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Host-Pathogen Interactions of Porphyromonas Gingivalis

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Host-Pathogen Interactions of Porphyromonas Gingivalis Host-Pathogen Interactions of Porphyromonas gingivalis Jiamin Aw ORCID 0000-0002-0973-9376 Submitted in total fulfilment of the requirement of the degree of Doctor of Philosophy April 2018 Melbourne Dental School The University of Melbourne Declaration This is to certify that: (i) the thesis comprises only my original work towards the PhD, except when indicated in the Preface, (ii) due acknowledgment 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. Jiamin Aw Melbourne Dental School The University of Melbourne April 2018 i Preface In accordance with the regulations of The University of Melbourne, I acknowledge that some of the work presented in this thesis was collaborative. Specifically: (i) In Chapter 6, cryo-electron microscope images of P. gingivalis W50 and P. gingivalis ΔPG0382 were acquired by Dr. Yu-Yen Chen (The University of Melbourne). The remainder of this thesis comprises only my original work. ii Publications The work presented in this thesis has given rise to the following publication: Aw, J., Scholz, G.M., Huq, N.L., Huynh, J., O’Brien‐Simpson, N.M., Reynolds, E.C. (2018), “Interplay between Porphyromonas gingivalis and EGF signalling in the regulation of CXCL14”, Cellular Microbiology, e12837 The work done during my PhD also contributed to the following publications: Scholz, G.M., Heath, J.E., Aw, J. and Reynolds, E.C. (2018), “ Regulation of the petidoglycan amidase PGLYRP2 in epithelial cells by IL-36”, Infection and Immunity, doi: 10.1128/IAI.00384-18 Huynh, J., Scholz, G.M., Aw, J. and Reynolds, E.C. (2017), “Interferon Regulatory Factor 6 Promotes Keratinocyte Differentiation in Response to Porphyromonas gingivalis”, Infection and Immunity, Vol. 85 No. 5, pp. 1-12. Huynh, J., Scholz, G.M., Aw, J., Kwa, M.Q., Achuthan, A., Hamilton, J.A. and Reynolds, E.C. (2016), “IRF6 Regulates the Expression of IL-36 by Human Oral Epithelial Cells in Response to Porphyromonas gingivalis”, The Journal of Immunology, Vol. 196 No. 5, pp. 2230–2238. Kwa, M.Q., Huynh, J., Aw, J., Zhang, L., Nguyen, T., Reynolds, E.C., Sweet, M.J., Hamilton, J.A., Scholz, G.M. (2014), “Receptor-interacting protein kinase 4 and interferon regulatory factor 6 function as a signalling axis to regulate keratinocyte differentiation”, Journal of Biological Chemistry, Vol. 289 No. 45, pp. 31077-31087. iii Abstract Periodontal health is supported by various host immune defence mechanisms, which act in concert to maintain host-microbe homeostasis. However, breakdown of homeostasis can lead to the development of chronic periodontitis, an inflammatory disease that causes the destruction of periodontal tissues. Pattern recognition receptors, including Toll-like receptors (TLRs), enable the detection of microorganisms and subsequent activation of the host immune response. The modular intracellular Toll/Interleukin-1 receptor (TIR) domain of TLRs forms heterotypic interactions with intracellular adaptor proteins, such as MAL and MYD88, to activate downstream signalling pathways to regulate the transcription of inflammatory genes (e.g. cytokines and chemokines). P. gingivalis is a major periodontal pathogen and can disrupt homeostasis between the host and tooth-accreted subgingival biofilm (plaque) by dysregulating the immune response. The extracellular gingipain proteases (Kgp and RgpA/B) produced by P. gingivalis are potent virulence factors, which can stimulate as well as proteolytically degrade host immunomodulatory factors. Consequently, an otherwise host-protective immune response can become destructive and cause tissue pathology when dysregulated by P. gingivalis. This thesis has characterised two facets of the interaction between the host and P. gingivalis. The first part of this thesis investigated the molecular regulation of the orphan chemokine, CXCL14, by P. gingivalis in oral epithelial cells (e.g. OKF6 cells). By using an isogenic P. gingivalis gingipain (Kgp/Rgp) protease-deficient mutant and a cysteine protease inhibitor, the stimulation of CXCL14 expression was shown to be mediated by the gingipain proteases. Given this finding, a role for protease-activated receptors (PARs) in the regulation of CXCL14 expression was investigated. Gene silencing experiments revealed that P. gingivalis-stimulated CXCL14 expression occurs in a PAR-3-dependent manner. Notably, CXCL14 expression was found to be transcriptionally repressed in response to epidermal growth factor-induced activation of the MEK-ERK1/2 pathway. However, P. gingivalis can overcome the repression of CXCL14 via the gingipain protease-mediated degradation of EGF. Therefore, P. gingivalis not only directly stimulates CXCL14 expression