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The Effect of Treponema Denticola on Porphyromonas Gingivalis Phenotypes and Transcriptome

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The Effect of Treponema Denticola on Porphyromonas Gingivalis Phenotypes and Transcriptome The Effect of Treponema denticola on Porphyromonas gingivalis Phenotypes and Transcriptome Lin Xin Kin ORCID identifier 0000-0003-2378-1711 Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy October 2018 Oral Health CRC Melbourne Dental School Faculty of Medicine, Dentistry and Health Sciences The University of Melbourne Abstract Chronic periodontitis is an inflammatory, bacterial biofilm-associated disease resulting in destruction of the tooth’s supporting tissues. The imminent progression of chronic periodontitis can be predicted by the levels of Porphyromonas gingivalis and Treponema denticola in subgingival plaque. Living in a complex oral polymicrobial community, these two bacterial species display close association via physical interaction and metabolic cooperativity in the biosynthesis and cross-feeding of growth substrates. These interspecies interactions result in the coordination of their physiological activities, some of which exhibit complementary and combinatory effects in enhancing their growth and virulence factors. A previous study demonstrated that coculture of T. denticola and P. gingivalis in a continuous system led to upregulation of T. denticola glycine utilisation systems. Likewise, P. gingivalis increased the production of free glycine by proteolytic hydrolysis of peptide-bound glycine during growth in T. denticola conditioned medium (TdCM), suggesting cross-feeding of glycine from P. gingivalis to T. denticola. Free glycine is an important nutrient source for T. denticola, contributing to a dramatic increase in T. denticola growth rate and biomass. Given that P. gingivalis glycine release was stimulated in TdCM, this study aimed to determine and characterise the T. denticola stimulatory factors that had been released into TdCM. TdCM was fractionated by size filtration and reversed-phase high-performance liquid chromatography (RP-HPLC) to determine the most active stimulatory fractions. The free glycine produced by P. gingivalis in different TdCM fractions was then quantified using a glycine enzyme-linked immunosorbent assay (ELISA) kit and liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS). As several RP-HPLC fractions of TdCM appeared to stimulate the release of free glycine by P. gingivalis, this indicated that there was no specific signalling molecule that stimulated P. gingivalis to produce glycine. Instead, the release of glycine by P. gingivalis was likely due to P. gingivalis proteinases that further digested the peptides that had been partially processed by T. denticola proteases during growth in the medium. Candidate peptidases of P. gingivalis that could be involved in the hydrolysis of glycine-containing peptides into free glycine were selected by bioinformatic predictions of the localisation and specificities of P. gingivalis putative peptidases. Interestingly, inactivation of PG0753 and PG1788 that encode putative PrtQ collagenase and C1 family cysteine peptidase respectively, showed a reduction in the rate of free glycine production relative to P. gingivalis I calculated cell number in OB:CM, relative to wild type. These results indicated that these two peptidases might play a role in the release of free glycine by P. gingivalis in the presence of T. denticola stimulatory factors. Other potential multimodal interactions of T. denticola and P. gingivalis were examined by investigating the differential gene expression profiles of P. gingivalis during growth in TdCM, compared to oral bacterial growth medium (OBGM). There were a total of 132 genes that showed differential expression, which included transcripts related to metabolic pathways, signal transduction systems, transcription regulatory systems, nucleic acid interacting protein encoding genes, transporters and hypothetical protein encoding genes. This work generated more testable hypotheses of the molecular effects of T. denticola on P. gingivalis gene expression and as well as potential mechanisms that might contribute to P. gingivalis and T. denticola physiological interactions. 