INVESTIGATION INTO THE PATHOGENIC POTENTIAL OF THE EMERGENT GASTROINTESTINAL PATHOGENS CAMPYLOBACTER UREOLYTICUS AND CAMPYLOBACTER CONCISUS
By Jose Burgos-Portugal
A thesis submitted as partial fulfillment
of the requirement for the degree of
Doctor of Philosophy
Supervisor: Professor Hazel Mitchell
Co-supervisor: Dr Nadeem O. Kaakoush
School of Biotechnology and Biomolecular Sciences
Faculty of Science
The University of New South Wales
June 2015
THE llNIVI<:I{BITY OF NEW SOlJTH WALES
Thcsis/DisscJ·tation Sheet
Surname or Family name: BlJRGOS-PORTUGAL
First name: JOSE Other namc/s: AUGUSTO
Abbreviation for degree as given in the University calendar: Ph.D
School: Biotechnology and Biomolccular Sciences Faculty: Science
Title: Investigation into the pathogenic potential of the emergent gastrointestinal pathogens CameJllobacter tl~'£()f)ltic;us_ _[lll Campylobacter ureo/yticus and Campylobacter concisus arc emerging pathogens that have been associated with gastrointestinal disease. Detection of these bacteria in faecal samples of patients with gastroenteritis, and intestinal biopsies and faecal samples of patients with Crohn's disease, led us to investigate the pathogenic mechanisms used by hoth C. ureolyticus and C. concisus to infect host cells and cause gastrointestinal disease. Investigation of the ability of C ureolyticzis to interact with intestinal epithelial cell lines showed that it wa~ able to adhere to both Caco-2 and I-IT-29 cell lines. This was confirmed using scanning electron microscopy (ScFM), showing that C. ureolyticus UNSWCD employed a "sticky-end" mechanism to attach to the microvilli of Caco-2 cells resulting in cellular damage and microvilli degradation. In contrast, gentamicin protection assays revealed that C. ureolyticus was unable to invade either cell line, a finding confirmed by SeEM. Furthermore, addition of pro-inflammatory cytokines TNF-o: and IFN-y to these cell lines prior to infection had no effect on attachment or invasion. Investigation of the secretome of C. ureo61ticus UNSWCD revealed the presence of putative virulence and colonisation factors. Given that previous studies had demonstrated varying abilities of C. concisus strains to invade intestinal cell lines, this thesis explored the potential of C. concisus strains to survive within host cells by manipulating the autophagy process. Gentamicin protection assays revealed that autophagy inhibition resulted in 2-4 fold increases in intracellular levels of C. condsus UNSWCD within Caco-2 cells, whereas autophagy induction resulted in a reduction in intracellular levels or bacterial clearance. Confocal laser scanning microscopy showed co~ localisation of the bacterium within autophagosomes, while an optimised transmission electron microscopy procedure identified intracellular bacteria persisting within autophagic vesicles. Furthermore, qPCR showed that 13 genes in the autophagy process were significantly regulated following C. concisus infection. Overall, these findings revealed that C. concisus UNSWCD elicits a dampening effect on the autophagy process. The studies from this thesis have significantly increased our understanding of the pathogenic mechanisms used by these bacteri<~l- to interact with and manipulate host. intestinal ~el_l~--~.~J~Otei~!J-~~--£au~~J1~~g~C?_intcsf!!Ial Qi?~-~~~~:-~ Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all t 00 ..1:!:.~?. ~r..·~~~:" 00 00 .. 00 00 " 00 00 00 ,,;, ioo ;/;;, 00 < •M Q u39oo 00 "00 000 00 00 o "" ..J.5../fz/c.). c; .. o.~ s c.J..A'\ Signature Witness ) Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional _circumstances and rc 'l hereby declare that this submission is my own work and to the best of my knowledge it contains no material previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other dress or diploma at UNSW or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to the research by others, with whom l have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, expect to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.' c:{;p 2Yv / ;:;:~;f)'"v~~'i'"l'';;c_9L; SIGNl•~D ...... ':. .. :::::-:;: ...... /.1...... ~ 15/ac{/2015' DA1 E ...... 11 COPYRIGHT STATEMENT 'I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all f(mns of media, now or here after known, subject to the provisions of the Copyright Act I 968. I retain all proprietary rights, such as patent rights. I also retain the right to usc in Jhturc works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to usc the 350 word abstract of my thesis rn Dissertation Ahstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to usc copyright material; where permission has not been granted J have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' . ?"f):>' .;,,j3~~) SlGNEI) ... .1.. S...... AUTIU~NTICITY STATEMENT 'I certify that the Library deposit digital copy is a direct equivalent ofthc final ofl1cially approved version of my thesis. No emendation of content has occurred and if there are any mmor variations in formatting, they arc the result of the conversion to digital t(m1lat.' , , ,, ,«:~;9,~f~~;yv,/Z~c!> · SIGN~, I) ....)~ ...... "' /S.jOCf/20!5' DAlE ...... Ill ACKNOWLEDGEMENTS (i) “The bigger the mountain the harder the climb, the better the view from the finish line”- Anonymous. (ii) “Focus on the journey not the destination. Joy is found not in finishing an activity but in doing it”- Greg Anderson. In dedication to my future self and my entire family to serve as a lasting testament in knowing that anyone can persevere and accomplish anything. As long as they set their mind to that task with the right dedication and attitude to move forward and prevail. Fulfilling a Ph.D has been a dream of mine since I started as an Undergraduate student at UNSW. It has been a long journey and I have had many challenges and rewards along the way. I have been through physical as well as mental challenges, which have taught me a lot about my own perseverance and will to keep on moving forward and learn from the experience that life throws at you. When you face a life threatening illness it teaches you that there is more to life than you would have originally thought. Hence, wisely someone told me that life does not give you a challenge that you cannot handle. It goes without saying that my accomplishment in completing my Ph.D thesis would not have been possible without the help of so many people who I count very fortunate to have in my own life. These special people are: my family, Supervisors Hazel and Nadeem, plus my friends and colleagues. You have all been there for me throughout the toughest moment in my life, and thankfully I’ve come out of it stronger than ever. To my supervisor Prof Hazel Mitchell, it has been a pleasure to be your Ph.D student. I could not have asked for a more supportive and caring Supervisor. You have nurtured me from a novice research scientist to one with more experience. Thank you for allowing me to pursue my passions and research interests in microscopy. Additionally, you have my gratitude for giving the opportunity to be part of your research group, and for instilling the passion to pursue investigations in Campylobacter species. My gratitude to you for been there for me when I really needed you most, and for taking the time to advice me and support me through conference meetings, travel grants, seminar presentations, as well as my recovery post ailment. I am eternally thankful for the support and advice you have given me Hazel, which I will carry throughout the rest of my life. iv To my co-supervisor Dr Nadeem Kaakoush, what can I say you have been behind me from the very beginning since I commenced my Honours year in the Mitchell lab. I know you will do great things in the future, and I am glad I helped contribute to the intriguing research done in the Mitchell lab. I will always cherish the time and chats we had about Campylobacter research, our love for Football (the round ball world game), Dexter and Batman amongst other things. You were never too busy to take the time and teach me the skills I needed in the laboratory to get my experiments done. Importantly, you were always there when I needed to chat to you so I wouldn’t be confused. I could not have asked for a better mentor or friend. Thank you for inspiring and believing in me to become a better research scientist. To my colleagues and friends who have shared this journey with me, thank you for the memories: Natalia Castaño- Rodriguez, Nidhi Sodhi, Mahmoud Ruzayqat, Faisal Khattak, Karina Huinao, Peter Lavrencic, David Lynch, Emily Bainbridge, Ashley Morgan, Alex Underwood and Johnny QV Hsu, I apologise if I have missed anyone in particular. I would like to mention a few people in particular. Natalia, we both started this research journey together and I’m so glad we could both finish it. Nati I will always cherish our random chats in Spanish and our memorable passion for football. Nidhi and Alex thank you kindly for sharing your love of good food, good wine and good movies with me. I feel I am a far more cultured person thanks to your fine tastes. I am especially blessed to have shared this journey with all the numerous lab members at 301A who have come and gone and you have all left a special mark in my life. To my other friends who have given inspiration and a helping hand to helping me accomplish my Ph.D: Si Ming Man, Heather Schmidt, Vikneswari Mahendran, Christopher Fife, Jose Rodrigo Vargas, Andrea Sirianni, Pejhman Keshvardoust, Marwan Majzoub, Vipra Kumar, and so many more, you know who you are. To Clara Cheah thank you for inspiring me and for the time we shared performing our numerous gentamicin protection assays together. I have to give a huge thank you to the wonderful administration and research staff at BABS: Michelle Potter, Kylie Jones and Jani O’Rouke. Also, a massive thank you to my Ph.D research Panel, which consisted of Dr Nicodemus Tedla and Prof Marc Wilkins. Thank you for been so understanding and supportive throughout my Ph.D Candidature. To all the people who helped me review and edit my Ph.D Thesis thank you for the helpful feedback, discussions and advise. To all the UNSW Students I had the pleasure of teaching as a demonstrator. I hope I left you with inspiration to be the best scientists you can be with an open and analytical mind. You have all taught me a thing or two, and it was a pleasure to guide you through your journey as scientists. This Ph.D would also not have been possible without the financial support I received from my APA scholarship as well as grant funding obtained by the Mitchell lab from the NHMRC and the UNSW gold star award, which provided me with the financial support to carry out my experiments in this intriguing area of research. I would also like to extend my gratitude to all the collaborators I have had the pleasure of working with at the BMIF and EMU. Many thanks to the entire staff at the EMU, especially to Jennifer Norman the awesome EM guru. Jenni you taught me a lot about EM and scientific research from my Honours year right throughout my Ph.D candidature. Thank you for v been my confidant and a friendly face to chat too when I’ve faced my challenges. Thank you for encouraging my passions for health and safety and in dong also nurtured my passion in electron microscopy. To the entire staff at the BMIF, especially to Renae Wan and Michael Carnell the fluorescence microscopy gurus. Thank you for been so supportive throughout my Immuno-fluorescence investigations at the BMIF and for showing my how to use the Fiji software. Also, a big thanks to the staff at the UNSW Learning Centre, especially to Linda Burnell for their support in helping to proof read my thesis. To my lovely family, I am eternally very thankful for the positive influence and wonderful support. My wonderful parents Jose Carlos Burgos-Macedo and Beatriz Portugal De Burgos who cared and supported me so much. You cared for me so much while I suffered through my arduous illness and it’s thanks to your persistence in helping me find the best medical care that I am alive today and finishing my Ph.D candidature. Thank you for your sacrifice and for continuing to motivate and support me in my dream of completing my Ph.D candidature. Dad this accomplishment is one we can both share, as a testimony of the extent of a person’s will power to accomplish their dreams. To my sister Giuliana Burgos-Portugal, you have been my anchor throughout my Ph.D candidature. Your advice, excellent editing skills and our discussions helped me realise how important my research was and the benefits it could bring. To my family overseas, may my achievement serve as a testament to how much I care about you and how important it is to dream and complete a task until it is accomplished. In loving memory of my Grandparents may my work serve in your memory to help others suffering from devastating Gastrointestinal diseases around the world. vi ABSTRACT Campylobacter ureolyticus and Campylobacter concisus are emerging pathogens that have been associated with gastrointestinal disease. Detection of these bacteria in faecal samples of patients with gastroenteritis, and intestinal biopsies and faecal samples of patients with Crohn’s disease, led us to investigate the pathogenic mechanisms used by both C. ureolyticus and C. concisus to infect host cells and cause gastrointestinal disease. Investigation of the ability of C. ureolyticus to interact with intestinal epithelial cell lines showed that it was able to adhere to both Caco-2 and HT-29 cell lines. This was confirmed using scanning electron microscopy (ScEM), showing that C. ureolyticus UNSWCD employed a “sticky-end” mechanism to attach to the microvilli of Caco-2 cells resulting in cellular damage and microvilli degradation. In contrast, gentamicin protection assays revealed that C. ureolyticus was unable to invade either cell line, a finding confirmed by ScEM. Furthermore, addition of pro-inflammatory cytokines TNF-α and IFN-γ to these cell lines prior to infection had no effect on attachment or invasion. Investigation of the secretome of C. ureolyticus UNSWCD revealed the presence of putative virulence and colonisation factors. Given that previous studies had demonstrated varying abilities of C. concisus strains to invade intestinal cell lines, this thesis explored the potential of C. concisus strains to survive within host cells by manipulating the autophagy process. Gentamicin protection assays revealed that autophagy inhibition resulted in 2-4 fold increases in intracellular levels of C. concisus UNSWCD within Caco-2 cells, whereas autophagy induction resulted in a reduction in intracellular levels or bacterial clearance. Confocal laser scanning microscopy showed co-localisation of the bacterium within autophagosomes, vii while an optimised transmission electron microscopy procedure identified intracellular bacteria persisting within autophagic vesicles. Furthermore, qPCR showed that 13 genes in the autophagy process were significantly regulated following C. concisus infection. Overall, these findings revealed that C. concisus UNSWCD elicits a dampening effect on the autophagy process. The studies from this thesis have significantly increased our understanding of the pathogenic mechanisms used by these bacteria to interact with and manipulate host intestinal cells and potentially cause gastrointestinal disease. viii PUBLICATIONS AND CONFERENCE PROCEEDINGS LIST OF PUBLICATIONS 1. Kaakoush NO, Deshpande NP, Man SM, Burgos-Portugal JA, Khattak FA, Raftery MJ, Wilkins MR, Mitchell HM. 2015. Transcriptomic and Proteomic Analyses Reveal Key Innate Immune Signatures in the Host Response to the Gastrointestinal Pathogen Campylobacter concisus. Infect Immun 83:832-845. 2. Jose Burgos-Portugal, Hazel Mitchell, Natalia Castano-Rodriguez, and Nadeem Kaakoush. The role of autophagy in the intracellular survival of Campylobacter concisus. FEBS Open Bio. 2014. (4): 301–309. 3. Jose A. Burgos-Portugal, Nadeem O. Kaakoush, Mark J. Raftery & Hazel M. Mitchell. The pathogenic potential of Campylobacter ureolyticus. Infect Immun. 2012. 80(2): 883-890. 4. Nadeem O. Kaakoush, Nandan P. Deshpande, Marc R. Wilkins, Chew Gee Tan, Jose A. Burgos-Portugal, Mark J. Raftery, Andrew S. Day, Daniel A. Lemberg, and Hazel Mitchell. The Pathogenic Potential of Campylobacter concisus Strains Associated with Chronic Intestinal Diseases. PLoS One. 2011. 6(12): e29045. LIST OF CONFERENCE PROCEEDINGS 1. Jose A. Burgos-Portugal, Nadeem O. Kaakoush. Natalia. C. Rodriguez and Hazel. M. Mitchell. The role of autophagy in the pathogenic potential of the emerging gastrointestinal pathogen Campylobacter concisus. 15th International EMBL Ph.D symposium in Heidelberg, Germany. 2013. Oral Presentation. Recipient of Oral Presentation Award. 2. Jose A. Burgos-Portugal, Nadeem O. Kaakoush. Natalia. C. Rodriguez and Hazel. M. Mitchell. The role of autophagy in the pathogenic potential of the emerging gastrointestinal pathogen Campylobacter concisus. 1st Annual BABS Research ix Symposium, The University of New South Wales, Sydney, 2013. Australia. Oral Presentation and Poster Presentation. 3. Jose A. Burgos-Portugal, Nadeem O. Kaakoush. Natalia. C. Rodriguez and Hazel. M. Mitchell. The role of autophagy in the pathogenic potential of the emerging gastrointestinal pathogen Campylobacter concisus. Campylobacter, Helicobacter and Related Organisms. Aberdeen, Scotland, 2013. Poster Presentation. 4. Jose A. Burgos-Portugal, Nadeem O. Kaakoush, Mark J. Raftery & Hazel M. Mitchell. The pathogenic potential of the emerging intestinal pathogen Campylobacter ureolyticus. Lorne Infection and Immunity, Victoria, 2012. Poster Presentation. 5. Nadeem O. Kaakoush, Nandan P. Deshpande, Marc R. Wilkins, Chew Gee Tan, Jose A. Burgos-Portugal, Mark J. Raftery, Andrew S. Day, Daniel A. Lemberg, and Hazel Mitchell. The Pathogenic Potential of Campylobacter concisus Strains Associated with Chronic Intestinal Diseases. Campylobacter, Helicobacter and Related Organisms (CHRO), Vancouver, Canada, 2011. Poster Presentation. x TABLE OF CONTENTS ABSTRACT ...... VII PUBLICATIONS AND CONFERENCE PROCEEDINGS ...... IX LIST OF FIGURES ...... XXI LIST OF TABLES ...... XXV ABBREVIATIONS ...... XXVII CHAPTER 1 ...... 31 1. INTRODUCTION ...... 31 1.1. THE CAMPYLOBACTER GENUS ...... 31 1.1.1. Campylobacter species associated with gastroenteritis ...... 32 1.1.2. Campylobacter jejuni and Gullain Barré Syndrome ...... 32 1.1.3. Campylobacter and Irritable Bowel Syndrome ...... 33 1.2. CAMPYLOBACTER UREOLYTICUS ...... 34 1.2.1. Historical background of Campylobacter ureolyticus ...... 34 1.2.2. Reclassification of Bacteroides ureolyticus to Campylobacter ureolyticus ...... 35 1.2.3. Campylobacter ureolyticus (previously known as B. ureolyticus) and disease ...... 36 1.2.4. C. ureolyticus and Inflammatory Bowel Disease ...... 36 1.2.5. C. ureolyticus and gastroenteritis ...... 38 1.2.6. Sources and transmission of C. ureolyticus infection ...... 40 1.2.7. Genetic heterogeneity ...... 41 1.2.8. The secretome and virulence factors of C. ureolyticus ...... 43 1.3. BACKGROUND CAMPYLOBACTER CONCISUS ...... 46 xi 1.3.1. Historical background of Campylobacter concisus ...... 46 1.4. DISEASES ASSOCIATED WITH CAMPYLOBACTER CONCISUS ...... 47 1.4.1. C. concisus and oral cavity diseases ...... 47 1.4.2. Campylobacter concisus and Barrett’s oesophagus ...... 49 1.4.3. Campylobacter concisus as a possible cause of acute gastroenteritis ...... 51 1.4.4. Campylobacter concisus and Inflammatory Bowel Disease ...... 57 1.4.5. The invasive potential of C. concisus in host intestinal epithelial cells ...... 61 1.4.6. Genetic and phenotypic variation in Campylobacter concisus spp .. 63 1.5. PATHOGENESIS OF C. CONCISUS ...... 66 1.5.1. The pathogenic potential of Campylobacter concisus revealed via in vitro model studies ...... 66 1.5.2. C. concisus toxin production and other virulence and colonisation factors ...... 68 1.5.3. C. concisus pathotypes ...... 70 1.5.4. C. concisus and the activation of the host’s innate immune system . 71 1.5.5. Immune response to Campylobacter infections ...... 74 1.5.6. The genome of C. concisus UNSWCD ...... 76 1.6. INTRACELLULAR SURVIVAL OF ENTERIC PATHOGENS ...... 80 1.6.1. The intracellular survival of Campylobacter spp residing in intestinal cells ...... 81 1.6.2. A key intracellular survival mechanism: The C. jejuni Campylobacter containing vacuole ...... 82 1.7. AUTOPHAGY ...... 85 1.7.1. The process of autophagy ...... 85 xii 1.7.2. Autophagy and the innate immune system: A plausible link between autophagy and Crohn’s disease ...... 87 1.7.3. The role of autophagy in C. jejuni infection ...... 88 1.8. HYPOTHESIS ...... 90 1.8.1. C. ureolyticus specific aims ...... 90 1.8.2. C. concisus specific aims ...... 91 CHAPTER 2 ...... 92 2. MATERIALS AND METHODS ...... 92 2.1. BACTERIAL CULTURES ...... 92 2.1.1. Campylobacter concisus strains ...... 92 2.1.2. Campylobacter ureolyticus strains ...... 92 2.1.3. Other bacterial species utilised ...... 93 2.2. CULTURE MEDIA ...... 93 2.2.1. Horse Blood Agar ...... 93 2.2.2. Nutrient Agar ...... 94 2.2.3. Brain heart infusion ...... 94 2.2.4. Brucella Broth ...... 94 2.2.5. Brain Heart Infusion Broth Plus Glycerol ...... 94 2.2.6. Brucella Broth with Fetal Bovine Serum ...... 95 2.2.7. Brain Heart Infusion Broth with Fetal Bovine Serum ...... 95 2.3. BUFFERS ...... 95 2.3.1. Phosphate buffered Saline 0.1 M ...... 95 2.4. BACTERIAL PRESERVATION, CULTURE AND ENUMERATION ...... 95 2.4.1. Cryoperservation ...... 95 2.4.2. Resuscitation and culture of bacterial strains ...... 96 2.4.3. Bacterial harvesting from solid and liquid media ...... 96 xiii 2.4.4. Determination of bacterial cell concentration ...... 96 2.4.5. Liquid culture for time course experiments ...... 97 2.5. MAMMALIAN CELL CULTURE ...... 97 2.5.1. Growth and maintenance of Caco-2 cells ...... 97 2.5.2. Growth and maintenance of HT-29 cells ...... 98 2.6. LIGHT MICROSCOPY FOR MAMMALIAN AND BACTERIAL INSPECTION ...... 98 2.7. BACTERIAL GROWTH AND MORPHOLOGY CONFIRMATION OF CULTURES ... 99 2.7.1. Gram staining and morphological characterisation of bacterial cultures ...... 99 2.7.2. Biochemical identification of C. ureolyticus and C. concisus ...... 99 CHAPTER 3 ...... 101 3. THE INVESTIGATION OF THE PATHOGENIC POTENTIAL OF CAMPYLOBACTER UREOLYTICUS IN HOST INTESTINAL EPITHELIAL CELLS ...... 101 3.1. BACKGROUND ...... 101 3.1.1. Aims ...... 104 3.2. MATERIALS AND METHODS ...... 105 3.2.1. C. ureolyticus strains and other bacterial species ...... 105 3.2.2. Bacterial cultivation ...... 105 3.2.3. Cultivation of the human intestinal Caco-2 cell line ...... 106 3.2.4. Cultivation of the human intestinal HT-29 cell line ...... 107 3.3. ADHERENCE AND GENTAMICIN PROTECTION (INVASION) ASSAYS USING CACO-2 AND HT-29 CELLS ...... 108 3.3.1. Adherence assay ...... 108 3.3.2. Gentamicin protection (invasion) assay ...... 109 xiv 3.3.3. Examination of the effect of pre-existing inflammation on C. ureolyticus UNSWCD adherence and invasion ...... 110 3.3.4. Determination of percentage adherence and invasion ...... 110 3.3.5. Translocation of C. ureolyticus across HT-29 cells ...... 111 3.4. DETERMINATION OF THE ADHERENCE PATTERN OF C. UREOLYTICUS UNSWCD USING SCANNING ELECTRON MICROSCOPY ...... 111 3.4.1. Sample preparation ...... 111 3.4.2. Critical point drying, gold coating and imaging ...... 112 3.5. CACO-2 CELL VIABILITY AND IL-8 PRODUCTION FOLLOWING C. UREOLYTICUS INFECTION, IN THE PRESENCE OR ABSENCE OF PRE-EXISTING INFLAMMATION ...... 112 3.5.1. Determination of Caco-2 cell viability following infection with C. ureolyticus UNSWCD, in the presence or absence of pre-existing inflammation ...... 112 3.5.2. Determination on the effect of C. ureolyticus UNSWCD infection and the addition of cytokines (TNF-α or IFN-γ) on interleukin-8 production . 113 3.6. THE SECRETOME OF C. UREOLYTICUS UNSWCD ...... 115 3.6.1. Bacterial cultures ...... 115 3.6.2. Isolation of the C. ureolyticus UNSWCD secretome ...... 115 3.6.3. Determination of protein content ...... 115 3.6.4. One-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis ...... 116 3.6.5. LTQ LC/MS-MS sample preparation and LTQ-MS ...... 116 3.6.6. Bioinformatics analysis ...... 117 3.6.7. Determination of the effect of the C. ureolyticus UNSWCD secretome on HT-29 cell viability and interleukin-8 production ...... 118 xv 3.6.8. Statistical analysis ...... 118 3.7. RESULTS ...... 120 3.7.1. Adherence, invasive, and translocation abilities of C. ureolyticus UNSWCD ...... 120 3.7.2. Effect of pre-existing inflammation on the adherence and invasive ability of C. ureolyticus UNSWCD ...... 121 3.7.3. Visualisation of attachment by C. ureolyticus UNSWCD to Caco-2 cells using Scanning Electron Microscopy ...... 124 3.7.4. Visualisation of the effect of pre-existing inflammation on the attachment of C. ureolyticus UNSWCD to Caco-2 cells using Scanning Electron Microscopy ...... 127 3.7.5. Viability of Caco-2 cells following infection with C. ureolyticus UNSWCD at different MOIs in the absence and presence of pre-existing inflammation ...... 129 3.7.6. Interleukin-8 production following C. ureolyticus UNSWCD infection of HT-29 cells in the absence and presence of inflammation ...... 132 3.7.7. The secretome of C. ureolyticus UNSWCD ...... 134 3.7.8. Effect of the secretome of C. ureolyticus UNSWCD on cell viability and IL-8 production in HT-29 cells ...... 140 3.8. DISCUSSION ...... 143 3.9. CONCLUSION ...... 153 CHAPTER 4 ...... 154 4. THE ROLE OF AUTOPHAGY IN THE INTRACELLULAR SURVIVAL OF CAMPYLOBACTER CONCISUS ...... 154 4.1.1. Aims ...... 157 4.2. MATERIALS AND METHODS ...... 158 xvi 4.2.1. Bacterial strains and growth ...... 158 4.2.2. Cell culture ...... 158 4.2.3. Determination of intracellular levels of C. concisus using gentamicin protection assays ...... 159 4.2.4. The effect of aerobic and microaerobic growth conditions on C. concisus UNSWCD adherence and invasion of Caco-2 cells ...... 160 4.2.5 Determination of the effect of autophagy inhibition and induction on intracellular levels of C. concisus ...... 160 4.2.6. Determination of the effect of Chloroquine di-phosphate on the ability of Caco-2 cells to exocytose C. concisus ...... 161 4.2.7. Determination of the effect of re-invasion of C. concisus UNSWCS on intracellular survival within Caco-2 cells ...... 161 4.3. THE EFFECT OF CHLOROQUINE DI-PHOSPHATE ON C. CONCISUS ...... 162 4.3.1. Determination of the effect of Chloroquine di-phosphate on C. concisus viability ...... 162 4.3.2. Determination of the effect of Chloroquine di-phosphate and 3- Methyladenine on Caco-2 cell viability following C. concisus UNSWCD infection ...... 162 4.4. DETECTION OF AUTOPHAGOSOMES AND BACTERIAL CO-LOCALISATION USING CONFOCAL MICROSCOPY ...... 163 4.4.1. The confocal microscopy imaging procedure ...... 165 4.5. DETECTION OF CACO-2 CELL MEMBRANE ALTERATIONS UPON C. CONCISUS INFECTION USING SCANNING ELECTRON MICROSCOPY ...... 166 4.6. QUANTITATIVE PCR FOR ANALYSIS OF GENES WITHIN THE AUTOPHAGY PATHWAY ...... 168 4.7. RESULTS ...... 170 xvii 4.7.1. Effect of autophagy inhibition on intracellular levels of Campylobacter concisus ...... 170 4.7.2. The effect of Chloroquine di-phosphate and 3-Methyladenine on Caco-2 cell viability following C. concisus UNSWCD infection ...... 174 4.7.3. The effect of Chloroquine di-phosphate on C. concisus UNSWCD bacterial cell viability ...... 175 4.7.4. The effect of Chloroquine di-phosphate on the ability of Caco-2 cells to exocytose C. concisus UNSWCD ...... 176 4.7.5. Visualisation of the effect of autophagy induction on intracellular levels of C. concisus ...... 177 4.7.6. Investigation of autophagosomes in Caco-2 cells ...... 178 4.7.7. Co-localisation of C. concisus with autophagosomes ...... 179 4.7.8. Modulation in the expression of genes involved in autophagy by C. concisus ...... 181 4.8. DISCUSSION ...... 183 4.9. CONCLUSION ...... 193 CHAPTER 5 ...... 194 5. DEVELOPMENT OF A TRANSMISSION ELECTRON MICROSCOPY PROCEDURE TO INVESTIGATE THE MODULATION OF THE AUTOPHAGY PROCESS BY CAMPYLOBACTER CONCISUS 194 5.1. BACKGROUND ...... 194 5.1.1. Aims ...... 207 5.2. MATERIALS AND METHODS ...... 208 5.2.1. Biological sample preparation using the agar embedment method 208 5.2.2. Biological sample preparation without agar embedment ...... 212 xviii 5.2.3. Ultramicrotomy ...... 214 5.2.4. Semi-thin sectioning and staining ...... 214 5.2.5. Ultra-thin sectioning ...... 215 5.2.6. Staining of ultra-thin sections ...... 215 5.2.7. Transmission Electron Microscopy imaging procedure ...... 216 5.3. RESULTS ...... 217 5.3.1. Optimisation of Transmission Electron Microscopy sample preparation ...... 217 5.3.2. Visualisation of semi-thin sections prepared using agar embedment, as an assessment for progression to ultra-thin sectioning ...... 217 5.3.3. Visualisation of semi-thin sections without agar embedding, as an assessment for progression to ultra-thin sectioning ...... 219 5.3.4. Visualisation of ultra-thin sections prepared using Caco-2 cells and C. concisus UNSWCD agar embedded samples under Transmission Electron Microscopy ...... 221 5.3.5. Visualisation of C. concisus using Transmission Electron Microscopy without agar embedment ...... 223 5.4. DISCUSSION ...... 228 5.5. CONCLUSION ...... 235 CHAPTER 6 ...... 236 6. GENERAL DISCUSSION AND FUTURE DIRECTIONS ...... 236 6.1. GENERAL DISCUSSION ...... 236 6.1.1. C. ureolyticus ...... 236 6.2. SUMMARY OF MAJOR FINDINGS ON THE PATHOGENESIS OF C. UREOLYTICUS ...... 236 xix 6.2.1. The putative pathogenic mechanism of infection used by C. ureolyticus ...... 240 6.3. C. CONCISUS ...... 243 6.4. SUMMARY OF THE MAJOR FINDINGS ON THE PATHOGENESIS OF C. CONCISUS ...... 243 6.5. THE PUTATIVE PATHOGENIC MECHANISMS USED BY C. CONCISUS ...... 248 6.6. FUTURE DIRECTIONS ...... 251 6.6.1. Future directions for C. ureolyticus investigations ...... 251 6.6.2. Future directions for C. concisus investigations ...... 255 REFERENCES ...... 259 APPENDICES ...... 289 xx LIST OF FIGURES FIGURE 1.1. REPRESENTATIVE AMPLIFIED POLYMORPHISM OF SELECTED C. CONCISUS STRAINS...... 65 FIGURE 1.2. THE PATHOGENIC DIFFERENCE BETWEEN AICC AND ATOCC PATHOTYPES AND THEIR EFFECT ON HOST INTESTINAL CELLS ...... 71 FIGURE 1.3. THE HOST CELLS PROPOSED IMMUNE RESPONSE TO C. CONCISUS UNSWCD . 73 FIGURE 1.4. THE C. CONCISUS UNSWCD 30 KB PLASMID ...... 79 FIGURE 1.5. THE SYNTHETIC CONSERVATION OF THE SEVEN GENES WITHIN THE UNSWCD PLASMID FOUND ONLY IN THE HIGHLY INVASIVE C. CONCISUS STRAINS ...... 79 FIGURE 1.6. MODEL FOR C. JEJUNI INTERNALISATION WITHIN INTESTINAL EPITHELIAL CELL ...... 84 FIGURE 1.7. THE MACROAUTOPHAGY PROCESS OCCURRING WITHIN THE HOST CELL ...... 86 FIGURE 1.8. THE XENOPHAGY PROCESS OCCURRING WITHIN THE HOST CELL ...... 86 FIGURE 3.1. INFECTION OF CACO-2 CELLS WITH THE AFLAGELLATE C. UREOLYTICUS UNSWCD FOR 6 H ...... 125 FIGURE 3.2. INFECTION OF CACO-2 CELLS WITH THE AFLAGELLATE C. UREOLYTICUS UNSWCD FOR 1 H AND 3 H ...... 126 FIGURE 3.3. INFECTION OF CACO-2 CELLS WITH THE AFLAGELLATE C. UREOLYTICUS UNSWCD IN THE PRESENCE OF PRE-EXISTING INFLAMMATION ...... 128 FIGURE 3.4. CELL VIABILITY RESULTS OF VIABLE CACO-2 CELLS UPON INOCULATION WITH AND WITHOUT C. UREOLYTICUS UNSWCD AT VARIANT MOIS ...... 130 FIGURE 3.5. COMPARISON OF THE VIABILITY OF C. UREOLYTICUS INFECTED AND UNINFECTED CACO-2 CELLS, WITH AND WITHOUT THE PRESENCE OF THE PRO- INFLAMMATORY CYTOKINES TNF-ALPHA AND IFN-GAMMA ...... 131 xxi FIGURE 3.6. IL-8 PRODUCTION EXHIBITED BY HT-29 CELLS INFECTED WITH C. UREOLYTICUS UNSWCD, WITH OR WITHOUT PRE-EXISTING INFLAMMATION INSTIGATED BY TNF-ALPHA AND IFN-GAMMA ...... 133 FIGURE 3.7. A ONE-DIMENSIONAL SDS PAGE GEL OF C. UREOLYTICUS UNSWCD SECRETED PROTEINS...... 135 FIGURE 3.8. CELL VIABILITY OF HT-29 CELLS INOCULATED WITH C. UREOLYTICUS UNSWCD SECRETED PROTEINS OVER A 6 H PERIOD ...... 141 FIGURE 3.9. IL-8 PRODUCTION BY HT-29 CELLS EXPOSED TO INCREASING CONCENTRATIONS OF C. UREOLYTICUS SECRETED PROTEINS ...... 142 FIGURE 4.1. WORKFLOW SHOWING THE PREPARATION OF SPECIMENS FOR CONFOCAL MICROSCOPY ...... 165 FIGURE 4.2. WORKFLOW OUTLINING THE SAMPLE PREPARATION PROCESS FOR SCEM INVESTIGATIONS ...... 167 FIGURE 4.3. THE AUTOPHAGY QRT-PCR PROCESS USED TO DETERMINE EXPRESSION LEVEL OF AUTOPHAGY GENES FOLLOWING INFECTION OF CACO-2 CELLS WITH AND WITHOUT C. CONCISUS UNSWCD ...... 169 FIGURE 4.4. INTRACELLULAR SURVIVAL OF C. CONCISUS UNSWCD FOLLOWING AUTOPHAGY INHIBITION AND INDUCTION ...... 172 FIGURE 4.5. INTRACELLULAR SURVIVAL OF C. CONCISUS UNSWCS, ATCC 51562 AND BAA-1457 FOLLOWING AUTOPHAGY INHIBITION USING 3-MA...... 173 FIGURE 4.6. VIABILITY OF C. CONCISUS UNSWCD TREATED WITH CHLOROQUINE DI- PHOSPHATE…...... 175 FIGURE 4.7. SCANNING ELECTRON MICROSCOPY IMAGES OF CACO-2 CELLS WITH AND WITHOUT CHLOROQUINE DI-PHOSPHATE TREATMENT ...... 177 FIGURE 4.8. VISUALISATION OF AUTOPHAGOSOMES USING CONFOCAL MICROSCOPY TARGETING LC3B ...... 178 xxii FIGURE 4.9. VISUALISATION OF THE CO-LOCALISATION OF C. CONCISUS WITH AUTOPHAGOSOMES USING CONFOCAL MICROSCOPY ...... 180 FIGURE 5.1. TEM IMAGE SHOWING (A) AUTOPHAGIC COMPARTMENTS FOUND IN A MOUSE FIBROBLAST AND (B) AN IMAGE OF A MOUSE HEPATOCYTE SHOWING THE MISCLASSIFICATION OF AUTOPHAGIC ULTRASTRUCTURES AS AN AUTOPHAGOSOME ...... 200 FIGURE 5.2. AGAR EMBEDMENT AND TRIMMING OF THE BIOLOGICAL SAMPLES TO PRODUCE SMALLER SAMPLE FRAGMENTS...... 210 FIGURE 5.3. AN OUTLINE OF SAMPLE PREPARATION USING THE AGAR EMBEDMENT METHOD FOR TEM...... 211 FIGURE 5.4. AN OUTLINE OF THE TEM SAMPLE PREPARATION PROCEDURE WITHOUT AGAR EMBEDMENT ...... 213 FIGURE 5.5. AGAR EMBEDDED SEMI-THIN SECTION (1 MICRON) OF UNINFECTED CACO-2 CELLS AND C. CONCISUS INFECTED CACO-2 CELLS ...... 213 FIGURE 5.6. NON-AGAR EMBEDDED SEMI-THIN SECTIONS (1 ΜICRON) SHOWING UNINFECETD CACO-2 CELLS AND C. CONCISUS UNSWCD INFECTED CACO-2 CELLS ...... 215 FIGURE 5.7. TRANSMISSION ELECTRON MICROSCOPY IMAGES OF ULTRA-THIN SECTIONS OBTAINED FROM SAMPLES PREPARED USING AGAR EMBEDMENT ...... 217 FIGURE 5.8. TRANSMISSION ELECTRON MICROSCOPY ULTRA-THIN IMAGES OF C. CONCISUS UNSWCD WITHOUT AGAR ...... 218 FIGURE 5.9. TRANSMISSION ELECTRON MICROSCOPY ULTRA-THIN IMAGES OF CACO-2 CELLS WITHOUT AGAR EMBEDMENT, SHOWING THE INITIAL STAGE OF C. CONCISUS UNSWCD INFECTION ...... 220 FIGURE 5.10. VISUALISATION OF THE INTERNALISATION OF C. CONCISUS INTO CACO-2 CELLS WITHOUT AGAR EMBEDMENT USING TRANSMISSION ELECTRON MICROSCOPY ...... 221 xxiii FIGURE 6.1. THE PUTATIVE PATHOGENIC MECHANISMS USED BY C. UREOLYTICUS TO INTERACT WITH HUMAN INTESTINAL EPITHELIAL CELLS IN THE GASTROINTESTINAL SYSTEM ...... 242 FIGURE 6.2. THE SCHEMATIC REPRESENTATION OF THE DAMPENING EFFECT EXERTED ON THE AUTOPHAGY PATHWAY BY C. CONCISUS UNSWCD INFECTION ...... 246 FIGURE 6.3. THE PUTATIVE PATHOGENIC MECHANISMS USED BY C. CONCISUS TO ATTACH TO HUMAN INTESTINAL CELLS AND CAUSE DISEASE...... 245 APPENDIX FIGURE 1. PIE CHART OUTLINING THE FUNCTIONAL CLASSIFICATION OF C. UREOLYTICUS UNSWCD BIOINFORMATICALLY ANALYSED AND PREDICTED TO BE NON- SECRETED PROTEINS (N = 82), WITH VALUES EXPRESSED AS PERCENTAGES ...... 289 xxiv LIST OF TABLES TABLE 1.1. A COMPARISON OF THE PERCENTAGE INVASION AND ADHERENCE OF EIGHT C. CONCISUS STRAINS THAT ARE ABLE TO INVADE AND ADHERE TO CACO-2 CELLS...... 62 TABLE 2.1. C. CONCISUS STRAINS EMPLOYED IN THIS THESIS AND THEIR ISOLATION SOURCE...... 92 TABLE 2.2. C. UREOLYTICUS STRAINS EMPLOYED IN THIS STUDY AND THEIR ISOLATION SOURCE...... 93 TABLE 2.3. AN OUTLINE OF OTHER STRAINS EMPLOYED IN THIS STUDY AND THEIR RELEVANT ISOLATION ORIGINS...... 93 TABLE 2.4. THE MORPHOLOGICAL CHARACTERISTICS AND BIOCHEMICAL TESTS USED FOR THE IDENTIFICATION OF C. CONCISUS AND C. UREOLYTICUS STRAINS...... 100 TABLE 3.1. C. UREOLYTICUS AND OTHER BACTERIAL SPECIES EMPLOYED IN THIS STUDY AND THEIR ISOLATION SOURCE...... 105 TABLE 3.2. THE PERCENTAGE RELATIVE ATTACHMENT AND INVASION OF C. UREOLYTICUS STRAINS TO CACO-2 CELLS IN THE ABSENCE AND PRESENCE OF PRE- EXISTING INFLAMMATION...... 122 TABLE 3.3. THE PERCENTAGE RELATIVE ATTACHMENT OF C. UREOLYTICUS STRAINS TO HT-29 CELLS IN THE ABSENCE AND PRESENCE OF PRE-EXISTING INFLAMMATION...... 123 TABLE 3.4. THE FUNCTIONAL CLASSIFICATION OF C. UREOLYTICUS UNSWCD SECRETED PROTEINS, BIOINFORMATICALLY ANALYSED AND PREDICTED TO BE SECRETED (N = 29) WITH THE OPEN READING FRAME (ORF) OF EACH INDIVIDUAL PROTEIN ALSO PROVIDED….…...... 136 TABLE 4.1. GENES WITHIN THE AUTOPHAGY PATHWAY REGULATED UPON INFECTION WITH C. CONCISUS UNSWCD...... 181 APPENDIX TABLE 1. PROTEINS IDENTIFIED WITHIN THE SECRETOME FRACTION OF CAMPYLOBACTER UREOLYTCIUS UNSWCD (N = 111)...... 283 xxv APPENDIX TABLE 2. REGULATION OF (N = 84) GENES WITHIN THE AUTOPHAGY PATHWAY UPON INFECTION WITH C. CONCISUS UNSWCD...... 290 xxvi ABBREVIATIONS 1DE One-Dimensional Gel Electrophoresis 1D SDS PAGE One-Dimensional Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis 3-MA 3-Methyladenine AFLP Amplified Fragment Length Polymorphisms AHL Acyl Homoserine Lactone AI-2 Autoinducer-2 AICC Adherent Invasive C. concisus ANOVA Analysis Of Variance ATG Autophagy Related ATG16L1 Autophagy Related 16-like 1 AToCC Adherent Toxigenic C. concisus BB Brucella Broth BCA Bicinchoninic Acid BCV Brucella Capsular Vesicle BHI Brain Heart Infusion BHIG Brain Heart Infusion plus Glycerol BLAST Basic Local Alignment Search Tool BO Barrett’s Oesophagus CCV Campylobacter Containing Vacuole CD Crohn’s Disease cDNA Complementary Deoxyribonucleic Acid CFU Colony Forming Units CLSM Confocal Laser Scanning Microscopy CO2 Carbon Dioxide CQD Chloroquine Di-Phosphate CTSD Cathepsin D CTSS Cathepsin S DNA Deoxyribonucleic Acid DMSO Dimethyl Sulfoxide DPBS Dulbecco’s Phosphate Buffered Saline DTT Dithiothreitol ECM Extracellular Matrix xxvii EDTA Ethylenediaminetetraacetic Acid EEA-1 Early Endosomal Antigen-1 EGFP Enhanced Green Fluorescent Protein ELISA Enzyme linked Immunosorbent Assay ESEM Environmental Scanning Electron Microscopy FBS Foetal Bovine Serum f-CP Calprotectin FHA Filamentous Haemagglutin Adhesin g Grams g Gravitational Force GBS Guillain–Barré syndrome GFP Green Fluorescent Protein GIT Gastrointestinal Tract GORD Gastro-Oesophageal Reflux Disease h Hours HBA Horse Blood Agar HCl Hydrochloric acid IBD Inflammatory Bowel Disease IBS Inflammatory Bowel Syndrome IC Immuno-competent IL Interleukin IFN-γ Interferon Gamma IgG Immunoglobulin-G IRGM Immunity related GTPase M Kb Kilo Bases KEGG Kyoto Encyclopedia of Genes and Genomes kV Kilo Volts l Litre LAMP-1 Lysosomal Associated Membrane Protein-1 LC3 Light Chain Three LDH Lactate Dehydrogenase LPS Lipopolysaccharide LR London Resin LTQ-FT-MS/MS Linear Trap Quadruple-Fourier Transformer Tandem Mass Spectrometry xxviii LTQ LC-MS/MS Linear Trap Quadruple Liquid Chromatography- Mass Spectrometry MAMP Microbe-associated Molecular Patterns Mb Mega Bases MEM Minimal Essential Medium min Minutes miR MicroRNA ml Milliliters MLC Myosin Light Chain MLST Multilocus Sequence Typing MOI Multiplicity of Infection mRNA Messenger Ribosomal Ribonucleic Acid MS Mass Spectrometry mTOR Mammalian Target of Rapamycin NA Nutrient Agar NaOH Sodium Hydroxide NCBI National Centre for Biotechnology Information NF-κB Nuclear Factor Kappa B NLR NOD-like Receptor NS Non Significant OA Oesophageal Adenocarcinoma OD Optical Density PAGE Polyacrylamide Gel Electrophoresis PAMP Pathogenic Associated Molecular Patterns PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PD-IBS Post-Dysenteric Irritable Bowel Syndrome PFGE Pulsed Field Gel Electrophoresis PI3P Phosphatidylinositol 3-phosphate PI-IBS Post-infectious Irritable Bowel Syndrome PMN Polymorphonuclear PNS Peripheral Nervous System PRR Pattern Recognition Receptor qPCR Quantitative Polymerase Chain Reaction qRT-PCR Quantitative Real Time Polymerase Chain Reaction xxix PV Parasitophorus Vacuole QS Quorum Sensing RAPD Random Amplified Polymorphic DNA rDNA Ribosomal Deoxyribonucleic Acid RLC Reynolds Lead Citrate rRNA Ribosomal Ribonucleic Acid RTX Repeats in Toxin SDS Sodium Dodecyl Sulfate sec Seconds SEM Standard Error Mean ScEM Scanning Electron Microscopy SNP Single Nucleotide Polymorphism SPB Sorensen’s Phosphate Buffer spp Species STRING Search Tool for the Retrieval of Interacting Proteins T4SS Type IV Secretion System T6SS Type VI Secretion System TCA Tricholoracetic Acid TEM Transmission Electron Microscopy TER Trans-Membrane Epithelial Resistance TFP Type Four Pilli TJ Tight Junctions TLR Toll-like Receptor TLR-4 Toll-like Receptor-4 TPS Two-Partner Secretion TNF-α Tumor Necrosis Factor Alpha TSU Tris-HCl, SDS, Urea UA Uranyl Acetate UC Ulcerative Colitis UNSW University of New South Wales VCC Vibrio cholera cytolysin ZOT Zona-occludens Toxin xxx CHAPTER 1 1. INTRODUCTION 1.1. The Campylobacter genus Members of the Campylobacter genus are Gram-negative, spiral / s-shaped, curved or rod-shaped bacilli. Campylobacter spp have been reported to be between 0.2 to 0.9 µm wide and 0.5 to 5 µm long (1-3). Campylobacters are also known to be nutritionally fastidious microaerophilic organisms, which reside in the gastrointestinal tracts of humans and animals. Further, Campylobacter spp also have a low G + C content (1-3). The Campylobacter genus was initially described by Sebald and Véron in 1963. At that time the Campylobacter genus contained only two Campylobacter species, Campylobacter fetus and Campylobacter bubulus, previously named Vibrio fetus and Vibrio bubulus (4, 5). Since 1963 the development and application of molecular typing techniques such as 16S rRNA sequencing, has resulted in many bacteria being reclassified into the Campylobacter genus. For example based on the use of these techniques, Vandamme et al. reported the reclassification of Bacteroides gracilis to Campylobacter gracilis (2). At the current time the Campylobacter genus encompasses 16 species and 6 subspecies of Campylobacter including: C. jejuni, C. coli, C. concisus, C. lari, C. hominis, C. fragilis, C. showae and the reclassified bacterium C. ureolyticus (5). 