides have been suggested to play a

role in the pathogenesis of RA; however, the exact nature of this role is unclear. Whether the

instigator of citrullination in RA is of host or prokaryotic origin is also unknown. It is interesting

to note that P. gingivalis DNA has been found in the synovial fluid from RA patients (Moen et al.

2006). It seems plausible that, in patients with chronic PD, the increased levels of P. gingivalis,

frequently detected at diseased sites, might play a triggering or amplification role in RA, via its

ability to promote peptide citrullination. This mechanism might explain the over representation

of RA patients in the population suffering from PD.

It is for these reasons, it was decided to investigate the conditions under which P. gingivalis

modifies peptide-linked arginine residues to citrulline. A preliminary investigation of the links

between PD and polyarthritis in an animal model was also undertaken, with special reference

to the presence of inflammatory cells and citrulline in areas of inflammation.

4

1.2 Periodontal Disease

1.2.1 Introduction

Periodontal diseases are a group of inflammatory disorders of the hard and soft tissues

surrounding the teeth and are associated with plaque accumulation. The uncontrolled

inflammatory response leads to the destruction of both the periodontal ligament attachment and

the adjacent alveolar bone (Page and Schroeder 1976) as shown in Figure 1.1. Chronic

periodontitis, one of the more severe forms of PD, is a major cause of tooth loss in adults

(Niessen and Weyant 1989; Page and Schroeder 1976; Petersen 2003).

In the diseased state, inflammation of the gingival tissues involves the clinical signs of redness,

warmth, swelling and pain as its normal response to infection, trauma, or non-microbial

irritation. As disease progresses, epithelial attachment to the tooth surface is lost, leading to

migration of the junctional epithelium and the development of a gingival pocket (Loesche et al.

1985). Moreover, if left untreated, the destruction of supporting tissue will eventually result in

tooth loss. In recent years, it has been suggested that the inflammation associated with

periodontitis affects not only the dentition but may impact on general health. PD has been

suggested as a possible risk factor in systemic diseases such as myocardial infarction,

cardiovascular disease (Genco et al. 2002), pneumonia, pre-term low birth weight (Lopez et al.

2002) and RA (Mercado et al. 2003; Ribeiro et al. 2005).

5

Figure 1.1: Surgical exposure of bone loss (arrow) resulting from periodontitis as adapted from

(Pihlstrom et al. 2005).

It is now widely recognised that PD has a multi-factorial aetiology that can be genetically

related and environmentally involved. Bacterial colonization of the gingival tissues and the

host’s response to this challenge are thought to contribute to the disease severity (Pihlstrom et

al. 2005). Genetic disorders such as Chédiak-Higashi, Ehlers-Danlos, Kindlers, Cohen

syndromes, Haim-Munk and Papillon-Lefèvre syndromes have been linked to the onset of PD.

The disease prevalence is seen to increase with age (Horning et al. 1992), although loss of

periodontal attachment and alveolar bone with age is dependent on the presence of plaque as

shown on the presence of sub-gingival calculus, over-hanging restorations (Lang et al. 1983)

and/or crowded teeth are also factors thought to contribute to the disease process.

NOTE: This figure is included on page 5 of the print copy of the thesis held in the University of Adelaide Library.

6

A report from the United States 3rd National Health and Nutrition Examination Survey revealed

that approximately 42% of periodontitis cases in the adult population were associated with

cigarette smoking. The study indicated that more than one-half of the periodontitis affecting

adult cases maybe promoted by this habit (Tomar and Asma 2000). Similar findings were

reported in a recent survey of the Australian population, where 23% of adults were classified as

having moderate or severe periodontitis, with more than half the sufferers are former or current

cigarette smokers. In the same study, Do et al. (2008) estimated that one third of the

periodontitis cases can be prevented by elimination of cigarette smoking (Do et al. 2008).

Smoking has been suggested as a potential risk factor as it promotes the accumulation of oral

pathogens and progression of PD. Intensive exposure to tobacco smoke was found to affect

the rate of colonization by pathogenic bacteria in periodontitis-free young smokers (Shiloah et

al. 2000). Shiloah and co-workers also reported that seven of eight subjects with elevated

levels of PD associated pathogens were cigarette smokers. In addition to these findings,

smokers also have increased attachment loss, deeper periodontal pockets and more missing

teeth than non-smokers (Haffajee and Socransky 2001). Smoking may influence PD

progression by affecting the host control of bacteria as well as facilitating a more favourable

habitat for the establishment of pathogens such as P. gingivalis and A.

actinomycetemcomitans, often isolated from the shallow sites of gingival crevice (Eggert et al.

2001). Other potential effects of smoking on PD include the increased levels of carbon

monoxide on gingival tissues. This toxic by-product damages cells, such as neutrophils,

involved in the protection of the periodontal environment. It is thought that this cell damage, in

turn, provides nutrition for the pathogenic Gram-negative anaerobes, enhancing the growth of

these bacteria.

7

1.2.2 Types of periodontal disease

Gingivitis and periodontitis are recognised as the most prominent of the PDs. Gingivitis is an

inflammatory response to the presence of the dental plaque biofilm that accumulates at the

gingival margin. It is often associated with poor oral hygiene and can be reversed when good

oral hygiene is applied (Loe et al. 1965). Gingivitis has been reported to be highly prevalent

throughout the developing world (Pilot 1998). It is thought that prolonged chronic gingivitis may

lead to periodontitis (Criswell et al. 2002; Marc et al. 2003; Page et al. 1997).

During early stages of gingival inflammation, redness of the gingival tissues can be observed,

resulting from the enlargement of blood vessels in sub-epithelial connective tissue and during

disease progression swelling increases. If left untreated, chronic periodontitis may lead to

progressive loss of collagen attachment of the tooth to the underlying alveolar (jaw) bone and

the teeth become loose. Bleeding of gingival tissue can be triggered upon probing with blunt

instruments. These changes are rather vague, and usually painless.

