Investigation of the role of Treponema denticola motility and uncharacterized protein TDE0659 in synergistic biofilm formation with Porphyromonas gingivalis Hong Min Ng ORCID 0000-0002-4957-1424 Doctor of Philosophy May 2018 Oral Health CRC Melbourne Dental School Faculty of Medicine, Dentistry and Health Sciences The University of Melbourne A thesis submitted in total fulfilment of the degree ABSTRACT Chronic periodontitis has a polymicrobial biofilm etiology and interactions between key oral bacterial species such as Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia contribute to disease progression. P. gingivalis and T. denticola both have a set of virulence factors that are believed to contribute to the initiation and development of disease. It has been previously shown that P. gingivalis and T. denticola exhibit strong synergy in growth, biofilm formation and virulence in an animal model of disease. The motility of T. denticola, although not considered as a classic virulence factor, is likely to be involved in synergistic biofilm development between P. gingivalis and T. denticola. In order to investigate this, an optimized protocol for the transformation of T. denticola was developed and used to produce a number of T. denticola mutants targeting the motility machinery. The resulting mutants lacked periplasmic flagella (∆flgE) or possessed periplasmic flagella that were either non-functional (∆motA and ∆motB) or non-regulatable (∆cheY). ∆cheY contained a large genomic excision and was thus omitted from the study, however analyses of ∆flgE, ∆motA and ∆motB showed that they were impaired in motility and growth. Quantitative proteomic analyses of mutant strains showed that the inactivation of these motility-associated genes, especially motA and motB, have far reaching effects beyond motility. The inactivation of motA and motB activated a cellular stress response in the mutants and directly or indirectly impacted the growth of the mutants through the change in abundance of a number of proteins. T. denticola motility mutant and WT strains were grown as mono- and dual-species biofilms with P. gingivalis. Results showed that T. denticola motility and/or spiral morphology are required for monospecies biofilm formation and T. denticola periplasmic flagella are essential for synergistic biofilm formation with P. gingivalis. Zones of clearing were observed between T. denticola sibling colonies grown close to one another on agar, similar to the sibling killing phenomenon observed in Paenibacillus dendritiformis. Given that a sibling killing phenomenon could have a considerable effect on biofilm formation, this phenomenon was investigated further in T. denticola. Although T. denticola was found to possess a homologue (TDE0659) of the dendritiformis sibling bacteriocin (DfsB) produced by P. dendritiformis, the protein did not undertake the same function in T. denticola. Construction and analysis of a T. denticola mutant strain lacking TDE0659 showed that the loss of this gene product from T. denticola did not prevent i adjacent sibling colonies from forming zones of clearing on agar, further the mutant was able to form monospecies biofilms similar to WT. However, the lack of TDE0659 prevented the incorporation of P. gingivalis in a dual-species static biofilm with T. denticola, indicating that TDE0659 is essential for promoting synergistic biofilm formation with P. gingivalis. Together, the findings from this study will further our understanding about how P. gingivalis and T. denticola interact with one another and proliferate during disease. ii DECLARATION This is to certify that: i. the thesis comprises only my original work towards the PhD except where indicated in the Preface, ii. due acknowledgement has been made in the text to all other material used, iii. the thesis is fewer than 100,000 words in length, exclusive of figures, tables, maps, bibliographies and appendices. Hong Min Ng iii PREFACE Several members of the Oral Health CRC, Melbourne Dental School and Bio21 Institute, The University of Melbourne contributed to the work contained within this thesis. Cloning and propagation of plasmid pCF382, a kind gift from Dr Christopher Fenno (School of Dentistry, University of Michigan), in E. coli hosts were carried out by Dr Kheng Hui Tan. Genome sequencing of T. denticola ATCC 33520, ∆flgE33520, ∆motA33520 and ∆motB33520 using the Ion Torrent Personal Genome Machine (PGM) was performed by Ms. Brigitte Hoffmann and the resulting sequencing reads were analyzed by Dr Catherine Butler (Section 2.12.1). Samples of T. denticola ATCC 35405, ATCC 33520, HL51, ∆flgE33520, ∆motA33520 and ∆motB33520 were prepared for cryo-EM and imaged by Dr Yu-Yen Chen (Section 2.12.4). Label-free proteomics by MaxQuant was carried out by Dr Paul Veith (Section 2.12.8). Statistical analysis of the data from the static biofilm assays of T. denticola ATCC 33520, ∆flgE33520, ∆motA33520 and ∆motB33520 using Kruskal-Wallis with Conover-Imam test was performed by Mr. Yong Kai Wong from RMIT University (Section 2.12.9). Real-time PCR for the enumeration of bacterial