31 Chapter 1: Introduction 1.1.1. Campylobacter species associated with gastroenteritis Campylobacters species and in particular C. jejuni and C. coli, have been shown to be major causative agents of acute gastrointestinal infection (6). C. jejuni is reported to be one of the most common Campylobacter isolates detected in patients suffering from gastroenteritis (7), with C. coli being the second most prevalent and C. upsaliensis and C. lari being reported to being responsible for the remainder of cases (6). More recent studies have shown, that in addition to the above Campylobacter species, C. concisus and C. ureolyticus are increasingly being detected in patients with gastroenteritis worldwide (8-12). Campylobacteriosis is the general term given to infections caused by Campylobacter species, with gastroenteritis being the most common outcome. Intestinal Campylobacterosis manifests itself as a form of enteritis in the terminal region of the ileum and colon (13). Signs and symptoms of gastroenteritis occur following a 2 to 5 day incubation period. These include fever, vomiting, headache, abdominal cramping, nausea and severe diarrhoea including bloody or watery diarrhoea depending on the gravity of the infection (6, 13, 14). The symptoms typically last for one week and in moderate to severe infections may be treated with antibiotics such as Azithromycin and Ciprofloxacin (6, 13, 14). Following gastroenteritis caused by Campylobacter jejuni a percentage of patients develop Guillain Barré syndrome. 1.1.2. Campylobacter jejuni and Gullain Barré Syndrome Guillain-Barré syndrome (GBS) is a neurological autoimmune disorder brought on by immune-mediated demyelination of the peripheral nervous system (PNS). Based on the current literature the annual incidence of GBS is approximated to be in the range of 1.2 to 2.3 cases per 100,000 people. The number of Campylobacter cases resulting in GBS 32 Chapter 1: Introduction has been reported to be 0.07% (95% CI, 0.03% to 0.15%) (15, 16), with higher incidence rates being associated with increasing age and male gender (15, 17). Importantly, epidemics of GBS have been linked with occurrences of C. jejuni infection (15, 18) with evidence suggesting that C. jejuni linked GBS cases are rising in certain countries (15, 19). GBS causes motor weakness, areflexia (absent reflexes) and mild-to- moderate sensory abnormalities, which can lead to paralysis (20-22). Among numerous microbial infections, only C. jejuni a leading cause of gastroenteritis worldwide (23, 24) is firmly established as a causative agent of GBS (25, 26). 1.1.3. Campylobacter and Irritable Bowel Syndrome Studies would suggest that approximately 25% of patients who have suffered Campylobacter enteritis may potentially develop post-dysenteric irritable bowel syndrome (PD-IBS) (27). The onset of gastroenteritis can potentially lead to the development of post-infectious irritable bowel syndrome (PI-IBS), as a consequence of the establishment of a chronic Campylobacter infection (28). The clinical manifestations of PI-IBS include the development of chronic abdominal pain, an altered bowel habit and potentially the development of severe diarrhoea (28). However, the risk factors for Irritable Bowel Syndrome (IBS) following Campylobacter infection are not well understood. According to Everest et al. following the resolution of acute infection caused by C. jejuni infection, chronic inflammation may occur (6). For example Spiller et al. have demonstrated that post dysenteric IBS develops in up to 25% of patients following acute Campylobacter infection (27). Indeed, one of the most common risk factors for post-infectious IBS is Campylobacteriosis (6). More recently the development of PI-IBS following gastroenteritis caused by Campylobacter concisus has been investigated by Nielsen et al. (29). In this 33 Chapter 1: Introduction community-based investigation carried out in the Northern region of Denmark. Adult patients diagnosed with C. concisus infection (n = 106) and C. jejuni / C. coli infection (n = 162) were asked to participate in a questionnaire detailing IBS symptoms and psychometric scores at baseline and after 6 months. The results of this study indicated that 56/268 (21%) of Campylobacter positive patients reported symptoms of IBS at 6 months. In patients with confirmed C. concisus induced gastroenteritis, 26/106 (25%) suffered IBS symptoms 6 months following infection, a percentage comparable to that reported in patients suffering IBS following C. jejuni / C. coli infection 30/162 (19%) (29). Based on their questionnaire high baseline anxiety scores were found to be a significant risk factor for the development of PI-IBS following C. concisus infection. Additionally, these affected patients had high risk ratio scores for anxiety, depression and somatisation, which were all identified in this study as considerable risk factors for the development of PI-IBS in all patients with persistent Campylobacter induced gastroenteritis (29). Thus, these findings suggest that psychological factors may play a significant role in development of PI-IBS. 1.2. Campylobacter ureolyticus 1.2.1. Historical background of Campylobacter ureolyticus In 1948, Henriksen first described the isolation of an anaerobic Gram-negative bacillus from the genital tract of a patient suffering from endometriosis and from pus collected from pulmonary and perineal abscesses. In their study Henriksen reported this bacterium to produce corroding pits upon growth on agar-based media (30). Two years later both Holm and Eiken isolated a similar anaerobic bacterium, and named it the “corroding bacillus” (31-33). Following the isolation of a similar anaerobic organism from patients with buccal abscesses in 1958, Eiken proposed to name this clinical 34 Chapter 1: Introduction isolate Bacteroides corrodens (33). In 1978, Jackson and Goodman renamed a subgroup of B. corrodens, which were anaerobic and urease positive, as Bacteroides ureolyticus, meaning urea dissolving (34). 1.2.2. Reclassification of Bacteroides ureolyticus to Campylobacter ureolyticus In 1991 Vandamme et al. proposed that B. ureolyticus should be reclassified as a member of the Campylobacter genus based upon sequencing of the 16S ribosomal RNA (rRNA) gene of B. ureolyticus (35). According to Vandamme et al. B. ureolyticus fell into rRNA cluster I of the rRNA super family VI, which contains members of the Campylobacter genus (35). A follow-up study by the same group, which used polyphasic taxonomic analyses showed that B. ureolyticus shared similar features including respiratory quinone content, a similar DNA base ratio and similar phenotypic characteristics to other Campylobacter species, including C. jejuni (2). A subsequent study by Vandamme et al. acknowledged that B. ureolyticus had a number of properties similar to Campylobacter species (1). However, differences in fatty acid composition, proteolytic metabolism, hydrolysis of gelatin, casein and the ability to hydrolyse urea between B. ureolyticus and Campylobacter species were observed (1). As a result of the differences identified by Vandamme et al. B. ureolyticus was not formally reclassified as a Campylobacter for a number of years (1). To ensure that re- classification of B. ureolyticus as a member of the Campylobacter genus was valid, isolation and thorough taxonomic characterisation of additional B. ureolyticus-like bacteria were undertaken. In the interim, B. ureolyticus was reported to be incertae sedis (of uncertain placement). In 2010, a publication by Vandamme et al. finally acknowledged and verified the reclassification of B. ureolyticus to C. ureolyticus comb. nov, and it became a newly designated member of the Campylobacter genus (36). 35 Chapter 1: Introduction 1.2.3. Campylobacter ureolyticus (previously known as B. ureolyticus) and disease Prior to its re-classification, the identification of B. ureolyticus from patients with a wide range of diseases, including superficial ulcers, soft-tissue infections, non- gonococcal urethritis, perianal abscess and gangrenous lesions, as well as the genital tract and amniotic fluid were reported in the literature (37-40). Further, B. ureolyticus was reported to be commonly isolated from the oral cavity of patients suffering from periodontal disease, a disease which involves inflammatory and destructive conditions of the tissues surrounding the teeth (40). 1.2.4. C. ureolyticus and Inflammatory Bowel Disease Furthermore, C. ureolyticus has been isolated from biopsy samples of children with newly diagnosed Crohn’s disease (CD) (41). CD, is one of the two major subtypes of Inflammatory Bowel Diseases (IBD), a chronic relapsing idiopathic inflammatory disease of the gastrointestinal tract that is a significant cause of morbidity worldwide (42). CD is characterised by deep submucosal ulcerations, which can occur anywhere throughout the gastrointestinal tract, although the latter part of the small intestine and the first part of the colon (ileo-colonic) are the regions most commonly involved (43). In contrast Ulcerative Colitis (UC) is characterised by inflammation that occurs throughout the colon, and is typified by the presence of superficial and fine ulcerations. The histological features of UC include the presence of an elevated number of polymorphonuclear cells within the lamina propria as well as in the intestinal crypts where micro-abscesses may form. Although currently the aetiology of IBD is not well understood, it is well accepted that microorganisms, as well as host genetic predisposition, host immune responses and environmental factors play an important role (43). 36 Chapter 1: Introduction Two recent studies have investigated the prevelance of C. ureolyticus in adult patients with IBD (44, 45). In the first of these studies Mahendren et al. examined the prevalence of C. ureolyticus in biopsy samples collected from the ileum, caecum, descending colon and rectum of 15 patients with CD, 13 with UC and 33 non-IBD controls with no pathology, using both PCR and sequencing analysis (44). The results of this study showed C. ureolyticus to be present in 13% of CD patients, 8% of UC cases and 6% of healthy controls with no pathology. However, in this study no significant difference was detected in the prevalence of C. ureolyticus between CD or UC patients and controls (44). In a second study Mukhopadhya et al. determined the prevalence of Campylobacter species in biopsy specimens collected from patients undergoing diagnostic colonoscopy. Of these, 69 were shown to have histologically proven UC and 65 were healthy controls (45). Following collection and storage of the biopsies at -80oC, DNA was extracted and subjected to Campylobacter genus specific and Campylobacter concisus specific polymerase chain reaction (PCR) and sequencing. Based on the Campylobacter genus specific primers, a significantly higher prevalence of Campylobacters were detected in UC patients 73.9% (51/ 69) as compared with the controls 23.1% (15/65) controls (P = 0.0001). Sequencing of the PCR products of the Campylobacter positive samples, showed C. ureolyticus to be present in 15/69 UC patients and in 2/65 controls. Based on these results the prevalence of C. ureolyticus in UC patients was found to be significantly higher (21.7%) than that in controls (3.08%) (P = 0.0013). Based on their results Mukhopadhya et al. concluded that C. ureolyticus could play a role in the chronic inflammation observed in UC (45). 37 Chapter 1: Introduction 1.2.5. C. ureolyticus and gastroenteritis In recent years C. ureolyticus has been increasingly detected in patients with gastroenteritis worldwide, suggesting that C. ureolyticus may be a causative agent of gastroenteritis. In a recent study Bullman et al. used the EntericBio multiplex PCR system to analyse faecal samples collected from 7194 Southern Irish patients presenting with symptoms of diarrhoea over a 12-month period (46). Of these samples, 373 samples were Campylobacter positive, 72.4% being identified as C. jejuni, 24.4% as C. ureolyticus, 6.7% as C. coli, 2.1% C. fetus, 1.3% C. hyointestinalis, 1.1% C. upsaliensis and 0.5% C. lari (46). Interestingly, the results of this study showed that in Southern Ireland the prevalence of C. ureolyticus in faecal samples of patients with gastroenteritis was significantly higher than C. coli, which previously was believed to be the second most common causative agent of Campylobacter related gastroenteritis (46). Additionally, analysis of the data based on age group revealed a high prevalence of C. ureolyticus in patients across all age groups (46). However, in the (< 5 years and > 70 years) age groups the prevalence of C. ureolyticus was more frequently detected. Further, re-screening of the 349 Campylobacter positive samples using 16S rRNA sequencing and PCR analysis using primers targeting of the hsp60 gene of C. ureolyticus revealed that 83 (23.8%) of these Campylobacter positive samples were C. ureolyticus (46). Based on their results, Bullman et al. suggested that C. ureolyticus could potentially be a gastrointestinal pathogen (46). The early findings obtained by Bullman indicating the presence of C. ureolyticus in patients with diarrhoea were re-confirmed by a later study carried out a year later, by the same group (47). This study investigated the presence of Campylobacter spp in 204 samples that were culture negative for Campylobacter spp using a combination of Campylobacter specific uniplex PCRs and 16S rRNA sequencing to confirm the 38 Chapter 1: Introduction presence or absence of Campylobacter DNA. This showed 191 (93.6%) of the culture negative samples to be Campylobacter DNA positive. Based on species-specific PCR C. ureolyticus was identified in 41.9% of these samples (47). Interestingly, C. ureolyticus accounted for 1 of the 3 most prevalent Campylobacter spp alongside C. jejuni (50.8%) and C. coli (5.7%). This study again highlights that non-culturable Campylobacter spp could potentially be responsible for a large proportion of human enteritis and that the true incidence level is likely to be significantly underestimated (47). The detection of C. ureolyticus has not only been limited to findings obtained in studies in Southern Ireland, but also from studies of patients suffering from gastroenteritis in Chile, South America. For instance, Collado et al. used a combination of traditional culture and PCR restriction fragment length polymorphism (RFLP) analysis, based on previous RFLP methods (48, 49), to determine the presence of C. ureolyticus in faecal samples from 140 patients with diarrhoea as well as 116 asymptomatic controls (9). Based on PCR restriction fragment length polymorphism analysis, C. ureolyticus was detected in 5/140 (3.6%) of Chilean patients with diarrhoea and in 1.7% of healthy controls (9). However, statistical analysis showed no significant difference in C. ureolyticus detection between patients with diarrhoea and healthy controls (P > 0.05) (9). However, in healthy patients with no reported pathology the most prevalent species was C. concisus (3.4%), followed by C. ureolyticus (1.7%), while C. jejuni and C. coli showed the same prevalence (0.9%). Additionally, this study highlighted that C. ureolyticus was the third most prevalent species detected in diarrhoeic samples behind C. jejuni (10.7%) and C. concisus (11.4%) (9), which is in accord with similar findings from studies in both Europe (47, 50) and Africa (51). 39 Chapter 1: Introduction While some studies have reported high detection rates between non-jejuni Campylobacter spp like C. ureolyticus in patients with gastroenteritis (8, 46, 47). Other studies in contrast have also argued that no other Campylobacter species apart from C. jejuni and C. coli are involved in gastroenteritis as a result of their findings (52-54). One such example is a study by Cornelius et al. that examined the prevalence of epsilon-proteobacteria in faecal samples of diarrhoeic patients from New Zealand, in which a specific pathogen was not identified (52). This study showed that C. concisus (46.9%), C. ureolyticus (10.9%), C. hominis (8.6%), and C. gracilis (14.0%) were detected in 128 patients with diarrhoea (52). Moreover the frequency of these pathogens detected in diarrhoeic patients was not significantly different to that in healthy controls, a finding that suggested a lack of association between emerging Campylobacter species like C. ureolyticus and C. concisus in gastroenteritis cases (52). Additionally, the identification of C. ureolyticus in 12 (24.5%) of 49 healthy volunteers in the same study led Cornelius et al. to argue that C. ureolyticus and C. hominis are unlikely causes of diarrhoea (52). This argument is in contrast to previous studies indicating a strong association between C. ureolyticus and gastroenteritis (8, 46, 47). While a possible explanation for these variations in the observed association between C. ureolyticus and gastroenteritis could relate to differences in the sensitivity and specificities of the molecular methods used, or to geographic differences in the prevalence of C. ureolyticus (55). Further, studies in other regions are required to support the role of C. ureolyticus in gastroenteritis, and remove any discrepancies that would refute such associations. 1.2.6. Sources and transmission of C. ureolyticus infection The source of C. ureolyticus infections in humans remains unknown. Given the isolation of C. ureolyticus from a high percentage of diarrhoeal clinical samples 40 Chapter 1: Introduction obtained from Irish patients with diarrhoea (46, 47). Koziel et al. investigated whether C. ureolyticus was a zoonotic infection (56). In their study the presence of C. ureolyticus was investigated in a range of domestic animals in Ireland. While chicken and cattle are thought to be animal reservoirs of Campylobacter spp, particularly for C. jejuni and C. coli (57), other animals including pigs, sheep and dogs have also been associated with human infections (56). In their study Koziel et al. investigated over a six month period the presence of C. ureolyticus in 164 samples collected from dogs, cats and pigs using molecular methods based on the detection of the C. ureolyticus specific gene hps60. C. ureolyticus was detected in 32% (10/31) of feline faeces, 9% (4/44) of canine faeces and 18% (16/89) of porcine faeces (56). Based on their findings Koziel et al. concluded that the emerging pathogen C. ureolyticus in a similar manner to C. jejuni, also has zoonotic potential (56). In a second study Koziel et al. investigated using molecular-based detection methods whether cattle or un-pasteurised milk could be a source of C. ureolyticus. This showed that C. ureolyticus was present in 1 of 20 bovine faeces and in 6/47 un-pasteurised milk samples. In contrast, unlike C. jejuni and C. concisus, C. ureolyticus was not detected in poultry (58). Importantly, these studies were the first articles to report the presence of C. ureolyticus in animals, suggesting a possible route for its transmission to humans (58). 1.2.7. Genetic heterogeneity Recent studies suggest that C. ureolyticus could be made up of a number of genomospecies similar to those observed in C. concisus (58,59). While it is thought that phenotypically an isolate may resemble another isolate of the same species. On the contrary it is also possible that these resembling isolates could also exhibit a great 41 Chapter 1: Introduction degree of genetic heterogeneity at the genomic level (61,62). This concept of intra- species variation provides a logical perspective to a differing opinion on the true pathogenic nature of C. ureolyticus (18,63), and warrants further investigation. A recent study by Bullman et al. revealed 75–79.5% of proteins to be highly conserved (70%) between the C. ureolyticus strains C. ureolyticus DSMZ 20703 and ACS-301-V- Sch3b (59). The genomes of both DSMZ 20703 and ACS-301-V-Sch3b were shown to be roughly similar in size being 1.74 Mb and 1.66 Mb respectively (59). However, bioinformatic analysis indicated only 18.8% (341/1810) and 17.1% (290/1700) of the protein coding sequence in these species were unique (59). Using the same parameters with C. jejuni NCTC11168 based as the reference genome, individual and multiple comparisons to 3 other C. jejuni strains, revealed that 92% and 87% of proteins are highly conserved (59). Such data provides compelling evidence of considerable variation existing within these two C. ureolyticus genomes. Interestingly, average percentage identities for all homologs revealed that C. ureolyticus strains had a higher variation when compared with the phylogenetically related species C. jejuni (94% against 98%) (59). Furthermore, this same study reported that analysis of whole genome comparisons focusing on the protein sequences of the two C. ureolyticus strains (DSMZ 20703 and ACS-301-V-Sch3b) against other members of the same genus, resulted in genetic conservation across the different Campylobacter spp with 9–22% of gene products been conserved (59). In addition to the significant degree of variation observed between the genomes of DSMZ 20703 and ACS-301-V-Sch3b strains, protein profiles of additional 6 C. ureolyticus isolates, were observed to cluster in accordance with banding patterns (59). Subsequently, the observation of these characteristic banding patterns was also 42 Chapter 1: Introduction thought to contribute to the high degree of heterogeneity between the C. ureolyticus strains analysed (59). Bullman et al. conducted whole genome analysis of at least 12 C. ureolyticus strains (CIT01–CIT13), which were isolated from animal reservoirs, asymptomatic patients, and patients with diarrheal illness (59). The initial findings of this analysis revealed considerable heterogeneity to exist between the different C. ureolyticus strains analysed (59). These findings were also supported by comparisons of whole genome coding sequences between these strains, which also showed signs of heterogeneity. It was demonstrated through whole genome comparison that 2–20% of C. ureolyticus proteins are distinctively based on paired genome comparison of the coding sequences of 14 C. ureolyticus strains (59). Moreover, it is becoming increasingly evident that a single strain rarely typifies an entire bacterial species (60). One perspective that explains the heterogeneity in the virulence potential of the C. ureolyticus genomospecies is the “eco-evo” perspective on host-pathogen interactions (60). The “eco-evo” perspective is based on genomic evidence that postulates that commonly used laboratory strains have undergone considerable changes, producing genotypic and phenotypic variants during their conversion from environmental to virulent agents (61, 62). Recent studies suggest that C. ureolyticus could also potentially be made up of a number of genomospecies (60), as has been reported in C. concisus (63, 64). 1.2.8. The secretome and virulence factors of C. ureolyticus Bullman et al. recently published the first whole genome analysis of two C. ureolyticus isolates DSMZ 20703 (an isolate from amniotic fluid and ACS-301-V-sch3b (a vaginal isolate) in silico (59). Using comparative analysis, hybridisation and gene ontology searches against other Campylobacter species, this study reported a high degree of 43 Chapter 1: Introduction heterogeneity amongst C. ureolyticus isolates (59). Based on the results of their analyses 75–79.5% of proteins were shown to be highly conserved having a 70% identity between C. ureolyticus DSMZ 20703 and C. ureolyticus ACS-301-V-Sch3b (59). Additionally, both genomes are currently available on the databases such as NCBI. Further, whole genome comparisons of the protein encoding sequences of the 2 C. ureolyticus strains (DSMZ 20703 and ACS-301-V-Sch3b) against other members of the same genus, exhibited a degree of conservation across the different species of Campylobacter, with 9–22% of gene products being conserved (59). The largest number of conserved protein homologs were found in C. concisus with the lowest identified within C. upsaliensis (59). In the same study Bullman et al. investigated the secretomes of DSMZ 20703 and ACS- 301-V-Sch3b through in silico analyses (59). This identified 106 recognised virulence associated factors, of which 52 were presumed to be secreted (59). These included key established virulence factors used by other pathogenic Campylobacter spp (59). This study will be discussed in detail in Chapter 3. These included virulence factors associated with adhesion and colonisation (CadF, PEB1, IcmF and FlpA), invasion (ciaB and 16 virB-virD4 genes) and toxin production (S-layer RTX and Zot) (59). Further this study (59) showed that, as has been reported in C. concisus spp, heterogeneity to exist amongst C. ureolyticus spp (59). Additionally, Bullman et al. also identified 13 hemolytic cytotoxins and cytolysin-related proteins, from which 8 of these toxins were predicted to be secreted (59). Importantly, these pore-forming toxins could potentially be a vital constituents of the virulence potential of C. ureolyticus, as their purpose is to increment the availability of iron during the infection process and could potentially promote human disease considering the availability of iron in the human bloodstream (65). 44 Chapter 1: Introduction The secretome represents the totality of secreted proteins excreted from an organism (66) and is an essential component of the pathogenic repertoire and virulence of a pathogen (67). In a recent study Bullman et al. examined the secretome of C. ureolyticus and found its secretome contained a number of adhesins, which are known play an important role in host cell-pathogen interactions and support the infection process by mediating the interaction of pathogens to host cells. One such adhesin is HecA protein, a member of the filamentous haemagglutinin adhesin (FHA) family (59). The protein products of these genes form a two-partner secretion (TPS) system, in which a TpsA family exoprotein is recognised by the associated TpsB family channel-forming transporter permitting passage through the membrane (68). Subsequently, in support of the “eco–evo” theory previously mentioned, there has been reports of lateral gene transfer of HecA in other organisms (69-71). Thus, it is possible that this adhesin could have been acquired from another bacteria that was not a member of Campylobacter genus, probably while they were residing in the same ecological niche (69-71). Subsequently, a potential microbial source of this gene could well be the Fusobacterium species, which like C. ureolyticus has been linked to the cases of gastroenteritis and periodontitis (40, 45, 72). A PEB1 homolog has also been identified in C. ureolyticus strains. Interestingly PEB-1 been shown to play a role in virulence and colonisation in C. jejuni infection (73). For example a study using in vitro epithelial cell line cultures demonstrated that mutations in the PEB1 gene of C. jejuni resulted in a 50 to 100-fold decrease in adherence to epithelial cells. In a later study a 15-fold decrease in invasion of the same epithelial cells by C. jejuni was also reported. (74). The intracellular multiplication factor, IcmF was also identified in C. ureolyticus (59). Moreover, it is thought that this protein is a constituent of a Type VI secretion system (T6SS), which has recently been identified as 45 Chapter 1: Introduction having a role in bacterial virulence within eukaryotic host cells and may contribute to human pathogenesis (75, 76). Recently, Lucid et al. sequenced the genome C. ureolyticus CIT007, a strain isolated form the faecal sample of an elderly female presenting with diarrheal illness and end- stage chronic renal disease (77). As a result of this study, Lucid et al. reported that the genome of C.ureolyticus CIT007 genome (77), comprised a number of virulence factors previously identified in the genomes of C. ureolyticus DSMZ 20703 and ACS-301 V- Sch (59). These included a VirB/D4 Type IV secretion system (T4SS), adhesion associated factors (fibronectin-fibrinogen binding protein) and invasion-associated factor (CiaB) (59). The identification of these adhesion-associated factors in the genome of C. ureolyticus CIT007 further highlights their functional relevance in the pathogenicity of this bacterium. Subsequently, the findings previous mentioned by Lucid et al. (77) were in accordance with the findings of Bullman et al. who previously sequenced the genomes of the strains DSMZ 20703 and ACS-301 V-Sch C. ureolyticus (59). 1.3. Background Campylobacter concisus 1.3.1. Historical background of Campylobacter concisus Campylobacter concisus (Latin translation meaning concise) is described as a Gram- negative curved to spiral rod which is 0.5 × 4 µm in size with rounded ends (1). It is motile due to a single polar flagellum (1). In 1981 Tanner et al. first isolated C. concisus from the gingival crevices of patients with gingivitis and periodontitis (78). In their study Tanner et al. reported the isolation 46 Chapter 1: Introduction of six isolates from individuals suffering from periodontitis and gingivitis, which they described as Gram-negative, non-corroding, microaerobic, predominantly curve shaped rods, with a deoxyribonucleic acid guanine plus cytosine content of 34-38% (78). Based on the characteristics of these isolates, Tanner et al. suggested that these isolates represented a new species, which they named “Campylobacter concisus spp. nov.” (Strain type, ATCC 33237) (78). The first isolation of C. concisus from a non-oral origin was reported in 1985, by Johnson and Finegold who isolated C. concisus from a foot ulcer of a patient with diabetes mellitus (79). Since that time C. concisus has been implicated in a range of other diseases including oral disease such as oesophageal disease, gastroenteritis and inflammatory bowel disease (80-83). 1.4. Diseases associated with Campylobacter concisus 1.4.1. C. concisus and oral cavity diseases Following the initial isolation of C. concisus from the oral cavity by Tanner et al. an increasing focus of research into C. concisus centred on investigating whether C. concisus was associated with disease or was simply a member of the normal microbiota (78). Initial studies in this area focussed upon investigation of the role of C. concisus in periodontal disease (84). Such studies showed that within the oral cavity C. concisus could be isolated from both healthy sites (85) and diseased sites including gingivitis (86) and periodontitis (87, 88). Early associations between C. concisus and periodontitis were made by Ebersole et al. who showed in adult patients with periodontitis undergoing subgingival scaling, that antibodies to C. concisus were elevated as compared with that in the normal population (89). This initial finding by 47 Chapter 1: Introduction Ebersole et al. was supported by a follow up study from Taubman et al. who found higher antibody levels to C. concisus in subjects with periodontal disease when compared to healthy subjects (90). Additionally, a study by Macuch and Tanner, which compared the presence of Campylobacter spp in healthy subjects with that in patients with gingivitis, initial periodontitis and established periodontitis (91). Findings from this study showed that C. concisus was more frequently isolated from subjects with initial periodontitis (68%) than healthy individuals (35%) (91). Moreover, given reports that the enzyme aspartate aminotransferase (AST) are elevated in gingival crevicular fluid (GCF), Kamma et al. determined the association between AST activity in GCF from periodontal sites of individuals with early onset periodontitis (92). This study showed that seven bacterial species including C. concisus were significantly higher in AST positive sites than in AST negative sites, with C. concisus being reported to have the fourth strongest positive association with AST activity (92). In 2010 Zhang et al. compared the prevalence of C. concisus in the saliva of 59 healthy individuals of different ages and patients with IBD using culture and polymerase chain reaction (PCR) (93). Based on a PCR targeting the 16S rRNA gene, C. concisus was detected in 97% (57/59) of saliva samples collected from healthy individuals aged from 3-60 years (93) and in 8/8 (100%) of IBD patients. In contrast using a filtration culture method C. concisus was cultured from 75% (44/59) of saliva samples (93). Determination of the prevalence of C. concisus by age showed the C. concisus culture positivity rate in children 3 - 5 years of age to be significantly lower than that in 12-18 years olds (P < 0.01) as well as all other age groups (P < 0.05) (93). Based on the study by Zhang et al. 33% (4/12) children aged 3-5 years were positive for C. concisus while 48 Chapter 1: Introduction 82% (9/11) of children aged 6-11 years were also positive (93). Based on their study Zhang et al. concluded that in healthy individuals C. concisus may be part of the normal human oral microflora (93). In a study by Ismail et al. the housekeeping genes of 70 oral and enteric C. concisus isolates obtained from 8 patients with IBD and 6 controls were compared using multilocus sequence typing (MLST) (94). Based on MLST analysis, 87.5% of individuals whose C. concisus isolate belonged to Cluster I had enteric disease, 6/8 having IBD and one bloody diarrhoea (94). This was significantly higher than that in the remaining individuals (28.6%) (P < 0.05) (94). A high prevalence of C. concisus has also been reported in the human oral cavity by Petersen et al. who detected C. concisus in 100% of saliva samples (11/11) collected from healthy individuals using a PCR targeting 16S rRNA gene (95). Given that C. concisus appears to be highly prevalent in healthy individuals, whether C. concisus is an opportunistic pathogen in inflamed areas or simply a commensal bacterium of the oral cavity remains unclear. 1.4.2. Campylobacter concisus and Barrett’s oesophagus Barrett’s oesophagus (BO) is a condition that results from inflammation and scarring of the lower oesophagus. The major predisposing factor for BO is gastro-oesophageal reflux disease (GORD), a condition in which gastric acid and bile are refluxed into the lower part of the oesophagus (96). BO occurs as a result of long-term damage to the oesophagus, which leads to the transformation of squamous oesophageal cells into columnar epithelial cells that share similar characteristics to those found in the intestines (82). BO is an important precursor to the development of oesophageal adenocarcinoma (OA) (97). 49 Chapter 1: Introduction Recent studies have suggested that the microbiota of the upper gastrointestinal tract might play a role in the development of BO (96). The lower oesophagus of healthy subjects has been reported to be colonised by a range of bacteria, including the Gram- positive bacteria Streptococci and Lactobacilli (98). Interestingly, in a recent study by MacFarlane et al. which compared the composition of the oesophageal mucosa in patients with BO with that in healthy controls, showed that in patients with BO a number of bacterial genera normally present in the oesophagus including Lactobacillus species were absent (82). In contrast a number of new microorganisms including, yeasts and Neisseria spp, were detected in the mucosa of 4 out of 7 subjects (57%) with BO (82). Interestingly, in their study MacFarlane et al. also showed the presence of a high abundance of C. concisus and C. rectus in four of the seven patients (57%) with BO which were not present in the control patients (82). Interestingly, C. concisus was shown to be the most prevalent bacterium isolated from both aspirate and mucosal samples (82). As a result of their study MacFarlane et al. hypothesised that “pathogenic and putative toxin generating Campylobacter species could potentially play a role in the initiation, continuation, or exacerbation of oesophageal disease” (82). Furthermore, in a study aimed at defining the oesophageal microbiota of patients with GORD, BO and OA as compared with controls, Blackett et al. compared the microbiota of 45 patients with BO, 37 with GORD and 34 with OA with that in 39 healthy controls using a combination of culture and molecular techniques (96). This showed that in patients with both GORD and BO the abundance of Campylobacter species increased, with C. concisus being detected in 19 of 37 of GORD patients (51.4%) and 19 of 45 BO patients (42.2%) as compared with 5 of 39 controls (12.8%) (96). In contrast the prevalence of C. concisus in OA patients was 8.8% (96). Based on their findings the authors suggested that a strong relationship existed between C. concisus and refluxate in 50 Chapter 1: Introduction the oesophagus, a condition that is minimal in most controls and reduced in most cancers. In a study published by members of the same group in abstract format submitted in Gut in 2013, Ho et al. investigated in patients with BO (n = 92), severe dysplasia (n = 39) and OA (n = 64) whether C. concisus may contribute to the malignant progression of BO to OA (99). This study reported no association to exist between C. concisus status in BO and altered expression of molecular markers of cancer development, phospho-p53 and Ki-67. In contrast the presence of C. concisus in BO was found to be associated with reduced COX-2 expression as compared with non-colonised subjects, a finding that led the authors to suggest that C. concisus may induce a phenotype protective against the development of OA (99). 