Numerous bacterial species, such as Fusobacterium nucleatum and Eikenella corrodens, have

been isolated in elevated numbers in plaque from individuals suffering from gingivitis. However,

pathogenic species, such as P. gingivalis, commonly isolated in increased numbers and

proportions in plaque from periodontitis sufferers, were absent or not significantly increased

when compared with plague from healthy individuals (Moore et al. 1987).

Periodontitis is characterised by the formation of a significant gingival pocket, varying degrees

of gingival inflammation (swelling and redness) and bleeding upon probing (Lang et al. 1996).

As the periodontal pocket deepens, the environmental redox potential decreases and the

composition of the microbial community shifts, as the changed environment encourage the

growth of anaerobic Gram-negatives. In turn the host response becomes more destructive and

chronic disease ensues.

8

Host defence mechanisms against microbial infection are thought to contribute significantly to

the symptoms and severity of periodontitis. The oral pathogens involved are thought to promote

host tissue damage and activate inflammatory and immune responses. Various inflammatory

molecules such as proteases, cytokines, prostaglandins and enzymes are released from

leukocytes and fibroblasts once inflammation is initiated. The periodontal tissues become

loose, swollen and inflamed, there will be tissue inflammatory infiltrate present and moreover,

osteocytes begin the destruction of bone. The destruction of deeper tissues results, leading to

the loss of alveolar bone and periodontal ligament. Eventually, the connective tissue

attachment to the tooth may be destroyed leading to tooth loss.

1.2.3 Microorganisms linked to the aetiology of periodontal disease

In a recent study, the most frequently detected species in gingival crevicular epithelial cells

from chronic periodontitis lesions were P. gingivalis (42%), Treponema denticola (38%),

Prevotella intermedia (37%), Streptococcus intermedius (36%), Campylobacter rectus (35%),

Streptococcus sanguinis (35%) and Streptococcus oralis (34%) (Colombo et al. 2006).

The presence of elevated numbers of microbes is believed to be an important factor in the

progression of PD. However, the increased proportions of specific microbial pathogens that is

likely to be the crucial factor in the progression of PD. Several Gram-negative anaerobes,

including A. actinomycetemcomitans, P. gingivalis, Prevotella intermedia, Tannerella forsythia,

Fusobacterium nucleatum and Peptostreptococcus micros, have been associated with the

onset of periodontitis (Carlsson et al. 1984; van Winkelhoff et al. 2002). Most of these species

are found in very low numbers in plaque collected from the healthy gingival crevice (Griffen et

al. 1998). However, in the onset and progression of disease, these Gram-negatives are

frequently found in significantly higher numbers and proportions in sub-gingival plaque from the

gingival pockets that develop in PD sufferers.

9

Many species of bacteria isolated from diseased plaque are thought to generate much of their

energy from amino acid metabolism and some are highly proteolytic. This activity enables the

bacterial community in the subgingival plaque biofilm to break down host proteins and

glycoproteins, supplying nutrition for their growth. The battery of proteases liberates a mixture

of peptides and amino acids that are able to be assimilated and metabolised by the bacterial

cell to produce ATP. The ammonia that arises as a product of amino acid metabolism, leads to

the environment in the pocket becoming increasingly alkaline, compared to that of the healthy

gingival crevice. It is thought that the significant change in the sub-gingival ecology facilitates

the shift in dominant organisms present, by providing a more favourable environment for the

growth of the significant periodontopathogens, all of which have optimal growth pH above

neutrality.

The gingival crevice differs from the other environments in the mouth. It provides a relatively

anaerobic environment and the organisms present rely on the supply of gingival crevicular fluid

(GCF) for their nutrition. At diseased sites, the secretion of GCF will increase to continuously

flush the gingival sulcus. The increased flow of GCF is an inflammatory response to the

increased microbial load, facilitating the removal of non-adherent microbial cells and also

contains host defence components. GCF is a serum-like fluid, containing neutrophils and also

immunoglobulins, predominantly IgG at levels higher than salivary IgA. In addition, GCF

provides a range of proteins, including haem-containing proteins and nutrients such as iron that

nourish the oral microbes present, especially the black-pigmented anaerobic bacteria.

10

1.3 Porphyromonas gingivalis and Periodontal Disease

P. gingivalis is a key member of the group of microbes described above, releasing peptides

and other nutrients via the expression of multiple proteases that target host proteins in GCF

and epithelial tissues. In addition, P. gingivalis is also able to obtain essential factors for growth

such as haemin by producing haemolysin toxin. Interestingly, McGraw and colleagues

suggested that this microbe has the ability to stimulate an increased flow of GCF in order to

maintain their existence in a periodontal pocket (McGraw et al. 1999). Numerous studies have

proposed this species to be a predominant pathogen in PD (Colombo et al. 2006; Lamont et al.

1995; Shiloah et al. 2000). P. gingivalis survives in subgingival plague because it produces

numbers of virulence factors that play a role in tissue colonization and destruction in addition to

the perturbation of host defences (Holt et al. 1999). Cell surface structures such as fimbriae

and lectin-type adhesions, a polysaccharide capsule, lipopolysaccharide and outer membrane

vesicles as shown in Figure 1.2 enable the organism to adhere to the oral epithelium at buccal

and sub-gingival sites as well as co-aggregate with other oral bacteria. P. gingivalis and

Fusobacterium sp. have the ability to adhere to each other and also to crevicular epithelial cells

(Kolenbrander 2000).

