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The Interaction of Porphyromonas Gingivalis with Host Epithelial Cells and Its Relevance to Periodontal Disease

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The Interaction of Porphyromonas Gingivalis with Host Epithelial Cells and Its Relevance to Periodontal Disease The interaction of Porphyromonas gingivalis with host epithelial cells and its relevance to periodontal disease By: Firas Bashir Hashim Al-Taweel A thesis submitted for the degree of Doctor of Philosophy in Periodontics June 2017 The University of Sheffield Faculty of Medicine, Dentistry and Health School of Clinical Dentistry Abstract Periodontitis is one of the most prevalent bacterial diseases affecting man with up to 90% of the global population affected. Its severe form can lead to the tooth loss in 10-15% of the population worldwide. The disease is caused by a dysbiosis of the local microbiota and one organism that contributes to this alteration in the bacterial population is Prophyromonas gingivalis. This organism possesses a range of virulence factors that appear to contribute to its growth and survival at a periodontal site amongst which is its ability to invade oral epithelial cells. Such an invasion strategy provides a means of evasion of host defence mechanisms, persistence at a site and the opportunity for dissemination to other sites in the mouth. However, previous studies have demonstrated that invasion of the mammalian cells in a population by P. gingivalis is heterogenous, with some cells becoming heavily invaded while others harbour no or only a few bacteria. An understanding of this heterogeneity may throw light on the mechanisms involved and we hypothesised that the phase of the host cell cycle may explain this phenomenon. In an attempt to study the factors influencing P. gingivalis invasion and the cell response to that invasion, a standard antibiotic protection assay was employed and an oral keratinocyte cell line, H357. The results showed that P. gingivalis NCTC 11834 invasion was significantly increased with increasing time of exposure to the cells and the cell density. This may reflect an increased host cell surface area available for bacterial attachment. No effect on invasion of P. gingivalis invasion was observed by the bacterial growth phase, H357 cell passage number or whether cells were pre- incubated with P. gingivalis lipopolysaccharide. Epithelial cells did, however, respond to the presence of P. gingivalis in a number of ways. For example, the mRNA expression of endothelin-1 and urokinase receptor were upregulated with increasing P. gingivalis infection time, suggesting that these proteins could act as inflammatory mediators and possibly as useful markers of the severity of periodontal disease or in the diagnosis and treatment of periodontitis. ii Secondly, in an attempt to investigate the reason for the observed heterogeneous P. gingivalis invasion of H357 cell populations, the effect of cell cycle phase on P. gingivalis invasion was investigated. H357 cells were synchronized by serum starvation. On re-introduction of serum, characterisation of cell cycle phase distribution was performed by flow cytometry following staining with propidium idodide (PI) or by immunofluorescence using bromodeoxyuridine (BrdU), which specifically identifies cells in S-phase. The effect of cell cycle phases on P. gingivalis invasion was measured using the antibiotic protection assay, immunofluorescence and flow cytometry and these were correlated with gene and surface expression of the urokinase receptor and the α5-integrin subunit, which is thought to mediate P. gingivalis invasion. Results showed that the percentage invasion was enhanced with increasing serum re-introduction time, and positively correlated with the number of cells in S-phase. In addition, flow cytometry data showed that the highest association of fluorescent P. gingivalis was with PI positive S-phase cells. Moreover, BrdU positive S-phase cells were 3 times more likely to be invaded and contained 10 times more P. gingivalis than cells in other phases. Also, α5-integrin was more highly expressed in cells in S-phase than other phases, which could explain the mechanism underlying this enhanced invasion. Data presented here have suggested that P. gingivalis targeting of cells in S- phase could, in vivo, allow preferential invasion of the junctional epithelial cells which turns over rapidly. The data presented in this thesis suggest that P. gingivalis invasion is greatly dependent on several factors attributed to the host, the bacteria itself, and to the environment which the bacteria reside in. The invasion occurs within a population of host cells in a heterogeneous fashion, and is dependent on the cell cycle phase, specifically S-phase. This novel finding, in addition to the previously reported mechanisms of P. gingivalis invasion, increases our understanding of this virulence trait and suggests