1.4.3. Campylobacter concisus as a possible cause of acute gastroenteritis The first study to demonstrate convincing evidence that C. concisus was present in the gastrointestinal tract (GIT) of humans was by Vandamme et al. (84). In this study Vandamme et al. used protein analysis, immunotyping, DNA hybridisation, and DNA base analysis to determine the species identity of Campylobacter species previously isolated from blood cultures, 2 antral biopsies, a duodenal aspirate, 2 oesophageal biopsies and 8 faecal samples of patients with persistent diarrhoea (84). Based on the above tests these isolates and a range of Campylobacter reference strains were shown to be C. concisus, a finding that led Vandamme et al. to conclude that faecal carriage of C. concisus is common and might be associated with gastrointestinal disease (84). In light of these studies Van Etterijck et al. (54) used a filter isolation method to culture C. concisus from the faeces of children with enteritis (n = 174) and a healthy control population (n = 958) without diarrhoea (54). This study showed the isolation rate of 51 Chapter 1: Introduction C. concisus in children with diarrhoea (13%) to be higher than that in the control children (9%). However, statistical analysis revealed no significant difference (P = 0.15) between C. concisus isolation rates for children with enteritis and healthy controls. This led Van Etterijck et al. to conclude that C. concisus should not be considered a primary pathogen associated with gastrointestinal disease (54). More recently, Inglis et al. investigated the presence of Campylobacter species in stools from diarrhoeic (n = 442) and healthy (n = 58) humans living in South-western Alberta using PCR (53). In contrast to other studies, in this population the prevalence of C. concisus DNA as detected by two primer sets was significantly higher (P < 0.001) in healthy subjects (52%), as compared with patients with diarrhoea (34.8%), a finding that further questioned the involvement of C. concisus in gastroenteritis (53). A major problem in relation to determining the association between C. concisus gastroenteritis has been that the growth conditions required to isolate C. concisus differ significantly from that of other common Campylobacter species such as C. jejuni and C. coli (76). This relates to the fact that C. concisus is a fastidious bacterium that fails to grow under the atmospheric conditions commonly used for the culture of C. jejuni and C. coli. It is now well established that C. concisus requires a hydrogen rich (3-7%) anaerobic or microanaerobic environment for optimal growth conditions (76), which in general are not conditions used by the majority of clinical laboratories. Given this, the extent to which C. concisus contributes to gastrointestinal disease is likely to have been extremely underestimated (80). Over recent years the introduction of the ‘Cape Town Protocol’ (100) which employs membrane filtration onto antibiotic free culture medium and incubation in an H2-enhanced microaerobic atmosphere has increased the success rate of culture from faecal samples (93). For example, Lasticova reported that through 52 Chapter 1: Introduction the use of the ‘Cape Town Protocol’ stool cultures positive for Campylobacteria rose to 21.8% from the 7.1% previously obtained when using Skirrow's medium and other selective media in conjunction with conventional microaerobic incubation (101, 102). Apart from improvements in culturing methods, PCR detection procedures are becoming widely used in clinical microbiology laboratories worldwide (80). In recent years following the introduction of improved cultural and molecular techniques for the detection of C. concisus the association between C. concisus and gastroenteritis has become much clearer. Indeed, since 2012 a series of studies have provided evidence that C. concisus is likely to be important cause of gastroenteritis. One such study conducted in Denmark by Nielsen et al. used appropriate conditions for the culture for C. concisus in their routine culture protocols (12). Importantly, this study reported the isolation of C. concisus from 400 of 8302 (4.82%) of patients with gastroenteritis (12). In contrast C. concisus was not isolated from healthy control patients without pathology (12). Interestingly, the isolation rates of C. jejuni / C. coli from these patients 489/8302 (5.8%) was shown to be very similar to that of C. concisus (12). Interestingly, this study also showed that around 10% of the patients with C. consisus had co-infections dominated by Clostridium difficile and Salmonella enterica, whereas co-infections occurred in about 5% of C. jejuni / coli patients (12). Based on their findings Nielsen et al. concluded that there is a high incidence of C. concisus in Denmark, and their findings add to our understanding of the epidemiology of Campylobacteria in this region. Further, considering that there was little knowledge concerning the impact of C. concisus infection on children a subsequent study by Nielsen et al. determined the clinical manifestations in C. concisus positive children with gastroenteritis (83). Nielsen 53 Chapter 1: Introduction et al. findings showed that C. concisus was detected in 85 of 2372 diarrhoeic stool samples and C. jejuni / C. coli in 109 of 2372 diarrhoeic stool samples (83). Nielsen et al. concluded that C. concisus infection in children seems to have a milder course of acute gastroenteritis compared with C. jejuni / coli infection, but is associated with more prolonged diarrhoea. Also, children with C. concisus have the same degree of late gastrointestinal complaints as children diagnosed with C. jejuni / coli infection (83). Considering that limited information was known about the short-term and medium-term clinical impacts of C. concisus infection (103). A clinical study was performed during a 2-year period to determine the clinical manifestations in C. concisus-positive adult patients (103). This study by the Nielsen group conducted an extensive survey of Danish patients who registered a C. concisus positive stool sample (n = 139) as well as patients who registered a C. jejuni / C. coli positive stool sample (n = 189) (103). This study found that patients infected with C. concisus suffered prolonged acute diarrhoea, with 80% reporting diarrhoea, which lasts for more than 14 days, as compared to 32% of those infected with C. jejuni or C. coli (103). Further, individuals with C. concisus infection experienced milder acute gastroenteritis, as indicated by a lower number of individuals reporting fever, chills, weight loss, and mucus and blood in stools as compared with those infected with C. jejuni and C. coli (103). In conclusion, Nielsen et al. showed that C. concisus infection in children appears to have a milder course of acute gastroenteritis compared to C. jejuni or C. coli infection but alternatively it is associated with an onset of more prolonged diarrhoea (103). Interestingly, it has been reported that children with C. concisus infection have the same degree of late gastrointestinal complaints as children diagnosed with C. jejuni or C. coli infection (103). 54 Chapter 1: Introduction Interestingly, as a follow up from Nielsen et al. previous findings reporting a high incidence of C. concisus in a Danish population (12). Further, Nielsen et al. aimed to assess the faecal calprotectin (f-CP) levels, as a hallmark for the intestinal inflammation in C. jejuni, C. coli and C. concisus infected patients (104). Importantly, f-CP was used as a protein indicator for intestinal inflammation, as its concentration in faeces reflects the migration of neutrophils in the gut lumen (104). Nielsen et al. reported that 6 months following diagnosis of gastroenteritis, 12% of patients infected with C. concisus had microscopic colitis (104). In contrast, no patient previously diagnosed with C. jejuni / C. coli had microscopic colitis (104). C. concisus infection has also been associated with lower levels of f-CP compared to C. jejuni or C. coli infections (104). Interestingly, based on these findings it would be worthwhile considering that the low level of intestinal inflammation induced by C. concisus could potentially facilitate persistent colonisation and chronic infection in the intestine. Nielsen et al. study raised awareness for the use of f-CP levels to determine C. concisus infection of gastroenteritis cases, which should be compared to viral gastroenteritis with a prolonged clinical outcome (104). In conclusion, Nielsen et al. mentioned that clinicians should be especially aware of C. concisus infection, especially in patients with prolonged mild diarrhoea, in the overall differential diagnosis to IBD. A further study by Ferreira et al. conducted in Portugal, which examined the presence of gastrointestinal pathogens in patients with diarrhoea, reported Campylobacter spp. to be present in 31.9% of samples (11). Of these, C. jejuni and C. concisus were found to be the most prevalent being present in 13.7% and 8.0% of samples respectively (11). Further, a study by de Boer et al., which examined the presence of Campylobacter species and Arcobacter butzleri in 493 faecal samples obtained from Dutch patients with gastroenteritis using a range of PCR approaches, reported that 71.4% of 493 cases 55 Chapter 1: Introduction to test positive for Campylobacter DNA (10). Of these Campylobacter positive samples PCR and/or sequencing results revealed C. jejuni accounted for 4.1%, C. concisus for 4.1%, C. concisus or C. curvus for 0.8% and C. ureolyticus for 0.6% (10). Moreover, comparison of the prevalence rates of C. jejuni and C. concisus showed the detection frequency of C. concisus (4.1%) was identical to that of C. jejuni (4.1%) (10). Further, real time PCR analysis of a subset of samples revealed that the bacterial loads present in C. concisus positive samples were suggestive of symptomatic infection (10). Further, a recent study conducted in Chile by Collado et al. assessed the diversity of Campylobacter and Arcobacter in human faecal samples from 140 patients with diarrhoea as well as 116 asymptomatic controls using a combination of traditional culture and molecular methods via PCR RFLP analysis (9). Based on the results of this study the most prevalent species associated with gastroenteritis was shown to be C. concisus 16/140 (11.4%) followed by C. jejuni 15/140 (10.7%) and C. ureolyticus 5/140 (3.6%) (9). In healthy persons, the most prevalent species detected was C. concisus 4/116 (3.4%), followed by C. ureolyticus 2/116 (1.7%), while the prevalence of both C. jejuni and C. coli were 0.9% (1/116) (9). Amongst all detected species, only C. jejuni and C. concisus were shown to be significantly associated with diarrhoea (P = 0.003 and P = 0.033, respectively). Overall, Collado et al. findings indicate that both C. concisus and C. ureolyticus are emerging gastrointestinal species that have an underestimated clinical importance in gastroenteritis (9). Moreover, Collado et al. study was the first to report on the detection of both C. ureolyticus and C. concisus from diarrhoeic patients in Chile. Importantly, their findings add to our understanding of the epidemiology of Campylobacteria in this region (9). The above studies conducted in Denmark, Portugal, Netherlands and Chile, have 56 Chapter 1: Introduction provided important evidence that supports the role of C. concisus as a gastrointestinal pathogen. Advancements in the methodologies used for the culture and molecular detection of C. concisus have the potential to allow the introduction of Campylobacter surveillance programs to monitor the prevalence of C. concisus induced gastroenteritis cases worldwide. 1.4.4. Campylobacter concisus and Inflammatory Bowel Disease The possible role of C. concisus in IBD was first reported by the Mitchell group (41, 105) who, as part of an investigation on Helicobactericeae in children with newly diagnosed CD (106), also isolated Campylobacter concisus from a biopsy sample of a child with CD (41). Based on this previous finding, further investigation by Zhang et al. aimed to understand the association between C. concisus and newly diagnosed paediatric CD (41). Zhang et al. examined the prevalence of C. concisus in patients with CD and controls by taking intestinal biopsy samples collected from (n = 85) children undergoing colonoscopic diagnosis (41). Importantly, the screening for CD diagnosis was carried out by using standard endoscopic, histologic and radiologic examinations, which separated these biopsy samples into two categories; those taken from CD patients (n = 33) and healthy patients (n = 52) showing no pathology, serving as the control in this investigation (41). Moreover, the presence of C. concisus was examined using a C. concisus species-specific PCR and sequencing analysis, which detected the prevalence of C. concisus in children with CD (51%), which was significant higher than in controls (2%) (P < 0.0001) (41). Additionally, antibody production was also examined and Zhang et al. findings revealed that C. concisus specific IgG levels were significantly elevated (P < 0.001) in CD patients (0.991 ± 0.447) as opposed to levels observed in the control patients (0.329 ± 0.303) (41). Further, in this study C. concisus UNSWCD was isolated from a biopsy sample, indicating that C. concisus was viable 57 Chapter 1: Introduction within the intestinal tract (41). In a follow-up study Man et al. investigated the presence of C. concisus in faecal samples collected from (n = 54) children diagnosed with CD and two control groups consisting of non-IBD control (n = 27) and a healthy control group with no pathology (n =33) (105). Moreover, using C. concisus species-specific PCR that targeted the 16S rRNA gene of C. concisus, Man et al. findings showed that 65% (35/54) of faecal samples from CD children were positive, a prevalence significantly higher than that in the healthy (33%, 11/33, P = 0.008) and non-IBD controls (37%, 10/27, P = 0.03) (105). Importantly, Man et al. findings provided a link between C. concisus and paediatric IBD (105), which warranted further investigation. Despite associations from Man et al. study linking C. concisus with paediatric IBD (105). In 2011 a further study by Mukhopadhya and colleagues (45) detected Campylobacter spp including C. concisus from biopsy samples obtained from adult patients suffering from UC. Based on Campylobacter genus specific and a Campylobacter concisus specific PCR, Mukhopadhya and colleagues (45) showed a significantly higher prevalence 73.9% (51/69) of Campylobacter species in patients with UC as compared with controls 23.1% (15/65) (45). Further, based on nested PCR a significantly higher prevalence of C. concisus DNA (33.3%) was detected in the UC adult patients as compared with healthy controls (10.8%) (P = 0.0019) (45). Ultimately, based on their findings Mukhopadhya and colleagues (45) suggested that the higher prevalence of Campylobacter genus in particular C. concisus and C. ureolyticus detected in biopsy samples from adult UC patients may suggest that Campylobacters play a role in chronic inflammation, a characteristic hallmark observed in patients suffering from UC. 58 Chapter 1: Introduction Further, evidence to support the role of C. concisus in UC comes from a study by Mahendran et al. who determined the prevalence of C. concisus in adult patients suffering from UC and CD using both PCR and sequencing analysis (44). Mahendran et al. study examined 301 biopsies collected from ileum, caecum, descending colon and rectum of 28 patients IBD (15 CD and 13 UC) and 33 controls with normal intestinal histology (44). In their study Mahendran et al. detected the presence of C. concisus positivity in 69% (9/13) of UC patients, which was significantly higher (P < 0.05) compared with 36% (12/33) obtained from the control non-UC patients (44). Mahendran and colleagues also detected DNA of C. concisus in 67% (10/15) of adult patients with CD as opposed to 36% (12/33) obtained from healthy control patients, which proved to be non-significant (P > 0.05) (44). Mahendran et al. concluded by indicating that a high intestinal prevalence of C. concisus found in patients with IBD particularly in the proximal large intestine, suggests that future studies are needed to investigate the possible involvement of C. concisus in IBD (44). More recently, Hansen et al. in contrast to Mukhopadhya and Mahendran’s findings disputed the involvement of C. concisus and other microaerophilic bacteriums such as Helicobacter in cases of paediatric IBD (107). In their study Hansen et al. study recruited 100 Scottish children undergoing colonoscopy of whom 44 received treatment for naïve de novo IBD, (29 with CD and 13 UC) and 42 were normal colon controls (107). Moreover, prevalence of Campylobacter spp amongst other microaerophilic bacteria was examined. Colonic biopsies were collected from all children and cultured under microaerophilic growth conditions. In cases where Gram-negative rods were cultured, they were also identified by sequencing. Further biopsy specimens were screened by PCR screened for Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis (107). Interestingly, attempts to culture C. concisus from colonic 59 Chapter 1: Introduction biopsies resulted in the isolation of 3 C. concisus isolates. These isolates originated from 2 patients with CD and 1 from a patient with UC (107) and were confirmed as C. concisus through 16S rDNA sequencing (107). In addition C. curvus, C. lari and C. rectus were each cultured from 1 patient and C. showae from 3 patients. Investigation of the presence of Campylobacter in DNA extracted from colonic biopsies of paediatric patients diagnosed with either CD or UC patients and controls, using PCR showed 22 CD colonic biopsies (75.9%), 9 UC biopsies (69.2%) and 32 normal colon biopsies (76.2%) to be positive for Campylobacter. Further, 16S rDNA sequencing analysis revealed C. concisus DNA to be present in 13 colonic biopsies (44.8%) from CD children as compared to 16 (38.1%) from children with no pathology. Moreover, C. concisus DNA was detected in 4 (30.8%) colonic biopsies from children with UC, which was lower than that observed in biopsies of children with no pathology (38.1%) (107). Based on this study, Hansen et al. refuted previous claims of a possible association between the Campylobacter and IBD (107). However, this investigation showed a high prevalence and variety of Campylobacter spp as well as S. wadsworthensis (a common intestinal commensal) in paediatric colon biopsies, albeit with no significance to IBD pathology. Moreover, in the opinion of Hansen et al. their findings showed “surprising results’ upon the application of targeted culture and molecular microbial techniques to detect these atypical microorganisms (107). Thus, these findings by Hansen et al. highlights the importance of further studies to dispel any uncertainties in associating C. concisus with IBD (107). Clearly further research in this area is warranted. 60 Chapter 1: Introduction 1.4.5. The invasive potential of C. concisus in host intestinal epithelial cells Initial studies into the attachment and invasive potential of C. concisus were reported by Man et al. (108) who used in vitro attachment and gentamicin protection assays as well as scanning electron microscopy (ScEM) to determine the adherence and invasion ability of C. concisus. This study showed that C. concisus UNSWCD was able to attach to and invade both Caco-2 and HT-29 intestinal epithelial cells. Based on ScEM analysis these authors showed that C. concisus UNSWCD used it polar flagellum to wrap itself around the tip of the microvilli for adherence and induced a membrane ruffling like effect on the apical membrane surface of the cells to induce invasion (52). To determine if C. concisus strains differed in their ability to attach to and invade intestinal cells, Kaakoush et al. examined strains of C. concisus from colonic biopsies associated from Crohn’s disease (n = 3), from human faecal samples of patients with acute gastroenteritis (n = 3) and a strain from a healthy control patient with no pathology (Table 1.1) (109). This study showed that all C. concisus strains attached to Caco-2 cells, although differences in attachment levels were observed (109). In relation to invasion major differences were observed among C. concisus strains obtained from different disease states. Specifically strains from patients with chronic gastroenteritis (UNSWCD, UNSW1-3) exhibited higher levels of invasion than strains obtained from patients with acute gastroenteritis, one patient with gastroenteritis and the strain from a healthy control strain showing no invasion of Caco-2 cells (109). These results suggest that cellular invasion by C. concisus may be important for the development of chronic disease including CD (109). Such differences in the invasive ability of C. concisus strains is likely to relate to the high level of genetic variance reported in C. concisus strains. Given this, it is possible that specific strains of C. concisus may be associated with disease severity. 61 Chapter 1: Introduction Table 1.1. A comparison of the percentage invasion and adherence of eight C. concisus strains that are able to invade and adhere to Caco-2 cells. The in vitro adherence and invasion levels obtained following infection of Caco-2 intestinal epithelial cells with four C. concisus strains isolated from colonic biopsy samples of patients with chronic disease, three C. concisus strains from faecal samples of patients with acute gastroenteritis and a C. concisus strain isolated from a healthy control patient sample. Table adapted from Kaakoush et al. (109). C. concisus Invasion ± Attachment ± SEM Disease strain SEM (%) (%) UNSWCD Crohn’s Disease 0.47 ± 0.04 4.51 ± 0.81 UNSW2 Crohn’s Disease 0.24 ± 0.04, 4.27 ± 1.31, P = 0.01 P = 0.87 UNSW3 Crohn’s Disease 0.34 ± 0.01, 4.50 ± 0.83, P = 0.03 P = 0.99 UNSW1 Chronic 0.49 ± 0.04, 2.27 ± 0.81, Gastroenteritis P = 0.80 P = 0.08 ATCC 51561 Healthy Control 0, P < 0.01 0.11 ± 0.03, P < 0.01 ATCC 51562 Acute 0.00048 ± 0.16 ± 0.02, Gastroenteritis 0.00016, P < 0.01 P < 0.01 UNSWCS Acute 0.00059 ± 4.6 ± 1.5, Gastroenteritis 0.00015, P = 0.89 P < 0.01 BAA-1457 Acute 0, P < 0.01 3.6 ± 1.2, Gastroenteritis P = 0.48 62 Chapter 1: Introduction 1.4.6. Genetic and phenotypic variation in Campylobacter concisus spp Considerable genotypic and phenotypic variation has been shown to present in C. concisus strains. For example, in 1989 Vandamme et al. using DNA-DNA hybridisation studies compared the genetic make-up of C. concisus strains isolated from the faeces and and oral cavity (84). The results of this study showed that isolates obtained from patients with diarrhoea, that phenotypically matched the description of C. concisus, had only 42-50% DNA-DNA hybridisation levels with strains from the oral cavity and the C. concisus reference strain (84). Further, evidence of the genetic diversity of C. concisus was reported in 2002 by Matsheka et al. who used pulsed field gel electrophoresis (PFGE) based macrorestriction profiling to examine the profiles of C. concisus strains obtained from children with diarrhoea (110). The results from this study showed that of the 53 isolates examined, 51 had distinct NotI macrorestriction fragments, while 2 strains were resistant to NotI digestion. The patterns of these strains comprised between 1 and 14 restriction fragments, while the type and reference strains of two well-defined genomospecies of oral and faecal origin were found to contain 6 and 12 fragments, respectively. Based on their findings Matsheka et al. concluded that C. concisus included at least two genomospecies that were phenotypically indistinguishable, but genetically divergent (110). In a subsequent study, Matsheka et al. typed 100 C. concisus isolates obtained from 98 children with diarrhoea and 2 dental isolates from adult patients using DNA fingerprinting (111). Based on random amplified polymorphic DNA (RAPD) fingerprinting, 86% of 100 isolates were found to be genotypically diverse (111). Of these heterogeneous isolates, 25 had, in their previous study (110), been shown to have unique profiles based on PFGE (111). The remaining 14 strains were reported to have 5 RAPD profiles, with their results confirming the high level of 63 Chapter 1: Introduction heterogeneity observed in these C. concisus strains (111), were in accord with Matsheka’s previous study (110). In contrast examination of the genetic profiles of 62 C. concisus strains [56 strains from diarrhoeic patients, 4 oral C. concisus strains and 2 reference strains (oral isolate CCUG 13144T and the intestinal isolate CCUG 19995)] by Aabenhus et al. using amplified fragment length polymorphisms (AFLP) showed all strains examined to have a unique profiles. However, numerical analysis of the AFLP profiles indicated that these strains were distributed across four distinct clusters, that were defined at the 21% similarity level (112). Cluster 1 contained the type strain of oral origin (CCUG 13144) and 22 other clinical isolates, 27% of which were isolated from immune-competent (IC) patients Cluster 2 contained the reference strain CCUG 19995 originally isolated from a human diarrhoea sample, as well as 32 other clinical isolates, 59% of which were isolated from IC patients. AFLP cluster 3 consisted of a single diarrheal isolate from an IC patient, whereas cluster 4 contained five strains that had been isolated from severely IC patients, a finding that led Aabenhus et al. to suggest that the strains in cluster 4 may be less invasive. Based on their findings Aabenhus et al. concluded that at least four distinct C. concisus genomospecies existed (GS1-4), (Figure 1.1), a genomospecies being defined as a phenotypically indistinguishable, yet genetically distinct species, and that these four genomospecies exhibited differences in their spectra of virulence potential (112). Based on their findings Aabenhus et al. postulated that specific subtypes of C. concisus may be associated with gastrointestinal disease (112). Importantly, such differences could explain why C. concisus spp may differ in their pathogenic potential (112) (Figure 1.1). 64 Chapter 1: Introduction Figure 1.1. Representative amplified polymorphism of selected C. concisus strains. Image obtained from Aabenhus et al. (112). To investigate whether differences in the genetic make-up of C. concisus strains may relate to their ability to attach to and invade intestinal cells Kaakoush et al. investigated the genome and proteome of UNSWCD (64). Additionally, this particular strain C. concisus UNSWCD was isolated from a child with newly diagnosed CD, which as described above had an increased ability to invade intestinal cells and compared this to the only available reference genome BAA-1457 that was published at that time (64). Comparison of the genomes, the 16S rRNA gene, the internal transcribed region sequence and the 23S rRNA genes between both these strains revealed that 76% of the proteins in UNSWCD and BAA-1457 were homologous, which was lower than the homology of several C. jejuni strains whose proteins were reported to be approximately 90% homologous (64). Based on this, it was concluded that C. concisus had a highly variable genome as compared to other species of Campylobacter (64). In a follow-up study, protein profile analysis of a further 6 strains of C. concisus revealed that the strains showed an average of 64.7% similarity. In contrast the reference genome strain 65 Chapter 1: Introduction C. concisus BAA-1457 had only 56.8% protein similarity, and was identified as been highly divergent (64). This degree of genetic and proteomic disparity may explain the disparity in conclusions regarding the pathogenic potential of C. concisus. Further, given the high level of heterogeneity between the majority of C. concisus strains and strain BAA-1457, Kaakoush et al. concluded that C. concisus strain BAA-1457 was genetically atypical as compared with other C. concisus strains and was not a good candidate reference strain (64). 1.5. Pathogenesis of C. concisus Until recently, the pathogenic potential of C. concisus was largely unknown and unexplored due to the controversy regarding its role in gastrointestinal disease given its reported detection in both healthy and diseased patients, as well as the difficulty of culturing C. concisus. However, following the association of C. concisus with cases gastroenteritis and IBD, studies have increasingly investigated the pathogenic potential of C. concisus with the bacterium now considered an emerging gastrointestinal pathogen. 1.5.1. The pathogenic potential of Campylobacter concisus revealed via in vitro model studies A study by Man et al. investigated for the first time the ability of C. concisus UNSWCD to attach to and invade intestinal epithelial cells using in vitro attachment, invasion gentamicin protection assays and scanning electron microscopy (ScEM) (108). These results revealed that C. concisus UNSWCD was able to adhere to and invade both Caco- 2 and HT-29 cells on the apical membrane surface. Moreover, upon closer of the transcellular invasion process by C. concisus UNSWCD, the ScEM images revealed that C. concisus UNSWCD was able to induce a membrane ruffling like effect 66 Chapter 1: Introduction comparable to S. Typhimurium (108). Subsequently in light of these results Man et al. determined whether C. concisus UNSWCD could disrupt barrier function in these cell types by inducing the movement of tight junction proteins from the membrane to the cytosol in these particular cell types (108). This study revealed that mechanistically C. concisus UNSWCD uses a microtubule dependent, and an actin independent mode of internalisation. Interestingly microtubule dependent internalisation is also utilised by the enteric pathogen C. jejuni (108). This study also showed that C. concisus UNSWCD preferentially adhered to intercellular tight junctional (TJ) spaces, a finding that led Man et al. to suggest that C. concisus could possibly translocate across the epithelium via a paracellular route through the TJs (108). Furthermore, Man et al. suggested that this intracellular mechanism resulted from a decrease in trans-membrane epithelial resistance (TER) as shown by the use of transwell assays (108). Moreover, Man et al. revealed via confocal laser scanning microscopy that the loss of trans-membrane epithelial resistance was associated with the internalisation of ZO-1 and occludin from the Caco-2 monolayers, disrupting TJ integrity following C. concisus UNSWCD infection (108). Moreover according to Man et al. the process of adherence may result in a rapid disruption of TJs either by direct C. concisus host contact or by the release of secretory proteins directly to the target sites, which causes damage to the structural integrity of the epithelial cells (108). Subsequently, the precise signal for the activation of this system is unknown. However, it is possible that internalisation of ZO-1 and occludin from the cell membrane to the cytosol induced by C. concisus UNSWCD may be attributed to the activation of a Zot toxin (108), which has been identified in the complete genome of C. concisus strain 13826 (48). Although the mechanism of Zot toxin in C. concisus is yet to be confirmed, studies in Vibrio cholerae have shown that 67 Chapter 1: Introduction Zot toxin significantly increases tissue permeability, actin polymerisation, and intestinal secretion (113, 114). In a later study C. concisus strains isolated from 6 oral cavity and 8 from faecal samples were investigated by Nielsen and colleagues (115) who examined the apoptotic effect of C. concisus on epithelial tight junctions (115). This study revealed that these C. concisus strains were able to internalise into HT-29/B6 colonic cells and that both oral and faecal C. concisus strains induced epithelial barrier dysfunction as a result of apoptotic leaks, caused by a leak-flux mechanism, which is a hallmark manifested in clinical diarrhoea cases. Epithelial necrosis and apoptosis induction are mechanisms that can potentially contribute to a leak-flux type of diarrhoea. Thus, this event leads to a greater quantity of antigens passing the mucosa that can increment the inflammatory mechanism (115). 1.5.2. C. concisus toxin production and other virulence and colonisation factors It has been shown that C. concisus have been shown to produce several toxins and virulence factors. Istivan and colleagues, reported isolates of C. concisus cultured from faecal samples of children with gastroenteritis to have both cell-bound and haemolytic phospholipase A2 activity against Chinese hamster ovary cells (65, 116). Engberg et al. study was one of the first to investigate the ability of C. concisus to interact with host cells (117). In their study Engberg et al. showed that C. concisus cell- free infiltrates induced pathology in a monkey kidney epithelial cell line, with the cytopathic effects induced being similar to that shown by other Campylobacter cytolethal distending toxins (117). However, while this study presented interesting findings no disease association was determined. Interestingly, in a follow up study 68 Chapter 1: Introduction Nielsen et al. provided further evidence of the cytotoxicity of C. concisus isolates by examining the cytotoxicity effect induced by oral and faecal C. concisus strains on HT- 29/B6 cells in vitro (115). Nielsen et al. observed a significant increase in the level of lactate dehydrogenase (LDH) released from HT-29/B6 cells infected with C. concisus 3.1 ± 0.3% as opposed to a lower LDH release observed in the control 0.7 ± 0.1% without C. concisus infection (P < 0.001) (115). Moreover, Nielsen et al. also revealed via TUNEL staining that 48 h post infection of HT-29/B6 cells by C. concisus showed a 5-fold increase in apoptosis 5.2 ± 0.9%, which was significantly higher then those observed in the control 1.0 ± 0.3% (P < 0.01). Conjunctively, these findings show that C. concisus could potentially induce cytotoxic effects on HT-29/B6, with both the necrotic and apoptotic mechanisms contributing to clinical manifestation of a leak-flux type of diarrhoea. In contrast a study by Kalischuk and Inglis reported none of their C. concisus isolates to induce significant epithelial cytotoxicity, as measured through lactate dehydrogenase release (118). However, these authors also reported that 64.3% (9 out of 14) of their C. concisus isolates induced epithelial DNA fragmentation and that this correlated with an increase in host cell metabolic activity (118). Studies by the Mitchell group have also identified an invasion factor InvA, a haemolysin TlyA and Zot within the genome of C. concisus BAA-1457, isolated from a patient with acute enteritis (67). Interestingly determination of the presence of Zot protein in 8 C. concisus strains showed only 1 of 8 strains examined had the Zot protein (64). In comparison, Kalishuck and lnglis detected the zot gene in 42.8% (6 out of 14) of their C. concisus isolates, which could suggest that these 6 isolates were genetically associated to C. concisus BAA-1457 strain (119). Given that the secretome of a pathogen reflects its pathogenic repertoire, Kaakoush et al. investigated the secretome of C. concisus UNSWCD an isolate, obtained from an intestinal biopsy of a CD patient 69 Chapter 1: Introduction (67). This study showed that the C. concisus UNSWCD secretome comprised 201 proteins, of which 86 were bioinformatically predicted to be secreted based on the C. concisus 13628 reference genome (67). Identification of these secreted proteins revealed the presence of a Zot toxin, a toxin used by pathogens to increase tissue permeability, an S-layer RTX toxin a pore-forming toxin synthesised by a diverse group of gram-negative pathogens, the surface antigen CjaA, and the colonisation factor CadF, as well as an outer membrane fibronectin binding protein (67). 1.5.3. C. concisus pathotypes The heterogeneity observed amongst C. concisus spp in relation to their pathogenicity has led to the differentiation of C. concisus spp into pathotypes based on their virulence characteristics (81). Specifically distinction of these pathotypes was based on the ability of C. concisus strains to adhere to and invade host cells, secrete toxins. Based on differences in the genetic and pathogenic characteristics of C. concisus it has recently been proposed that C. concisus can be divided into two distinct pathotypes: adherent and invasive C. concisus (AICC) and adherent and toxigenic C. concisus (AToCC), which are genetically different to non-pathogenic strains (76). While both pathotypes have the ability to adhere to host cells, AICC pathotypes possess a greater ability to survive intracellularly, while AToCC pathotypes can produce the zonula occludens toxin, which potentially targets tight junctions of host cells (76). Figure 1.2 highlights the differences between the virulence mechanisms of AICC and AToCC pathotypes. 70 Chapter 1: Introduction Figure 1.2 has been removed due to copyright resrictions. Figure 1.2. The pathogenic difference between AICC and AToCC pathotypes and their effect on host intestinal cells. AICC is able to adhere and transcellularly invade through the cell causing a membrane like ruffling effect. Once inside the cell C. concisus is able to envelop itself in a Campylobacter containing vacuole (CCV), which blocks lysosomal fusion and degradation. Infection with AICC induces the secretion of IL-8, IL-12 and IFN-γ, which results in inflammation. In contrast AToCC, is able to adhere to the host cell and paracellularly translocate through the cell tight junction using its virulence factor, the Zot toxin. This Zot toxin targets the tight junctions of host cells, resulting in tight junction barrier disintegration, and facilitates bacterial translocation into the host cell. This targeted action by the Zot toxin, results in the induction of IL-8 and IL- 12, driving inflammation of the host cell. Image adapted from Kaakoush et al. (81). 1.5.4. C. concisus and the activation of the host’s innate immune system Research into the immune response to C. concisus has revealed that this bacterium is able to induce activation of the host’s immune system. For example, an early in vivo study by Man et al. reported that infection of the THP-1 monocytic cell line with C. concisus UNSWCD led to the production of both IL-8 and TNF-α, but not IL-1β (108). In contrast when primary human macrophages were infected with C. concisus UNSWCD, significantly higher levels of TNF-α were induced as compared with an unstimulated control, however relatively lower levels of not IL-1β were produced. Whilst C. concisus UNSWCD induced increased levels of IL-8 in primary human macrophages, the levels observed were not significantly higher than that in the un- stimulated control (108). A study by Kalischuk and Inglis (118) further examined the ability of C. concisus to induce IL-8 by comparing the pathogenic and genotypic properties of C. concisus faecal 71 Chapter 1: Introduction isolates from diarrhoeic and healthy individuals residing in South Western Alberta, Canada. Analysis via AFLP revealed a cluster (cluster 1), which was predominately associated with healthy individuals and an oral C. concisus isolate (LMG7788). Examination of IL-8 production using qRT-PCR revealed that this cluster induced greater expression of epithelial IL-8 mRNA (P = 0.04) when compared to cluster 2. Interestingly, cluster 1 was commonly associated with genes encoding for the Zot toxin and the S-layer RTX toxin. In a later study by Kaakoush et al. showed that eight C. concisus strains isolated from subjects with chronic and acute infection, as well as a healthy control produced high levels of IL-12 (109), a cytokine reported to induce intestinal mucosal inflammation through an IFN-γ dependent manner. Although all strains of C. concisus produced high levels of IL-12, only C. concisus strains that could invade host cells led to increased levels of IFN-γ (109). Thus it would appear that invasive C. concisus strains adhere to and invade the host cells inducing both IL-12 and IFN-γ, the latter leading to activation of the immunoproteosome (57). Further, C. concisus infection up-regulates ubiquitinating and down-regulates de-ubiquitinating enzymes, an event that leads to the ubiquitination of NF-κB inhibitors. The immunoproteosome then targets these inhibitors, which leads to the activation of NF-κB and further inflammation (57), as shown in (Figure 1.3). A further study has investigated, by Ismail et al. has examined toll-like receptor 4 (TLR4) expression following infection of HT-29 cells with C. concisus (120). Based on flow cytometry, all eleven C. concisus strains examined were shown to up-regulate both surface and total expression of TLR4, nine strains being shown to induce greater than a two fold increases in surface expressed TLR4. Further, this study showed that the 72 Chapter 1: Introduction standard level of surface TLR4 stimulated by C. concisus strains acquired from patients with IBD was significantly greater than that stimulated by C. concisus strains acquired from healthy control patients (3.70 ± 0.46 vs.1.93 ± 0.05, P < 0.05) (120). In contrast, the level of total TLR4 stimulated by C. concisus strains acquired from IBD patients was not statistically different from the levels stimulated by C. concisus from healthy control patients (1.81 ± 0.08 against 1.63 ± 0.05, P > 0.05) (120). Despite these findings, the physiological relevance of the up-regulation in TLR4 is not entirely clear, given that up-regulation of TLR4 did not correlate with the production of pro- inflammatory cytokines (120), further research is required to elucidate stronger findings. Figure 1.3. The host cells proposed immune response to C. concisus UNSWCD. A: Non-invasive C. concisus are capable of adhering to the host cell but not invade them, which leads to the stimulation and production of IL-12. B: Highly invasive C. concisus strains adhere to and invade the host cell inducing production of IL-12 and IFN-γ, which inturn activate the immunoproteosome to ubiquitinate and subsequently degrade NF-κB inhibitors and activate NF-κB production. Image from Kaakoush et al. (109). 