P. gingivalis also possesses other factors associated with tissue damage such as

haemagglutinating factors that have also been identified as important adhesion molecules

(DeCarlo et al. 1999). These allow P. gingivalis cells to adhere to gingival tissue cells in

addition to attachment and lysis of erythrocytes, facilitating the uptake of essential iron (Olczak

et al. 2005). Metabolic end-products, such as an array of volatile fatty acids that include

butanoate, propionate, and isobutanoate may also be cytotoxic to the gingival tissue (Jeng et

al. 1999) in addition to the release of numerous enzymes (Cutler et al. 1995; Mayrand and Holt

1988; Sundqvist 1993). These virulence factors lead to chronic inflammation and ultimately to

11

degradation of host periodontal tissues and, in turn, result in the release of nutrient molecules

for their growth. These factors are also likely to play important roles in manipulating the host

defence system for its own advantage.

Figure 1.2: Electron microscopic image of Porphyromonas gingivalis taken from http://www.microbiologybytes.com.

This rod-like Gram negative bacterium has vesicles at its outer membrane.

1.3.1 Porphyromonas gingivalis virulence factors

1.3.1.1 Fimbriae

The invasion of epithelial cells by oral bacteria is facilitated by the expression of surface

proteins known as fimbriae. In advanced periodontitis, 5% of plasma cells taken from lesion

sites formed antibody to the fimbriae of P.gingivalis (Ogawa et al. 1991). These structures act

as adhesion molecules, capable of binding specifically to and activating various host cells such

NOTE: This figure is included on page 11 of the print copy of the thesis held in the University of Adelaide Library.

12

as human epithelial cells, endothelial cells, spleen cells and peripheral blood monocytes

(reviewed in Amano et al. 2004). A unique class of Gram-negative fimbriae, they consist of

protein subunits called fimbrillins with sizes varying between 41 to 49 kDa (Dickinson et al.

1988; Lee et al. 1991). These proteinaceous hair-like appendages are 2 to 8 nm in diameter

and range between 0.3 and 3 μm in length. The fimbrillin polypeptide binds to proline-rich

proteins, statherin, lactoferrin, fibrinogen and fibronectin, oral epithelial cells and other oral

bacterial species cells (e.g. A. naeslundii), (Amano et al. 1996; Lamont and Jenkinson 1998;

Murakami et al. 1996). A 48 kDa surface protein on human gingival epithelial cells has also

been reported to interact with fimbriae of P. gingivalis (Weinberg et al. 1997).

The expression of P. gingivalis fimbriae appears environmentally influenced. Changes in

temperature altered the regulation of fimbriae gene expression and fimbriae mediated

adherence to oral epithelium and to other oral microbes (Amano et al. 1994). The production of

fimbriae is reduced by 54% when subgingival temperature is elevated from 37oC to 39oC

(Amano et al. 1994).

Recent studies have shown that a fimbriae-deficient P. gingivalis mutant had decreased

binding to whole saliva-coated oral surfaces and caused periodontal bone loss in an animal

model of PD when compared with wild type P. gingivalis (Malek et al. 1994). Hamada et al.

also reported the inability of this P. gingivalis mutant to bind human gingival fibroblasts and

epithelial cells (Hamada et al. 1994). These findings provide evidence that suggest P. gingivalis

fimbriae are important virulence factors, aiding adherence and play a role in the pathogenesis

of human periodontal disease.

13

1.3.1.2 The gingipain proteinases

The gingipains are a group of cysteine endo-proteinases that have long been associated with

P. gingivalis virulence (Imamura 2003; Potempa et al. 1995). The gingipains are classified

according to their specificity of peptide bond cleavage; the R-gingipains (Rgp) hydrolyse

peptide bonds C-terminal to arginine residues and K-gingipain (Kgp) C-terminal to lysine

residues (Imamura 2003). The gingipains can degrade collagens, important structural

components of periodontal connective tissue and other extracellular matrix proteins, including

fibronectin and laminin, are also targeted by these enzymes. The gingipains are resistant to

host proteinase inhibitors such as cystatins, serpins and tissue inhibitors of metalloproteinases

as they exhibit significant proteolytic activity in their presence (Abe et al. 1998; Kadowaki et al.

1994). This suggests that these enzymes are significant virulence factors in P. gingivalis.

There are compounds that inhibit the activity of gingipains, such as the serine proteinase

inhibitor, tosyl-L-phenylalanine that inhibits both Kgp and Rgp, metal chelators (EDTA, EGTA)

which inhibit Rgp but not Kgp. Leupeptin can also be used as an Rgp specific inhibitor (Abe et

al. 1998; Holzhausen et al. 2006).

1.3.2 Peptidylarginine deiminases

Peptidylarginine deiminase (PAD) activity is possessed by P.gingivalis and also by many

mammalian cells. The first mammalian PAD was identified in 1977 (Rogers et al. 1977) and the

PAD enzymes were eventually grouped as guanidino-group modifying enzymes by Shirai et al.

(Shirai et al. 2001). Subsequently mammalian PADs have been extracted from human, rat,

rabbit, guinea pig, chicken and sheep tissues. There are five PAD isotypes produced by a

variety of human cells. PAD-1 is mainly expressed in the uterus and epidermal tissue (Guerrin

et al. 2003; Rus'd et al. 1999; Terakawa et al. 1991). PAD-2 is a widely expressed PAD, and is

most abundant in the cells of tissues such as skeletal muscle, brain, uterus, salivary glands and

14

pancreas (Foulquier et al. 2007; Terakawa et al. 1991). PAD-3 is located in hair follicles and

epidermis (Kanno et al. 2000; Rogers et al. 1997; Rus'd et al. 1999; Terakawa et al. 1991).

PAD-4, formerly known as PAD-5, is found primarily in haematopoietic cells such as

eosinophils, neutrophils, lymphocytes and monocytic cells (Asaga et al. 2001; Foulquier et al.

2007). The most recently discovered PAD enzyme is PAD-6 expressed in ovary and testis

tissues, small intestine, spleen, lung, liver and skeletal muscle cells (Chavanas et al. 2004;

Zhang et al. 2004).

The first purification of P. gingivalis PAD (PgPAD) was reported in 1999 by McGraw et al.