via PAR-3, but also promotes its expression by antagonising EGF signalling. The functions of CXCL14, including its ability to regulate inflammatory gene expression and oral epithelial cell migration, were also investigated. No evidence was obtained to indicate that CXCL14 regulates inflammatory gene expression in oral epithelial cells (e.g. OKF6 cells) or macrophages (e.g. RAW 264.7 cells), or oral epithelial cell migration. Furthermore, CXCL14 did not induce changes in the expression levels of inflammatory genes when injected into the mouse gingiva. However, CXCL14 was shown in vitro to potently kill oral Streptococcus species. iv Significantly, P. gingivalis was resistant to CXCL14 killing, most likely because the gingipain proteases can degrade CXCL14. Therefore, the dysregulation of CXCL14 by P. gingivalis may potentially destabilise the proportions of bacterial species in the tooth-accreted biofilm and thereby promote biofilm dysbiosis. In the second part of this thesis, the expression and function of TIR domain-containing proteins (Tcps) by P. gingivalis was investigated. Other bacterial pathogens have been shown to express Tcps as a mechanism of molecular mimicry to subvert TLR-mediated immune responses. Bioinformatics analysis identified eleven putative Tcps (e.g. PG0382) in nine different strains. Further analyses revealed that the putative TIR domain in the C-terminal half of PG0382 contains sequence features similar to TLR adaptor proteins and bacterial Tcps. Homology modelling of PG0382 suggests that the domain may adopt a TIR-like structure. Like other bacterial Tcps, PG0382 is predicted to also contain a coiled-coil motif in the N-terminal half of the protein, which may facilitate homodimerisation. Functional characterisation of PG0382 by transient expression in human HEK293T cells and analysis by Western blotting and immunofluorescence confocal microscopy revealed that the N-terminal half of PG0382 appears to largely dictate its subcellular localisation. Moreover, co-expression of PG0382 strongly reduced MAL and MYD88 protein levels. Therefore, P. gingivalis PG0382 may have potential immunomodulatory functions. An isogenic P. gingivalis PG0382-deficient mutant (P. gingivalis ΔPG0382) was created to establish a role for PG0382 in modulating the host immune response to P. gingivalis. Phenotypic characterisation of the mutant indicates that PG0382 is not important for P. gingivalis growth, formation of an electron-dense surface layer, or gingipain protease activity. The absence of PG0382 did not affect the inflammatory gene response of oral epithelial cells (e.g. OKF6 cells) towards P. gingivalis. However, P. gingivalis ΔPG0382 stimulated a weaker inflammatory response in macrophages (e.g. RAW 264.7 cells). A peritoneal infection model in mice was used to further investigate a role for PG0382 in modulating the host immune response to P. gingivalis. However, the absence of PGO382 did not affect the recruitment of neutrophils and inflammatory monocytes in response to P gingivalis infection. Further studies will be required to determine whether PG0382 has a role in P. gingivalis immune subversion. This thesis has identified and defined novel interactions between host-derived and P. gingivalis- derived factors in modulating the immune response. Moreover, it provides a molecular basis for exploring potential roles for CXCL14 and P. gingivalis PG0382 in the development and progression of chronic periodontitis. v Acknowledgements This thesis is the culmination of my PhD journey, which would not have been possible or as enjoyable without the support and encouragement of numerous people. I would like to start by expressing my deepest gratitude to my supervisor Associate Professor Glen Scholz, whose enthusiasm for scientific research knows no bounds. His unwavering belief in my abilities encouraged me to challenge myself as a researcher. Without his patience and knowledge, I would not have been able to achieve all that I have over the past few years. I would also like to extend my thanks to my co-supervisor Professor Neil O’ Brien-Simpson for his thought-provoking discussions. A special thanks to Professor Eric Reynolds for giving me the opportunity to undertake my PhD under the Oral Health CRC. My sincerest thanks go to my colleagues at the Melbourne Dental School who have offered their valued technical support and advice. I have greatly benefited the support of Christine Seers, who helped me devise a strategy for creating the P. gingivalis PG0382-deficient mutant. I would like to thank Yu-Yen Chen for acquiring the cryo-EM images of P. gingivalis. I would also like to express my gratitude towards Katrina Walsh and Alexis Gonzalez who have shared their insightful knowledge about flow cytometry. Thank you to Dhana Gorasia, Michelle
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