73 Chapter 1: Introduction Studies have also revealed that C. concisus can activate the host innate immune system through neutrophil activation. For example Sorensen et al. examined the effect of 5 C. concisus faecal isolates and 1 oral reference strain C. concisus ATCC33237 on the expression of the adherence molecule CD11b on neutrophils (121). The results of this study revealed that C. concisus activated the innate immune system by stimulating neutrophil cells resulting in both increased expression of the adherence molecule CD11b and their oxidative burst response, both of which are crucial components for acute inflammation (121). Further, these authors showed based on a chemiluminescence assay, that the opsonic activity of heat-treated serum from patients infected with C. concisus was not increased as compared with heat treated control serum, leading the authors to conclude that there was a weak systemic IgG response to infection with C. concisus (121). Ultimately, Sorensen et al. concluded that their findings showing pro-inflammatory stimulation of neutrophils by C. concisus, supports the potential and clinical relevance of C. concisus as an emerging pathogen (121). However, whether stimulation of innate immunity, correlated with clinical observations remains to be clarified. 1.5.5. Immune response to Campylobacter infections Innate immunity plays an important role in the recognition of pathogenic associated molecular patterns (PAMPs) of pathogens. Campylobacter spp results in distinct immune responses, with infection of the gastrointestinal tract resulting in infiltration of high numbers of neutrophils, loss of epithelial barrier integrity and watery or bloody diarrhoea (122). Based on in vitro studies it has been shown that upon infection of human tissue cell lines with Campylobacter spp. including C. jejuni and C. coli (122) strong innate immune responses are induced. Indeed infection of the intestinal cell lines INT407 and T84 with C. jejuni was shown to result in the stimulation of the cytokines 74 Chapter 1: Introduction IL-8 and TNF-α. Further, infection of THP-1 monocytes and dendritic cells with C. jejuni resulted in the induction of an even broader range of pro-inflammatory cytokines (122). Innate responses to C. jejuni are primarily initiated to protect the host against the invading pathogen. One vital innate immune response is IL-8 production given its ability to attract neutrophils to the site of infection (123). IL-8 production is not only triggered by C. jejuni, but also other enteric pathogens including Salmonella, Shigella and many other pathogens, which signal through the NF-κB system (123). While the innate immune response is an effective first barrier mechanism against infection, Campylobacter species can potentially evade these host defence mechanisms by means of enhanced bacterial virulence, depressed host defences, or both. This can result in C. concisus gaining access to the deeper tissues of the intestine and gain access to subcutaneous layers (123). Despite ongoing research on Campylobacter virulence mechanisms, limited information is available regarding the host immune response to C. concisus infection. A study by Kaakoush et al. (124) that utilised RNA-seq, qPCR, mass spectrometry and confocal microscopy techniques, has recently provided an insight into the immunological response following infection of differentiated THP-1 macrophages with adherent and invasive strains of C. concisus. Interestingly, this study showed differential expression of pattern recognition receptors and up-regulation of DNA and RNA sensing molecules (124). Of particular interest was the observation of the IFI16 inflammasome assembly in C. concisus-infected macrophages (124). Importantly, extensive characterisation of the transcriptome detected substantial regulation of 8,343 transcripts upon infection with C. concisus. These transcripts included the stimulation of key inflammatory pathways 75 Chapter 1: Introduction involving: CREB1, NF-κB, STAT and IRF signaling (124). Further thirteen microRNAs (miR) and (n = 333) non-coding RNAs were significantly regulated upon infection. Moreover, of particular interest was miR-221, which has been associated with colorectal carcinogenesis (124, 125). This study represented a substantial step forward in the understanding of host recognition and innate immune responses to C. concisus infection. 1.5.6. The genome of C. concisus UNSWCD Given, the difference shown in the invasive potential of C. concisus strains. A study by Deshpande et al. sequenced the genome of C. concisus UNSWCD, which has further aided in the interpretation of the genetic heterogeneity that exists within C. concisus isolates (63). In their study Deshpande et al. compared the genome of C. concisus UNSWCD to the previously sequenced genome of C. concisus BAA1457 (63). This study showed that the genome of C. concisus was 1.8 Mb in size, which was considerably smaller than the C. concisus BAA-1457 genome 2.1 Mb (63). Moreover, whilst genomic analysis revealed that 1593 genes were conserved across C. concisus UNSWCD and BAA-1457 strains, comparative analysis of these 1593 genes revealed that 138 genes from C. concisus UNSWCD and 281 from C. concisus BAA-1457 strains, differed from each other, which equated to significant functional differences amongst both strains (63). The study concluded by noting that many of the observed differences between both C. concisus strains, support the theory that C. concisus UNSWCD could have adjusted itself to increased surface interactions with host cells. As opposed to C. concisus BAA-1457, which could prefer an independent environment without host interactions (63). In addition to the above differences in the genetic makeup of C. concisus UNSWCD as 76 Chapter 1: Introduction compared with C. concisus BAA-1457, showed that C. concisus UNSWCD carried a unique 30 Kb plasmid (Figure 1.4), which differed from the 2 plasmids in C. concisus BAA-1457, previously reported (109). The C. concisus UNSWCD plasmid was shown carry a number of genes that encoded putative virulence determinants that the authors suggested could potentially be responsible for the heterogeneity in the invasive ability of C. concisus strains (109). Importantly, this plasmid was found to be present only in the highly invasive UNSWCD strains, which led Kaakoush et al. to suggest that this plasmid may play a role in the invasive ability of C. concisus strains (63, 109). In a subsequent study Deshpande et al. sequenced and annotated a total of 6 C. concisus genomes acquired from 2 isolates from CD patients (UNSW2 and UNSW3), 3 isolates from gastroenteritis patients (UNSW1, UNSWCS and ATCC 51562) and 1 isolate from a healthy individual (ATCC 51561) (126). Comparative analysis of the 6 genomes with the two genomes available on NCBI (C. concisus UNSWCD and BAA-1457) revealed 7 genes within the UNSWCD plasmid to be conserved within highly invasive strains. However, only 3 of these genes (exotoxin 9 / DnaI helicase, site specific recombinase, and a restriction endonuclease) were found to be specific to these strains (Figure 1.5). A DNA-cytosine methyltransferase, 1 of the 7 synthenic conserved genes present between the restriction endonuclease and the site-specific recombinase, was also found in strains with low invasive potential (UNSWCS and ATCC 51562), but were not found in non- invasive strains (BAA-1457 and ATCC 51561) (126). These 4 genes make up a putative restriction modification system exclusive to highly invasive C. concisus strains. Importantly, these findings reveals the need for further studies to elucidate the role of these 4 genes in mediating intracellular survival (126). Moreover, the functional purpose of these genes encoding proteins could aid in providing insights into conserved patterns amongst the C. concisus genomes examined (126). The existence of the MobA- 77 Chapter 1: Introduction like protein and the StbD / E toxin antitoxin system in the other strains most likely relates to their widespread role in ensuring that daughter cells inherit DNA properly (127). In contrast, further BLAST searches revealed exotoxin 9 was possibly a helicase DnaI. Moreover, its syntenic conservation with the restriction endonuclease, recombinase and DNA methyltransferase suggests a collective function for these proteins within the organism, that could be associated with the ability to survive within host cells than its entry into host cells (126). The study concludes by confirming the genetic heterogeneity observed amongst C. concisus isolates, but more importantly identified several factors concerning the pathogenic potential of C. concisus. Of particular interest was the peptidoglycan layer of this bacterium and conserved elements within highly invasive strains such as exotoxin 9, which could play an important role in C. concisus virulence (126). 78 Chapter 1: Introduction Figure 1.4. The C. concisus UNSWCD 30 Kb plasmid. This plasmid was present in C. concisus strains isolated from patients with chronic intestinal disease. Image adapted from Kaakoush et al. (109). Figure 1.5 has been removed due to copyright resrictions. Figure 1.5. The synthetic conservation of the seven genes within the UNSWCD plasmid found only in the highly invasive C. concisus strains. Only three genes (exotoxin 9, site specific recombinase and a restriction endonuclease) were identified in highly invasive C. concisus strains like C. concisus UNSWCD. Image taken from Deshpande et al. (126). 79 Chapter 1: Introduction In a recent study by Kaakoush et al. examined the presence of C. concisus and exotoxin 9 / DnaI levels in faecal samples of CD patients and healthy controls using real-time PCR (128). In addition these authors compared the levels of C. concisus and exotoxin 9 / DnaI levels with the composition and abundance of microbial taxa present in the faeces of a subset of subjects. Interestingly, this study revealed that both C. concisus and exotoxin 9 levels were higher in CD patients as compared with healthy controls, suggesting that CD patients not only had a greater abundance of C. concisus but that their strains were likely to be more virulent (128). Results extrapolated pertaining to the intestinal microbiota found that C. concisus levels correlated with Eubacterium, Subdoligranulum and Blautia (128). Subsequently, exotoxin 9 levels correlated with Dialister, Oscillospira, Lachnospira and Prevotella (128). This extrapolation implies that either the composition of the intestinal microbial flora has the capability to mediate levels of both virulent and non-virulent C. concisus strains, or alternatively infection with C. concisus may mediate the levels of specific bacterial taxa within the gastrointestinal tract (128). 1.6. Intracellular survival of enteric pathogens Intracellular survival is an important mechanism used by bacteria to survive within their host (129). For those particular bacteria that do survive and invade host cells, following invasion they are potentially phagocytosed or degraded via lysosomal degradation (129). Subsequently, following the invasion of host cells pathogens that have the capability can survive the autophagy process, multiply and evade the host immune system, increasing its chances for survival within the organism. However, the human body has natural immunological responses, which combats such actions caused by 80 Chapter 1: Introduction pathogens through uptake of the enteric pathogen by the phagosome or through lysosome fusion (129). For pathogens to survive intracellularly they require specific virulence mechanisms that allow them to evade lysosomal degradation. A key survival mechanism found in a number of pathogens is the ability to form a vacuole, which encases them in a protective coating that prevents them from being degraded. Importantly, pathogens that possess such virulence factors have increased ability to survive intracellularly. For example, Salmonella Typhimurium and Mycobacterium tuberculosis are pathogens that can exploit the host immune responses by residing within a vesicular compartment, which allows them to avoid phagosome-lysosome fusion and bacterial clearance (129). Both of these pathogens are able to alter the biosynthesis and dynamics of their vacuolar compartments, which allows them to evade and prevent phagosome-lysosome fusion, a process that is critical for the degradative action of lysosomal enzymes found in the autolysosome (129, 130). 1.6.1. The intracellular survival of Campylobacter spp residing in intestinal cells For a microorganism like C. concisus to survive intracellularly it must employ mechanisms to aid in the survival process otherwise it will be flushed away by the peristalsis motion in the GIT and destroyed by the hosts immune system. The employment of such a mechanism is crucial for the survival of the bacterium in its battle against the host’s immune system. One such key mechanism for Campylobacters in particular is the formation of a vacuole like compartment known as the Campylobacter containing vacuole (CCV). Thus following the adherence of C. jejuni it is endocytosed, a process that appears to be mediated by the use of host microtubules and or microfilaments (129). 81 Chapter 1: Introduction Upon entry into the host cells the survival of C. jejuni is dependent on its ability to circumvent the delivery of endosome to the lysosome, by deviating from the canonical endocytic pathway (131). Subsequently, C. jejuni translocates across the intestinal epithelium to the basolateral surface where it exocytoses into the underlying submucosa (132). In addition to this intracellular route of translocation C. jejuni may also transverse across the intestinal epithelium using a paracellular route found between- intercellular junctional spaces (132). 1.6.2. A key intracellular survival mechanism: The C. jejuni Campylobacter containing vacuole While the host factors required for C. jejuni internalisation into non-phagocytic cells remains unclear, it appears that manipulation of cytoskeletal components of the host cells are required for invasion (129). Many bacterial pathogens including Listeria monocytogenes, Shigella flexneri, and Salmonella Typhimurium utilise cytoskeletal rearrangement to gain intracellular access to host cells (129). However, what is known is that C. jejuni is internalised into intestinal epithelial cells in a microtubule dependent manner (129). In an important study by Watson and Galan, which investigated the fate of C. jejuni following internalisation into intestinal epithelial cells Watson and Galan showed that C. jejuni could survive within intestinal epithelial cells in a compartment, which they named the Campylobacter containing vacuole (CCV) that was distinct from lysosomes (Figure 6.1). Further, Watson and Galan showed that CCVs within intestinal cells were not accessible to endocytic tracers, suggesting that they were functionally separated from endocytic pathways leading to lysosomes. Further the CCV was shown to associate with early endosome components and markers early antigen-1 (EEA-1), Rab 82 Chapter 1: Introduction 5, Rab 4 and PI3P (129) (Figure 1.6). This interaction with early endosomal markers was however shown to be transient, as it did not progress down the path of canonical endocytosis (129). Interestingly, this study also reported the presence of markers of lipid-associated rafts and caveolae on the CCV, which led the authors to suggest that C. jejuni may reside in a compartment that is functionally unique from early endosomes (129). Furthermore, Watson and Galan showed 15 min following infection the majority (~65%) of the intracellular bacteria co-localised with the early endosomal marker EEA- 1, by 2 h the majority of CCVs co-localised with the late endosomal marker lysosomal associated membrane protein (LAMP-1). This compartment was clearly distinguished from lysosomes as it does not co-localise with the lysosomal protein marker Cathepsin B. Remarkably, the CCV has rarely been associated with the lysosomal marker Cathepsin B, which provides further support for the view that the CCV diverges from the classical endocytic pathway. However, the mechanism by which the CCV is able to alter its properties to prevent fusion with the lysosome are not yet known (129). Interestingly, S. Typhimurium also expresses LAMP-1, which resides within a vacuole that is apparently segregated from the canonical endocytic pathway and harbours LAMP-1, acquisition of this marker occurring via Rab 7 (129). Based on their findings Watson and Galan concluded that “C. jejuni had evolved specific adaptations to traffic within host cells and avoid delivery into lysosomes” (124). 83 Chapter 1: Introduction Figure 1.6. Model for C. jejuni internalisation within intestinal epithelial cells. Comparison of the endocytic pathway of C. jejuni, which deviates in comparison to the classical endocytic pathway outlined by S. Typhimurium mutated in the invA gene. Image obtained from Watson and Galan (129). 84 Chapter 1: Introduction 1.7. Autophagy 1.7.1. The process of autophagy The process of autophagy is defined as auto (self) and phagy (eating). Autophagy can be divided into three distinct pathways: macroautophagy, microautophagy, and chaperone- mediated autophagy (133, 134). Autophagy is principally a process aimed at controlling the digestion of internalised macromolecules such as organelles and cellular metabolites (135). This elaborate and ancient cytoplasmic process ultimately ensures a homeostatic balance controlling quantity and quality of intracellular biomass in eukaryotic cells via targeting them for auto-digestion (133). The mechanism of autophagy ensures the degradation of cytosolic components including bacteria, long-lived proteins and damaged organelles (135, 136). The role of macroautophagy (mentioned throughout this thesis as autophagy), functions by protecting host cells against invading bacteria. Autophagy is a conserved catabolic pathway that is preserved in eukaryotes, which functions by depositing cargo into a double-membraned vesicle designated the autophagosome, which mediates delivery of the cargo to the lysosome for degradation (137), as shown in (Figure 1.7). Autophagy mediates the removal of long-standing proteins, protein aggregates, and organelles (mitochondria and peroxisomes), as well as pathogens via a process termed xenophagy (137) (Figure 1.8). 85 Chapter 1: Introduction Figure 1.7. The macroautophagy process occurring within the host cell. Macroautophagy is responsible for the degradation of macromolecules by the autolysosome. Image adapted from (138, 139). Figure 1.8. The xenophagy process occurring within the host cell. Xenophagy is responsible for the degradation and bacterial clearance of invading pathogens by the lysosomal enzymes found in the autophagolysosome. Image adapted from (138, 139). 86 Chapter 1: Introduction Autophagy is stimulated in response to various stresses or stimuli including infection, with the process playing an essential defensive role in eliminating pathogens, as part of an innate immune response (140, 141). Antimicrobial autophagy also referred to as xenophagy targets microbes, including: viruses, protozoa and bacteria for elimination via lysosomal degradation (140, 142). Initially pathogens invading host cells are recognised by pattern recognition receptors (PRR), which include Toll-like receptors (TLR) on the surface of cells and interior of endosomes, and cytosolic NOD-like receptors (NLR) (143). PRRs recognise the presence of microbes through specific microbe-associated molecular patterns (MAMPs) including peptidoglycan and lipopolysaccharide, which are components of bacterial cell walls (143). The detection of MAMPs by PRRs triggers the activation of several signalling cascades, amongst them NF-κB, a vital mediator of inflammatory cytokine production, as well as antimicrobial mechanisms to clear the infection (144). 1.7.2. Autophagy and the innate immune system: A plausible link between autophagy and Crohn’s disease A possible link between genetic mutations in genes present in the autophagy process and IBD has been proposed, with a number of mutations in autophagy and autophagy related genes being identified as being associated with CD (145). For example it has been shown that a single nucleotide polymorphism (SNP) in ATG16L1 (autophagy- related 16 like 1 gene) is associated with CD (145). Additionally, a large 20.1 Kb deletion in the promoter of another autophagy related gene collectively known as IRGM (Immunity related GTPase M) has also been shown to confer susceptibility to CD (146). Further, numerous studies have reported the importance of ATG16L1 and IGRM in combating and destroying intracellular pathogens including S. Typhimurium and Mycobacterium tuberculosis (136, 146-148). 87 Chapter 1: Introduction 1.7.3. The role of autophagy in C. jejuni infection In recent years the importance of autophagy as an innate defence mechanism has received significant attention. In a recent study by Sun et al. determined the importance of mTOR (mammalian target of rapamycin) signalling, a pathway that mediates autophagic responses involved in bacterial killing and clearance, (145 146) following C. jejuni infection. In this study germ free (control) or conventionally-derived Interleukin 10−/− mice expressing the enhanced green fluorescent protein (EGFP) under the control of NF-κB (IL10−/−; NF-κBEGFP mice) were infected with 109 CFU C. jejuni for a period of 12 days. Following infection, their response to infection was determined using histologic, semi-quantitative reverse transcription PCR, fluorescence in situ hybridisation, transmission electron microscopy, and tissue culture analyses (149). The results of these studies showed that in IL10−/− mice, C. jejuni initiated intestinal inflammation, which was shown to correlate with the activation of mTOR signalling and neutrophil infiltration. To determine if mTOR signalling mediated the pro- inflammatory effect induced by C. jejuni infection, Sun et al. administered the mTOR signalling inhibitor rapamycin to IL10−/− mice before or after C. jejuni infection. This showed that treatment with rapamycin administered either before or after introduction of C. jejuni resulted in dramatically reduced levels of C. jejuni infiltration into the colon and spleen, which was shown to be independent of T-cell activation. Additionally, the effect of rapamycin on C. jejuni survival based on gentamicin protection assays in splenocytes, revealed that rapamycin promoted C. jejuni eradication and LC3-II (a marker of the autophagy process) generation, suggesting an interplay between C. jejuni and the autophagy process (149). While the above study indicates that autophagy may play an important role in C. jejuni infection further studies are required to definitively establish the role of 88 Chapter 1: Introduction autophagy in Campylobacteriosis (149). Given that some strains of C. concisus have the ability to invade host cells, determination of the interaction between C. concisus and the autophagy process is of considerable interest. 89 Chapter 1: Introduction 1.8. Hypothesis That the emerging gastrointestinal pathogens C. concisus and C. ureolyticus are associated with chronic and acute gastrointestinal disease and that their ability to cause disease relates to the presence of specific virulence factors and their ability to manipulate the host’s innate immune system. 1.8.1. C. ureolyticus specific aims The primary aims of these studies were: 1. To determine using adherence and gentamicin protection assays the ability of C. ureolyticus to attach to and invade the intestinal cell lines Caco-2 and HT-29. 2. To use scanning electron microscopy to visualise the interaction between C. ureolyticus and the intestinal cell lines. 3. To determine if pre-existing inflammation affects the pathogenic potential of C. ureolyticus. 4. To determine using HT-29 cells the ability of C. ureolyticus to translocate through intestinal epithelial tight junctions. 5. To use a one-dimensional gel electrophoresis (1DE) protein separation technique coupled with Linear Trap Quadruple-Fourier transformer tandem mass spectrometry (LTQ-FT-MS/MS) to characterise the secretome of C. ureolyticus UNSWCD. 6. To determine the effect of the C. ureolyticus secretome on intestinal cell viability and inflammation, using in vitro viability and IL-8 ELISA assays. 90 Chapter 1: Introduction 1.8.2. C. concisus specific aims The primary aims of these studies were: 1. To determine whether growth of C. concisus under aerobic or microaerobic conditions affects the ability of C. concisus to adhere to and/or invade Caco-2 cells. 2. To examine the effect of C. concisus infection on Caco-2 cell viability. 3. To determine, the ability of C. concisus to survive intracellularly within Caco-2 cells over time. 4. To examine, using transmission electron microscopy (TEM), any intracellular changes that may occur following invasion of Caco-2 cells by C. concisus. 5. To compare the invasive potential and intracellular survival of C. concisus in Caco- 2 cells following treatment of the cells with Chloroquine di-phosphate (autophagosome inducer), Rapamycin (autophagy inducer) and 3-Methyladenine, Wortmannin and Bafilomycin A1 (autophagy inhibitors) with that of untreated Caco-2 cells infected with C. concisus. 6. To determine, using confocal laser scanning microscopy, how C. concisus interacts with autophagosomes following infection of Caco-2 cells with C. concisus UNSWCD. 7. To develop and optimise TEM sample preparation for the visualisation of autophagy ultrastructures and C. concisus infection within Caco-2 cells. 8. To visualise and examine the process of autophagy within intestinal epithelial Caco- 2 cells following infection with C. concisus UNSWCD, using TEM. 9. To determine following infection of Caco-2 cells with C. concisus, the expression of genes associated with the autophagy pathway, using a quantitative RT-PCR array approach. 91 CHAPTER 2 2. MATERIALS AND METHODS 2.1. Bacterial cultures 2.1.1. Campylobacter concisus strains The C. concisus strains employed in this study were C. concisus UNSWCD, isolated from a child with Crohn’s disease, and three strains isolated from patients with acute gastroenteritis, C. concisus UNSWCS, BAA-1457 and ATCC 51562, (Table 2.1). Table 2.1. C. concisus strains employed in this thesis and their isolation source. Bacteria Strain Source of isolation C. concisus UNSWCD Human Colonic Biopsy C. concisus ATCC 51562 Human Faeces C. concisus UNSWCS Human Faeces C. concisus BAA-1457 Human Faeces 2.1.2. Campylobacter ureolyticus strains The C. ureolyticus strains employed in this study were C. ureolyticus UNSWCD, isolated from a child with Crohn’s disease, and strains C. ureolyticus UNSWE and C. ureolyticus UNSWR. These strains were isolated from human faecal samples of children with no pathology (Table 2.2). 92 Chapter 2: Materials and Methods Table 2.2. C. ureolyticus strains employed in this study and their isolation source. Bacteria Strain Source of Isolation C. ureolyticus UNSWCD Human Colonic Biopsy C. ureolyticus UNSWE Human Faeces C. ureolyticus UNSWR Human Faeces 2.1.3. Other bacterial species utilised Salmonella enterica serovar Typhimurium LT2 was obtained from the UNSW culture collection. This highly invasive bacterium was used as a positive control. Escherichia coli K-12, also obtained from the UNSW culture collection, was used as a negative control, which has been proven not to invade intestinal epithelium cells and is considered to be a commensal within the gastrointestinal tract (Table 2.3). Table 2.3. An outline of other strains employed in this study and their relevant isolation origins. Bacteria Strain Source of Isolation S. Typhimurium LT2 UNSW Collection Unknown E. coli K-12 UNSW Collection Human Faeces 2.2. Culture media 2.2.1. Horse Blood Agar Horse blood agar (HBA) was prepared by suspending 20 g of Blood Agar Base No.2 (Oxoid; Heidelberg, VIC, Australia) in 500 ml of distilled water. This was sterilised by 93 Chapter 2: Materials and Methods autoclaving at 121°C for 15 min. The agar was cooled to 56°C after which 30 ml (6% w/v) of sterile defibrinated horse blood (Oxoid) was added. Plates were stored at 4°C before they were used for microbial culturing. 2.2.2. Nutrient Agar Nutrient agar (NA) was prepared by suspending 14 g of NA Base (Oxoid) in 500 ml of distilled water and sterilised by autoclaving at 121°C for 15 min. Plates were stored at 4°C before they were used for microbial culturing. 2.2.3. Brain heart infusion Brain heart infusion (BHI) was prepared by suspending 3.7 g of BHI powder (Oxoid) in 100 ml of distilled water. The BHI broth was dissolved in water and then sterilised by autoclaving at 121°C for 15 min. The BHI broth was stored at 4°C before use. 2.2.4. Brucella Broth Brucella broth (BB) was made by suspending 3.7 g of Brucella powder (Becton Dickson; Sparks, USA) in 100 ml of distilled water. BB was sterilised by autoclaving at 121°C for 15 min. BB was stored at 4°C before use. 2.2.5. Brain Heart Infusion Broth Plus Glycerol Brain heart infusion broth plus glycerol (BHIG) was used as a preservation medium for the bacterial strains used in the investigations. Sterile BHI broth was prepared as outlined in section 2.2.3, and glycerol (Ajax Finechem; Tarren Point, Australia) was mixed at a ratio of 7:3 (v/v), BHI: glycerol, and stored at 4°C. 94 Chapter 2: Materials and Methods 2.2.6. Brucella Broth with Fetal Bovine Serum BB was made up according to section 2.2.4. Fetal bovine serum (FBS) (Bovogen Biologicals; Keilor East, Australia) was added to the broth at 10% (v/v) and filter- sterilised using a 0.4 µm filter (Milipore). 2.2.7. Brain Heart Infusion Broth with Fetal Bovine Serum BHI broth was prepared as outlined in section 2.2.3. FBS (Bovogen) was added to the broth at 10% (v/v) and filter-sterilised using a 0.4 µm filter (Milipore). 2.3. Buffers 2.3.1. Phosphate buffered Saline 0.1 M A solution of 0.1 M Phosphate buffered saline (PBS) was prepared using the following ingredients: 1.37 M NaCl (Ajax Finechem), 0.0268 M KCl (Ajax Finechem), 0.1 M Na2HPO4 (Ajax Finechem) and 0.0176 M KH2PO4 (Ajax Finechem). The constituents were then dissolved in 1 l of distilled water and the pH adjusted to 7.4 using 1 N HCl using a pH meter (Model pHi 570, Beckman Coulter, USA). The PBS solution was sterilised by autoclaving at 121°C for 15 min and then stored at room temperature. 2.4. Bacterial preservation, culture and enumeration 2.4.1. Cryoperservation Bacterial strains used in the study were stocked in aliquots of 1 ml in BHIG and stored at -80°C. 95 Chapter 2: Materials and Methods 2.4.2. Resuscitation and culture of bacterial strains C. ureolyticus and C. concisus were revived from frozen stocks and grown on HBA solid media under microaerobic conditions with H2 (generated using Campylobacter Gas Generating Kits (Cat. #. BR0056A, Oxoid; Adelaide, SA, Australia) at 37°C. 2.4.3. Bacterial harvesting from solid and liquid media Bacterial strains were harvested from HBA by adding 1 ml of PBS to the plate and using a sterile hockey stick to harvest all bacterial cells. Following harvesting, the cells were transferred into sterile eppendorf tubes and centrifuged at 14,160 × g and re- suspended in 1 ml PBS for further experimental use. For liquid media, C. concisus UNSWCD and C. ureolyticus UNSWCD cultures were initially grown on HBA plates for 48 h at 37°C under microaerobic growth conditions with H2 (generated using Campylobacter Gas Generating Kits (Cat. #. BR0056). Following incubation, the bacterial cells were harvested using 1 ml of BHI broth (Oxoid). The bacteria harvested from a single plate were then grown in a 75 cm2 flask containing 9 ml of BHI (Oxoid), incubated under microaerobic conditions for 48 h at 37°C. Following incubation, the content of each flask was centrifuged at 1,581 × g for 15 min and the bacterial cell pellet re-suspended in 1 ml of PBS. 2.4.4. Determination of bacterial cell concentration Bacteria were harvested from solid HBA agar media using 1 ml of sterile PBS, centrifuged at 14,160 × g for 1 min, and the cell pellet re-suspended in 1 ml of PBS. Eight eppendorf tubes were set up for each bacterial strain investigated as serial dilutions (1:10) prepared from 10-1 to 10-8 with PBS. Five microliters of each dilution was then dropped onto HBA plates in quadruplicate and incubated under 96 Chapter 2: Materials and Methods microaerophilic conditions for 48 h at 37°C. The bacterial cell concentration of each strain was calculated based on the number of colony forming units (CFU). 2.4.5. Liquid culture for time course experiments Prior to the time course experiments, bacterial strains were maintained in 24 well plates (Nunc Thermoscientific; Roskdile, Denmark) containing BHI or BB incubated under microaerophilic conditions for 24 h at 37°C. The bacterial cells were then centrifuged at 14,160 × g for 1 min and re-suspended in 1 ml of PBS for use in further experiments. 2.5. Mammalian cell culture 2.5.1. Growth and maintenance of Caco-2 cells Caco-2 cells (American Type Culture Collection No: HTB-37) were removed from liquid nitrogen stocks and immediately placed in a 37°C water bath until the cells were almost completely thawed. An equal volume of 1 ml heat-inactivated FBS (Bovogen) was then added and the cells centrifuged at 232 × g for 5 min to remove the DMSO solution in which they were stored in. The Caco-2 cells were maintained in 25 cm2 sterile tissue culture flasks (In Vitro Technologies; Noble Park, VIC, Australia) containing Minimal Eagle Medium (MEM) (Life technologies; Mulgrave, VIC, Australia) supplemented with 10% (v/v) heat inactivated FBS (Bovogen), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 U/ml penicillin and streptomycin (Life technologies) and 2.25 M sodium bicarbonate (Life technologies) and maintained at 37°C and 5% CO2. The Caco-2 cell culture were replenished with fresh supplemented media every 2-3 days and the cell monolayer passaged every 7 days using treatment with 1 ml of 0.25% (v/v) 97 Chapter 2: Materials and Methods Trypsin-EDTA digest (Life technologies) for 5 min. The trypsin was neutralised by the addition of an equivalent volume of 1 ml heat-inactivated FBS (Life technologies). The Caco-2 cells were then centrifuged at 232 × g for 5 min. The cell pellet was then resuspended in MEM supplemented media and diluted to 1.0 × 105 cells/ml in a sterile 25 cm2 sterile tissue flask. 2.5.2. Growth and maintenance of HT-29 cells HT-29 cells (American Type Culture Collection HTB-38) were revived fresh from liquid nitrogen in a similar fashion to the procedure outlined in section 2.5.1. HT-29 cells were maintained in 25 cm2 sterile tissue flasks (In Vitro Technologies) at 37°C and 5% CO2 in McCoy’s 5A medium (Life technologies) supplemented with 10% (v/v) heat inactivated FBS (Bovogen) and 100 U/ml penicillin and streptomycin (Life technologies). The HT-29 cells were replenished with fresh supplemented media every 2-3 days and the cell monolayer passaged every 7 days by treating with 0.25% (v/v) Trypsin-EDTA digest (Life technologies) for 5 min. The trypsin was then neutralised by adding an equivalent volume of heat-inactivated FBS (Life technologies). The HT-29 cells were then centrifuged at 232 × g for 5 min. The cell pellet was then resuspended in supplemented media and diluted to 1.0 × 105 cells/ml in a sterile 25 cm2 tissue flask. 2.6. Light microscopy for mammalian and bacterial inspection To monitor confluency levels, mammalian cell line monolayers were visualised using an Olympus CH2 (Olympus, Japan) inverted microscope. To check the morphology of all C. concisus and C. ureolyticus strains, an Olympus BH-2 light microscope was utilised. Bacterial colonies on the agar plates were removed and emulsified in 0.1 M PBS on a glass slide and a cover slip added. In the case of broth cultures, 0.1 ml of 98 Chapter 2: Materials and Methods culture was added directly to the slide and a cover slip added. The samples were then viewed via oil immersion at 100 × magnification and the morphology of the cells noted. 2.7. Bacterial growth and morphology confirmation of cultures 2.7.1. Gram staining and morphological characterisation of bacterial cultures To confirm that the bacterial cultures were Gram-negative rods and spiral curved, bacterial cultures were Gram stained. In brief, bacterial solutions were ‘heat fixed’, after which the slide was flooded with crystal violet solution (Sigma; St. Louis, USA) and allowed to remain for 1 min. This was then rinsed off with distilled water and iodine solution (Sigma) was added for 1 min, after which it was rinsed off with distilled water. The slide was then flooded with alcohol iodine, a decolourising solution (Sigma), and rinsed off with distilled water. The slide was then flooded with a carbolfuchsin solution (Sigma) for 30 s and then rinsed with distilled water. Once the slides were dry, they were viewed on an Olympus CH2 (Olympus, Japan) under 100 × magnification with oil immersion. 