(McGraw et al. 1999). The enzyme, although not evolutionarily related to the mammalian

PADs, catalyses the same chemical reaction, the modification of the guanidino group of

arginine residues from various peptides to produce ammonia (shown in Figure 1.3).

Figure 1.3: Citrullination activity by P. gingivalis PAD.

This was modified from Shirai et al., 2001

15

Although catalysing the same reaction, P. gingivalis and mammalian PADs do not share the

same catalytic mechanism. Mammalian PADs are metalloenzymes and require calcium ions for

activation, in contrast to PgPAD, which can modify arginine residues in the absence of calcium

(McGraw et al. 1999; Takahara et al. 1986). Furthermore, mammalian PADs are unable to

convert free arginine to citrulline, unlike PgPAD, that has been reported to catalyze conversion

of both peptide-bound arginine and free arginine to citrulline containing peptides and free

citrulline, respectively. As with all enzymes, PADs are influenced by environmental pH. The

activity of purified PgPAD was optimal at pH 9.3 (McGraw et al. 1999). Interestingly,

Nakayama-Hamada and colleagues reported that PAD-2 had an optimal activity between pH 6

and 10 while PAD-4 is active between pH 6.5 and 9 (Nakayama-Hamada et al. 2005)

suggesting that all PADs prefer an alkaline pH environment for maximal activity. Mammalian

PADs are located intracellularly whereas 90% of PgPAD activity has been reported to be cell or

membrane vesicle associated (McGraw et al. 1999).

1.3.3 Peptide metabolism by Porphyromonas gingivalis

The metabolism of energy-yielding peptides by P. gingivalis is vital for growth of the

microorganism as it is unable to utilize glucose as source of energy (Masuda et al. 2001; Shah

and Gharbia 1989; Shah and Williams 1987). Evidence of the asaccharolytic nature of P.

gingivalis is provided from a study showing that growth yields of the organism were constant in

the presence or absence of glucose (Takahashi and Schachtele 1990). A number of studies

have shown that P. gingivalis prefers to utilise peptides over free amino acids (Milner et al.

1996; Takahashi et al. 2000; Wyss 1992). P. gingivalis is reported to be able to generate ATP

via catabolism of amino acids and releases the ammonia it produces as a result of its

proteolytic metabolism to the environment (Shah and Williams 1987). This end product alters

16

the environment by elevating the local pH to a more favourable alkaline growth condition

(Takahashi and Schachtele 1990) as the bacterium showed a stable growth over a pH range

from 6.7 to 9 when cultured in haemin-excess conditions in a chemostat (McDermid et al.

1988).

The metabolism of arginine via the arginine deiminase pathway has been suggested as a

mechanism to produce energy from free arginine in P. gingivalis (Masuda et al. 2001). In

addition to arginine, glutamate- and aspartate-containing peptides are metabolised by P.

gingivalis and result in the production of a mixture of cytotoxic fatty acid metabolic end

products. Of these, butanoate is considered one of the most toxic to host tissues (Niederman et

al. 1997).

1.4 Citrullination

More than 70 years ago Fearon was the first to describe the conversion of arginine to citrulline

(Fearon 1939). Citrullination involves modification of the guanidino group of arginine,

specifically by the replacement of a nitrogen-containing group by an oxygen atom in the side

chain of the amino acid. This conversion also results in the loss of a positive charge, therefore

citrullinated proteins lose charge, are unfolded and interference in organized protein structure is

promoted (Tarcsa et al. 1996). The citrullination reaction is catalysed by PADs and occurs

when protein-bound arginine or free arginine are converted into protein-bound citrulline or free

citrulline, respectively. Ammonia is produced as by-product of the reaction.

17

Figure 1.4: The process of citrullination of arginine to citrulline catalysed by mammalian PAD.

The diagram was modified from Masson-Bessi`ere et al., 2001.

1.4.1 Citrullination and the immune system

The human immune response is highly complex and functions to protect the individual from

foreign cells and molecules detected in the body. Occasionally, this response can cause

damage to host tissues when the immune system misinterprets host tissue components as

foreign and initiates an inflammatory response. This type of response against host tissue is

known as an autoimmune disease.

Citrulline is classified as a non-coded amino acid that is not incorporated during the translation

of protein. Citrullination leads to post-translationally modified proteins and peptides as their

formation occurs subsequent to protein synthesis. Enzymatic citrullination abolishes positive

changes contained within native proteins, inevitably causing significant alterations in their

tertiary structures and loss of function. Although the conversion results in a relatively small

NOTE: This figure is included on page 17 of the print copy of the thesis held in the University of Adelaide Library.

18

chemical alteration to the protein involved, the reactivity of auto-antibodies specific for citrulline-

containing epitopes seems to be critically dependent on the presence of a citrulline residues.

Peptides that are post-translationally modified to contain citrulline will exhibit different epitopes

when compared to those of containing normal peptide-bound arginine residues (Masson-

Bessiere et al. 2001). It is possible that these modified proteins and peptides can be detected

as foreign antigens by the host’s immune system (Doyle and Mamula 2002).

The reaction catalysed by PADs, the modification of arginine residues to citrulline, has been

reported to trigger the host immune response (Girbal-Neuhauser et al. 1999; Schellekens et al.

1998). Accordingly, PAD activity has been linked to the initiation of a number of inflammatory

diseases, with citrullinated proteins proposed as a linking autoantigen (Masson-Bessiere et al.

2000; Nienhuis et al. 1964). The reactivity of auto-antibodies towards citrulline-containing

epitopes is critically dependent on the presence of a citrulline residue.

1.4.2 Citrullinated human proteins

Previous studies have shown the effect of citrullination on the immune response (Lundberg et

al. 2005; van Boekel et al. 2002). Recently, the presence of citrullinated peptides has been

reported to be associated with a number of inflammatory disorders (Chapuy-Regaud et al.