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 tables, maps, bibliographies and appendices. Lin Xin Kin III Preface This thesis is submitted for the degree of Doctor of Philosophy at the University of Melbourne. The research described herein was conducted as a continuation of Tan et al. (2014) work: Porphyromonas gingivalis and Treponema denticola Exhibit Metabolic Symbioses. This project was performed under the supervision of Dr. Nada Slakeski, Prof. Stuart Dashper and Dr. Catherine A. Butler in the Melbourne Dental School, from July 2014 until July 2018. Part of the contents of Chapter 1 have been published in a review article with Hong Min Ng as first co-author: *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. For Chapter 3, Dr. Kheng Tan guided me in the preparation of TdCMs and processing of samples for the metabolomic experiments. Ms. Komal Kanojia and Dr. Dedreia Tull from Metabolomics Australia provided service and assistance in the generation and analysis of data on metabolomics. Chapter 4, genomic library preparation and Ion Torrent sequencing were performed by Brigitte Hoffmann. The resulting sequencing reads were analysed by Dr. Catherine A. Butler. For Chapter 5, Micromon Monash and Monash Bioinformatics were paid to perform the RNA sequencing and analyse the results. RNA sequencing library preparation and sequencing were performed by Mr. Scott Coutts from Micromon Monash. The raw files were analysed by Mr. Kirill Tsyganov and Assoc. Prof. David Powell from the Monash Bioinformatics platform and output was shared on the Degust web tool. IV Acknowledgements To Yong Kai, my soulmate, companion, cheerleader, supporter, teacher and househusband for your support, encouragement and enthusiasm that have made our journeys in Australia fruitful and exciting. I would like to thank my mom, dad and family members for their love, understanding and moral support to me in the pursuit of this study and constantly reminding me to finish what I started. Thank you my supervisors, Dr. Nada Slakeski for accepting me as her PhD student, Prof. Stuart Dashper for entrusting me with this project and Dr. Catherine Butler for her warm hugs, patience and support in the laboratory. I am very appreciative for their dedication in proofreading and reviewing my thesis drafts. My sincere gratitude to Assoc. Prof Paul Veith, who has provided helpful suggestions and advice on the research direction. His guidance has been a valuable input in this work. I am grateful to all staff at Metabolomics Australia, especially Komal Kanojia and Dr. Dedreia Tull for their time and patience in helping me with the metabolomic data analysis. Mr. Scott Coutts and Mr. Mark Cauchi from Micromon Monash for performing the library preparation and RNA sequencing adeptly. Mr. Kirill Tsyganov and Assoc. Prof. David Powell from the Monash Bioinformatic platform for their quick response in answering questions I have on the RNA sequencing data. I would like to extend my gratitude to all past and present lab members. Dr. Kheng Tan for his suggestions and guidance in reproducing his work and continuing his research project. Mr. David Stanton for his guidance on the RP-HPLC. Ms. Brigitte Hoffmann for her contribution on the Ion Torrent sequencing. Dr. Alexis Gonzalez for tirelessly optimizing the protocol for bacterial cell enumeration using the flow cytometry. A great thank you to Ms. Deanne Catmull, Dr. Tanya D’Cruze, Dr. Christine Seers, Ms. Caroline Moore, Mr. Steve Cleal, Ms. Sze Wei Liu, Mr. William Singleton, Dr. Brent Ward and Dr. Jacqueline Heath, who have provided technical assistance and support in my studies. I am also truly thankful to other PhD students and friends, who provide me inspirations, emotional support and sweet memories. It is my pleasure to have both Hong Min and May Ju as my comrades in completing this PhD journey with me, and to have all the desserts they both made and shared with me. Thanks to Jia Min, Lilian, Mathew, Ros, Nidhi, Sayeed, Grace and Jason for all the lunch breaks, chats and laughter together. V Finally, I am grateful to the University of Melbourne for the Fee Remission Scholarship (MIFRS) and Melbourne International Research Scholarship (MIRS). I appreciated Melbourne Dental School, The University of Melbourne for the provision of studentship stipend support for the extension of my PhD candidature. Laureate Prof. Eric C. Reynolds AO and Oral Health Cooperative Research Centres (CRC) for the top-up scholarship and funding support in this research project. VI Table of Contents Abstract ...................................................................................................................................... I Declaration ............................................................................................................................... III Preface...................................................................................................................................... IV Acknowledgements ..................................................................................................................
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