2.7.2. Biochemical identification of C. ureolyticus and C. concisus Rapid urease was used as a chemical means to confirm the identity of cultured C. ureolyticus spp. Rapid urease solution comprises 0.33 M Urea, 0.14 M phenol red adjusted to 10 ml, 0.044 M Na2HPO4.12H2O, 0.005 M Na2HPO4.2H2O and 0.003 M NaN3 0.003 M, made up to 100 ml of Milli-Q water, and the pH adjusted to 6.3-6.5 using 1 N HCl using a pH meter (Beckman Coulter). The rapid urease test was performed by dispensing 200 µl of the prepared rapid urease solution into a 96 well microtitre plate, and emulsifying it with a loop-full of bacteria. The presence of urease was indicated by a colour change in the medium from yellowish orange to dark red. 99 Chapter 2: Materials and Methods Results with C. ureolyticus spp showed a positive (+) red colour change and results with C. concisus spp showed a negative (-) yellow orange colour. Apart from the rapid urease test, other biochemical tests were used. A combination of both catalase and oxidase were used to biochemically distinguish the formation of both C. concisus and C. ureolyticus colonies. The reaction of both of these bacteria to catalase and oxidase was examined by dispensing 200 µl of either catalase or oxidase (Sigma) onto a sterile glass slide, and emulsifying it with a loop-full of bacteria. The results of these biochemical examinations are provided in Table 2.4. Table 2.4. The morphological characteristics and biochemical tests used for the identification of C. concisus and C. ureolyticus strains. As outlined in Bergey’s Manual of systematic bacteriology (1). Characteristics / C. concisus C. ureolyticus Biochemical properties Flagella Single polar flagellum Aflagellate, non-motile Morphology Spiral or curved rod Predominantly curved or straight rod Gram-stain Gram-negative Gram-negative Urease Negative Positive Oxidase 60-93% strains Positive Positive Catalase Negative 14-50% strains Positive 100 CHAPTER 3 3. THE INVESTIGATION OF THE PATHOGENIC POTENTIAL OF CAMPYLOBACTER UREOLYTICUS IN HOST INTESTINAL EPITHELIAL CELLS 3.1. Background Bacteroides ureolyticus, an aflagellate Gram-negative bacterium, which may exhibit a twitching motility, has recently been reclassified as a member of the Campylobacter genus (1, 36). In 1991, Vandamme et al. first proposed that B. ureolyticus be reclassified as a member of the Campylobacter genus as it fell into the rRNA cluster that contained members of the Campylobacter genus (35). Using polyphasic taxonomic analyses, follow-up studies by the same group showed that B. ureolyticus shared a range of characteristics with other members of the Campylobacter spp., including: respiratory quinone content, DNA base ratio and phenotypic characteristics with Campylobacter species, including Campylobacter jejuni (150). However, in a subsequent study Vandamme et al. reported differences with respect to fatty acid composition, proteolytic metabolism, hydrolysis of gelatin and casein, as well as the ability to hydrolyse urea (1), which led to speculation as to the appropriateness of reclassifying B. ureolyticus as a new member of the Campylobacter spp. In 2010, a publication by Vandamme et al. (36) finally acknowledged and verified the reclassification of B. ureolyticus to Campylobacter ureolyticus comb. nov as a newly designated member of the Campylobacter genus. Prior to its reclassification, the isolation and detection of C. ureolyticus had been reported in patients with a range of diseases including: superficial ulcers, soft tissue 101 Chapter 3: Pathogenic potential of C. ureolyticus infections, non-gonococcal urethritis, peri-anal abscess, and gangrenous lesions, as well as in the amniotic fluid and genital tract of humans (37-40). C. ureolyticus has also commonly been isolated from patients suffering from periodontal disease, which involves inflammation and destruction of the tissues surrounding the teeth (40). Moreover, C. ureolyticus has been detected in faecal samples, with Bullman et al. (151) reporting the detection of C. ureolyticus in the faeces of 23.8% of patients suffering from gastroenteritis, leading them to suggest that this bacterium may be an emerging gastrointestinal pathogen (151). C. ureolyticus has also been isolated form patients with inflammatory bowel disease (IBD), which is categorised into two major subtypes, Crohn’s Disease (CD) and Ulcerative Colitis (UC) (43). For example, Mahendran et al. have reported the detection of C. ureolyticus in children with UC, as well as in biopsies of adults with UC (44). These findings were further supported by a study by Mukhopadhya et al. who determined the prevalence of Campylobacter species in biopsy specimens collected from patients undergoing diagnostic colonoscopy (45). Of these, 69 were shown to have histologically proven UC and 65 were healthy controls (45). Following collection and storage of the biopsies at -80oC, DNA was extracted and subjected to Campylobacter genus specific polymerase chain reaction (PCR) examination and sequencing. Based on the Campylobacter genus specific primers, a significantly higher prevalence of Campylobacters were detected in UC patients (73.9%, 51/69) as compared with the controls (23.1%, 15/65) (P = 0.0001). Sequencing of the PCR products of the Campylobacter positive samples, showed C. ureolyticus to be present in 15/69 UC patients and in 2/65 controls. Based on these results, the prevalence of C. ureolyticus in UC patients was found to be significantly higher (21.7%) than that in healthy controls (3.08%) (P = 0.0013) (45). C. ureolyticus has also been successfully isolated from 102 Chapter 3: Pathogenic potential of C. ureolyticus biopsy samples of children with newly diagnosed CD (41), leading researchers to question its role in intestinal disease. A previous study by Man et al. (132) investigating the pathogenic potential of C. ureolyticus demonstrated that the bacterium, when cultured with human monocytes and primary macrophages, initiated the production of inflammatory cytokines (132). While this suggests that C. ureolyticus may have the potential to cause intestinal disease, it does not identify the bacterial factors involved, or what effect contact with this bacterium has on human intestinal cells. Thus, the overall aim of this study was to assess the pathogenic potential of three C. ureolyticus strains, one strain (UNSWCD) isolated from an intestinal biopsy of a CD patient, and 2 strains (UNSWE and UNSWR) isolated from faecal specimens of patients with no pathology. The successful isolation of C. ureolyticus UNSWCD from a biopsy sample of a CD patient raises the question as to whether C. ureolyticus could potentially play a role in intestinal disease, or, alternatively, whether it is simply part of the normal flora of the gastrointestinal tract. Thus, this study aimed to determine the pathogenic potential of C. ureolyticus by quantifying and visualising the adherence and invasive abilities of these C. ureolyticus strains. In order to examine these properties, adherence assays, gentamicin protection (invasion) assays, scanning electron microscopy (ScEM) and translocation assays were undertaken. Further, given that the two prime pro- inflammatory cytokines associated with IBD include tumor necrosis factor alpha (TNF- α) and gamma interferon (IFN-γ) (43, 152). Moreover, the pathogenic potential of C. ureolyticus under the effect of pre-existing inflammation was also investigated. The sections in the thesis where inflammation is used in the context of an epithelial cell line exposed to cytokines, with the intent to modulate the activity and responsiveness of the 103 Chapter 3: Pathogenic potential of C. ureolyticus cells, refers to “changes of the epithelial cell line following cytokine modulation, as occurs in inflammatory reactions.” Additionally, given that proteins secreted by bacteria come into direct contact with host cells, the secretome (the proteome subset that is secreted from the cell) of isolate C. ureolyticus UNSWCD was determined using a similar methodological approach applied to the identification of the secretome of C. concisus (67). 3.1.1. Aims The aims of this chapter are: 1. To determine using adherence and gentamicin protection assays the ability of C. ureolyticus to attach to and invade the intestinal cell lines Caco-2 and HT-29. 2. To use scanning electron microscopy to visualise the interaction between C. ureolyticus and the intestinal cell lines. 3. To determine if pre-existing inflammation affects the pathogenic potential of C. ureolyticus. 4. To determine using HT-29 cells the ability of C. ureolyticus to translocate through intestinal epithelial tight junctions. 5. To use a one-dimensional gel electrophoresis (1DE) protein separation technique coupled with Linear Trap Quadruple-Fourier transformer tandem mass spectrometry (LTQ-FT-MS/MS) to characterise the secretome of C. ureolyticus UNSWCD. 6. To determine the effect of the C. ureolyticus secretome on intestinal cell viability and inflammation, using in vitro viability and IL-8 ELISA assays. 104 Chapter 3: Pathogenic potential of C. ureolyticus 3.2. Materials and Methods 3.2.1. C. ureolyticus strains and other bacterial species The C. ureolyticus strains used in this study were C. ureolyticus UNSWCD, isolated from an intestinal biopsy specimen of a child with newly diagnosed CD, and C. ureolyticus UNSWE and UNSWR isolated from faecal samples of children with no pathology. Salmonella serovar Typhimurium LT2 was obtained from the UNSW culture collection and was used as the positive control in invasion assays. Escherichia coli K-12 was obtained from the UNSW culture collection and was used as a commensal negative control in IL-8 ELISA assays as described in section 3.5.2. All strains used in the study are summarised in Table 3.1. Table 3.1. C. ureolyticus and other bacterial species employed in this study and their isolation source. Bacteria Strain Source of Isolation C. ureolyticus UNSWCD Human Colonic Biopsy C. ureolyticus UNSWE Human Faeces C. ureolyticus UNSWR Human Faeces S. Typhimurium LT2 Unknown E. coli K-12 Human Faeces 3.2.2. Bacterial cultivation C. ureolyticus UNSWCD, UNSWE and UNSWR stocks, stored at -80oC in Brain Heart Infusion (BHI) broth (Oxoid; Heidelberg, VIC, Australia) with glycerol, were removed from storage, thawed and cultured on Horse Blood Agar (HBA) [Oxoid Blood agar base No.2 containing 6% (w/v) sterile defibrinated horse blood] for 48 h at 37oC under 105 Chapter 3: Pathogenic potential of C. ureolyticus microaerobic conditions generated by an atmospheric gas generating system (Campylobacter Gas Generation Kit Catalogue # BR0056A; Oxoid; Hampshire, U.K). S. Typhimurium LT2 (UNSW culture collection) and Escherichia coli K-12, stored in BHI plus glycerol at -80oC, were thawed and cultured on Nutrient agar (Oxoid) for 24 h at 37oC under aerobic conditions. All bacterial strains used in the adherence and gentamicin protection assays section 3.3, were grown to log phase before being harvested for experiments. For the IL-8 ELISA studies, E. coli K12 was also grown to log phase (section 3.5.2). 3.2.3. Cultivation of the human intestinal Caco-2 cell line The human colorectal adenocarcinoma cell line Caco-2 (American Type Culture Collection No: HTB-37) was used in the study. Caco-2 cells which had been preserved with 5% Dimethyl Sulfoxide (DMSO) (Sigma; St Louis, U.S.A) in antibiotic free Minimal Essential Medium (MEM) (Invitrogen; Grand Island, N.Y, USA) and stored in liquid nitrogen were removed from cryostorage and the cells quickly thawed in a 37oC water bath. An equal volume of Foetal Bovine Serum (FBS) (Bovogen Biologicals; Keilor East, Australia) was then added to the cells and this was then centrifuged at 232 × g for 5 min. The cell pellet was then re-suspended in 10 ml of Caco-2 cell culture media comprising MEM supplemented with 10% heat-inactivated FBS, 0.1 mM non- essential amino acids, 1 mM sodium pyruvate, 100 µg/ml penicillin and streptomycin, and buffered with 2.25 mg/l sodium bicarbonate (Invitrogen), and maintained in a 25 2 o cm cell culture flask (T25) (Nunc; Roskilde, Denmark) at 37 C and 5% CO2 for 7 days. Following incubation the cells were detached using 0.25% Trypsin-EDTA (Invitrogen) for 5 min. The trypsin was then deactivated with 10% FBS (Invitrogen) and then centrifuged at 232 × g for 5 min after which the cell pellet was resuspended in 10 ml of cell culture media and passaged down to 1.0 × 105 cells/ml in 25 cm2 sterile tissue 106 Chapter 3: Pathogenic potential of C. ureolyticus flasks. The Caco-2 monolayers were then checked for confluence using an inverted microscope (CK2 Olympus; Tokyo, Japan). 3.2.4. Cultivation of the human intestinal HT-29 cell line The HT-29 colorectal adenocarcinoma cell line (American Type Culture Collection HTB-38), which had been preserved in antibiotic free McCoy’s 5A (Life technologies; Location) cell culture media containing 5% DMSO (Sigma) and stored in liquid nitrogen was removed from cryostorage and quickly thawed in a 37oC water bath. The cells were maintained in 25 cm2 sterile tissue flasks (In Vitro Technologies; Noble Park, o VIC, Australia) at 37 C and 5% CO2 in McCoy’s 5A medium (Life technologies; Mulgrave, VIC, Australia) supplemented with 10% heat inactivated FBS and 100 U/mL penicillin and streptomycin (Life technologies). HT-29 cells were supplemented with fresh media every 2-3 days and the cell monolayer passaged every 7 days by treating the cell line with 0.25% Trypsin-EDTA digest (Life Technologies) for 5 min. The trypsin was then neutralised with an equivalent volume of heat-inactivated FBS after which the cells were centrifuged at 232 × g for 5 min and the cell pellet resuspended in supplemented media and passaged down to 1.0 × 105 cells/ml in 25 cm2 sterile tissue flasks. The HT-29 monolayers were checked for confluence using an inverted microscope (CK2 Olympus). 107 Chapter 3: Pathogenic potential of C. ureolyticus 3.3. Adherence and gentamicin protection (invasion) assays using Caco-2 and HT- 29 cells To seed the 24-well tissue culture plates used for the attachment and invasion assays, confluent Caco-2 cell or HT-29 monolayers in tissue flasks were detached using 0.25% Trypsin-EDTA (Invitrogen) for 5 min, after which the trypsin was deactivated with 10% FBS (Invitrogen). The cells were then centrifuged at 232 × g for 5 min and the cell pellet resuspended in 10 ml of cell culture media. In order to determine cell viability a cell count was performed using a haemocytometer and 0.4% trypan blue (Sigma). The cells were then seeded at a concentration of 5 × 105 cells/ml into 24-well tissue culture plates, which had been pre-coated with 1 ml of 0.338 mg/ml collagen at 37oC for 30 min, in order to aid the formation of the cell monolayer. The 24-well plates were then incubated for 2 or 4 days for Caco-2 cell or HT-29 cell line, respectively, at 37oC and 5% CO2 to allow the formation of a confluent cell monolayer. 3.3.1. Adherence assay Prior to infection, Caco-2 and HT-29 cell monolayers were washed three times with antibiotic-free cell culture media. The monolayers were then infected with a multiplicity of infection (MOI) of 200 for C. ureolyticus UNSWCD, UNSWE and UNSWR and a MOI of 100 for S. Typhimurium LT2. Due to the highly invasive nature of S. Typhimurium LT2, a lower MOI was employed. The 24-well tissue culture plate was then centrifuged at 232 × g for 5 min using a 4K15C centrifuge (Sigma) in order to promote bacterial-mammalian cell contact. Infected monolayers were then incubated for o 6 h at 37 C and 5% CO2. The number of viable C. ureolyticus was determined after the 6 h incubation period, to avoid any bias that may arise due to bacterial growth or death during the incubation period. 108 Chapter 3: Pathogenic potential of C. ureolyticus After incubation for 6 h, the monolayers were gently washed 4 times with antibiotic-free media, after which the Caco-2 or HT-29 monolayer were lysed using 500 µl 1% Triton X-100 (Sigma) for 5 min. After collection of the cell lysate, ten-fold dilutions of the lysates were prepared up to 1 × 10-3 for C. ureolyticus UNSWCD, UNSWE and UNSWR, and 1 × 10-6 for S. Typhimurium LT2, and 5 µl drops of each dilution was then plated in quadruplicate onto appropriate agar plates. Three biological replicates and 2 technical replicates were conducted. 3.3.2. Gentamicin protection (invasion) assay The initial steps of the invasion assay (n = 6 per cell line) were conducted as described for the adherence assay section 3.3.1. Following the 6 h incubation period, the infected HT-29 or Caco-2 monolayers were washed 3 times in MEM or McCoy’s 5A cell culture media containing gentamicin (Invitrogen) at a concentration of 200 µg/ml to kill any extracellular bacteria. Following incubation with gentamicin for 1 h, the media was collected and 5 µl drops were plated in quadruplicate, both undiluted and at 1:10 dilution, to determine whether any viable extracellular bacteria remained. To determine the number of bacterial cells that had invaded the Caco-2 or HT-29 cells, each cell line was lysed by addition of 500 µl of 1% Triton X-100 (Sigma) for 5 min. The cell lysates of each monolayer were then diluted by a series of ten-fold series to 1 × 10-3 for C. ureolyticus UNSWCD, UNSWE and UNSWR, and to 1 × 10-5 for S. Typhimurium LT2, after which 5 µl drops were plated in quadruplicate onto the appropriate agar plates. Three biological replicates and 2 technical replicates were conducted. 109 Chapter 3: Pathogenic potential of C. ureolyticus 3.3.3. Examination of the effect of pre-existing inflammation on C. ureolyticus UNSWCD adherence and invasion To determine the effect of pre-existing inflammation on attachment and invasion, the Caco-2 and HT-29 cell monolayers were treated with 40 ng/ml TNF-α (Sigma) or IFN-γ (Sigma) 1 h prior to the commencement of the adherence and invasion assays. The monolayers were then infected with C. ureolyticus strains at a MOI of 200 and S. Typhimurium (positive control) at a MOI of 100. The 24 well culture plate was then centrifuged at 232 × g for 5 mins after which it was incubated for 6 h at 37oC and 5% CO2. The number of adherent or internalised bacteria was determined as previously described in sections 3.3.1 and 3.3.2, respectively. Three biological replicates and 2 technical replicates were conducted. 3.3.4. Determination of percentage adherence and invasion The percentage adherence and invasion of each bacterium were determined using the following formulas below: !"#$%& !" !"ℎ!"#$% !"!"#$%& − !"#$%& !" !"#$%!"& !"#$%&'" % !"ℎ!"!#$! ∶ × 100 !"#$% !"#$%& !" !"#$%&'" !""#" !" !"#$ − 2 !" !" − 29 !"##$ !"#$%& !" !"#$%!"& !"#$%&'" % !"#$%&'" ∶ × 100 !"#$% !"#$%& !" !"#$%&'" !""#" !" !"#$ − 2 !" !" − 29 !"##$ 110 Chapter 3: Pathogenic potential of C. ureolyticus 3.3.5. Translocation of C. ureolyticus across HT-29 cells To determine if C. ureolyticus could translocate across the HT-29 cells, transwell filter units (0.4 µm Nunc) were collagen coated as per the manufacturer’s instructions and then seeded with 5.0 × 105 cells/ml of HT-29 cells in a 24 well plate. The plate with the transwell inserts was then incubated for 14 days with the apical and basolateral media changed every 48 h. The transwell inserts were then washed three times with McCoy’s 5A antibiotic-free cell culture medium and inoculated with C. ureolyticus UNSWCD at an MOI of 200. The monolayers were then incubated for 6 h, after which 100 µl was removed from the basal compartment and serially diluted, and the cells counted using the drop plate count method. Three biological replicates and 2 technical replicates were conducted. 3.4. Determination of the adherence pattern of C. ureolyticus UNSWCD using Scanning Electron Microscopy 3.4.1. Sample preparation Caco-2 cells were grown on Poly-L-Lysine coated cover slips for 48 h in cell culture medium in 24-well plates at a cell density of 5 × 105 cells per well and incubated at o 37 C and 5% CO2. C. ureolyticus UNSWCD was harvested and added at a MOI of 200 to the corresponding wells for 1, 3 and 6 h. To examine the effect of pre-existing inflammation on attachment and invasion, 40 ng/ml of TNF-α and IFN-γ was added to the corresponding wells 1 h prior to co-incubation with C. ureolyticus UNSWCD. Caco- 2 cells were then inoculated with C. concisus UNSWCD at a MOI 200 and incubated for 6 h. For ScEM examination the infected monolayers were fixed in a fixative solution comprising of 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1M Sorensen’s Phosphate Buffer (SPB) (ProSciTech) pH 7.2, and left overnight at 4oC. Samples were 111 Chapter 3: Pathogenic potential of C. ureolyticus then carefully washed 3 times for 10 min in 0.1M SPB, pH 7.2 to avoid dislodgement of the monolayer. The sample coverslips were then carefully removed from the 24 well plate using fine forceps, after which they were loaded onto an autosamdri-815 sample holder with mesh coverings above and below each sample coverslip, allowing the even diffusion of each solution throughout the sample holder. The ethanol concentration gradient protocol was as follows. The biological samples contained within a sample holder were initially exposed to ethanol concentrations of 30% (10 min), 50% (10 min) and 70% (2 h). Following this, they were exposed to 80% and 90% ethanol concentrations, each for 10 min. In the final step samples were exposed three times to 100% ethanol for 10 min. 3.4.2. Critical point drying, gold coating and imaging The critical point drying stage was performed over a 3 h period employing a Critical point dryer CPD 030 (BAL-TEC AG, Balzers, Liechtenstein) using liquid CO2. The coverslips were then mounted onto carbon tabs. A sputter gold coat was cast on top of these coverslips using an EMITECH K-55OX gold coater (Emitech; Ashford, U.K). ScEM was performed using a Hitachi S3400-X model Scanning Electron Microscope (Hitachi High-Technologies Corporation; Tokyo, Japan), using a 32 kV voltage. 3.5. Caco-2 cell viability and IL-8 production following C. ureolyticus infection, in the presence or absence of pre-existing inflammation 3.5.1. Determination of Caco-2 cell viability following infection with C. ureolyticus UNSWCD, in the presence or absence of pre-existing inflammation To determine the effect of C. ureolyticus UNSWCD on Caco-2 cell viability, Caco-2 cells were seeded according to the procedure outlined in section 3.3 and C. ureolyticus 112 Chapter 3: Pathogenic potential of C. ureolyticus UNSWCD was harvested according to section 3.3.2. C. ureolyticus UNSWCD was then inoculated at MOIs of 100, 200 and 1000, and cell viability determined. A negative control with no addition of C. ureolyticus UNSWCD was included. In addition, to determine the effect pre-existing inflammation, the above procedure was repeated with the addition of 40 ng/ml of either TNF-α or IFN-γ to the Caco-2 cells as outlined in section 3.3.3. Following incubation with C. ureolyticus UNSWCD for 6 h, the Caco-2 monolayers of each well were detached using 250 µl of 0.25% Trypsin EDTA for 5 min, after which 250 µl of 10% FBS (Invitrogen) was added to deactivate the trypsin. In order to quantify the percentage of viable Caco-2 cells from the total cell population a cell count was performed on the trypsinised cells using a haemocytometer and 0.4% trypan blue (Sigma). 3.5.2. Determination on the effect of C. ureolyticus UNSWCD infection and the addition of cytokines (TNF-α or IFN-γ) on interleukin-8 production To determine the effect of C. ureolyticus UNSWCD and E. coli K-12 (negative control) on IL-8 secretion, HT-29 cells were seeded at a density of 5 × 105 cells per well and incubated for 4 days as outlined in section 3.3. The HT-29 monolayers were then washed five times using McCoy’s 5A media prior to the inoculation of bacteria remove any traces of antibiotics. The HT-29 cells were then inoculated with C. ureolyticus UNSWCD (MOI 200) and E. coli K-12 (MOI 100) and incubated for 24 hours at 37oC under 5% CO2. Uninfected HT-29 cells were used as a control. Following the inoculation of bacteria, the mammalian cultures were centrifuged at 232 × g for 5 min using a 4K15C centrifuge (Sigma) to promote bacterial-mammalian cell contact and adherence. To provide an estimate of the number of C. ureolyticus UNSWCD and E. coli K12 in the inoculum prior to inoculation, the OD levels of each bacterial inoculum were analysed by spectrophotometry (wavelength of 595 nm) using a Bio-Rad 113 Chapter 3: Pathogenic potential of C. ureolyticus 550 Microplate Reader. In addition viable drop plate counts on the appropriate agar plates were undertaken to determine the exact number of bacteria added. Following incubation of the HT-29 cells with C. ureolyticus UNSWCD and E. coli K-12, the supernatants were collected and the levels of interleukin-8 (IL-8) determined using a Human IL-8 ELISA kit (Invitrogen). Further, to test the effect of pre-existing inflammation on IL-8 production, 40 ng/ml of either TNF-α or IFN-γ were added to the HT-29 cells 1 h prior to infection. Monolayers were then infected with C. ureolyticus UNSWCD (MOI 200) and E. coli K-12 (MOI 100). Following the inoculation of bacteria the mammalian cultures were centrifuged at 232 × g for 5 min using a 4K15C centrifuge (Sigma) to promote bacterial-mammalian cell contact and adherence. The cultures were then incubated for 24 h at 37oC under 5% CO2. The concentration of IL-8 secreted into the supernatant was then measured using a Human IL-8 ELISA kit (Invitrogen) according to the manufacturer’s instructions. The above experiments were repeated to investigate the effect of heat-killed bacteria on inflammation based on IL-8 production. C. ureolyticus UNSWCD and E. coli K-12 were heat killed at 80oC for 20 min. To confirm that the bacterial cells had been killed they were plated onto appropriate agar plates and if no growth occurred following incubation they were used in the study. Each of the above investigations was performed independently at least three times and repeated in duplicate. 114 Chapter 3: Pathogenic potential of C. ureolyticus 3.6. The secretome of C. ureolyticus UNSWCD 3.6.1. Bacterial cultures C. ureolyticus UNSWCD cultures were grown on HBA plates for 48 h at 37oC under microaerobic conditions. Following incubation, cells were harvested using 1 ml of BHI broth (Oxoid). Bacteria were then grown in liquid media comprising BHI supplemented with 0.1% w/v β-cyclodextrin (Sigma). 3.6.2. Isolation of the C. ureolyticus UNSWCD secretome The secreted proteins were isolated as described previously by Bumann et al. (153). In brief, bacterial cultures were centrifuged at 7,230 × g for 15 min at 4oC. The supernatant was then filtered through a 0.45 µm pore sized membrane filter to remove residual bacteria. The secreted proteins were then precipitated using a previously described tricholoracetic acid (TCA) method (154). Briefly, 300 ml of filtrate was mixed with 95 ml of pre-chilled TCA and incubated on ice water for 15 min. The mixture was then centrifuged at 7,230 × g for 10 min at 4oC. The protein pellet was re-suspended in 10 ml of acetone and centrifuged at 7,230 × g for 10 min. This acetone washing step was repeated twice and the final protein pellet was air dried. The protein pellet was then dissolved in 0.5 ml TSU buffer [0.1% SDS, 2.5 M urea and 50 mM Tris-HCl pH 8.0], and stored at -80oC. 3.6.3. Determination of protein content The protein concentration of the isolated C. ureolyticus UNSWCD secreted proteins was determined using the Bicinchoninic acid (BCA) method microtitre protocol according to the manufacturer’s instructions (Pierce; Rockford, IL, USA). Optical 115 Chapter 3: Pathogenic potential of C. ureolyticus density absorbances were read at 595 nm using a Bio-Rad 550 Microplate Reader and the protein concentration was determined using a standard curve. 3.6.4. One-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis One-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (1-D SDS PAGE) was employed to separate C. ureolyticus UNSWCD secreted proteins. Forty micrograms of the secreted protein solution was resuspended in an equal volume of SDS-PAGE sample buffer [0.375 M Tris, pH 6.8, 0.01% SDS, 20% glycerol, 40 mg/ml SDS, 31 mg/ml dithiothreitol (DTT), 1 μg/ml bromophenol blue], and the proteins further denatured by heating at 100oC for 5 min. The proteins were then separated on a 12% SDS-PAGE gel, prepared using 40% acrylamide, 1.5M Tris (pH 8.8), 10% SDS, 10% APS, TEMED and H2O. The 12% SDS-PAGE gel was electrophoresed for 1.5 h at 100 V and stained using Coomassie Brilliant Blue G-250 (Bio-Rad). 3.6.5. LTQ LC/MS-MS sample preparation and LTQ-MS Protein spots or bands were excised from the gels and digested according to a previously published method described by Kaakoush et al. (155). Briefly, bands were washed twice for 10 min in 200 µl of 100 mM NH4HCO3, incubated at 37°C for 1 h with 50 µl of 10 mM DTT, alkylated for 1 h in 50 µl of 10 mM iodoacetamide, washed for 10 min with 200 µl of 10 mM NH4HCO3, dehydrated in acetonitrile, and trypsin- digested with 10 ng/µl of trypsin (Promega; Annandale, NSW, Australia). After digestion for 14 h at 37oC, peptides were extracted by washing the gel slice for 15 min in 25 µl 1% formic acid, which was followed by dehydration in acetonitrile. Digests were then dried in vacuo and the dried samples stored at -80oC until mass spectrometry analysis was performed, at which point they were resuspended in 10 µl of 1% formic acid. 116 Chapter 3: Pathogenic potential of C. ureolyticus Digests were separated by nano-LC using an Ultimate 3000 HPLC and auto-sampler system (Dionex; Amsterdam, Netherlands). An LTQ-FT Ultra mass spectrometer (Thermo Electron; Bremen, Germany) was used for the analysis of the C. ureolyticus UNSWCD secreted proteins. Positive ions were formed by electrospray, and the LTQ- FT Ultra was operated in the data-dependent acquisition mode. Peak lists were generated using Mascot Daemon/extract msn (Matrix Science, Thermo; London, England) using the default parameters, and submitted to the database search program Mascot (version 2.1, Matrix Science). Search parameters were conducted as follows: precursor tolerance 4 ppm and product ion tolerances ± 0.4 Da; oxidation (M) and carbamidomethyl (C) specified as variable modifications, enzyme specificity was trypsin, 1 missed cleavage was possible and the NCBInr database (May, 2010) searched. A false positive significance threshold was set at P < 0.01 to eliminate any false- positive identification. Mass spectrometry analyses were performed at the Bio-analytical Mass Spectrometry Facility, UNSW. 3.6.6. Bioinformatics analysis BLASTP searches were performed using complete protein sequences available from the NCBI database (http://www.ncbi.nlm.nih.gov) to standardise and remove repetitive proteins obtained against the genome of C. concisus strain 13826, since the genome of C. ureolyticus UNSWCD has not been sequenced. Additionally, any other proteins that were unable to be standardised against C. concisus 13826 were standardised against the C. hominis ATCC BAA-381 genome. The presence and location of signal peptide cleavage sites in the amino acid sequences were predicted using the default settings for Gram-negative bacteria on the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SIGNALP/). 117 Chapter 3: Pathogenic potential of C. ureolyticus Non-classically secreted proteins were predicted by using the SecretomeP 2.0 server available at (http://www.cbs.dtu.dk/services/SecretomeP/). The Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg), was employed to determine the biochemical pathways to which genes were assigned. The Search Tool for the Retrieval of Interacting Proteins (STRING) (http://string.cmbl.d/), a known protein- protein interaction database, was employed to examine putative interactions between the identified secreted proteins. 3.6.7. Determination of the effect of the C. ureolyticus UNSWCD secretome on HT- 29 cell viability and interleukin-8 production HT-29 cells were seeded following the procedures outlined in section 3.3. To assess the toxicity and inflammatory effect of the secretome of C. ureolyticus UNSWCD on HT- 29 cells, HT-29 cell viability and IL-8 production levels were quantified following addition of a range of concentrations of the purified secretome (2.6 µg/ml, 13 µg/ml, 26 µg/ml and 130 µg/ml) and incubation at 37oC for 6 h. Cell viability was determined using a live/dead cell count as outlined in section 3.5.1. IL-8 secretion levels in the supernatant of the HT-29 cells following the 6 h incubation period with the secretome were determined using an IL-8 human ELISA kit (Invitrogen) as outlined in section 3.7.2. 3.6.8. Statistical analysis Data values followed a normal (Gaussian) distribution, thus the mean and standard error of the mean (SEM) were included. The statistical significance of the differences in the levels of adherence achieved by C. ureolyticus spp. with and without the addition of both pro-inflammatory cytokines was determined using a two-tailed paired T-test. The same statistical test was used to determine the statistical significance of the Caco-2 cell 118 Chapter 3: Pathogenic potential of C. ureolyticus viability percentages (%) achieved following C. ureolyticus UNSWCD infection. Two- tailed paired T-tests were also used to determine the statistical significance of percentage changes in the viability of HT-29 cells following incubation with the secretome of C. ureolyticus UNSWCD. A statistically significant value for each individual test was defined as P < 0.05. The statistical significance between the levels of IL-8 induced following treatment with the secretome of C.ureolyticus UNSWCD was determined using a one-way Anova with all values corrected using a Dunnett’s Multiple Comparison Test. The statistical software used was Prism GraphPad version 6.0 (GraphPad Software; San Diego, USA). 119 Chapter 3: Pathogenic potential of C. ureolyticus 3.7. Results 3.7.1. Adherence, invasive, and translocation abilities of C. ureolyticus UNSWCD To investigate the ability of C. ureolyticus UNSWCD to attach to and invade human intestinal epithelial cells, in vitro adherence and invasion assays were performed using two intestinal cell lines, the Caco-2 and HT-29 cell lines. The results of the adherence assay showed that C. ureolyticus UNSWCD was able to attach to both Caco-2 and HT- 29 cells, with attachment levels of 5.9 ± 0.5% and 1.4 ± 0.2% being observed, respectively (Table 3.2 and 3.3). Similar attachment levels were observed for C. ureolyticus UNSWE, with attachment of 6.1 ± 0.4% and 1.2 ± 0.2% on Caco-2 and HT-29 cells, respectively. Comparable levels of attachment were observed for C. ureolyticus UNSWR, with levels to Caco-2 and HT-29 being 5.4 ± 0.3% and 1.0 ± 0.4%, respectively (Table 3.2 and 3.3). The positive control, S. Typhimurium LT2 showed attachment levels of 16.6 ± 1.0% to Caco-2 cells and 14.1 ± 1.5% to HT-29 cells (Table 3.2 and 3.3). In contrast, C. ureolyticus UNSWCD, UNSWE, and UNSWR were unable to invade either cell line. As expected, the positive control, S. Typhimurium LT2 was able to invade both cell lines, with invasion levels of 1.3 ± 0.1% in Caco-2 cells and 1.3 ± 0.1% HT-29 cells respectively. No S. Typhimurium LT2 cells were recovered from the supernatant, suggesting that the S. Typhimurium LT2 invasion was not due to the growth of extracellular bacteria resistant to killing by gentamicin. In order to investigate the ability of C. ureolyticus strains to invade cells via a paracellular mode of translocation through cellular tight junctions, in vitro translocation transwell assays were conducted. The translocation assays showed that C. ureolyticus 120 Chapter 3: Pathogenic potential of C. ureolyticus UNSWCD was capable of translocating across the cell monolayer (0.018 ± 0.002%), indicating that this strain could invade through a paracellular route. 3.7.2. Effect of pre-existing inflammation on the adherence and invasive ability of C. ureolyticus UNSWCD To determine whether pre-existing inflammation affected the ability of C. ureolyticus spp to attach and invade human intestinal cells, Caco-2 and HT-29 cells were treated with the pro-inflammatory cytokines (TNF-α and IFN-γ) prior to infection with C. ureolyticus spp. The adherence level of C. ureolyticus UNSWCD to Caco-2 cells treated with pro-inflammatory cytokines TNF-α and IFN-γ was observed to be 4.2 ± 0.6% and 6.5 ± 0.6%, while their attachment to HT-29 cells treated with the same cytokines was 1.3 ± 0.4% and 1.4 ± 0.4%, respectively. These values were not significantly different from the attachment levels recorded for C. ureolyticus UNSWCD without the addition of the pro-inflammatory cytokines in Caco-2 cells (5.9 ± 0.5%) (TNF-α: P = 0.270 and IFN-γ: P = 0.235) and HT-29 cells (1.4 ± 0.2%) (TNF-α: P = 0.519 and with IFN-γ: P = 0.595). In summary these results and those for assays conducted with C. ureolyticus strains UNSWE, UNSWR and S. Typhimurium LT2 are summarised in Tables 3.2 and 3.3. 121 Chapter 3: Pathogenic potential of C. ureolyticus Table 3.2. The percentage relative attachment and invasion of C. ureolyticus strains to Caco-2 cells in the absence and presence of pre-existing inflammation. % Relative % Relative P - Statistically Bacteria MOI Attachment ± Invasion value significant SEM ± SEM C. ureolyticus 200 5.967 ± 0.489 0 - - UNSWCD C. ureolyticus 200 4.200 ± 0.600 0 0.207 No UNSWCD + TNF-α 40 ng/ml C. ureolyticus 200 6.500 ± 0.600 0 0.235 No UNSWCD + IFN-γ 40 ng/ml C. ureolyticus 200 6.133 ± 0.433 0 0.977 No UNSWE C. ureolyticus 200 5.390 ± 0.277 0 0.471 No UNSWR S. Typhimurium 100 16.600 ± 1.006 1.300 ± - - LT2 0.100 Three independent invasive and adherence assays were conducted in triplicate. Adherence and invasion levels are expressed as mean percentages ± SEM. 122 Chapter 3: Pathogenic potential of C. ureolyticus Table 3.3. The percentage relative attachment of C. ureolyticus strains to HT-29 cells in the absence and presence of pre-existing inflammation. % Relative % Relative P - Statistically Bacteria MOI Attachment Invasion value significant ± SEM ± SEM C. ureolyticus 200 1.389 ± 0.165 0 - - UNSWCD C. ureolyticus 200 1.313 ± 0.443 0 0.519 No UNSWCD + TNF-α 40 ng/ml C. ureolyticus 200 1.383 ± 0.370 0 0.595 No UNSWCD + IFN-γ 40 ng/ml C. ureolyticus 200 1.173 ± 0.135 0 0.322 No UNSWE C. ureolyticus 200 0.960 ± 0.428 0 0.404 No UNSWR S. Typhimurium 100 14.110 ± 1.300 ± - - LT2 1.482 0.100 Three independent invasive and adherence assays were conducted in triplicate. Adherence and invasion levels are expressed as mean percentages ± SEM. 123 Chapter 3: Pathogenic potential of C. ureolyticus 3.7.3. Visualisation of attachment by C. ureolyticus UNSWCD to Caco-2 cells using Scanning Electron Microscopy ScEM was used to complement the in vitro attachment and invasion assays. This approach was undertaken to not only confirm the results of these assays, but to provide important information regarding the manner by which C. ureolyticus UNSWCD interacts with the Caco-2 cell line (Figures 3.1 and 3.2) and any potential damage that may have occurred to the infected cell lines. Following infection of Caco-2 cells with C. ureolyticus UNSWCD, the process of attachment was viewed using ScEM after 1 h, 3 h and 6 h periods (Figures 3.1 and 3.2). Analysis of ScEM images revealed that the aflagellate C. ureolyticus UNSWCD (Figure 3.1 A) appeared to employ a “sticky end” flagellum-independent mechanism for attachment to the microvilli and to Caco-2 cells directly (Figures 3.1 E-F). Further, the addition of C. ureolyticus UNSWCD at a MOI of 200 to Caco-2 cells resulted in degradation of the filamentous structure of microvilli (Figure 3.1 C-D and insets), with cellular debris of these remnant microvilli structures being observed (Figure 3.1 G and insets). As a result of this degradation the Caco-2 cell apical membrane surface appeared to lack the highly dense accumulation of microvilli when compared with the negative control sample (no addition of C. ureolyticus UNSWCD), which showed highly dense microvilli (Figure 3.1 B). Moreover, extensive microvilli degradation was observed following incubation with C. ureolyticus for 1 h (Figure 3.2 A and B) and 3 h (Figure 3.2 C and D). 124 Chapter 3: Pathogenic potential of C. ureolyticus Figure 3.1. Infection of Caco-2 cells with the aflagellate C. ureolyticus UNSWCD for 6 h. A: C. ureolyticus UNSWCD is represented as a rod-shaped aflagellate bacterium 1 to 2 µm long (Panel A scale bar, 2.5 µm 7,000 × magnification). B: The human intestinal cell line Caco-2 microvilli are seen at a high density on the apical membrane surface (Panel B scale bar, 10 µm 2,000 × magnification). No degradation of the microvilli was seen in this uninfected control sample. C and D: Show the effect of microvillus degradation. The presence of C. ureolyticus UNSWCD as an aggregated mass appears to induce microvillus degradation, as shown in greater magnification in the insets (Panel C scale bar, 10 µm 2,000 × magnification; inset scale bar, 2.5 µm 10,000 × magnification, Panel D scale bar, 10 µm; inset scale bar, 2.5 µm 6,000 × magnification). E: C. ureolyticus UNSWCD was also involved in the attachment process by attracting nearby microvilli (arrow) onto the bacterial surface (Panel E scale bar, 2 µm 12,000 × magnification). F: The aflagellate C. ureolyticus UNSWCD can employ a sticky-end mechanism of attachment (arrow) (Panel F scale bar, 2 µm 14,000 × magnification). G: The extent of microvillus degradation (arrow) (Panel G scale bar, 10 µm 2,000 × magnification). The effect of cellular damage can be viewed more closely in the higher-magnification inset image (scale bar, 2.5 µm 9,000 × magnification). 