2005; Makrygiannakis et al. 2006; Wanchu et al. 2001). The presence of citrullinated peptides

in inflamed tissues has been proposed as a general sign of inflammation (Kinloch et al. 2008)

as they can be detected in synovial membranes in patients with rheumatoid arthritis (Chapuy-

Regaud et al. 2005), systemic lupus erythematosus (SLE)(Wanchu et al. 2001), and Sjögren

syndrome (Nissinen et al. 2003).

Anti-citrullinated cyclic protein (anti-CCP) antibodies are produced by rheumatoid arthritis

patients against citrulline-containing epitopes (Girbal-Neuhauser et al. 1999; Schellekens et al.

1998). Masson-Bessi`ere and colleagues reported the presence of citrulline in joint synovium

19

and suggested it becomes a potential target for IgG antibodies (Masson-Bessiere et al. 2001).

Citrullinated proteins can be detected in the leukocyte infiltrate in inflamed synovium and fibrin,

along with other proteins, is citrullinated during the inflammation of mouse synovium

(Vossenaar et al. 2003). Interestingly, the antibody response to citrullinated proteins is highly

specific for RA and may be involved in the perpetuation of the human disease. Their detection

has also provided hope for better diagnosis of the disease.

There are several citrullinated proteins that are present naturally in the body. Filaggrin is a 40

kDa protein that is found the in human epidermis and was identified as a neutral/acidic isoform

of pro-filaggrin (Simon et al. 1993). Tarcsa reported that filaggrin is mostly β-turn in structure,

but after complete citrullination, the shape became flat, which is indicative of the loss of

organized structure that can arise following modification mentioned previously (Tarcsa et al.

1996). The presence of filaggrin in humans can be detected by anti-perinuclear factor and anti-

keratin antibody tests (Vincent et al. 1999). In addition to filaggrin, vimentin, a structural

component of the intermediate filaments, is also citrullinated in synovial sites (Bang et al.

2007). The antigenic properties of vimentin are substantially activated by citrullination and are

thought to be the target of anti-Sa antibodies (Vossenaar et al. 2004).

20

1.5 Rheumatoid Arthritis

1.5.1 Introduction and history

Rheumatoid arthritis is a well-known autoimmune chronic inflammatory disorder that affects

humans of all ages and races with a prevalence of 1% in the world population (Silman and

Pearson 2002). The first clinical description of RA was reported by Augustin-Jacob Landre-

Beauvais in 1800 (Landre-Beauvais 2001). RA is believed to have existed more than 3,000

years ago with the finding of ancient skeletons that’s showed evident of RA in North America.

There are certain ethnic groups that suffer increased prevalence of RA, for example Pima

(5.3%) and Chippewa (6.8%) Indians; two Native American tribes exhibit the highest rates of

disease compared to other groups. In Europe, the evidence of RA-like symptoms can be seen

in 17th century art as shown in a family portrait of a Dutch artist dated 1622 (Figure 1.5).

21

Figure 1.5: La Familia de Jordaens en un Jardín Jacob Jordaens

Note from this portrait the maid’s fingers showed the evident of features of RA; swelling of the metacarpal-phalangeal and proximal interphalangeal joints (Firestein 2003).

A review of literature shows that, the incidence of RA is two-fold higher in women than men,

with Gabriel reporting that females had a prevalence of RA of 1.37% compared to 0.74% in

22

males (Gabriel 2001). In addition, there is approximately double the risk of RA for

postmenopausal women (Criswell et al. 2002). This finding has led some to suggest sex

hormones play a role in the development of RA.

Genetic inherence has been proposed as an important factor, as the disease tends to cluster in

families. There are several environmental factors associated with an increased incidence of

RA, including smoking (Heliovaara et al. 1993; Klareskog et al. 2006) and obesity (Voigt et al.

1994). In contrast, alcohol consumption, which was previously reported as one of the causes of

RA (Hazes et al. 1990), has now been shown to be assiociated with decreased risk for the

disease (Kallberg et al. 2009; Voigt et al. 1994).

Smoking has been suggested as an environmental trigger and a risk factor for numerous

illnesses such as myocardial infarction (Prescott et al. 1998), diabetes mellitus (Will et al. 2001)

and PD. A Finnish study suggested that exposure to tobacco smoke, or factors associated with

smoking, may trigger the production of rheumatoid factors in males (Heliovaara et al. 1993) and

in women who had smoked more than 15 cigarettes per day with a 2.5-fold increase in the risk

of contracting RA (Vessey et al. 1987). Interestingly, there is evidence that shows smoking

promote the production of citrullinated peptides which may activate the host immune system

(Klareskog et al. 2006). Although these findings demonstrate the increased risk of RA in

individuals exposed to cigarette smoke, further investigation is needed to determine its cause

as RA is a multi-factorial disease.

No aetiological agent has been identified and there are no unique clinical or laboratory features

that can be used to define this disease clearly. In order to more accurately estimate the

prevalence of RA, American Rheumatoid Association provided classification criteria in 1956

and revised them in 1987, 1988 and 2010 (Aletaha et al. 2010; Arnett et al. 1988).

23

Table 1.1: The 2010 American College of Rheumatology/ European League against rheumatism classification criteria for rheumatoid arthritis.

NOTE: This table is included on page 23 of the print copy of the thesis held in the University of Adelaide Library.

24

1.5.2 Features of rheumatoid arthritis

RA presents as a series of symptoms including redness; symmetrical joint swelling that is warm to

touch (Arnett et al. 1988), joint deformity and weight loss (Munro and Capell 1997). The sufferer may

commonly experience fatigue, malaise, joint pain and morning stiffness. This debilitating chronic

inflammatory disorder primarily has a focus on the synovial joints, leading to joint swelling,

progressive joint erosions eventually leading to disability and changes to the synovium, as shown in

Figure 1.6. The synovial membrane in RA patients is characterized by hyperplasia, increased

vascularity, and an infiltrate of inflammatory cells. Individuals with RA also demonstrate moderate to

severe periodontal bone loss (Mercado et al. 2001).

