PPAD, Porphyromonas gingivalis and the Subgingival Microbiome in Periodontitis and Autoantibody-positive Individuals at Risk of Rheumatoid Arthritis Zijian Cheng Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Dentistry September, 2018 The candidate confirms that the work submitted is his own and that appropriate credit has been given where reference has been made to the work of others. - ii - Acknowledgements It is a pleasure to thank a number of people who have supported me through my PhD study. This thesis could not have been completed without their support. Firstly, I would like to express my sincere gratitude and appreciation to my supervisors, Dr. Thuy Do, Dr. Josephine Meade, Prof. Paul Emery and Prof. Deirdre Devine for their valuable guidance, support and encouragement throughout my study. I am grateful to my colleagues and all staff in the Division of Oral Biology who are friendly and supportive, for making an enjoyable environment during this study period. Special thanks are extended to Shabnum Rashid from Oral Microbiology group and David Sharples from Faculty of Biological Science for the training and help with my lab work. Additionally, my appreciation is extended to Prof. Phil Marsh for his valuable help and suggestions. This research has been carried out by a team and I would like to thank all the clinicians, nurses and technicians from School of Dentistry, Leeds Dental Institute and Leeds Musculoskeletal Biomedical Research Unit, who have contributed to this project. I also wish to extend my special appreciation to the volunteers and patients who participated in this study. Most importantly, I am indebted to the Chinese Scholarship Council for financial support. I would also like to thank my friends in UK for their friendship that has made my life enjoyable. Finally, I would like to show my gratitude to my parents and family for their continuous encouragement and support with patience and love - iii - during this period. I would not have ventured to a foreign country for PhD study without their support. - iv - Abstract There is an epidemiological association between periodontitis and rheumatoid arthritis (RA). The subgingival microbiota may play an important role in the link between the two diseases. Porphyromonas gingivalis, which produces a peptidyl- arginine deiminase (PPAD) capable of citrullinating proteins, is considered a key organism inducing the production of antibodies against citrullinated proteins systemically and may initiate the pathogenic autoimmune responses associated with RA. The overall aim of this study was to explore the role of PPAD in P. gingivalis physiology and to better understand the links between P. gingivalis, periodontitis and risk of developing RA. P. gingivalis W83 and the corresponding Δppad mutant were grown in batch and continuous culture, to assess pH regulation, bacterial growth, gene expression and arginine gingipain (Rgp) and dipeptidyl-peptidase (DPP) activities. In a collaborative clinical study, the shotgun metagenomic approach was used to observe subgingival microbial profiles in individuals with and without periodontitis, with and without RA, and in those with autoantibodies against citrullinated peptides (CCP) at risk of developing RA. Based on in vitro studies, PPAD may citrullinate Rgp and DPP11, impair their activities and subsequently affect the alkali-promoting activity of P. gingivalis. Furthermore, both environmental pH and PPAD deficiency were able to regulate P. gingivalis gene expression, promoting adaptation to environmental changes and facilitating bacterial growth. In the clinical study, periodontitis occurred more often in anti-CCP positive at-risk individuals than in healthy controls and the subgingival - v - microbiomes of those individuals were perturbed, indicating that periodontitis and related microbial dysbiosis precede the onset of RA. P. gingivalis and its PPAD in established periodontitis conditions may play an important role in the initiation of RA. Moreover, PAD or PAD-like enzymes present in bacterial species other than P. gingivalis, e.g. Prevotella spp. exhibited some citrullination activity in vitro in a similar manner to PPAD. - vi - Table of Contents Acknowledgements .......................................................................................... ii Abstract ............................................................................................................ iv Table of Contents ............................................................................................ vi List of Tables .................................................................................................... x List of Figures .................................................................................................. xi Chapter 1 Introduction ................................................................................... 1 1.1 Overview of periodontal disease ......................................................... 2 1.2 Microbiology of periodontal disease .................................................. 4 1.2.1 Dental plaque and microbial communities ............................. 4 1.2.2 Identification of periodontal pathogens .................................. 6 1.2.3 Keystone pathogens ................................................................ 8 1.2.4 Aggressive periodontitis and Aggregatibacter actinomycetemcomitans .......................................................... 9 1.2.5 Porphyromonas gingivalis .................................................... 10 1.2.5.1 Capsule ...................................................................... 10 1.2.5.2 Fimbriae .................................................................... 11 1.2.5.3 Lipopolysaccharide (LPS) ........................................ 12 1.2.5.4 Gingipains ................................................................. 13 1.2.5.5 Exopeptidases ........................................................... 17 1.2.5.6 P. gingivalis peptidylarginine deiminase (PPAD) .... 19 1.3 Roles of neutrophils in periodontal diseases ..................................... 22 1.4 Rheumatoid arthritis (RA) ................................................................ 23 1.4.1 HLA-DR ............................................................................... 23 1.4.2 Smoking ................................................................................ 24 1.4.3 Autoantibodies associated with RA ...................................... 24 1.4.4 Individuals at risk of developing RA .................................... 26 1.5 The relationship between periodontitis and RA ................................ 27 1.5.1 P. gingivalis, RA and autoantibody production .................... 28 1.5.2 Other oral pathogens and multiple mechanisms underlying the link .................................................................................. 30 1.6 Aims of this study ............................................................................. 31 1.6.1 The aim of the in vitro study ................................................. 32 1.6.2 The aims of study of clinical samples ................................... 32 - vii - Chapter 2 Materials and Methods ................................................................ 33 2.1 Bacterial strains, storage and batch culture conditions ..................... 34 2.2 Continuous culture of P. gingivalis W83 and its Δppad mutant using a chemostat ..................................................................................... 36 2.2.1 Assembly of the chemostat ................................................... 36 2.2.2 Operation of the chemostat ................................................... 37 2.2.3 Sampling and analyses of the chemostat culture .................. 38 2.3 Measurement of bacterial enzyme activities ..................................... 38 2.3.1 P. gingivalis peptidylarginine deiminase (PPAD) ................ 38 2.3.2 Citrullination activity in different species ............................. 40 2.3.3 Dipeptidyl-peptidase (DPP) activity ..................................... 42 2.4 Analysis of gene expression .............................................................. 43 2.4.1 P. gingivalis DPP 5 and DPP 11 ........................................... 43 2.4.1.1 RNA extraction ......................................................... 43 2.4.1.2 DNase treatment ........................................................ 44 2.4.1.3 Precipitating RNA ..................................................... 44 2.4.1.4 Assessment of genomic DNA contamination ........... 44 2.4.1.5 cDNA synthesis and qRT-PCR ................................. 45 2.4.1.6 Relative quantitation ................................................. 46 2.4.2 RNA sequencing of P. gingivalis W83 and its Δppad mutant growing in chemostat ............................................................ 47 2.4.2.1 RNA extraction from stored samples ........................ 47 2.4.2.2 Library preparation ................................................... 47 2.4.2.3 Reads preprocessing and data analysis ..................... 48 2.5 Metagenomic study of subgingival plaques in relation to RA .......... 49 2.5.1 Ethical approval .................................................................... 49 2.5.2 Study participants .................................................................. 49 2.5.3 Collection and processing of subgingival dental plaque samples .................................................................................