125 Chapter 3: Pathogenic potential of C. ureolyticus Figure 3.2. Infection of Caco-2 cells with the aflagellate C. ureolyticus UNSWCD for 1 h and 3 h A and B: The effect of microvillus degradation by C. ureolyticus UNSWCD can be observed at 1 h (Panel A scale bar, 15 µm 1,500 × magnification; Panel B scale bar, 5 µm 5,000 × magnification). C and D: The effect of microvillus degradation by C. ureolyticus UNSWCD can be observed at 3 h (Panel C scale bar, 15 µm 1,500 × magnification; Panel D scale bar, 2.5 µm 7,000 × magnification). B and D: The presence of C. ureolyticus UNSWCD as an aggregated mass appears to induce microvillus degradation, as shown in greater magnification. 126 Chapter 3: Pathogenic potential of C. ureolyticus 3.7.4. Visualisation of the effect of pre-existing inflammation on the attachment of C. ureolyticus UNSWCD to Caco-2 cells using Scanning Electron Microscopy The effect of pre-existing inflammation on the attachment of C. ureolyticus UNSWCD to Caco-2 cells was also investigated using ScEM. This showed that pre-existing inflammation did not affect the ability of C. ureolyticus to attach to the microvilli (Figure 3.3 A-C) or to use the same “sticky end” mechanism to adhere to Caco-2 cell microvilli on the apical membrane surface (Figure 3.3 D-E). In the presence of inflammation, C. ureolyticus UNSWCD took on a distinct cauliflower appearance with aggregated masses attaching to microvilli, which appeared to be present at a lower density (Figures 3.3 F-H). The presence of C. ureolyticus UNSWCD on the apical membrane surface of Caco-2 cells also appeared to induce cellular damage causing degradation of microvilli, observed surrounding the aggregated mass of C. ureolyticus UNSWCD (Figure 3.3 I). 127 Chapter 3: Pathogenic potential of C. ureolyticus Figure 3.3. Infection of Caco-2 cells with the aflagellate C. ureolyticus UNSWCD in the presence of pre-existing inflammation. A: In the presence of inflammation induced by TNF-α, C. ureolyticus UNSWCD showed signs of bacterial aggregation (Panel A scale bar, 10 µm 2,000 × magnification). B-C: The marked boxes highlight the areas of aggregation, with higher magnification of these areas shown in (Panel B scale bar, 2 µm 10,000 × magnification) and (Panel C scale bar, 2.5 µm 12,000 × magnification). D-E: C. ureolyticus UNSWCD was still able to attach to microvilli and to the Caco-2 cells directly (arrows) (Panel D scale bar, 2 µm 12,000 × magnification) and (Panel E scale bar, 2 µm 12,000 × magnification). F-H: demonstrate that in the presence of inflammation induced by IFN-γ C. ureolyticus UNSWCD also showed mass aggregation on the Caco-2 apical membrane surface inducing cellular damage, which took on a distinct cauliflower-like appearance. (Panel F Scale bars, 10 µm 2,000 × magnification; inset scale bar, 2.5 µm 10,000 × magnification), (Panel G scale bar 10 µm 2,000 × magnification), and (Panel H scale bar 5 µm; inset scale bar, 2.5 µm 10,000 × magnification). I: The full extent of microvillus degradation can be viewed (Panel I scale bar, 5 µm 3,000 × magnification) and is shown at a higher magnification in the inset (scale bar, 2.5 µm 10,000 × magnification). 128 Chapter 3: Pathogenic potential of C. ureolyticus 3.7.5. Viability of Caco-2 cells following infection with C. ureolyticus UNSWCD at different MOIs in the absence and presence of pre-existing inflammation Given that the ScEM studies had shown cellular damage and microvilli degradation at the apical membrane surface of Caco-2 following infection with C. ureolyticus UNSWCD (Figures 3.1 G and 3.3 I), further studies were conducted to quantify the extent of Caco-2 cell damage. In vitro infection assays were performed with Caco-2 cells lines and C. ureolyticus UNSWCD at MOIs of 100, 200 & 1000, and the percentage of viable Caco-2 cells (viable cells/total cells) was determined based on the results of the trypan blue exclusion method. As shown in Figure 3.4, Caco-2 cells infected with C. ureolyticus UNSWCD at a MOI of 1000 showed decreased viability (61.50 ± 2.17%) as compared with cells infected with C. ureolyticus UNSWCD MOI of 100 (88.83 ± 2.79%) and a MOI of 200 (83.63 ± 5.097%). Paired T-test analysis revealed that when compared with the uninfected control (no infection) (95.5 ± 5.9%), the viability decreased significantly with increasing MOI of C. ureolyticus UNSWCD MOI 100 (P = 0.047), MOI 200 (P = 0.0014) and MOI 1000 (P = 0.0001). Assessment of the effect of pre-existing inflammation, induced by TNF-α or IFN-γ on Caco-2 cells following infection with a C. ureolyticus UNSWCD MOI of 200 showed the viability of Caco-2 cells treated with TNF-α prior to infection to be 82.5 ± 0.6%, while those pre-treated with IFN-γ had a viability of 85.6 ± 0.6%. Similarly, Caco-2 cells infected with C. ureolyticus UNSWCD MOI of 200 in the absence of inflammation had a viability of 83.63 ± 5.10%. Paired T-test statistical analysis revealed that Caco-2 cell viabilities obtained between C. concisus UNSWCD and C. concisus UNSWCD + TNF-αwas not significant (Figure 3.5). In contrast, statistical analysis showed a statistical significance to exist between C. concisus UNSWCD and C. concisus UNSWCD + IFN-γ (P ≤ 0.05). In addition, the viabilities of Caco-2 cells 129 Chapter 3: Pathogenic potential of C. ureolyticus infected at an MOI of 200 with and without cytokines were relatively similar thus verifying the observation that preexisting inflammation had no severe effect on the bacterium’s pathogenic potential. !!!! !! ! 100 90 80 70 % 60 y t i l i b a 50 i V l l e 40 C 30 20 10 0 MOI 200 UNSWCD Caco-2 cells alone UNSWCD MOI 100 UNSWCD MOI 1000 C.ureolyticus C. ureolyticus C.ureolyticus Figure 3.4. Cell viability results of viable Caco-2 cells upon inoculation with and without C. ureolyticus UNSWCD at variant MOIs. The data shown in this graph is representative of 2 independent experiments carried out in triplicate with values expressed as mean cell viability percentages ± SEM. Viable Caco-2 cells were determined by trypan blue exclusion. (P ≤ 0.05 ★); (P ≤ 0.01 ★★); (P ≤ 0.0001 ★★★★). 130 Chapter 3: Pathogenic potential of C. ureolyticus ! 100 NS 80 60 40 Cell viability (%) 20 0 ! " MOI 200 Caco-2 cells aloneUNSWCD UNSWCD MOI 200 UNSWCD + TNF- MOI 200 + IFN- C.ureolyticus C. ureolyticus C. ureolyticus Figure 3.5. Comparison in the viability of Caco-2 cells alone, infected with C. ureolyticus UNSWCD, and infected with C. ureolyticus UNSWCD and the pro-inflammatory cytokines TNF-α and IFN-γ. In order to examine the effect of pre-existing inflammation on Caco-2 cell viability following C. concisus UNSWCD infection, 40 ng/ml of both TNF-α and IFN–γ were added prior to infection. The viability of the Caco-2 cells was examined using the trypan blue exclusion method. The data shown in this graph is representative of 2 independent experiments, each carried out in triplicate. The values are expressed as mean percentage cell viability ± SEM. NS: Non-significant; (P ≤ 0.05 ★). 131 Chapter 3: Pathogenic potential of C. ureolyticus 3.7.6. Interleukin-8 production following C. ureolyticus UNSWCD infection of HT- 29 cells in the absence and presence of inflammation To confirm that the addition of TNF-α and IFN-γ induced inflammation in host cells, the secretion of IL-8 in cells exposed to these cytokines was measured. Cells exposed to TNF-α (687.5 ± 3.1 pg/ml, P < 0.0001) and IFN-γ (58.2 ± 2.0 pg/ml, P < 0.01) produced significantly greater quantities of IL-8 when compared to the negative control (33.8 ± 1.5 pg/ml) indicating that the addition of these cytokines resulted in an inflamed state (Figure 3.6). Cells infected with C. ureolyticus UNSWCD (58.2 ± 2.8 pg/ml, P < 0.01) produced significantly higher levels of IL-8 when compared to uninfected controls (33.8 ± 1.5 pg/ml). Interestingly, heat-killed C. ureolyticus UNSWCD produced similar levels of IL-8 to that observed for viable bacteria (62.3 ± 3.5 pg/ml) (Figure 3.6). Upon addition of TNF-α, cells infected with C. ureolyticus UNSWCD produced a similar level of IL-8 (681.7 ± 6.2 pg/ml, P = 0.12) to that observed in uninfected cells exposed to TNF-α (Figure 3.6). In contrast, addition of IFN-γ to cells infected with C. ureolyticus UNSWCD produced a significantly higher level of IL-8 (249.0 ± 9.1 pg/ml, P = 0.002) to that observed in non-infected cells exposed to IFN-γ (Figure 3.6). Infection of cells with commensal E. coli K-12 resulted in the production of significantly lower levels of IL-8 (12.8 ± 1.1 pg/ml, P < 0.01) when compared to the uninfected control. However, heat-killed E. coli K-12 induced significantly higher levels of IL-8 (78.7 ± 3.4 pg/ml, P < 0.001) (Figure 3.6). 132 Chapter 3: Pathogenic potential of C. ureolyticus Figure 3.6. IL-8 production exhibited by HT-29 cells infected with C. ureolyticus UNSWCD, with or without pre-existing inflammation instigated by TNF-α and IFN-γ. A: IL-8 production by HT-29 cells alone, infected with C. ureolyticus UNSWCD and with and without pre-existing inflammation (TNF-α and IFN-γ) and viable and heat-killed E. coli K12. The statistical analysis compares uninfected HT-29 cells with all treatment samples. B: The comparison of IL-8 production by HT-29 cells exposed to pre-existing inflammation with and without the addition of C. ureolyticus UNSWCD. The statistical analysis compares HT-29 cells with pre-existing inflammation against HT-29 cells infected with C. ureolyticus UNSWCD exposed to pre-existing inflammation. The data shown in these graphs are representative of 2 independent experiments each carried out in triplicate. The values are expressed as mean percentage cell viability ± SEM. NS: Non-significant; (P ≤ 0.05 ★); (P ≤ 0.01 ★★); (P ≤ 0.001 ★★★); (P ≤ 0.0001 ★★★★). 133 Chapter 3: Pathogenic potential of C. ureolyticus 3.7.7. The secretome of C. ureolyticus UNSWCD The secretome of C. ureolyticus UNSWCD was determined in order to elucidate its possible pathogenic potential. Since the genome of C. ureolyticus was unknown at the time of this study, the genomes of C. concisus 13826 and C. hominis ATCC BAA-381 were used as the reference genomes. The secreted proteins were run on a 1D-SDS PAGE gel (Figure 3.7), trypsin digested to peptides, ionised and analysed by mass spectrometry using an LTQ-FT Ultra MS/MS. Following bioinformatic analyses, 111 proteins were identified from a BLASTP analysis against the C. concisus 13826 and C. hominis ATCC BAA-381 genomes (Appendix Table 1). Additionally, SignalP and SecretomeP bioinformatic analysis resulted in the identification of 82 putatively non-secretory proteins, and 29 proteins predicted to be secreted (Table 3.4). Moreover, functional classification revealed the identification of three putative virulence and colonisation factors: surface antigen, CjaA, the outer membrane fibronectin binding protein and the S-layer RTX toxin. The remaining non-secretory proteins consisted of enriched fractions involved in amino acid metabolism (22/82, 20%), nucleotide metabolism (18/82, 16%) and carbohydrate metabolism (18/82, 16%) (Appendix Figure 1). 134 Chapter 3: Pathogenic potential of C. ureolyticus Figure 3.7. A one-dimensional SDS Page gel of C. ureolyticus UNSWCD secreted proteins. Albumin was used as the protein size marker (M lane). C. ureolyticus UNSWCD secreted protein bands (S lane) observed in a 1-D SDS PAGE gel stained with Commassie blue brilliant dye. The proteins in this lane were cut into 24 gel slices and processed for mass spectrometry analysis. 135 Chapter 3: Pathogenic potential of C. ureolyticus Table 3.4. The functional classification of C. ureolyticus UNSWCD secreted proteins, bioinformatically analysed and predicted to be secreted (n = 29) with the open reading frame (ORF) of each individual protein also provided. Functional ORF GI number Protein name classification CCC13826_ 157164945 DNA-binding protein HU 1 DNA- DNA/RNA 0021 binding protein II HB CCC13826_ 157165109 L-asparaginase Protein synthesis 0029 CCC13826_ 157164709 Peptidoglycan associated lipoprotein Membrane and cell 0131 wall synthesis CCC13826_ 157165495 50S ribosomal protein L11 Protein synthesis 0170 CCC13826_ 157165207 Fumarate hydratase Glyoxylate and 0208 Dicarboxylate metabolism Methane metabolism CCC13826_ 157165747 Fumarate reductase flavoprotein Citrate Cycle (TCA 0425 subunit cycle) Butanonate metabolism 136 Chapter 3: Pathogenic potential of C. ureolyticus Oxidative phosphorylation CCC13826_ 157164277 Glyceraldehyde-3-phosphate Glycolysis / 0516 dehydrogenase, type I Gluconeogenesis CCC13826_ 157164063 3-oxoacyl-acyl carrier protein Fatty acid synthesis 0560 synthase II CCC13826_ 157164484 3-ketoacyl-acyl-carrier-protein Fatty acid 0562 reductase Synthesis CCC13826_ 157164747 ADP-glyceromanno-heptose 6- Lipopolysaccharide 0576 epimerase biosynthesis Lipopolysaccharide biosynthesis proteins CCC13826_ 157164816 Surface antigen, CjaA Virulence 0664 CCC13826_ 157164953 NAD-FAD-utilising dehydrogenase Nitrogen 0702 metabolism CCC13826_ 157164740 Outer membrane fibronectin-binding Virulence 0739 protein CCC13826_ 157165691 C4-dicarboxylate-binding Soluble transport 0764 periplasmic protein 137 Chapter 3: Pathogenic potential of C. ureolyticus CCC13826_ 157165732 Nitrate reductase Nitrogen 0868 metabolism CCC13826_ 157165742 N-terminal methylation domain- Methylation 1110 containing protein CCC13826_ D-methionine-binding lipoprotein Soluble transport 158604975 1248 MetQ CCC13826_ Radical SAM domain-containing Electron transfer 157165471 1395 protein CCC13826_ 157163991 Molybdenum cofactor biosynthesis GTP binding 1396 protein A Catalytic activity Metal ion binding CCC13826_ 157165286 Phosphoenolpyruvate carboxykinase Pyruvate 1509 metabolism Glycolysis / Gluconeogenesis Citrate Cycle (TCA cycle) CCC13826_ 157165243 S-layer-RTX protein Virulence 1838 CCC13826_ 157165649 DNA-binding response regulator DNA-binding 2064 response regulator CCC13826_ 154149442 Holo-acyl-carrier-protein synthase Fatty acid synthesis 138 Chapter 3: Pathogenic potential of C. ureolyticus 2069 CCC13826_ 157165707 Fructose-bisphosphate aldolase Pentose phosphate 2070 pathway Fructose and mannose metabolism Glycolysis / Gluconeogenesis CCC13826_ 157165134 2-acylglycerophosphoethanolamine Peptidases 2319 acyltransferase CHAB381_ 154149442 Hypothetical protein Unknown 0277 CHAB381_0277 [Campylobacter hominis ATCC BAA-381] BACEGG_ 218129864 Hypothetical protein Unknown 01445 BACEGG_01445 [Bacteroides eggerthii DSM 20697] 139 Chapter 3: Pathogenic potential of C. ureolyticus 3.7.8. Effect of the secretome of C. ureolyticus UNSWCD on cell viability and IL-8 production in HT-29 cells To assess the toxicity of the secretome of C. ureolyticus UNSWCD, the viability of HT- 29 cells inoculated with 2.6 µg, 13 µg, 26 µg and 130 µg of the purified secretome was quantified. Additionally, the effect of inflammation through IL-8 secretion was evaluated directly using a human IL-8 ELISA (Figure 3.9). After 6 h incubation, the viability of the HT-29 cells inoculated with the secretome decreased (2.6 µg: 96.5 ± 0.3%, 13 µg: 93.7 ± 0.3%, 26 µg: 87.5 ± 2.0% and 130 µg: 81.0 ± 0.1%) as compared to uninoculated cells (98.7 ± 0.2%). Paired T-test analysis revealed that cell viability significantly decreased upon inoculation of the secretome as compared with the uninoculated controls (2.6 µg: P = 0.037, 13 µg: P = 0.007, 26 µg: P = 0.026 and 130 µg: P = 0.0003) (Figure 3.8). These results confirm that the secretome is indeed toxic to host cells. Quantification of IL-8 production in HT-29 cells exposed to the same concentrations of the C. ureolyticus secretome, found that all concentrations produced similar quantities of IL-8 (2.6 µg: 102.7 ± 12.9 pg/ml, 13 µg: 88.0 ± 5.8 pg/ml, 26 µg: 96.1 ± 5.9 pg/ml and 130 µg: 90.5 ± 2.4 pg/ml), which was significantly higher (P < 0.0001) than both the uninoculated control (33.8 ± 1.5 pg/ml) and cells exposed to C. ureolyticus (58.2 ± 2.8 pg/ml) (Figure 3.9). 140 Chapter 3: Pathogenic potential of C. ureolyticus !!! ! !! ! 100 90 80 70 ) % ( 60 y t i l i b 50 a i v l l e 40 C 30 20 10 0 g secretomeg secretomeg secretomeg secretome 2.6 µ 13 µ 26 µ HT-29 cells alone 130 µ Figure 3.8. Cell viability of HT-29 cells inoculated with C. ureolyticus UNSWCD secreted proteins over a 6 h period. The data shown in this graph is representative of 2 independent experiments carried out in triplicate. The values are expressed as mean percentage cell viability ± SEM. (P ≤ 0.05 ★); (P ≤ 0.01 ★★); (P ≤ 0.001 ★★★). 141 Chapter 3: Pathogenic potential of C. ureolyticus 150 ! ! 125 ! ! ) l m 100 / g p ( n o i t a 75 r t n e c n o c 50 8 - L I 25 0 g g g g µ µ µ µ HT-29 cells alone SecretomeSecretome 2.6 Secretome 13 26 UNSWCD MOI 200 Secretome 130 UNSWCD MOI 200 Heat Killed C. ureolyticus C. ureolyticus Figure 3.9. IL-8 production by HT-29 cells exposed to increasing concentrations of C. ureolyticus secreted proteins. The data shown in this graph is representative of 2 independent experiments carried out in triplicate. The values are expressed as mean percentage cell viability ± SEM. (P ≤ 0.0001 ★). 142 Chapter 3: Pathogenic potential of C. ureolyticus 3.8. Discussion As a result of the isolation of C. ureolyticus UNSWCD from a patient with CD (32) and the observation that other Campylobacter species associated with intestinal disease, including C. jejuni, C. coli and C. concisus, are able to colonise and invade intestinal epithelial cells, in vitro adherence, invasion, and translocation assays were conducted to determine whether C. ureolyticus UNSWCD was able to adhere to and invade Caco-2 and HT-29 cells. While C. ureolyticus UNSWCD attached to both of these cells lines, it was unable to invade these intestinal cell lines. Investigation of two further strains of C. ureolyticus, UNSWE and UNSWR isolated from faecal samples of children with no pathology, showed similar results. Interestingly, the attachment of C. ureolyticus UNSWCD to Caco-2 and HT-29 cells was similar to that for both C. ureolyticus UNSWE and UNSWR. Given that C. ureolyticus UNSWCD, UNSWE and UNSWR all showed similar levels of attachment, it is likely that attachment to intestinal cells may be universal for all C. ureolyticus strains. While attachment to host cells is an important virulence mechanism for most gastrointestinal pathogens, it is unlikely that attachment alone leads to gastrointestinal disease. The finding that C. ureolyticus strains were unable to invade Caco-2 or HT-29 cells would suggest that the ability to invade intestinal cells may not be an essential virulence mechanism in C. ureolyticus pathogenesis. Given that studies by our group had previously shown strains of the closely related species C. concisus to be able to internalise into both Caco-2 and HT-29 cells (108, 132), suggests that this failure to invade is unlikely to be due to the human cell lines. Further, this study demonstrated that C. ureolyticus UNSWCD was capable of translocating across the cell monolayer in vitro, indicating that as previously reported for C. concisus this bacterium may potentially invade epithelial cells via the paracellular route (156). As only one 143 Chapter 3: Pathogenic potential of C. ureolyticus C. ureolyticus strain was investigated, examination of further C. ureolyticus strains is required to strengthen the current findings suggesting that C. ureolyticus utilises a paracellular mode of translocation. To complement the in vitro attachment and invasion assays, ScEM was performed in order to visualise the manner in which C. ureolyticus UNSWCD interacted with the Caco-2 cell line. This revealed that the aflagellate C. ureolyticus UNSWCD (Figure 3.1 A) appeared to employ a “sticky end” mechanism of attachment to the microvilli and to the Caco-2 cells directly (Figure 3.1 E & F). Interestingly, the same mode of attachment has been observed in another aflagellate Campylobacter species, C. hominis UNSWCD, as well as in flagellate members of the Campylobacter genus, including Campylobacter showae UNSWCD and C. concisus ATCC 51562, both of which have been reported to attract neighbouring microvilli onto the bacterial surface (108, 132). ScEM also revealed that following infection of Caco-2 cells with C. ureolyticus UNSWCD, degradation of the filamentous structure of the microvilli occurred [Figure 3.1 C and D (results following 6 h of incubation) and Figure 3.2 A-D, results following 1 h and 3 h’s incubation)]. Cellular debris from these remnant microvillus structures was also observed (Figure 3.1 G and insets). As a result, the Caco-2 cell apical membrane surface appeared to lack the highly dense accumulation of microvilli observed in non-infected cells (Figure 3.1 B). Interestingly, Ganan et al. (157) reported C. jejuni strain 118, in the exponential phase of growth to adhere to Caco-2 cells at a level of 2.56%. Given that C. jejuni is an established gastrointestinal pathogen, the fact that C. ureolyticus UNSWCD has a higher percentage attachment than C. jejuni would support the pathogenic potential of this bacterium. However, unlike C. jejuni, C. ureolyticus does not have the ability to 144 Chapter 3: Pathogenic potential of C. ureolyticus invade intestinal epithelial cells, which would suggest that their pathogenic mechanisms differ. The ScEM studies presented in this Chapter suggest that C. ureolyticus may form biofilms, C. ureolyticus UNSWCD being observed to form aggregates on both an inert material (Figure 3.1 A) and on a living surface (Figure 3.1 C). This behaviour is a typical feature observed in biofilm formation, which results in an exo-polymeric matrix surrounding the bacteria, forming a structured community of cells that are adherent to each other and/or to the inert or living surface. Biofilms are known to assist in cell-to- cell communication and also function in virulence (158). Previous studies by the Mitchell group have shown that C. concisus strains also adhere to the surface of Caco-2 cells in an aggregative pattern, a finding that led Lavrencic et al. to postulate that this aggregation may actually represent biofilm formation (159). Given that within the intestinal tract C. concisus and C. ureolyticus are continually subjected to peristalsis and mucus turn-over, the ability to produce biofilms may allow these bacteria to remain within their niche (159). Biofilm formation has also been reported for C. jejuni, and has been shown to contribute to the survival of this pathogen in a range of environmental niches (6). For example C. jejuni has been reported to form biofilms on a range of inert surfaces, including glass, plastics and stainless steel (160), suggesting that C. jejuni can maintain itself in the environment through biofilm formation (160) and that the environment biofilms may aid the survival of C. jejuni, suggesting that this adaptation may contribute to its zoonotic lifestyle (161). Determination of the effect of pre-existing inflammation on the adherence and invasive ability of C. ureolyticus UNSWCD by treating Caco-2 and HT-29 cells with pro- inflammatory cytokines (TNF-α and IFN-γ) prior to infection with C. ureolyticus 145 Chapter 3: Pathogenic potential of C. ureolyticus UNSWCD, showed that in the presence of these pro-inflammatory cytokines the attachment levels of C. ureolyticus UNSWCD to Caco-2 and HT-29 cells were not significantly different from those without the addition of the pro-inflammatory cytokines. Examination of the effect of pre-existing inflammation on the attachment of C. ureolyticus UNSWCD to Caco-2 cells using ScEM showed that the presence of C. ureolyticus UNSWCD on the apical membrane surface of Caco-2 cells induced cellular damage, leading to degradation of the microvilli around the aggregated mass of C. ureolyticus UNSWCD (Figure 3.3 A–C). Furthermore, these studies showed that pre- existing inflammation did not appear to affect the ability of C. ureolyticus to attach to the microvilli (Figure 3.3 D & E). While the presence of inflammation has been shown to increase the ability of other Campylobacter species such as C. concisus UNSWCD to facilitate invasion into intestinal cells (132), C. ureolyticus UNSWCD does not appear to take advantage of possible inflammation-induced morphological changes in host cells to exert its ability to attach to or invade Caco-2 cells. As a result, it would appear that the mechanism by which C. ureolyticus UNSWCD exerts its pathogenic effect is not affected by pre-existing inflammation. The observation of Caco-2 microvillus degradation by C. ureolyticus UNSWCD is supported by an early molecular study by Fontaine et al. (162) who showed that cell- free filtrates of C. ureolyticus caused a loss of ciliary activity through disruption and sloughing off of epithelial cells from the mucosal epithelium of human fallopian tubes and bovine oviduct organ cultures (162). In a later study, Fontaine et al. suggested that these cytopathic effects might be due to the existence of an unidentified membrane- bound endotoxin in the form of a lipopolysaccharide (LPS) (37, 162, 163). Interestingly, a number of studies have suggested that the proteolytic activity from proteases could be a contributing factor to the C. ureolyticus tissue damage observed in ulcerative and 146 Chapter 3: Pathogenic potential of C. ureolyticus gangrenous lesions such as genital and perineal ulcers, decubitus and varicose lesions, and diabetic gangrene (164, 165). However, even though the effect of proteolytic enzymes as described in these studies has been speculated to induce this form of tissue damage, as yet no study has identified proteolytic enzymes in C. ureolyticus. To confirm that addition of the pro-inflammatory cytokines TNF-α and IFN-γ induced inflammation in host cells, IL-8 secretion was determined in cells following exposure to these cytokines. This showed that cells exposed to both TNF-α and IFN-γ produced significantly greater quantities of IL-8 than the negative control confirming that addition of these cytokines resulted in an inflamed state. Interestingly, heat-killed C. ureolyticus UNSWCD produced IL-8 levels similar to that observed for viable bacteria, while upon addition of TNF-α, they produced IL-8 levels similar to that observed in uninfected cells exposed to TNF-α. In contrast, addition of IFN-γ to cells infected with C. ureolyticus UNSWCD produced a significantly higher levels of IL-8 than uninfected cells exposed to IFN-γ. As expected infection of cells with the commensal E. coli K-12 resulted in the production of significantly lower levels of IL-8 than that in the negative control. Interestingly, heat-killed E. coli K-12 induced significantly higher levels of IL- 8, which may relate to the presence of endotoxins released upon cellular lysis. Based on these results it would appear that C. ureolyticus UNSWCD elicits a mild inflammatory response from epithelial cells; however, this response is substantially elevated upon exposure to IFN-γ. The observation that cellular damage and microvillus degradation was present on the apical membrane surface of Caco-2 cells, led us to quantify the extent of Caco-2 cell damage caused by C. ureolyticus UNSWCD infection at three different MOI’s (100, 147 Chapter 3: Pathogenic potential of C. ureolyticus 200 and 1000). This showed that following 6 h incubation the viability of Caco-2 cells alone (not infected with C. ureolyticus UNSWCD) retained a high level of viability (95.5 ± 5.9%). In contrast, upon infection with C. ureolyticus UNSWCD, Caco-2 cell viability, as compared with the uninfected control, decreased significantly with increasing MOIs (MOI 100, P < 0.047; MOI of 200, P < 0.0014; MOI of 1,000, P < 0.0001). These results confirmed that C. ureolyticus did indeed damage Caco-2 cells, as observed in the ScEM study. Furthermore, the finding that the viabilities of the Caco-2 cells infected at an MOI of 200 with and without cytokines were relatively similar consolidates the observation that pre-existing inflammation did not have a severe effect on the bacterium’s pathogenic potential. The observed degradation of Caco-2 cell microvilli using ScEM led to the hypothesis that C. ureolyticus UNSWCD may potentially have proteolytic enzymes or degradative toxins capable of causing this effect. As the secretome is believed to be a vital part of a bacterium’s pathogenic repertoire. Thus, an investigation was undertaken to determine the secretome of C. ureolyticus UNSWCD. Moreover, this study provided a novel approach to identify the putative proteins that make up the secretome of C. ureolyticus UNSWCD. Investigation of the secreted proteins of C. ureolyticus resulted in the identification of 29 proteins predicted to be secreted (Table 3.4), as well as 82 putative non-secretory proteins. The predicted non-secretory proteins identified in the C. ureolyticus UNSWCD secretome consisted of enriched fractions involved in amino acid metabolism (22/82, or 26.8%), nucleotide metabolism (18/82, or 22.0%), and carbohydrate metabolism (18/82, or 22.0%). The finding of non-secreted proteins in the secretome may relate to the fact that these metabolic proteins are highly abundant in the cell and may actually be present as contaminants within the secretome. Indeed, it has been reported that cellular lysis and degradation during the growth of bacterial cultures 148 Chapter 3: Pathogenic potential of C. ureolyticus may result in contamination by high-abundance non-secretory proteins (67). This view is supported by studies of the secretome of Helicobacter pylori that also identified non- secreted proteins such as thioredoxin, co-chaperone GroES, 3-keto acyl carrier acyl synthase, and fumarate reductase flavoprotein subunit in the secretome (153, 154). Further, it is possible that owing to the high stringency of the bioinformatic prediction processes employed in our study that some true positives may have been overlooked. As a result, the possibility that some of these 82 non-secretory proteins may actually be secretory proteins cannot be ruled out. Functional classification of the secreted proteins resulted in the identification of three putative virulence and colonisation factors: the surface antigen CjaA, the outer membrane fibronectin binding protein, and the S-layer RTX (for repeats in toxin) toxin. The surface antigen, CjaA protein is a surface-exposed protein, which is homologous to ABC transport proteins and has been shown to be highly immunodominant in C. jejuni (166). The ABC transport system transports substrates across the periplasm to maintain lipid asymmetry, a function that is critical to the cell membrane of Gram-negative bacteria. An outer membrane fibronectin binding protein (CadF homolog) was also identified. This particular protein is widely known to mediate adhesion to the host cells (167). Fibronectin is a large glycoprotein, which is a component of the extracellular matrix (ECM) of the human intestinal epithelium. Interestingly, a study by Konkel et al. (167) has reported that C. jejuni binds to fibronectin on the basolateral surface of human colonic cells. The secretion of an extracellular binding protein that is specific to receptors on the intestinal epithelium is significant in terms of C. ureolyticus UNSWCD and may potentially play a pathogenic role in adhesion to and subsequent colonisation of host cells (167). According to Patti et al. (168) the ability of bacteria to effectively attach to ECM components is a vital phenomenon and in some bacterial species may be 149 Chapter 3: Pathogenic potential of C. ureolyticus directly related to virulence. In addition, the S-layer RTX protein (for repeats in toxin) was also secreted by C. ureolyticus UNSWCD. RTX proteins are characterised by a domain located generally in the C-terminal part of the protein, consisting of a variable number of highly conserved glycine rich repeats. This domain has a high Ca2+ binding capacity and is involved in the binding of the toxin to the target cell (169). A study by Lally et al. (170) has revealed that RTX proteins are pore-forming toxins synthesized by a diverse group of Gram-negative pathogens. For instance, uropathogenic E. coli strains secrete an α-hemolysin toxin (HlyA) belonging to the RTX protein family, which contributes to this bacterium’s role as a genitourinary pathogen (171). Primarily the two forms of host cell death associated with this type of toxin include apoptosis and necrosis (170). RTX toxins act by creating pores in eukaryotic cell membranes with binding of calcium by the toxin being essential for pore formation. High levels of toxin have been shown to lyse cells as the pores formed by the toxin allow the cytoplasmic contents of the bacterium to leak out (171). Also of interest was the identification of a hypothetical protein belonging to Bacteroides eggerthii, a member of the Bacteroides genus that has been isolated from human faeces (172, 173). The relevance of identifying this protein in C. ureolyticus relates to the fact that it was previously classified as a member of the Bacteroides genus. Indeed detection of such proteins could provide insights into why C. ureolyticus is able to hydrolyse gelatin and casein, given that this particular activity is not common among Campylobacter species (1, 36). Following the publication of the study outlined in this chapter, Bullman et al. (59), investigated the secretome of two C. ureolyticus clinical isolate strains, DSMZ 20703 (Type strain; Amniotic Fluid isolate) and ACS-301-V-Sch3b (Vaginal isolate). In 150 Chapter 3: Pathogenic potential of C. ureolyticus contrast to this study, Bullman et al. applied an in silico analysis to predict the secretome of these strains (59). Using this approach Bullman et al. (59) predicted 288 proteins to be secreted by C. ureolyticus DSMZ 20703 which included at least 25 proteins with putative virulence roles. The secretome of ACS-301-V-Sch3b was predicted to contain 269 secreted proteins, of which 28 were known to play a role in virulence. The number of predicted secreted proteins in these two C. ureolyticus strains is significantly higher than that identified in the secretome of C. ureolyticus UNSWCD (59). This is not surprising given that Bullman et al. employed in silico secretome prediction based on the published genome of C. ureolyticus rather than in vitro studies as used in the current chapter (59). While in our study it would have been interesting to determine the predicted secreted proteins of C. ureolyticus, at the time of this study a C. ureolyticus genome was not available. Interestingly, the number of predicted secreted proteins of the C. ureolyticus strains reported by Bullman et al. (59) is very similar to that reported by Kaakoush et al. in a study of the secretome of C. concisus UNSWCD in which the secretome was shown to comprise 201 proteins, of which 86 were bioinformatically predicted to be secreted based on the C. concisus 13628 reference genome (67). Additionally, a number of the virulence and colonisation proteins identified in the secretome of C. concisus UNSWCD showed similarity to the virulence and colonisation factors identified in the predicted C. ureolytics secretome by Bullman et al. (59). Bullman et al. identified virulence factors pertaining to haemolysins, fibronectin binding protein CadF, Zot, surface antigen CjaA and twitching motility proteins (59). In comparison these virulence factors identified in Bullman et al. (59) were also identified in Kaakoush et al. study, which included: the Zot toxin, S-layer RTX toxin, the surface antigen CjaA, and the outer membrane fibronectin binding protein CadF, all of which pertained to the secretome of C. concisus UNSWCD with the exception of no twitching motility proteins been detected (67). Moreover, these findings 151 Chapter 3: Pathogenic potential of C. ureolyticus could indicate that virulence factors of Campylobacter spp. may be conserved within the species. Also these conserved virulence factors could equate to an affinity of particular strains to become host adapted, using these virulence factors to enhance pathogen-host interactions. Comparison of the putative secreted proteins associated with virulence identified in the study of C. ureolyticus UNSWCD with those predicted by Bullman et al. showed 3 secreted virulence proteins, the S-layer RTX toxin, the CadF outer membrane fibronectin-binding protein and the Surface antigen CjaA, to also be present in the secretomes identified in Bullman et al. study (59). Interestingly, Bullman et al. found only 83% of the predicted proteins to be homologous between C. ureolyticus DSM 20703 and ACS-301-V-Sch3b strains (59). Subsequent comparison of the protein coding regions of 12 C. ureolyticus isolates against C. ureolyticus DSM 20703 and C. ureolyticus ACS-301-V-Sch3b showed that 13–19% and 9–16% of proteins identified in these two strains respectively were unique, suggesting that potential heterogeneity could exist within C. ureolyticus spp (59). Based on the results of their study, Bullman et al. suggested that C. ureolyticus might potentially be composed of several genomospecies (phylogenetically distinct taxa that have an approximately 70% or greater DNA-DNA relatedness) (174) similar to that observed in C. concisus (63, 64). Clearly further investigations are required to evaluate the extent of genomic variation among C. ureolyticus strains of different origins, Based on the studies by Bullman et al. such studies are highly likely to confirm the existence of genomospecies in C. ureolyticus. Determination of the effect of the secretome of C. ureolyticus UNSWCD on HT-29 cell viability showed that upon inoculation with increasing concentrations of the secretome 152 Chapter 3: Pathogenic potential of C. ureolyticus (2.6 µg, 13 µg, 26 µg, and 130 µg), the viability of host cells decreased significantly as compared with the uninoculated control (P < 0.037; P < 0.007, P < 0.026 and P < 0.0003) respectively, a finding that confirmed that the secretome was indeed toxic to host cells. Quantification of the production of IL-8 in HT-29 cells exposed to the same concentrations of C. ureolyticus secretome showed that IL-8 production was similar across all concentrations of the secretome and were significantly higher (P < 0.0001) than that in both the uninoculated control and cells exposed to C. ureolyticus. Based on the results of this study, it is highly likely that C. ureolyticus secreted proteins play a role in the initiation of IL-8 in HT-29 cells. The finding that IL-8 production did not vary significantly across the different concentrations of the secretome would suggest that a threshold level may exist over which IL-8 induction does not increase. Further investigation is clearly required to characterise and determine the role of these virulence factors in mediating inflammatory immune responses. 3.9. Conclusion The findings presented in this Chapter have added significantly to our understanding of the pathogenesis of C. ureolyticus. These studies have demonstrated that C. ureolyticus UNSWCD has the ability to attach to and translocate through, but not invade, the human intestinal epithelial cell lines Caco-2 and HT-29, both in the presence and absence of pre-existing inflammation. Further, isolation and characterisation of the C. ureolyticus UNSWCD secretome revealed that this bacterium possesses putative virulence and colonisation factors, which may contribute to its pathogenic potential, including up regulation of inflammation through IL-8. 