Figure 1.6 Changes that occur in the synovial joint as a result of RA.

25

Early erosion of cartilage and bone is occupied predominantly by activated macrophages and T

cells. The T cells stimulate monocytes, macrophages, and synovial fibroblasts to produce the

cytokines such as interleukin-1, interleukin-6, and TNF- . The cells also secrete matrix

metalloproteinases. Cytokines enhance the expression of adhesion molecules on endothelial cells

and increase the recruitment of inflammatory cells such as macrophages, lymphocytes, fibroblasts

and neutrophils into the joints. Neutrophils release proteases, which degrade proteoglycan in the

superficial layer of cartilage. Vascularity is increased in the synovium of patients with RA by means

of the stimulation of angiogenesis.

This disease shares similar features with adult periodontitis (reviewed by (Bartold et al. 2005;

Mercado et al. 2000)). Mercado (2000) reported that RA patients are more likely to experience more

significance periodontitis and vice versa. It is of interest to note that the presence of bacterial DNA

and peptidoglycan has been reported in the joints of RA patients (Van Der Heijden et al. 2000). As in

the PD, bacteria may play an important role in determination of the disease severity. Mercado (2001)

has proposed an underpinning commonality of dysregulation of the immune system between

periodontitis and RA (Mercado et al. 2001).

1.6 Rheumatoid Arthritis and Citrullination

RA affected individuals have been found to express PAD-4 and citrullinated peptides can be

detected in their synovial tissue. Chang and colleagues reported in their findings that PAD-4 is

expressed by T cells, B cells, macrophages, neutrophils, fibroblast-like cells and endothelial cells in

the lining and sub-lining areas of the RA synovium (Asaga et al. 2001; Chang et al. 2005). The

production of post-translationally citrullinated peptides may trigger the body’s immune system, to

attack what appear to be foreign epitopes and thus cause inflammation of the synovium. Hence,

26

detection of citrullinated peptides which are specifically present in the sera of RA patients might help

in the early detection of RA.

Previous studies have shown the effect of citrullination on the autoimmune response (Lundberg et al.

2005; van Boekel et al. 2002). Anti-citrullinated cyclic protein (anti-CCP) antibodies are produced by

RA patients against citrulline-containing epitopes (Girbal-Neuhauser et al. 1999; Schellekens et al.

1998). Masson-Bessi`ere and colleagues reported the presence of citrullinated peptides in joint

synovium and their potential as targets for IgG antibodies (Masson-Bessiere et al. 2001).

1.6.1 Citrullination in RA patients and clinical markers

Cytoplasmic PADs can be activated by stimulation of cells with a calcium ionophore (Asaga et al.

1998). In the inflamed synovium, many cells undergo apoptosis or necrosis and as the membrane

integrity is lost during cell death, Ca2+ can easily enter and activate the PAD. Alternatively, PAD

enzymes may also be released from dying cells and become activated as the extracellular Ca2+

concentration is approximately 1 mM, and thus induce the citrullination of extracellular proteins such

as fibrin (Bongartz et al. 2007).

Rheumatoid factor (RF) is an antibody that binds to the Fc portion of immunoglobulin and was first

identified by Eric Waaler in 1939. Historically, RF has been used as a marker in the early detection

of RA. However, approximately 25% of RA patients are RF negative, and reports have shown that

RF can also be found in other diseases in addition to 3-5% of the healthy population (Mageed et al.

1997). Due to this lack of combined specificity and sensitivity, many studies have been undertaken

in search of a more accurate indicator for RA.

Recently, the presence of autoantibodies against citrullinated proteins have being used to aid in the

diagnosis of RA with 80% higher specificity and sensitivity than diagnosis using RF (Suzuki et al.

2007; van Boekel et al. 2002). When using citrulline-containing peptide variants in ELISA there are

autoantibodies detected in 76% of RA sera with a specificity of 96% (Schellekens et al. 1998). The

27

antibodies showed reactivity towards citrullinated peptides and grouped in anti-citrullinated

protein/peptide antibodies (ACPAs). There are several types of ACPAs that have been determined,

such as anti-perinuclear factor (APF) (Nienhuis et al. 1964), anti-keratin antibody (AKA) (Young et al.

1979), anti-Sa (Despres et al. 1994) and anti-CCP antibodies (Schellekens et al. 1998). All of these

have a high specificity for citrullinated peptides.

1.7 Research Questions

There has been much recent interest in possible links between in the inflammatory diseases PD and

RA. A review of the literature has shown that citrullination appears to be an important factor in the

inflammatory process. As P. gingivalis is a significant pathogen in the aetiology of PD and

possesses the ability to citrullinate via PAD activity the following research questions were posed.

Is PgPAD able to citrullinate arginine containing peptides and RA relevant host proteins?

Does exposure to PgPAD exacerbate inflammation in mammalian tissues via host protein

citrullination?

28

CHAPTER 2

29

2 Investigation of Porphyromonas gingivalis

Peptidylarginine Deiminase

P. gingivalis is the only prokaryote known to express PAD activity to date. PgPAD was investigated

by McGraw and colleagues at the end of last decade and this group was the first to partially

characterise the purified enzyme (McGraw et al. 1999). As discussed in the previous chapter the oral

anaerobe P. gingivalis has long been implicated as one of the key pathogens in the aetiology of

human PD, however little work has been undertaken to investigate the specific involvement of

PgPAD in the inflammatory disease process. Due to the recent interest in citrullination in relation to

RA and the suggested links between this disease and PD, it was decided to further investigate the

enzyme and its potential role as a virulence factor in inflammatory disease, RA in particular.