cells was conducted with the help of Ms. Lin Xin Kin (Section 2.12.10). Flow cell biofilm assays, imaging of biofilms and analysis of the confocal data sets with COMSTAT software were performed by Ms. Sze Wei Liu (Section 2.12.11 and Section 2.12.12). T. denticola HL51 was obtained from Dr Howard Kuramitsu. Part of the Introduction (Chapter 1) to this thesis and Figure 1.3 have been published as the review article, “Ng, H. M., Kin, L. X., Dashper, S. G., Slakeski, N., Butler, C. A. and Reynolds, E. C. (2016) 'Bacterial interactions in pathogenic subgingival plaque', Microbial Pathogenesis, 94, 60- 69”. The work contained within this thesis was supported by the NHMRC, the Oral Health CRC (established and supported under the Australian Government’s Cooperative Research Centers Program) and Melbourne Dental School, The University of Melbourne. iv ACKNOWLEDGEMENTS First and foremost, I would like to thank God for giving me the opportunity to undertake this PhD. I thank God for His abundant grace and mercy to me throughout this program. I acknowledged that without God, it is impossible for me to complete this PhD, for it is indeed a marathon that needs much perseverance and patience. God has sustained, encouraged and comforted me when all things seem to be not working. He has granted me peace that surpasses all understanding when I was worried and anxious. He has always provided me with a way out at His perfect timing and helped me to truly experience His grace and power. God is indeed the One who is in control of everything and “with God all things are possible” (Matthew 19:26, King James Version). I am grateful for the provisions of God over the past few years and I have lacked nothing under the care of my God Almighty. He has provided me with the financial support from the Research Training Program Scholarship (The University of Melbourne) and Oral Health CRC stipend top- up scholarship to help me with my tuition fees and allowance. God has also blessed me abundantly by enabling me to attend several local and international conferences with the financial support of both the Oral Health CRC and Melbourne Dental School. I thank God for the many people that He has sent to help me throughout this PhD program. My appreciation goes to my supervisors Dr Nada Slakeski, Dr Catherine Butler and Professor Stuart Dashper. They have supported and guided me through my PhD journey with much patience and kindness. Their commitment, knowledge and enthusiasm for research are much valued. Their willingness to spend time to discuss with me about my project and their efforts in guiding me to prepare the thesis are much appreciated. I would also like to thank Melbourne Laureate Professor Eric Reynolds AO FICD FTSE FRACDS for his support in this program. I am grateful for the kindness, support and advice from the fellow staff and students of the Oral Health CRC and Melbourne Dental School over the past four years. I would like to thank Ms. Caroline Moore, Mr. Steven Cleal, Ms. Brigitte Hoffmann, Ms. Deanne Catmull, Ms. Yan Tan and Dr. Kheng Hui Tan who had shared their expertise and knowledge in the laboratory work. Particular thanks go to Ms. Sze Wei Liu who has provided me with much help in the experiments especially near the end of the PhD. I am appreciative of the commitments of Dr Yu-Yen Chen, who has spent much time and effort, in obtaining the cryo-EM images. I am thankful also for the kindness and help from Dr. v Paul Veith and Ms. Dina Chen in mass spectrometry data acquisition and analysis and Dr Lianyi Zhang in helping me to get access to the spectrofluorometer in Bio21 Institute. Not forgetting Ms. Lin Xin Kin who is always ready and willing to lend a hand and whose friendship has made working in the lab a pleasure. Finally, I would like to thank God for the support and care from my family and relatives to undertake this PhD. A special appreciation goes to my housemate, friend and sister-in- Christ, Ms. May Ju Gui, who has undertaken her PhD together with me. I am grateful for her unceasing encouragement and support over the past four years. I thank God for sending me this faithful companion who has walked side by side with me, picked me up when I fell and shared the highs and lows of this PhD journey. Her company and sharing have made this PhD journey an enjoyable one. Most importantly, I thank God for drawing me to the saving knowledge of my Lord and Saviour Jesus Christ during the course of this PhD. His saving grace is the greatest gift that I have obtained throughout this course. vi Table of Contents Abstract…………………………………………………………………………………..i Declaration……………………………………………………………………………...iii Preface…………………………………………………………………………………..iv Acknowledgements……………………………………………………………………...v Table of contents……………………………………………………………………….vii Table of tables………………………………………………………………………….xv Table of figures……………………………………………………………………….xviii List of abbreviations………………………………………………………………….xxiii List of units…………………………………………………………………………..xxvii Chapter 1 Introduction .....................................................................................................