153 CHAPTER 4 4. THE ROLE OF AUTOPHAGY IN THE INTRACELLULAR SURVIVAL OF CAMPYLOBACTER CONCISUS 4.1. Background Autophagy is a mechanism by which cells degrade unnecessary or dysfunctional components using the lysosome (133). It can be broadly divided into three categories: macroautophagy, microautophagy, and chaperone-mediated autophagy (133), of which macroautophagy (referred to throughout as autophagy) can mediate and control bacterial clearance through lysosomal degradation. Following the induction of autophagy, the omegasome, a cup-shaped structure, is formed from the endoplasmic reticulum (130). The isolation membrane subsequently grows and matures to engulf cytoplasmic components in a process known as elongation. The membrane then closes to form the autophagosome, a structure characterised as having a double-layered membrane. The outer membrane of the autophagosome subsequently fuses with a lysosome to form an autolysosome (130). Finally after the fusion process is complete, lysosomal hydrolytic enzymes, including cathepsins (proteases) and lipases, degrade the intra- autophagosomal constituents in the inner membrane of the autophagosome (130, 140, 175). Little information is known about the intracellular survival of Campylobacter species and their interactions with the autophagy process of host cells. One mechanism of survival that has been identified for Campylobacter jejuni is the uptake by a vacuole- like compartment (176), now known as the Campylobacter-containing vacuole (CCV), 154 Chapter 4: Autophagy in the intracellular survival of C. concisus which diverges from the regular endocytic pathway within epithelial cells (177). The CCV has been found to avoid delivery into lysosomes; however, its ability to do so is reliant upon the entry mechanism of C. jejuni (131). More recently, Buelow et al. determined that the Campylobacter invasion antigen (Cia) responsible for intracellular survival of C. jejuni is CiaI, which is secreted by a type III secretion system, prevents the transfer of the CCV to lysosomes (178). In regard to interactions with the autophagy process, Sun et al. have shown that mTOR (mammalian target of rapamycin) signaling, which controls autophagy, mediates C. jejuni induced colitis in IL-10-/- mice independent of T-cell activation (149). Significantly, upon exposure to the autophagy inducer rapamycin, the authors showed that C. jejuni infiltration in the colon and spleen was dramatically reduced. Campylobacter concisus is an emerging gastrointestinal pathogen, which has been associated with acute gastroenteritis, inflammatory bowel diseases (IBD) and Barrett’s esophagus (41, 44, 45, 82, 108, 179). A recent study by Nielsen and colleagues reported a high incidence of C. concisus, almost as high as that of C. jejuni and C. coli, in patients with gastroenteritis from a mixed urban and rural community in Denmark (12). Moreover, a further study by Nielsen and colleagues (103), reported 80% of C. concisus infected patients to have suffered diarrhoea for a period greater than 2 weeks, as compared with only 32% of C. jejuni or C. coli infected patients. Significantly, 6 months post diagnosis, 12% of patients infected with C. concisus were diagnosed with microscopic colitis (12). In contrast, no patient previously diagnosed with C. jejuni or C. coli developed microscopic colitis, providing further support for an association between C. concisus and chronic intestinal diseases (41, 105). 155 Chapter 4: Autophagy in the intracellular survival of C. concisus Studies have shown that the invasive potential of C. concisus strains isolated from chronic intestinal diseases (including IBD), are greater than 500-fold higher than that of C. concisus strains isolated from acute intestinal diseases and a healthy subject (108, 109). Investigation of the genetic make-up of C. concisus, later showed that a plasmid containing several virulence determinants was present in only highly invasive strains, a finding that the authors suggested could potentially be responsible for the heterogeneity in the invasive ability of C. concisus strains (109). A more recent in depth investigation of this plasmid has revealed that only a conserved fragment containing four genes are specific to the invasive strains, and that this fragment is more likely to be involved in intracellular survival rather than internalisation into host cells (126). Given that many intestinal pathogens including C. jejuni have been shown to be able to manipulate the autophagy process to facilitate their survival in host cells, in the current study we investigated the ability of C. concisus to survive intracellularly in intestinal cells through the manipulation of the autophagic system using standard and modified gentamicin protection assays, confocal microscopy, scanning and transmission electron microscopy and qPCR. 156 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.1.1. Aims The aims of this chapter are: 1. To determine whether growth of C. concisus under aerobic or microaerobic conditions affects the ability of C. concisus to invade and/or adhere to Caco-2 cells. 2. To examine the effect of C. concisus infection on Caco-2 cell viability. 3. To determine, the ability of C. concisus to survive intracellularly within Caco-2 cells over time. 4. To examine, using transmission electron microscopy (TEM), any intracellular changes that may occur following invasion of Caco-2 cells by C. concisus. 5. To compare the invasive potential and intracellular survival of C. concisus in Caco- 2 cells following treatment of the cells with Choloroquine di-phosphate (autophagosome inducer), Rapamycin (autophagy inducer) and 3-Methyladenine, Wortmannin and Bafilomycin A1 (autophagy inhibitors) with that of untreated cells infected with C. concisus. 6. To determine, using confocal laser scanning microscopy, how C. concisus interacts with autophagosomes following infection of Caco-2 cells with C. concisus UNSWCD. 7. To determine following infection of Caco-2 cells with C. concisus the expression of genes associated within autophagy pathway, using a quantitative RT-PCR array approach. 157 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.2. Materials and Methods 4.2.1. Bacterial strains and growth Four C. concisus strains: UNSWCD, UNSWCS, ATCC 51562 and BAA-1457 were used in this study. UNSWCD and UNSWCS were isolated from biopsy specimens collected from patients with newly diagnosed CD and patients with gastroenteritis. These C. concisus strains were isolated as part of previous studies (41, 105) approved by the Research Ethics Committees of the University of New South Wales and the South East Sydney Area Health Service-Eastern Section, Sydney (Ethics No.: 06/164). ATCC 51562 and BAA-1457 were purchased from the American Type Culture Collection. C. concisus strains were grown on Horse Blood Agar (HBA) plates [Blood Agar Base No. 2 supplemented with 6% defibrinated horse blood (Oxoid)], and incubated at 37°C under microaerobic conditions with H2 [generated using Campylobacter Gas Generating Kits (Cat. # BR0056A, Oxoid; Adelaide, SA, Australia)] for 48 h. 4.2.2. Cell culture The human intestinal epithelial Caco-2 cell line (American Type Culture Collection; HTB-37) was used in this study. Cells were cultivated in 10 ml cell culture media comprising Minimum Essential Medium (MEM) (Life technologies; Mulgrave, VIC, Australia) supplemented with 10% FBS, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2.25 mg/1 sodium bicarbonate and 100 µg/ml penicillin and streptomycin (Life technologies) in 25 cm2 tissue culture flasks (In Vitro Technologies; Noble Park, VIC, Australia) at 37°C with 5% CO2. Cell viability was determined by performing a cell count on the trypsinised cells using a haemocytometer and 0.4% trypan blue (Sigma Aldrich; Castle Hill, NSW, Australia). 158 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.2.3. Determination of intracellular levels of C. concisus using gentamicin protection assays Caco-2 cells were seeded at a concentration of 5 × 105 cells per ml into 24-well plates and incubated for 2 days at 37°C with 5% CO2. Prior to seeding, the wells were coated with 1 ml collagen (0.338 mg/ml) and incubated for 20 min at 37°C with 5% CO2. Prior to infection, Caco-2 were washed 3 times with MEM antibiotic-free cell culture media. Monolayers were then infected with C. concisus strains (UNSWCD, UNSWCS, 51562, and BAA-1457) at a Multiplicity of Infection (MOI) of 200. Following addition of the bacteria, the 24-well plates were centrifuged at 232 × g for 5 min (Sigma) to promote bacterial-human cell contact. The infected monolayers were then incubated for 6 h at 37°C with 5% CO2 to allow adherence and invasion to occur. Following the 6 h incubation period, Caco-2 monolayers were washed 3 times in MEM cell culture media containing gentamicin (Invitrogen) at a concentration of 200 µg/ml to kill any extracellular bacteria. Following incubation with gentamicin for 1 h, the media was collected and 5 µl drops were plated in quadruplicate, both undiluted and at 1:10 dilution, to determine whether any viable extracellular bacteria remained. To determine the number of bacterial cells that had invaded the Caco-2 cells each monolayer was lysed by addition of 500 µl of 1% Triton X-100 (Sigma) for 5 min. The cell lysates of each monolayer were then diluted by a series of ten-fold series, after which 5 µl drops were plated in quadruplicate onto the appropriate agar plates. Three biological replicates and 2 technical replicates were conducted. 159 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.2.4. The effect of aerobic and microaerobic growth conditions on C. concisus UNSWCD adherence and invasion of Caco-2 cells As a result of an in vitro study by Mills et al. (180), which showed an increase in C. jejuni invasion upon incubation in an aerobic environment that mimics the environment found in the gastrointestinal tract, an investigation was carried out to investigate whether an aerobic or microaerobic co-culture growth system between C. concisus UNSWCD and Caco-2 cells produced optimal adherence and invasion levels of C. concisus UNSWCD. Thus, to examine this effect Caco-2 monolayers were seeded as mentioned in Section 4.2.3 and infected with C. concisus UNSWCD at MOI 200. The infected Caco-2 monolayers were incubated for 6 h at 37°C under either aerobic or microaerobic (5% CO2) conditions rich in H2 (generated using Campylobacter Gas Generating Kits). The adherence assay and gentamicin protection invasion assay was performed as previously described by in Sections 3.3.1, and 4.2.3. 4.2.5 Determination of the effect of autophagy inhibition and induction on intracellular levels of C. concisus To examine the effect of autophagy inhibition on intracellular levels of C. concisus UNSWCD, 5 mM or 10 mM 3-methyladenine (3-MA), 10 nM bafilomycin A-1 or 100 nM wortmannin (Sigma Aldrich) were added to the monolayer 1 h prior to the addition of the bacteria. To examine the effect of autophagy induction on intracellular levels of C. concisus UNSWCD, rapamycin or chloroquine di-phosphate (CQD) (Sigma Aldrich) were added to the monolayer at concentrations of 200 nM and 50 µM respectively, 14 h prior to commencement of the assay. Additionally, in order to investigate whether autophagy inhibition could also influence the invasive level of other C. concisus strains, three additional C. concisus strains previously shown to have naturally low intracellular percentages (ATCC 51562, UNSWCS and a strain with no invasion BAA-1457) were 160 Chapter 4: Autophagy in the intracellular survival of C. concisus employed. The effect of autophagy inhibition on the invasive levels of these strains was examined by the addition of 10 mM 3-MA (Sigma Aldrich), which was added 1 h prior to the addition of the bacteria. Furthermore, statistical analyses were performed using a one-way or two-way ANOVA with a post hoc Dunnett’s test employing GraphPad Prism version 5.0 (GraphPad Software; San Diego, CA, USA). 4.2.6. Determination of the effect of Chloroquine di-phosphate on the ability of Caco-2 cells to exocytose C. concisus To examine the effect of CQD on the ability of Caco-2 cells to exocytose C. concisus, Caco-2 cells were seeded as previously described (Section 4.2.3), and infected with C. concisus UNSWCD at a MOI of 200 for 6 h. Following incubation, the monolayers were washed with media without antibiotics three times and then exposed to 200 µg/ml gentamicin for 1 h to remove any extracellular bacteria. After gentamicin treatment, the cells were washed with media without antibiotics three times, and 50 µM CQD was added to the respective wells and incubated for 1 h. The supernatant was then collected and extracellular bacteria were quantified using the drop plate count method on HBA plates (as described in section 2.4.4). The intracellular bacteria were also quantified using the same method. 4.2.7. Determination of the effect of re-invasion of C. concisus UNSWCS on intracellular survival within Caco-2 cells C. concisus UNSWCS was grown under microaerobic conditions as outlined in section 4.2.1. Caco-2 cells were grown and seeded at 5.0 × 105 cells according to procedures stated in sections 4.2.2. Caco-2 cells were infected with C. concisus UNSWCS at a MOI o of 200 and incubated for 6 hours at 37 C with 5% CO2. A gentamicin protection assay was performed according to procedures outlined in section 4.2.3. Caco-2 cell lysates 161 Chapter 4: Autophagy in the intracellular survival of C. concisus containing intracellular C. concisus UNSWCS were grown on HBA plates. The invading CFU/ml obtained from the Caco-2 infected lysates were quantified using the drop plate count method. Intracellular C. concisus UNSWCS were then subcultured onto HBA plates and grown for 24 h under microaerobic conditions after which C. concisus UNSWCS cells were harvested from the HBA plate using 1 ml PBS. The bacterial cells were washed in PBS and then used in another gentamicin protection assay (MOI 200). The procedures above were repeated a further time however on this reinfection the invading C. concisus UNSWCS strain became resistant to gentamicin. 4.3. The effect of Chloroquine di-phosphate on C. concisus 4.3.1. Determination of the effect of Chloroquine di-phosphate on C. concisus viability C. concisus was grown in a broth medium consisting Brain heart infusion (BHI) (Oxoid), 10% FBS and CQD at concentrations of 0 µM, 25 µM, 50 µM, and 100 µM, and these were incubated for 24 h under microaerobic conditions. Bacterial viability following incubation was quantified on HBA by preparing serial dilutions in PBS and using the drop plate count method. 4.3.2. Determination of the effect of Chloroquine di-phosphate and 3- Methyladenine on Caco-2 cell viability following C. concisus UNSWCD infection In order to investigate the effect of chemical autophagy induction and autophagy inhibition on Caco-2 cell viability, Caco-2 cells were seeded at 5.0 × 105 cells onto 24- well tissue culture plates and incubated for 48 h. Prior to C. concisus UNSWCD infection 50 µM of CQD was added 14 h before and 5 mM 3-MA was added 1 h before, respectively. Plates were then incubated aerobically for 6 h, after which the cell 162 Chapter 4: Autophagy in the intracellular survival of C. concisus monolayers were washed three times with DPBS (Life Technologies). The cells were then detached using 0.25% Trypsin EDTA (Life Technologies) for 5 min, after which FBS (Bovogen) was added to deactivate the trypsin. Cell viability was determined by performing a cell count on the trypsinised cells using a haemocytometer and 0.4% trypan blue (Sigma). 4.4. Detection of autophagosomes and bacterial co-localisation using confocal microscopy o Caco-2 cells were grown at 37 C with 5% CO2 on 13 mm Poly-l-lysine glass coverslips (BD-Bioscience; Two Oak Park, Bedford, MA, USA) in 24-well plates at a concentration of 5.0 × 105 cells per well for 48 h. As a positive control for autophagosome formation, prior to commencing the assay Caco-2 cells were treated with 50 µM CQD (Sigma) for 14 h. To investigate autophagy inhibition, 5 mM and 10 mM concentrations of 3-MA (Sigma) were added directly 1 h prior to C. concisus infection. C. concisus UNSWCD was inoculated at MOI 200 onto the apical membrane o surface of the Caco-2 cell monolayer and incubated for 6 h at 37 C and 5% CO2. Caco-2 cells were then washed three times with DPBS (Life technologies) and fixed in 3.7% formaldehyde (Ajax Finechem, Seven Hills, NSW) and left overnight at 4oC. The samples were then washed three times with DPBS (Life technologies) and permeabilised using 0.2% Triton-X 100 (Sigma) in PBS for 15 min at room temperature to allow the Light Chain 3 Beta (LC3B) primary antibody to internalise into the cell. To identify the key autophagy marker LC3B, autophagosomes examined in the Caco-2 cell monolayers on growing glass cover slips, were labeled with anti-LC3B mouse antibody at a concentration of 0.5 µg/ml (Sigma) and C. concisus UNSWCD was labeled with anti-rabbit C. concisus sera at a concentration of 1:30 and both antibodies 163 Chapter 4: Autophagy in the intracellular survival of C. concisus were incubated at room temperature for 1 h. The concentration of anti-rabbit C. concisus sera at 1:30 was based on optimisations made prior to this investigation, which differed from a previous study in which a concentration of 1:40 was employed (108). Following the 1 h incubation, 10 drops of an image enhancing solution (Life technologies) was added to each sample to prevent non-specific binding between the primary and secondary antibodies. The secondary antibodies, anti-mouse antibody Alexa Fluor 594 (5 µg/ml) and anti-rabbit antibody Alexa Fluor 488 (5 µg/ml) (Life technologies), were then added and incubated at room temperature for 1 h. All antibody and secondary antibody concentrations were optimised prior to the study to ensure both adequate binding and fluorescent signals at 594 nm and 488 nm. To avoid photo bleaching of the fluorophores, samples were incubated at room temperature for 1 h in an aluminum foil covered container. Following the incubation period the individual coverslip samples were removed from the 24 well plates using fine forceps. Following this procedure one drop of Prolong gold, an anti-fade reagent (Life technologies; Oregon, USA) was added to each coverslip to prevent photo bleaching of the fluorescent antibodies. A square coverslip (LabServ; NSW, Australia) was used to cover the round glass coverslip containing the monolayer (BD-Bioscience). This was placed face down and sealed onto a glass slide (Livingstone; Rosebery, Australia) and labeled appropriately. Additionally, clear nail polish (Mavala; Geneva, Switzerland) was used to seal the square coverslip in place on top of the glass slide to ensure that no air pockets and gaps remained on the specimen. This procedure was conducted to ensure that each sample remained optimal for examination under confocal laser scanning microscopy (CLSM). 164 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.4.1. The confocal microscopy imaging procedure Specimens were visualised using an Olympus FluoviewTM FV1000 Confocal Laser Scanning Microscope (Olympus; North Ryde, NSW, Australia). Co-localisation was measured by calculating the Pearson’s correlation coefficients (RTotal and Rcoloc values > 0.5, which are indicative of co-localisation) using the Fiji software (http://fiji.sc/Fiji). The workflow of the procedure is outlined in Figure 4.1. Figure 4.1. Workflow showing the preparation of specimens for confocal microscopy. After chemical fixation with formaldehyde and antibody labeling with anti-LCB3 and anti- rabbit C. concisus sera, specimens were labeled with the secondary antibodies, anti-mouse antibody Alexa Fluor 594 and anti-rabbit antibody Alexa Fluor 488. The Texas red channel was used to excite the red Alexa Fluor 594 fluorophore, while the Alexa Fluor 488 channel was used to excite the green Alexa Fluor 488 fluorophore. The images were obtained and viewed using the FV1000 imaging software (Olympus). Images obtained and used for co-localisation analysis were captured from z-stacked images. 165 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.5. Detection of Caco-2 cell membrane alterations upon C. concisus infection using Scanning Electron Microscopy Caco-2 cells were grown on Poly-L-Lysine coated cover slips (BD-Bioscience) in cell culture media in 24-well plates at a cell density of 5 × 105 cells per well incubated at o 37 C and 5% CO2 for 48 h. C. concisus UNSWCD was harvested and a drop plate count method was used to determine the growth viability of C. concisus UNSWCD. Caco-2 cells were then inoculated with C. concisus UNSWCD at a MOI 200 for 6 h. As a positive control of autophagosome formation, 50 µM of CQD was added to Caco-2 cells 14 h prior to commencing the assay. Following incubation the samples were fixed in 1 ml of fixative solution comprising of 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1M Sorensen’s Phosphate Buffer (SPB) (ProSciTech) pH 7.2 and left overnight at 4oC. Samples were then washed carefully three times for 10 min using 0.1M SPB at a pH of 7.2 after which the sample coverslips were carefully removed from the 24 well- plate using fine forceps. Sample coverslips were then carefully loaded onto an autosamdri-815 sample holder that had mesh coverings above and below each sample coverslip to allow the even diffusion of each solution throughout the sample holder. The samples were then ethanol dehydrated, which was conducted as follows. The biological samples contained within an autosamdri-815 sample holder were initially exposed to ethanol concentrations of 30% (10 min), 50% (10 min) and 70% (2 h). Following this they were, exposed to 80% and 90% ethanol concentrations, each for 10 min. In the final step samples were exposed to 100% ethanol for 3 × 10 min time periods. Following dehydration the samples underwent critical point drying over a 2 h period using a Critical point dryer autosamdri-815 (Tousimis Research Corporation, Rockville, USA). The coverslips were then mounted onto carbon tabs and a sputter gold coat was cast on top of the coverslips using an EMITECH K-55OX gold coater (Emitech; Ashford, U.K). Scanning electron microscopy (ScEM) was performed using a Hitachi 166 Chapter 4: Autophagy in the intracellular survival of C. concisus S3400-X model Scanning Electron Microscope (Hitachi High-Technologies Corporation; Tokyo, Japan) at 32 kV. Figure 4.2. Workflow outlining the sample preparation process for ScEM investigations. This preparation process involved a series of procedures from cell seeding, bacterial infection, chemical fixation, ethanol dehydration at 50%-100% concentrations, critical point drying to final mounting and coating for sample visualisation using the Hitachi S3400-X ScEM microscope. 167 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.6. Quantitative PCR for analysis of genes within the autophagy pathway Caco-2 cells were grown at 37°C with 5% CO2 in 6-well plates at a concentration of 1 × 106 cells per well for 48 h. Cells were then infected with C. concisus UNSWCD at MOI 200 for 6 h. RNA was extracted from the cells using the Isolate I RNA extraction kit (Bioline; Alexandria, NSW, Australia). cDNA was prepared using the RT2 First strand cDNA synthesis kit, according to the manufacturer’s instructions (Qiagen; Chadstone Centre, VIC, Australia). The cDNA samples were then mixed with RT2 SYBR Green Fast Master mix (Qiagen) and 20 µl aliquoted into each well of a Human Autophagy RT2 ProfilerTM (PAHS-084) array (Qiagen), which targets 84 genes related to the autophagy pathway. Transcription profiles were obtained from three independent experiments of C. concisus UNSWCD infected (test) and non-infected (control) samples. The threshold cycle (Ct) of each gene was determined, and subsequently analysed by the RT2 Profiler PCR Array data analysis software. ΔΔCT values were determined following normalisation of the data using a relevant combination of house-keeping genes provided within the array. Statistically significant values were defined as (P < 0.05). The workflow of this assay is outlined in Figure 4.3. 168 Chapter 4: Autophagy in the intracellular survival of C. concisus Figure 4.3. The autophagy qRT-PCR process used to determine expression level of autophagy genes following infection of Caco-2 cells with and without C. concisus UNSWCD. The Caco-2 cells were seeded at a density of 1 × 106 cells per well and grown for 48 h to establish a confluent monolayer. The monolayer was then infected with C. concisus UNSWCD at a MOI of 200 as described above. Non-infected cells were included as a negative control. Following incubation RNA was extracted from UNSWCD infected samples and from the non- infected samples (control) using an RNA extraction kit (Bioline). cDNA was then prepared using the RT2 First strand Synthesis kit. cDNA samples were then mixed with the RT2 SYBR Green Fast Master mix (Qiagen) and aliquoted in equal volumes (20 µl) to each well of the human autophagy array on a loading block. The arrays were then heat-sealed and run on a Rotor gene Q RT-PCR cycler to determine CT expression results based on a defined threshold. 169 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.7. Results 4.7.1. Effect of autophagy inhibition on intracellular levels of Campylobacter concisus A range of MOIs were tested to determine the MOI that would give an optimal level of bacterial entry into host cells with minimal host cell death. The viability of host cells exposed to C. concisus at a MOI of 100, 200, 500 and 800 were 94.0 ± 0.2%, 88.2 ± 2.0%, 63.8 ± 3.3% and 50.9 ± 0.3%, respectively as compared with a viability of 99.0 ± 0.6% for the non-infected control. The effect of atmospheric conditions on C. concisus adherence and internalisation was assessed by incubating the infection plate containing Caco-2 cells infected with C. concisus UNSWCD at MOI 200 under both aerobic and microaerobic conditions. The results for this assay showed that C. concisus UNSWCD produced an adherence level of 4.11 ± 0.11% under aerobic growth conditions and 3.54 ± 0.56% under microaerobic growth conditions. Moreover, the invasion results revealed that C. concisus UNSWCD invaded Caco-2 cells at an invasive level of 0.26 ± 0.08% under aerobic growth conditions and 0.18 ± 0.02% under microaerobic growth conditions. Statistical analysis employing paired T-test analysis against the results obtained for both aerobic and microaerobic conditions showed no significant difference in both adherence (P = 0.32; P > 0.05) and invasion levels (P = 0.44; P > 0.05). C. concisus UNSWCD grown under aerobic conditions exhibited similar adherence and invasion levels to those observed under microaerobic growth conditions. Based on these findings subsequent experiments were conducted under aerobic conditions. Importantly, it is worthwhile mentioning that Man et al. (108) optimised conditions for the gentamicin protection 170 Chapter 4: Autophagy in the intracellular survival of C. concisus assay under aerobic conditions. Thus, these factors provided compelling evidence to continue the use of gentamicin protection assays under aerobic conditions. The intracellular levels of C. concisus UNSWCD 6 h post-infection were similar to previous results obtained for this strain (108, 109), with the intracellular percentage (number of internalised bacteria / number of bacteria added) of the bacterium being 0.44 ± 0.04% (Figure 4.4) and the total number of intracellular bacteria 5.1 × 105 ± 1.0 × 105 CFU/mL within the 5 × 105 host cells seeded onto the plate. In order to test the effect of autophagy inhibition and induction on C. concisus UNSWCD invasion, Caco-2 cells were exposed to three different autophagy inhibitors and 2 different inducers of autophagy, prior to C. concisus addition. Exposure to the autophagy inhibitor 3-MA at concentrations of 5 mM and 10 mM resulted in increased intracellular levels of C. concisus UNSWCD of approximately 2.2 and 3.5-fold respectively, the latter showing an intracellular percentage of 1.54 ± 0.008% in intracellular bacteria (Figure 4.4). Inhibition of autophagy using the alternative autophagy inhibitors, bafilomycin A-1 (10 nM) and wortmannin (100 nM) showed 2.0 and 2.8-fold increases in intracellular levels of C. concisus UNSWCD, respectively (Figure 4.4). Statistical analysis using a one-way ANOVA with Dunnett’s post hoc tests showed statistically significant increases (P < 0.05) for 5 mM and 10 mM 3-MA, 10 nM bafilomycin and 100 nM wortmannin when compared with the untreated controls. Moreover, the addition of the autophagy inducer rapamycin (200 nM) and the autophagy modulator chloroquine di–phosphate (CQD) (50 µM) resulted in significant decreases (P < 0.0001) in intracellular levels of C. concisus UNSWCD, with the intracellular percentages being 0.0007 ± 0.0001% and 0%, respectively (Figure 4.4). 171 Chapter 4: Autophagy in the intracellular survival of C. concisus Figure 4.4. Intracellular survival of C. concisus UNSWCD following autophagy inhibition and induction. The figure represents the relative intracellular levels of C. concisus in the presence of the autophagy inhibitors (3-MA: 5mM and 10mM; Wortmannin: 100nM and Bafilomycin A: 10nM) and the autophagy inducers (CQD: 50 µM and Rapamycin: 200 nM) as compared with the untreated controls (No treatment). The values are expressed as mean percentage cell viability ± SEM based on a minimum of four biological replicates. NS: Non-significant; (P ≤ 0.05 ★); (P ≤ 0.0001 ★★★★). 172 Chapter 4: Autophagy in the intracellular survival of C. concisus Further, the effect of autophagy inhibition on 3 C. concisus strains previously shown to have naturally low intracellular survival (ATCC 51562: 0.00048 ± 0.00016%; UNSWCD: 0.00059 ± 0.00015%) or no invasion (BAA-1457) (109) was also investigated. Upon addition of 10 mM 3-MA to Caco-2 cells prior to inoculation with C. concicus strains, the intracellular levels of these strains significantly increased (P < 0.0001) to levels observed for UNSWCD (ATCC 51562: 0.51 ± 0.06%; UNSWCD 0.72 ± 0.14%; BAA-1457: 0.66 ± 0.09%) (Figure 4.5). Figure 4.5. Intracellular survival of C. concisus UNSWCS, ATCC 51562 and BAA-1457 following autophagy inhibition using 3-MA. The figure represents the relative intracellular levels of C. concisus strains with the addition of the autophagy inhibitor 3-MA as compared to that in the untreated control (No treatment). Errors are presented as SEM based on a minimum of four biological replicates. 3-MA: 10 mM 3-methyladenine. 173 Chapter 4: Autophagy in the intracellular survival of C. concisus To determine whether host adaptation may have a similar effect on the invasion levels, intracellular levels of C. concisus UNSWCS a strain with normally low levels of invasion (0.00059 ± 0.00015%) was examined by conducting assays in which C. concisus UNSWCS bacteria that had internalised into host cells were used for the invasion assay (re-invasion). Upon re-invasion of host-adapted isolates of UNSWCS as outlined in 4.2.7, the intracellular levels increased approximately 8.6-fold to 0.0051 ± 0.0009%. Isolates from this re-invasion assay were then used for a second re-invasion. However, this did not alter the intracellular levels (0.0045 ± 0.0011%), and a third re- invasion resulted in isolates with gentamicin resistance. 4.7.2. The effect of Chloroquine di-phosphate and 3-Methyladenine on Caco-2 cell viability following C. concisus UNSWCD infection Investigation of the effect of chloroquine di-phosphate (CQD) and 3-Methyladenine (3-MA) on Caco-2 cell viability following C. concisus UNSWCD infection based on the trypan blue exclusion method showed that Caco-2 cell viability upon treatment with 50 µM CQD was 75.190 ± 1.956%, compared with the untreated control 98.790 ± 0.564%. Statistical analysis based on a one-way ANOVA with a Dunnetts post hoc test revealed a significant difference (P < 0.0001) in cell viability. In contrast, the same analysis showed no significant difference in viability was observed between Caco-2 cells treated with 5 mM 3-MA (96.700 ± 0.092%) compared to the untreated control (98.790 ± 0.564%) (P = 0.055; P > 0.05). 174 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.7.3. The effect of Chloroquine di-phosphate on C. concisus UNSWCD bacterial cell viability To determine the toxicity of CQD on C. concisus UNSWCD, the bacteria were incubated with a range of CQD concentrations (0 µM, 25 µM, 50 µM, and 100 µM) for 24 h under microaerobic conditions. Statistical analysis via a one-way ANOVA with a Dunnetts post hoc test, revealed that there was no statistical difference in cell viability between the untreated C. concisus UNSWCD with no CQD (P > 0.05) compared to C. concisus UNSWCD treated with increasing concentrations of CQD (Figure 4.6). NS NS 1.0 NS 0.8 0.6 0.4 0.2 Log 10 (Viable CFu/mL/ Bacterial cells added) cells Bacterial CFu/mL/ (Viable Log 10 0.0 BHI + UNSWCD no CQD BHI+ UNSWCD + CQD 25 µM BHI + UNSWCDBHI+ + CQDUNSWCD 50 µM + CQD 100 µM Figure 4.6. Viability of C. concisus UNSWCD treated with Chloroquine di-phosphate. C. concisus UNSWCD in BHI alone and in the presence of three different concentrations of CQD (25µM, 50µM and 100µM) was incubated for 24 h under microaerobic conditions. The viability of the cells is expressed as the Log 10 values of the number of viable bacterial cells present following incubation over the initial bacterial inoculum. The error bars are presented as SEM based on a minimum of three biological replicates. NS: no significant difference. 175 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.7.4. The effect of Chloroquine di-phosphate on the ability of Caco-2 cells to exocytose C. concisus UNSWCD Investigation of the effect of CQD on the ability of Caco-2 cells to exocytose C. concisus, showed no significant difference (P = 0.475) in extracellular levels of C. concisus UNSWCD bacteria obtained from Caco-2 cells exposed to antibiotic free media (0.010 ± 0.002%) as compared to Caco-2 cells treated with 50 µM of CQD for 1 h (0.013 ± 0.002%), based on a paired T-test analysis. These results obtained show that exposure to CQD did not have a significant influence on the extracellular number of C. concisus UNSWCD, brought on by exocytosis of C. ureolyticus UNSWCD from the Caco-2 cells. 176 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.7.5. Visualisation of the effect of autophagy induction on intracellular levels of C. concisus Visualisation of the effect of 50 µM CQD on Caco-2 cells using ScEM showed cellular protrusions (blebs) on the apical membrane surface (Figure 4.7 B, C) that were not observed in the negative control (Figure 4.7 A). The regions surrounding these protrusions showed a significant decrease in microvilli abundance (Figure 4.7 B & C). Figure 4.7. Scanning electron microscopy images of Caco-2 cells with and without Chloroquine di-phosphate treatment. A: Untreated Caco-2 cells showing clear and defined microvilli on the apical membrane surface. No cellular blebbing was observed on the apical membrane surface. B & C: Following treatment of Caco-2 cells with 50 µM CQD, cellular protrusions/blebs (indicated by an asterisk) were induced on the apical membrane surface of Caco-2 cells. 177 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.7.6. Investigation of autophagosomes in Caco-2 cells The formation of autophagosomes in Caco-2 cells was investigated using CLSM, with the presence of autophagosomes being detected based on the presence of the autophagy marker, LC3B. The results of this study showed that LC3B was distributed within the cytoplasm (Figure 4.9 A) and in close proximity to the membrane regions of the Caco-2 cells (Figure 4.8 A). Inhibition of the autophagy process using 10 mM 3-MA resulted in a decrease in autophagosome production (Figure 4.8 B) as compared with the untreated Caco-2 cells (Figure 4.8 A). In contrast, the density of LC3B detected following induction of the autophagy process with 50 µM CQD (Figure 4.8 C) was increased as compared with that observed in the Caco-2 cells treated with 10 mM 3-MA (Figure 4.8 B). Moreover, the density of LC3B detected following induction of autophagosomes with 50 µM CQD was higher than that observed in the Caco-2 cells without chemical treatment. Figure 4.8. Visualisation of autophagosomes using confocal microscopy targeting LC3B. A: Untreated Caco-2 cells, B: Caco-2 cells treated with 10 mM 3-MA, C: Caco-2 cells treated with 50 µM CQD. 178 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.7.7. Co-localisation of C. concisus with autophagosomes In order to investigate the formation of autophagosomes in Caco-2 cells, CLSM was used. Autophagosomes were detected through the presence of LC3B in Caco-2 cells distributed within the cytoplasm, and anti-rabbit AlexaFluor 488 was used to detect and observe C. concisus UNSWCD. The results showed that LC3B was distributed within the cytoplasm of untreated Caco-2 cells (Figure 4.9 A). Following infection of Caco-2 cells with C. concisus UNSWCD, the bacterium was found to co-localise with LC3B (Figure 4.9 B-I), indicated by the yellow regions. Moreover, some cells were found to be hyperinfected by C. concisus UNSWCD (Figure 4.9 F and J), as indicated by the green fluorescence. 179 Chapter 4: Autophagy in the intracellular survival of C. concisus Figure 4.9. Visualisation of the co-localisation of C. concisus with autophagosomes using confocal microscopy. A: Untreated Caco-2 cells, B-J: Caco-2 cells infected with C. concisus UNSWCD. LC3B was stained in red, C. concisus was stained in green. B-J: C. concisus UNSWCD was found to aggregate and adhere to Caco-2 cells, internalise into Caco-2 cells, and co-localise with the LC3B antibody. 180 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.7.8. Modulation in the expression of genes involved in autophagy by C. concisus Given the role of autophagy in modulating the intracellular levels of C. concisus and possible evasion of some strains of autophagy-mediated killing, we analysed the effect of C. concisus UNSWCD infection on the gene expression of 84 genes involved in autophagy. Out of the 84 genes analysed (Appendix Table 2), three genes were found to be significantly up-regulated and 16 genes were found to be down-regulated (10 significant, 6 borderline) (Table 4.1). Table 4.1. Genes within the autophagy pathway regulated upon infection with C. concisus UNSWCD. Fold Gene Gene name Refseq P – value 95% CI change BID BH3 interacting NM_001196 1.0876 0.025435 (1.04, domain death 1.14) agonist CDKN2A Cyclin-dependent NM_000077 1.0926 0.025666 (1.04, kinase inhibitor 1.15) 2A MAP1LC3B Microtubule- NM_022818 1.2697 0.018016 (1.12, associated protein 1.42) 1 light chain 3 beta AMBRA1 Autophagy/beclin- NM_017749 0.7142 0.016645 (0.59, 1 regulator 1 0.84) ATG4B ATG4 autophagy NM_178326 0.7888 0.0155 (0.69, related 4 homolog 0.88) B ATG7 ATG7 autophagy NM_006395 0.8017 0.012916 (0.72, related 7 homolog 0.88) ATG9B ATG9 autophagy NM_173681 0.7497 0.034192 (0.60, related 9 homolog 0.90) B 181 Chapter 4: Autophagy in the intracellular survival of C. concisus BAX BCL2-associated NM_004324 0.8035 0.030812 (0.70, X protein 0.91) CTSD Cathepsin D NM_001909 0.8572 0.011508 (0.80, 0.91) CTSS Cathepsin S NM_004079 0.6649 0.059751 (0.45, 0.88) FADD Fas (TNFRSF6)- NM_003824 0.6378 0.051529 (0.46, associated via 0.82) death domain GABARAPL-1 GABA(A) NM_031412 0.9124 0.021973 (0.87, receptor- 0.96) associated protein like 1 HDAC1 Histone NM_004964 0.8204 0.002512 (0.78, deacetylase 1 0.86) HSPA8 Heat shock 70kDa NM_006597 0.798 0.019944 (0.70, protein 8 0.89) IGF1 Insulin-like NM_000618 0.4878 0.088337 (0.15, growth factor 1 0.83) LAMP1 Lysosomal- NM_005561 0.8166 0.067864 (0.70, associated 0.93) membrane protein 1 MAPK14 Mitogen-activated NM_001315 0.8875 0.064372 (0.81, protein kinase 14 0.97) TGFB1 Transforming NM_000660 0.7209 0.029435 (0.57, growth factor, beta 0.88) 1 WIPI1 WD repeat NM_017983 0.8357 0.057777 (0.72, domain, 0.95) phosphoinositide interacting 1 Three biological replicates from each of the non-infected and infected cells were analysed 182 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.8. Discussion Following infection of intestinal epithelial cells, the intracellular levels of C. concisus have previously been shown to vary significantly between strains, with some strains having up to 500-fold more intracellular bacteria (108, 109). Numerous studies have detailed the importance of the autophagy genes ATG16L1 and IRGM in combating and destroying intracellular pathogens such as Salmonella enterica serovar Typhimurium and Mycobacterium tuberculosis (136, 146, 147). Thus, we studied the role of autophagy in governing the intracellular levels of C. concisus within intestinal epithelial cells. The intracellular levels of C. concisus UNSWCD 6 h post-infection were similar to previous results obtained for this strain (108, 109), with the intracellular percentage (number of internalised bacteria / number of bacteria added) of the bacterium being 0.44 ± 0.04% (Figure 4.4), and the total number of intracellular bacteria being 5.1 × 105 ± 1.0 × 105 CFU/ml within the 5 × 105 host cells seeded onto the plate. This level of invasion is similar to levels of invasion observed for C. jejuni (181). Upon addition of the autophagy inhibitor 3-MA at a concentration of 5 mM, the intracellular level of C. concisus UNSWCD increased approximately 2.2-fold, with the addition of 10 mM 3- MA an even greater increase (approximately 3.5-fold, intracellular percentage: 1.54 ± 0.08%) in intracellular bacteria was observed (Figure 4.4). These findings were confirmed by employing alternative autophagy inhibitors, with intracellular levels of C. concisus UNSWCD increasing 2.0 and 2.8-fold upon inhibition of autophagy with 10 nM bafilomycin A-1 and 100 nM wortmannin, respectively (Figure 4.4). Statistical analysis performed through a one-way ANOVA with a Dunnett’s post hoc test showed a statistically significant increase (P < 0.05) for 5 mM and 10 mM 3-MA, 10 nM bafilomycin A-1 and 100 nM wortmannin when compared to the non-treated control. 