that such a strategy is a highly organised process which the bacteria can follow to ensure its survival within the host. Furthermore, knowledge of these mechanisms could provide novel approaches to treatment of periodontal diseases. iii Acknowledgments Firstly, my sincerest gratitude and appreciation goes to my supervisors Dr. Simon Whawell and Prof. Ian Douglas for their attention to details, endless motivation and never ending scientific knowledge. Their kindness, unlimited support, guidance and encouragement throughout this study have been invaluable. I have been blessed to learn from the supportive staff of the Oral and Maxillofacial Department especially Dr. Dan Lambert. His scientific assistance with primer designing and the genetic statistical analysis was extremely helpful. My gratefulness also goes to Dr. Abigail Pinnock who has always supported me in the microbiology part of this study, and I will never forget her friendly fellowship and lovely moments that we spent together in the Department. My PhD journey would have been much more difficult without the amazing technical staff who are second to none. Particularly, the supportive role of Mr. Jason Heath and Mrs. Brenka McCabe has been indispensable regarding methodological problem solving and pleasant discussions. Also, the amusing talks with Mrs. Kirsty Franklin, who I have gained continuous guidance and support within the tissue culture lab, and the technical expertise from Mrs. Sue Newton and Mrs. Kay Hopkinson in the flow cytometry lab. I would also like to express my appreciation for all the advice, encouragement and funny moments I received throughout my PhD time from all the lovely friends I got to know throughout my study. My gratitude and love goes to my family especially my parents for supporting me spiritually throughout my life and giving me all the advice and encouragement I needed throughout my study. I would also like to thank my mother-in-law, Mrs. Suha for her support and wishing me all the best in my life, and I wish from the God Almighty to give her all the health she needs to recover quickly from her current illness. Ultimately, my life would be impossible including this PhD study without the support of my lovely wife, Albatool. Her patience, endless support, and continuous encouragement have been invaluable and greatly appreciated. For this, I like to dedicate this work to her and my gorgeous kids, Suha and Yousif. Last but not the least, I would like to express my gratitude for the Ministry of Higher Education and Scientific Research in Iraq for granting me this fantastic opportunity to finish my PhD study and excel academically, and I recognize that his research would not have been possible without it. iv Abbreviations ® Trade mark α Alpha β Beta γ Gamma μ Micro AAP American Academy of Periodontology APC Anaphase promoting complex BHI Brain heart infusion BrdU 5-bromo-2-deoxyuridine BSA Bovine serum albumin °C Centigrade CD Cluster of Differentiation CDKs Cyclin-dependent kinases cDNA Complementary Deoxyribonucleic Acid CKIs Cyclin-dependent kinase inhibitors CO2 Carbon dioxide CPS Capsular polysaccharide CR3 Complement receptor 3 CT Cycle Threshold DAPI 4', 6-diamidino-2-phenylindole DMEM Dulbecco’s Modified Eagle’s Medium DNA Deoxyribonucleic acid dNTP Deoxynucleotide Triphosphate ECM Extracellular matrix EDTA Ethylenediamine tetra-acetic acid ERK Extracellular signal-regulated kinases ET-1 Endothelin-1 FA Fastidious anaerobe FACs Fluorescence Activated Cell Sorting FAK Focal adhesion kinase FISH Fluorescence in situ hybridization FITC Fluorescein-isothiocyanate GCF Gingival crevicular fluid GECs Gingival epithelial cells GPI Glucosylphosphatidylinositol GTP Guanosine 5'-Triphosphate H2O2 Hydrogen peroxide Hag Haemagglutinin HGK Human gingival keratinocytes HIV Human Immuno-deficiency virus ICAM Intercellular adhesion molecules IFN Interferons IL Interluekin JAK/STAT Janus Kinase/Signal Transducer and Activator of transcription JNK Jun N-terminal Kinase KGM Keratinocyte growth medium v Kgp Lysine-specific proteinase LPS Lipopolysaccharide MAPK Mitogen-activated protein kinases MMPs Matrix metalloproteases MOI Multiplicity of Infection mRNA Messenger ribonucleic Acid NF-κB Nuclear factor-Kappa-B OMM Oral mucosal model OPG Osteoprotegerin PAMPs Pathogen-associated molecular patterns PARs Protein associated receptors PBS Phosphate buffered saline PCR Polymerase Chain Reaction PDLCs Periodontal ligament cells PI3K Phosphoinositide 3-kinase PMNs Polymorphonuclear cells PRRs Pattern recognition receptors qPCR Quantitative polymerase chain reaction Rgp Arginine-specific proteinases RANKL Receptor activator of nuclear factor-kappa B ligand rDNA Ribosomal Deoxyribonucleic Acid RNA Ribonucleic acid RNase Ribonuclease RT-PCR Reverse transcriptase-Polymerase chain reaction SCC Squamous cell carcinoma SD Standard Deviation SFM Serum free medium T3SS Type 3 secretion system
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