As this study proposed to survey scavenged tissues obtained from a parallel study investigating the

effect of P.gingivalis in the rat adjuvant arthritis model, the main focus of this chapter was to further

characterise the activity of the enzyme peptidylarginine deiminase in intact cells of the organism.

Accordingly, the following hypotheses were proposed;

� P. gingivalis cells express surface-associated PAD activity

� The enzyme is active over a biologically relevant pH range and is heat stable

� PgPAD is highly specific for arginine

� PgPAD preferentially citrullinates C-terminal arginine residues in peptides

� PgPAD is able to citrullinate arginine residues in RA relevant proteins; and

� The gingipains influence the rate of citrullination of proteins by PgPAD.

30

It should be noted that, to ensure reliability and consistency of results, all analyses in this chapter

were performed at least in triplicate and the data points are expressed as means of these

determinations. The primary results for each the following experiments can be found in Appendix 2.

2.1 Citrulline Assay

In order to investigate the activity of PgPAD, a reliable method for the detection and quantifying of

citrulline in assay samples was required. Therefore, the initial work conducted in this study was to

develop such a method. Accordingly, a citrulline assay to detect and measure citrulline in samples

was adapted and modified from that employed by McGraw (Boyde and Rahmatullah 1980; McGraw

et al. 1999). This method was further developed to facilitate the measurement of citrulline in the

small reaction volumes of microtitre plates.

2.1.1 Colour detection reagent

The carbamino detection reagent, used in this assay was prepared daily, prior to use, for the

detection of citrulline. Briefly, one part of solution A, consisting of 0.5% (v/v) diacetyl monoximine

(Sigma, USA) and 0.01% (w/v) thiosemicarbazide (Sigma, USA), was added to two parts of solution

B, consisting of 0.25 mg.mL-1 of FeCl3 (Sigma, USA), in a solution containing 24.5% (v/v) sulphuric

acid (Ajax Chemicals) and 17% (v/v) phosphoric acid (Ajax Chemicals). During the method

development process it was discovered that the resulting colour detection reagent was unstable,

changing from a colourless to a yellowish liquid over time and so, it was necessary to use this

reagent within 1 hour of the preparation of Part A.

31

2.1.2 Standard curve to determine citrulline concentration

In order to measure citrulline concentrations in unknown samples, a standard curve was constructed

by the addition 100 μL of varying amounts of L-citrulline (Sigma, USA), to microtitre plate wells using

2-fold dilution. The citrulline was dissolved in incubation buffer, containing 1 mM EDTA (Sigma,

USA), 10 mM cysteine (Sigma, USA), 1 μM FMN (Sigma, USA) in 0.2 M Tris-HCl (Sigma, USA) at

pH 8.0. To each well 100 μL of the colour detection reagent was added and the plates were

incubated at 100oC for 5 minutes to hasten colour development. The samples were allowed to cool

for 10 minutes following incubation and the absorbance of samples was determined at a wavelength

of 530 nm using a BIO-TEK Powerwave XS microtitre plate reader (Crown Scientific, NSW).

2.1.2.1 Results

The absorbance of samples was linear over the range of concentrations used, representing 0.5 to 16

nmoles of citrulline (Figure 2.1). Subsequently, this standard curve was used to quantify the amount

of citrulline produced by PAD in samples.

32

Figure 2.1: A standard curve for the colourimetric determination of citrulline concentration.

33

2.2 Citrullination by Mammalian Peptidylarginine Deiminase

2.2.1 Citrullination of BAEE by mPAD

Six mammalian PADs have been identified to date, and as with PgPAD, this family of enzymes are

characterised by their ability to citrullinate peptide-bound arginine residues (Raijmakers et al. 2007).

An early study by Takahara showed that rabbit muscle PAD (mPAD) (Sigma, USA) is a calcium

dependant enzyme (Takahara et al. 1986) and others have shown the enzyme is able to catalyze

the citrullination of the synthetic substrate benzoyl-arginine ethyl ester (BAEE) (Nakayama-Hamada

et al. 2005; Raijmakers et al. 2007). To ensure that BAEE was a valid substrate for the

measurement of PAD activity, mPAD, was used to confirm the suitability of this substrate for

subsequent enzyme characterisation experiments.

2.2.1.1 Method

The mPAD was stored in a buffered aqueous glycerol solution consisting of 20 mM Tris-HCl; pH 7.4

containing 10 mM 2-mercaptoethanol, 1 mM EDTA and 10% glycerol (v/v). Tris-HCl was included to

protect the enzyme against adverse pH change and the other ingredients conferred thermal stability,

which is important to prevent loss of activity from multiple freezes thawing cycles, or elevated

storage temperature. The manufacturer’s instructions indicated that the freezing and thawing

process would not normally adversely affect enzyme activity if the solution was routinely stored at -

80oC degrees.

To measure citrullination of BAEE by mPAD, a modified method from Takahara was used (Takahara

et al. 1986). Briefly, a working buffer was prepared containing 70 mM CaCl2 and 70 mM dithiothreitol

(Sigma, USA) in 350 mM Tris-HCl adjusted to pH 7.2. In order to confirm the previously reported

calcium dependence of the enzyme an additional working buffer was prepared excluding CaCl2.

34

To prepare the enzyme solution for the citrullination assay, immediately prior to commencing the

reaction, 2 μL of mPAD (0.3 Units) was mixed with cold 0.1% (w/v) bovine serum albumin (BSA).

(Sigma, USA). The solution was then pre-incubated in working buffer for 2 minutes at 55oC resulting

in a final volume of 900 μL. Subsequently, 100 μL of 50 mM BAEE was added to the reaction

solution, prior to incubation at 55oC for 60 minutes. BAEE in incubation buffer, in the absence of

enzyme, was used as a negative control. Over a one hour period, 25 μL aliquots were removed from

the reaction mixture at 10 minutes intervals and immediately placed into the wells of a microtitre

plate. All wells contained 20 μl of 10 μM EDTA in order to stop the reaction. To measure the

citrullination of BAEE, the colour detection reagent was prepared as reported in Section 2.1.1 and

100 μL of this solution was added to samples and incubated for 5 minutes at 100oC. Following

incubation, the absorbance of samples at a wavelength of 530 nm was measured using the

microtitre plate reader.

2.2.1.2 Results

The results of this experiment are displayed in Figure 2.2. As the change in absorbance over time

was linear, the rate of reaction was constant for the period of incubation. Of the two solutions used to

prepare mPAD for assay, only reaction mixtures containing calcium ions in the working buffer

exhibited a significant increase in absorbance over the time. A reaction mixture containing BAEE

without added enzyme also showed no increase in absorbance. Accordingly, the rate of citrullination

of BAEE by mPAD in the presence of CaCl2 was calculated to be 0.072 nmoles citrulline.unit-1.min-1.

35

Figure 2.2: The citrullination of BAEE by rabbit muscle PAD.

The solid blue line shows the reaction in the sample in the presence of calcium and the dashed red line represents the reaction in its absence.

36

2.2.2 Citrullination of free arginine by mPAD

Mammalian PADs have also been reported to be unable to citrullinate free arginine. In order to

confirm this finding, free L-arginine, dissolved in incubation buffer, was used as a substrate for

mPAD, replacing BAEE.

2.2.2.1 Method

Free arginine at a final concentration of 5 mM was used as the substrate for the reaction and

incubated with mPAD prepared in working buffer containing CaCl2. Control samples containing

BAEE (5 mM) were incubated with the enzyme in the presence and absence of calcium and the

method described in Section 2.2.1 was employed in order to measure the rate of citrullination.

2.2.2.2 Results

The change in absorbance at 530 nm of samples over time, for each test condition, is displayed in

Figure 2.3. A comparison of the rates of reaction of mPAD for the substrates BAEE and arginine, in

the presence of calcium, revealed only samples containing the former exhibited in significant rates of

citrullination. No activity was observed when mPAD was incubated with free arginine, thus

confirming the previous report that mPAD was unable to citrullinate the free amino acid, even in the

presence of calcium ions (Takahara et al. 1986).

37

Figure 2.3: The citrullination activity of mPAD against free arginine.

The blue solid line represents sample of arginine incubated with calcium while the red dashed line is for sample of BAEE without calcium (control), and BAEE incubated with calcium for green dashed dot lines.

38

2.2.3 Discussion

These preliminary experiments were carried out as confirmatory steps, required for the future

investigation of PgPAD activity. The assay used to detect citrulline was validated and it was also

determined that BAEE was a suitable substrate for measuring the rate of citrullination by PAD. The

results also confirmed the previously reported calcium ion dependency of mammalian enzymes and

that mPAD was unable to citrullinate free arginine (Kuhn et al. 2006; Raijmakers et al. 2007;

Vossenaar et al. 2003). These findings proved that the citrulline assay used was able to detect

citrullinated residues and could therefore be used for future experiments with P. gingivalis with

confidence.

2.3 Cultivation of Porphyromonas gingivalis

The Porphyromonas gingivalis strain W50 was employed in this study of PgPAD. The bacterium was

maintained by weekly subculture on anaerobic blood agar (Oxoid, Australia) by incubation at 37oC in

an anaerobic jar containing an atmosphere of 90% N2:5% H2:5% CO2. Following growth on solid

medium, several bacterial colonies from the agar plate were selected using a sterile wire loop and

cultured in brain heart infusion (BHI) broth(Oxoid, Australia) enriched with 5 mg.L-1 haemin (Sigma,

USA) and 0.5 g.L-1 cysteine (Sigma, USA), at 37oC for 48 hours in atmosphere of 90% N2:5% H2:5%

CO2. Following growth, the P. gingivalis culture was checked for purity by Gram staining prior to cell

harvesting (Figure 2.4).

39

Figure 2.4: A Gram stain of P. gingivalis W50 under light microscopy oil immersion at 1,000 x magnification.

40

2.4 Cell Harvesting and Preparation

P. gingivalis cells were prepared for the enzyme assay according to the following protocol. The

optical density of the cells in BHI broth was determined at a wavelength of 560 nm (OD560) using

Lambda 5 UV/Vis Spectrophotometer (Perkin-Elmer, USA) and adjusted to an OD560 of 1

absorbance unit (AU). Tubes containing 10 mL of cells were then centrifuged at 10,000 g for 10

minutes at 4oC and the resultant pellet suspended and washed in the incubation buffer. The resulting

cell suspension was centrifuged once again, employing the same conditions as described previously

and the washed cell pellet was suspended in 1 mL of fresh incubation buffer prior to the assessment

of PAD activity. A protein assay was utilised to determine the quantity of protein in cell samples.

2.5 Protein Assay

2.5.1 Method

An aliquot of cell suspension was sonicated using a SONIPROBE sonicator (DAWE Instrument,

England) for five cycles of 10 seconds to disrupt the cells prior to protein estimation. The samples

were kept on ice during processing and cell disruption was confirmed by Gram staining the

sonicated sample. The protein concentrations of sonicated samples were determined using a

Coomassie plus® protein assay kit (Thermo scientific, USA) using BSA as a protein standard (refer

for detailed method to Appendix 1.1).

2.5.2 Results

The absorbance of the protein standard solutions was measured at a wavelength of 595 nm and the

mean readings were calculated and a standard curve for protein concentration was plotted (Figure

2.5).

41

The absorbance of the concentrated P. gingivalis cell samples at a wavelength of 595 nm was 0.711

AU, a value equating to 0.67 mg.mL-1 of protein when calculated using the equation of the trend line

generated from the standard plot (Figure 2.5).

42

Figure 2.5: The standard curve of protein concentration at a wavelength of 595 nm.

The Coomassie protein assay is showing absorbance readings for a range of BSA concentrations.

43

2.6 Citrullination of