183 Chapter 4: Autophagy in the intracellular survival of C. concisus To investigate this further, three additional C. concisus strains previously shown to have naturally low intracellular percentages of bacteria (UNSWCS and ATCC 51562) or with no invasion (BAA-1457), were investigated. Interestingly, upon addition of 10 mM 3- MA, the intracellular percentages of these strains significantly increased (P < 0.0001) to levels observed for UNSWCD (ATCC 51562: 0.51 ± 0.06%; UNSWCS: 0.72 ± 0.14%; BAA-1457: 0.66 ± 0.09%) (Figure 4.5). Comparison of these results with that published for other bacterial pathogens shows that the addition of autophagy inhibitors results in a decline of bacterial invasion in host cells as opposed to increasing bacterial invasion observed with C. concisus spp. This difference could relate to the use of different species, different cell lines and concentrations of autophagy inhibitors. For example Dorn et al. used a modified antibiotic protection assay to examine the effects of the autophagy inhibitors 3- methyladenine and wortmannin on the survival of P. gingivalis 381 within HCAE cells (182). Treatment of the HCAE cells with 10 mM 3-methyladenine or 10 mM wortmannin resulted in a dramatic decrease of bacterial persistence, over an 8 h incubation period. Interestingly, the opposite occurred in the absence of autophagy inhibitors 3-MA and wortmannin, an increase of bacterial survival being observed over the 8 h incubation period. Further a study by Sajjan et al. which examined the ability of Burkholderia cenocepacia to survive within IB3 bronchial epithelial cells in the absence and in the presence of 1, 10 & 100 nM of wortmannin 24 h post infection has reported wortmannin to significantly inhibit the intracellular replication of B. cenocepacia in a dose-dependent process (183). Their results showed that in the absence of wortmannin B. cenocepacia intracellular levels were 4.8 x 104 ± 8.9 x 102 (CFU/ml), which upon the addition of wortmannin at 1 nM to 4.2 x 104 ± 1.5 x 103 (CFU/ml), at 10 nM to 3.6 x 103 ± 3.8 x 102 (CFU/ml), and at 100 nM to 1.2 x 103 ± 3.9 x 102 (CFU/ml) (183). 184 Chapter 4: Autophagy in the intracellular survival of C. concisus Statistical analysis showed that both 10 and 100 nM of wortmannin resulted in significant decreases (P < 0.05) in B. cenocepacia intracellular levels, as compared cells not treated with wortmannin (183). Additionally using bafilomycin A1 as a vacuolar inhibitor of autophagy Conte et al. noted a reduction in Listeria monocytogenes levels in Caco-2 cells following treatment with 0.1 µM bafilomycin A1 (184). Investigation of whether host adaptation of C. concisus may have a similar effect on the intracellular levels of C. concisus when bacteria that have internalised into host cells in the invasion assay are re-used to reinfect host cells. Results of this investigation showed that upon re-invasion of host-adapted isolates, C. concisus UNSWC a strain known to display low levels of invasion (0.00059 ± 0.00015%), showed increased percentage levels of intracellular C. concisus UNSWCS, with the levels increasing by approximately 8.6-fold to 0.0051 ± 0.0009%. A further invasion assay using these internalised isolates did not, however, alter the intracellular percentage levels of UNSWCS (0.0045 ± 0.0011%), and the third re-invasion assay resulted in isolates that displayed gentamicin resistance. Overall, these findings suggest that the autophagy process is one of the primary regulators of intracellular levels of C. concisus strains with low invasion capabilities. The compounds 3-MA and wortmannin act by blocking phosphotidyl-inositol-3-kinase activity, while bafilomycin A-1 inhibits vacuolor ATPase, all of which result in autophagy inhibition (185). Given the importance of autophagy in eliminating intracellular pathogens, it would be expected that if this pathway was involved in the pathogenesis of C. concisus, inhibition of autophagy would result in increased levels of intracellular C. concisus. Interestingly, while the highly invasive strain, C. concisus UNSWCD showed a 3.5-fold increase following inhibition of autophagy, using strains 185 Chapter 4: Autophagy in the intracellular survival of C. concisus with low or no invasion into host cells showed dramatic increases (> 1000-fold) in intracellular levels upon inhibition of autophagy. This finding is supported by a study by Lapaquette et al. who reported siRNA-mediated knock down of ATG16L1 or IRGM in HeLa cells to increase the proliferation of adherent and invasive Escherichia coli (147). A possible hypothesis to explain the observed autophagy inhibition is that the autophagy process is influenced by the interaction between the C. concisus CCV and the autophagosome. It is possible that the C. concisus CCV could disrupt the interaction between the autophagosome and the autolysosome. Other pathogens have evolved strategies that can block or delay the maturation of bacterium-containing autophagosomes into degradative autolysosomes, thus avoiding elimination. For example, infection with adherent-invasive Escherichia coli (AIEC) triggers the accumulation of AIEC-containing autophagosomes that acquire the lysosomal protein LAMP1, which eventually prevents the maturation of autolysosomes and results in AEIC evading lysosomal degradation (186, 187). Additionally, studies on another pathogen Mycobacterium marinum have shown that upon infection of macrophages the bacterium recruits LC3 positive M. marinum-containing phagosomes (186, 188). Moreover, reports have shown that these LC3-positive compartments have a single membrane, which has acquired the late endosomal proteins RAB7 and LAMP1, but not the lysosomal hydrolase cathepsin D, which is primarily a degradative enzyme found within the lysosome (186, 188). Moreover, this finding indicates that M. marinum blocks LC3-associated phagosome fusion with the lysosome to evade autophagy (186, 188). Interestingly, it has been shown that bacteria can directly inhibit autophagy. For example it has been shown that 4 h post infection, S. Typhimurium restores the activity of mTOR a known inhibitor of autophagy induction. Moreover, reports show that 186 Chapter 4: Autophagy in the intracellular survival of C. concisus mTOR then re-localises to late endosomes and the Salmonella-containing vacuole (SCV), which inhibits autophagy induction, promotes autophagy evasion and enables S. Typhimurium to replicate within SCVs (186, 189). Thus, as in the above described pathogens, C. concisus could employ similar subversion strategies to inhibit the autophagy process and survive intracellularly. However, further studies are required to determine how C. concisus could be inhibiting autophagy directly. Rapamycin is a known inducer of autophagy through its action in the inhibition of the mTOR raptor (regulatory associated protein of mTOR) complex (149, 190). Examination of the effect of autophagy induction on C. concisus intracellular levels using the autophagy inducer rapamycin (200 nM) and the autophagy modulator CQD (50 μM) revealed significant decreases (P < 0.0001) in the intracellular levels of C. concisus UNSWCD, to levels of 0.0007 ± 0.0001% and 0%, respectively (Figure 4.4). The finding that induction of autophagy by rapamycin resulted in the highly invasive strain UNSWCD displaying intracellular levels similar to those found in the low invasive strains ATCC 51562 and UNSWCS is in line with studies of other pathogens. For example, the intracellular survival of C. jejuni was shown to undergo a 75% reduction in murine embryonic fibroblasts and HT-29 cells induced to undergo autophagy with rapamycin (191). Further, a study by Yuan et al. examined the role of autophagy in the phagocytosis and clearance of Pseudomonas aeruginosa (192). This study used an alveolar macrophage cell line (MH-S) with and pre-treated these MH-S cells with rapamycin at 3 μM over 12 h (192). The results revealed that rapamycin improved bacterial clearance from ~5 P. aeruginosa per MH-S cell without rapamycin to ~2 P. aeruginosa per MH-S cell with rapamycin, according to data quantified via confocal z-stack images (192). Moreover, Wang et al. also reported that rapamycin, at a concentration of 200 nM in complete media, inhibited the multiplication of the 187 Chapter 4: Autophagy in the intracellular survival of C. concisus Helicobacter pylori at 6 and 12 h post infection of THP-1 cells (193). Importantly, based on these findings Wang et al. suggested that autophagosome formation seems to participate in the clearance of H. pylori, considering that 3-MA addition produced the opposite effect of bacterial clearance observed with rapamycin (193). CQD plays a more complex role in the cell, having been associated with stimulation of autophagosome formation. It has also been shown to accumulate inside lysosomes leading to inhibition of lysosomal enzymes (194). In the current study, addition of CQD resulted in total clearance of intracellular C. concisus. Interestingly, in a study by Oelschlaegear et al., which determined the effect of chloroquine on the intracellular survival of C. jejuni, E. coli, Citrobacter freundii and S. Typhimurium showed a reduction of their intracellular levels following the addition of chloroquine at 40 µg/ml to human epithelial cell lines (195). However, the authors did not follow up on this observation but deduced that a likely cause of this reduction was the intracellular accumulation of chloroquine to bacteriocidal concentrations within the cell (195). Further, a study by Yu et al. assessed the ability of Shigella flexneri to escape phagocytosis using murine J774 cells (196). This study also used chloroquine at a concentration of 2.5 mg/ml alongside gentamicin to assess the recovery of S. flexneri 1 h post infection of J774 cells. The results of this examination revealed a sharp decline in the recovery of S. flexneri CFU, with both S. flexneri sh4 and pWR800 infected J774 cells in the presence of chloroquine (196). To determine whether the bacterial clearance of C. concisus was attributed to a bacteriocidal effect caused by chloroquine, we investigated the effect of CQD on the viability of C. concisus UNSWCD. While the addition of CQD resulted in a decrease in C. concisus numbers of up to 1.2 log (25 µM CQD), 1.7 log (50 µM CQD) and 2.8 log 188 Chapter 4: Autophagy in the intracellular survival of C. concisus (100 µM CQD), based on statistical analysis these values were not significantly decreased in comparison with C. concisus alone (P > 0.05) (Figure 4.6). To investigate this further, we examined the effect of CQD on the exocytosis of C. concisus UNSWCD; however, no significant difference (P > 0.05) was observed between cells infected with C. concisus UNSWCD and cells treated with 50 µM CQD and infected with C. concisus UNSWCD. Determination of the effect of 50 µM CQD exposure for 14 h on the viability of the host cells resulted in a significant decrease in cell viability (75.2 ± 2.0%) as compared to the non-induced control (98.8 ± 0.6%). While this cell viability result may contribute to a certain extent to the reduction on C. concisus UNSWCD bacterial cell numbers. The cell viability result upon CQD addition cannot completely explain the lack of intracellular bacteria in Caco-2 cells exposed to CQD. The use of ScEM to further investigate the effects of CQD, led to the visualisation of cellular protrusions (blebs) on the apical membrane surface of the cells (Figure 4.7 B, C) that were not present in the negative control (Figure 4.7 A). Interestingly, the regions surrounding these protrusions showed a significant decrease in microvilli abundance (Figure 4.7 B, C). These findings are supported by a study by Fan et al., which reported exposure to high concentrations of CQD to result in vacuolation and blebbing in A549 lung cancer cells (33). Thus, although cellular alterations and toxicity as a consequence of CQD exposure will result in a significant decrease in intracellular levels of C. concisus, the effects that CQD exerts on autophagy are also likely to contribute to the complete bacterial clearance observed. Overall, these results collectively indicate that the host autophagy process controls the intracellular levels of C. concisus, and that some strains of this bacterium have the ability to overcome or manipulate this process. 189 Chapter 4: Autophagy in the intracellular survival of C. concisus To confirm the role of autophagy in the pathogenesis of C. concisus, we conducted confocal microscopy studies to allow us to determine interactions between C. concisus UNSWCD and the autophagy marker LC3B in Caco-2 cells. The results of these studies showed that autophagy is an active process that normally occurs within the cytoplasm and membrane regions of Caco-2 cells (Figure 4.9 A). As expected, inhibition of the autophagy process using 3-MA at 5 and 10 mM resulted in down-regulation of autophagosome formation in Caco-2 cells (Figure 4.8). In contrast, following induction of autophagosomes with 50 μM CQD, the density of LC3B detected was higher than that observed in Caco-2 cells without chemical treatment (Figure 4.8), a finding that is consistent with the activity of this compound. Further evidence supporting the role of autophagy was the finding that following infection of Caco-2 cells with C. concisus UNSWCD, the bacterium was found to co-localise with LC3B (Figure 4.9 B-I). Moreover, some cells appeared to be hyper-infected by C. concisus UNSWCD (Figure 4.9 F, J). The fact that C. concisus UNSWCD interacts with the autophagosome provides further evidence that autophagy is involved in the intracellular survival of C. concisus within host cells, and raises the possibility that C. concisus could employ the autophagy process to survive intracellularly. A similar observation has been made with C. jejuni, this bacterium being shown to co-localise with GFP-LC3-labeled structures consistent with autophagosomes (191). In a recent study, Lam et al. reported Listeria monocytogenes to co-localise with LC3, a finding that led them to suggest that this co-localisation develops into spacious Listeria-containing phagosomes, membrane- bound compartments that harbour slow-growing bacteria associated with persistent infection (197). Interestingly, analysis of autophagy gene expression following infection of Caco-2 cells with C. concisus UNSWCD gathered from the collective autophagy gene expression 190 Chapter 4: Autophagy in the intracellular survival of C. concisus data as shown in (Table 4.1), suggests that C. concisus exerts a weak inhibitory/dampening effect on the autophagy process (approximately 80% of normal levels). Additionally, this finding also indicates that the bacterium can exert some level of manipulation of the autophagy process. Although the fold changes may appear small they are consistent with other reports using similar Human Autophagy RT2 Profiler™ PCR Arrays from SA Biosciences (198) as well as other methods examining autophagy gene expression (199). Moreover, the down-regulation of both FADD (associated with caspase 8) (200) and BAX (accelerates apoptosis) (201), suggests that host cells infected with C. concisus down-regulate autophagy-related apoptosis. Genes involved in the regulation of autophagy by the host cell appeared to be down- regulated via the effect on the genes IGF1, MAPK14 and HDAC1 (Table 4.1). Both IGF1 and MAPK14 have been shown to be inhibitors of autophagy (202, 203). However, this down-regulation is not limited to inhibitors of autophagy. HDAC1 is a histone deacetylase that has also been shown to induce autophagic gene expression in mice (204), indicating that host regulation of the autophagy process as a whole is affected by the bacterium. In mammalian cells, autophagy-related proteins can be separated into five distinct subgroups: (i) the ULK1 protein-kinase complex, (ii) the ATG9-WIPI complex, (iii) the Vps34-beclin1 class III phosphoinositide 3 (PI3)-kinase complex, (iv) the ATG12 conjugation system, and (v) the LC3 conjugation system (130). ATG9B and WIPI1, which are two components of the ATG9-WIPI complex and are involved in the nucleation of autophagic vesicles (130), were shown to be down-regulated upon infection with C. concisus (Table 4.1). Moreover, AMBRA1, a positive regulator of beclin-1 that regulates autophagy through its association with the Vps34-beclin1 class 191 Chapter 4: Autophagy in the intracellular survival of C. concisus III PI3-kinase complex, was also down-regulated. This complex has also been associated with autophagosome nucleation (130). The LC3 conjugation system was also affected by C. concisus infection, through the down-regulation of both ATG4B and ATG7 (Table 4.1). ATG4B and ATG7 are involved in the conversion of proLC3 to LC3-I to LC3-II and back to LC3-I, a process that regulates autophagosome maturation and fusion of autophagosomes with lysosomes (130), suggesting that infection leads to inhibition or dampening of these processes. Notably, MAP1LC3B was significantly up- regulated, with fold level changes correlating to the decreases in ATG4B and ATG7 transcription (Table 4.1). The up-regulation of MAP1LC3B is considered a marker of autophagy inhibition (194). Further evidence to support the dampening effect on autophagosome-lysosome fusion comes from the down-regulation of LAMP1, CTSD and CTSS (Table 4.1). LAMP1, also known as CD107a (Cluster of Differentiation 107a), is a critical lysosomal adaptor protein that is involved in the interaction and fusion of the lysosomes with various cell components (130, 139). Moreover, deficiencies in both Cathepsin D (CTSD) and Cathepsin S (CTSS) have been shown to result in accumulation of autophagosomes and impairment of autolysosome degradation (205, 206). Interestingly, in a previous study investigating the changes in protein expression upon infection with C. concisus, cathepsin D was also identified to be down-regulated (109), indicating that these changes are reflected at the protein level. Collectively, these results show a dampening effect on autophagosome maturation and autophagosome-lysosome fusion, suggesting that C. concisus may utilise autophagic vesicles that have not matured for its intracellular survival. Similarly, the closely related pathogen C. jejuni forms the CCV, which associates with the early endosomal markers Rab 4 and Rab 7 to escape lysosomal degradation (129). 192 Chapter 4: Autophagy in the intracellular survival of C. concisus 4.9. Conclusion This study has shown for the first time that autophagy plays an important role in modulating the intracellular levels of C. concisus strains. Moreover, the results of this study would suggest that differences in the intracellular levels of C. concisus strains are associated with the ability of these strains to interact with the autophagy process, with some strains being able to evade destruction by autophagy more efficiently than others. Previous studies by our group have suggested that the increased ability of C. concisus to survive intracellularly might be due to four conserved genes on a plasmid within some strains (109, 126). Thus, it is plausible that these C. concisus genes may interact with the host autophagy process to enhance the intracellular survival of this bacterium. To determine whether the 4 conserved genes are interacting with the host autophagy process further studies could be undertaken in which a knock out system is developed to individually knockout the 4 conserved genes. The effect of knocking-out each of the genes on interaction with the autophagy process could then be assessed by determining if there is a change in the ability of the C. concisus KO strains to invade and survive intracellularly within host cells as compared with the C. concisus wild type strains. Interestingly, C. concisus has been associated with Crohn’s disease (CD) (41, 207). Given that polymorphisms in the autophagy genes ATG16L1 and IRGM are known to confer susceptibility to CD (145, 146) and that an increased number of autophagosomes are observed in CD patients (208), further investigation of the function of C. concisus as an initiator of this disease should be the focus of future studies. 193 CHAPTER 5 5. DEVELOPMENT OF A TRANSMISSION ELECTRON MICROSCOPY PROCEDURE TO INVESTIGATE THE MODULATION OF THE AUTOPHAGY PROCESS BY CAMPYLOBACTER CONCISUS 5.1. Background In Chapter 4, investigation of the role of autophagy following infection of the Caco-2 cell line with C. concisus demonstrated that while all strains of C. concisus could invade these cells, only some strains were able to evade the autophagy process and survive within Caco-2 cells (81). This finding was supported by the confocal microscopy studies that showed co-localistion of C. concisus UNSWCD with autophagosomes, and by the gene expression studies, which showed the regulation of genes involved in the autophagy process (81). While the results of these studies are of significant interest, they did not provide an in depth view of what is occurring within the cell during the autophagy process. Given that autophagy is such an active and ubiquitous process within host cells, it was considered important to gain a more in depth understanding of the interaction between the autophagy process and C. concisus (175, 209). Indeed, it is now considered essential that a range of methods be used to determine how the autophagy process functions within host cells following pathogen infection. Given that transmission electron microscopy (TEM) remains the optimal technique for the visualisation and characterisation of the morphological events of autophagy. Thus, in this chapter TEM 194 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus was employed to investigate the autophagy process following infection of Caco-2 cells with C. concisus. The human intestinal epithelial cell Caco-2 has been instrumental to in vitro studies examining the adherence and invasion of intestinal pathogens to host epithelial cells. Caco-2 cells, originally derived from a human colonic adenocarcinoma (210), have the ability to differentiate spontaneously in vitro into a highly polarised cell that has many features of small intestinal epithelium. These include microvilli on the apical surface, tight junctions, as well as a range of cell membrane receptors and brush border enzymes that are present in small intestinal epithelial cells (211-215); this process is very similar to the sequence of events that occurs in vivo when immature intestinal cells mature (210). Due to the above mentioned characteristics Caco-2 cells now serve as a recognised in vitro cell line model and have been used to investigate a range of research questions including drug trafficking across the intestinal epithelial barrier (216, 217), intestinal barrier function (216, 217), the ability of pathogens to attach to and invade intestinal cells (218) and investigation of xenophagy, a process similar to autophagy that mediates the removal of intracellular pathogens (219-221). Indeed the use of Caco-2 cells has been pivotal in investigations of xenophagy induction in pathogen infections (222, 223). For example, Kuballa et al. used a Caco-2 cell-line model to investigate if the host genetic polymorphism ATG16L1*300A was a protective factor in Crohn’s disease (CD) (223). This study showed that the ATG16L1*300A polymorphism in Caco-2 cells impaired the capture of internalised S. Typhimurium within autophagosomes. Given this, the authors hypothesised that an increased risk of CD may result from impaired ATG16L1 function, and suggested that this *300A polymorphism might lower the rate 195 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus of bacterial capture by the autophagic system (223). A further study by Gutierrez et al. utilised Caco-2 cells to determine the manner by which a Vibrio cholera haemolytic exotoxin, Vibrio cholera cytolysin (VCC), interacted with the autophagic pathway (224). Interestingly, this study showed that in several cell lines, including Caco-2 cells, that VCC-induced vacuoles co-localised with LC3, suggesting that an interaction had occurred between the vacuoles and autophagic vesicles. This was visually re-confirmed using TEM (224). TEM studies have significantly increased our knowledge in a range of fields, most notably Biology and Medicine. Unlike other approaches TEM allows the visualisation and characterisation of host cell morphology and cytoplasmic ultrastructures, including ribosomes, rough endoplasmic reticulum and mitochondria (225, 226). By the commencement of the 1950s the use of fixation in buffered osmic acid, embedding in hard plastic resin and ultra-thin sectioning via glass knives were key established elements used for morphological studies of biological samples at an ultrastructural level (227, 228). However, often the procedures outlined in early studies resulted in ultrastructural artifacts caused by inadequate sample preparation processes (227, 228). Nevertheless, in 1962 autolytic vacuoles were first observed in rat intestinal tissue using TEM (229, 230). This finding marked an important first step forward in the understanding of the role of autophagy in host cells. From these early pivotal studies the functional relevance of utilising TEM in biological applications has expanded considerably to include examination of host-pathogen interactions, including pathogen attachment and invasion, bacterial vesicle formation and for the morphological characterisation of pathogens (219, 231, 232). For example, TEM has been used to investigate the intracellular invasion of another member of the Campylobacter genus, the gastrointestinal pathogen C. jejuni (220, 231). 196 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Biological applications of TEM have the potential to allow the visualisation of molecules with near atomic resolution (233). The advantage of utilising the super resolution capacity of TEM in autophagy research is that it facilitates visualisation of the interaction between invasive pathogens and the autophagy process. An additional advantage is that TEM can clarify, for example, whether the pathogen remains within autophagic vesicles or, alternatively, is cleared away and degraded by the autophagy process (134, 194, 234). For example, the use of TEM to determine the outcome of the interaction between Helicobacter pylori and host gastric cells has shown that this interaction resulted in autophagy induction and the formation of autophagic vesicles (235). In addition, this study showed that H. pylori could replicate within double-layer vesicles either on the plasma membrane or in the cytoplasm, providing evidence that H. pylori was able to manipulate the autophagic system to avoid degradation. Moreover, TEM has been used effectively in vivo mouse studies to show that the autophagic process is effective in the bacterial clearance of Salmonella serovar Typhimurium in intestinal epithelial cells (236) and Pseudomonas aeruginosa in both lung aveolar macrophages and mouse mast cells (192, 237). Importantly, vital ultrastructures within the autophagy process have been identified through TEM by virtue of their morphological classification. These ultrastructures include: autophagosomes, amphisomes and autolysosomes, which are membrane- housed constituents that contain cytoplasmic material or organelles (233, 238, 239). In conventional TEM applications, autophagosomes have been observed to have a double or, occasionally, multiple membranes, which have the same morphology and electron density as the cytoplasm outside the autophagosome (238). TEM has also facilitated the visualisation of late degradative autophagic structures such as autolysosomes and autophagolysosomes (240), consisting of moderately degraded cytoplasmic, organelle 197 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus material (134, 194, 234) and in the case of autophagolysosomes degraded bacterial material from the process of xenophagy, resulting in the elimination of intracellular pathogens (240). While identification of these key autophagic compartments would not have been possible without the technical capability of TEM and the use of super resolution, achieving precise resolution is a rare occurrence, as the resolution itself is influenced by a number of factors, including sample preparation and TEM instrumentation limitations (233). Given these limitations, significant emphasis has been placed on achieving an optimal sample preparation method to ensure that the structure of the cells is preserved and that autophagy ultrastructures can be visualised (233). In order to achieve optimal resolution, studies have focused on developing methods to preserve, as best as possible, the in vivo nature of mammalian cells or tissues (228, 241- 244). The development of TEM sample preparation methodologies that have optimised the fixation, dehydration and resin embedding procedures for mammalian cells have all led to significant advances in the visualisation of ultrastructures (228, 241-244). These advances have provided improved visual quality for the characterisation and identification of autophagosomes and ribosomes within the cytoplasmic contents of autophagy ultrastructures that reside within host cells (227, 228) and have overcome previous problems associated with sub-optimal sample preparation procedures which resulted in misinterpretation of autophagosome compartments (238) including the incorrect classification of rough endoplasmic reticulum, mitochondria and translucent vacuoles being as autophagosomes (238) (Figure 5.1). 198 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus The sensitivity and specificity of TEM, has been shown to heighten the resolution of regions within the endoplasmic reticulum. Subsequently, these advantages have had profound effects on specific sites of the autophagosomes present in mammalian cells, which has resulted in a significant turning point in the general understanding of the autophagy process within mammalian cells (134, 194, 234). Moreover, the application of TEM has enabled the qualitative characterisation of initial autophagic compartments including autophagosomes, comprising of morphologically-integral cytosol or organelles (134, 194, 234). These qualitative characterisations have only been made possible through the continued development of optimal conventional sample preparation procedures focused on autophagy examination. 199 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Figure 5.1. TEM image showing (A) autophagic compartments found in a mouse fibroblast and (B) an image of a mouse hepatocyte showing the misclassification of autophagic ultrastructures as an autophagosome. A: Ribosomes can be visualised within an autophagosome-like compartment (1). In a second compartment (2), the ribosomes appear to have a greater electron density and are able to form dark granular masses denoted as (Ly), representing dense lysosomes. B: Misclassified autophagosomes are denoted as (?). Rough endoplasmic reticulum often surrounds organelles such as mitochondria indicated by (?). Sometimes these structures are mistakenly interpreted as autophagosomes. The insert shows the appearance of ribosomes on the endoplasmic reticulum membrane that show some granularity. This image also shows two visible autophagosomes (1). The autophagosome on the left is comprised of a ring-structured cistern made up of rough endoplasmic reticulum with the limiting membrane of this autophagosome indicated by arrowheads. Image taken from Eskelinen et al. (238). The preparation of larger sized TEM samples including tissues, mammalian cells or cellular organelles is based on the principle of turning larger and thicker specimens into 200 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus smaller thinner specimens, which can later be observed. Larger samples require fracturing or sectioning so they can be viewed by TEM (241), with sample thickness requiring considerable reduction to allow the electron beam to penetrate through the specimen (241). It is essential that the sample preparation and sectioning methods used are properly maintained to ensure that the sample closely resembles its prior live state without obvious structural damage, dimensional change, or marginal loss of biological material (241). Importantly, to minimise the chance of technical errors, conventional TEM methods for larger specimens requires a number of fundamental steps including: aldehyde fixation, post-fixation with osmium tetroxide, dehydration with alcohol from 30-100%, and embedding in resin that is polymerised by heat (238). The purpose of the fixation process is centered on the principle of preserving the structure of biological specimens, in order to avoid drastic distortions during later processes, including: dehydration, embedding, sectioning, staining and viewing (244, 245). Hence, the importance of fixation in TEM cannot be over-stated (246, 247). A range of fixation methods can be utilised that involve either physical or chemical procedures (242). Physical fixation methods such as cryopreservation were not applied in the current investigation as cryofixation is not recommended for mammalian cell monolayers, as its penetration depth is very limited (242). Moreover, there is also a risk of ice crystal build up that causes damage to larger sized specimens, as they cannot release heat quickly enough to avoid ice crystal build up (242). For these reasons chemical fixation was chosen as the fixation method in this investigation. 201 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Chemical fixation is the principal step in stabilising the cellular organisation of mammalian cells. In 1963, Sabatini et al. (248) was the first to recommend the use of glutaraldehyde, as a primary fixative, followed by secondary fixation with osmium tetroxide. This double fixation process is a regimen that is still commonly used in TEM (241) as it allows the sample to be stored for a period of time in buffer before further processing is established (241). The advantages of chemical fixation include the relatively stability of chemicals, the absence for the need of significant equipment and the relatively low cost of fixatives. Additionally, this also includes the fact that the specimen-handling technique can be simplified, and that materials can be stored in solution for some periods of time (241). Evidence of successful fixation procedure include the following: proof of granular material in the cytoplasm and nucleoplasm, the appearance of the nuclear envelope membrane of cells being in parallel without significant swelling. Additionally, this also includes the appearance of the mitochondria as intact and not collapsed, ultrastructure remnants not lacking electron density, with even parallel inner and outer membranes; a normal visualisation appearance of the endoplasmic reticulum without evidence of being swollen or shrunken; and all cellular membranes and cellular components appearing intact and not distorted (243). Taking these factors into consideration has enabled the development of fixation methods specific for biological specimens. One such fixation method is the immersion fixation procedure, which is most commonly used for cell monolayers, cell pellets or removed tissues. Apart from the use of primary chemical fixative procedures, it has also been shown that the combination of two chemical fixation procedures, termed double chemical fixation, functions as an enhanced fixative process. This double fixation method has been 202 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus integrated into the conventional TEM preparation of biological specimens. One such secondary chemical fixative is osmium tetroxide (OsO4), which acts as a slow penetrative fixative that interacts directly on lipids, proteins and lipoproteins (242, 246). In this fixative-reduced state, it turns the specimen black, becoming a key visual marker in distinguishing the location of the biological specimen during sample processing and sectioning procedures (242). A distinct advantage is that osmium tetroxide can preserve fine cellular structures, and is generally used after aldehyde fixation (242). It should be noted, however that the use of osmium tetroxide does have disadvantages, including being a slowly penetrating fixative and lacking the capacity to cross-link cellular components which reduces the ability of biological processes to be fixed in real time (242). Osmium tetroxide also serves as a fixative stain, which adds electron density to the sample. Moreover, the reduced form of osmium tetroxide is un-extractable and also has the added benefit of displaying electron density under an electron beam, due to its nature as a heavy metal fixative (242). This double fixation of glutaraldehyde with osmium tetroxide as part of conventional TEM sample preparation has been used effectively in the determination and monitoring of autophagosomes (228, 241, 245, 249, 250) adding credibility to its use in autophagy research. Furthermore, in conventional TEM samples the number and contrast of autophagosome limiting membranes may vary, which is believed to be due to limitations in the preservation of lipids during sample preparation. Typically, a mammalian cell autophagosome has two distinct double-layered cellular membranes. However, problems may arise in identifying autophagosomes as they may have only one electron dense membrane, or appear as several separate membranes (238). Furthermore, on rare 203 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus occasions the limiting membrane may not have contrast at all, which may result in the formation of lipid extraction during sample preparation (238). Many conventional TEM sample preparation methods have proven ineffective for accurate interpretation of autophagy ultrastructures like the autophagosome (238). Thus, improved optimised methods for the preservation of lipids has been recommended if the exact morphology of limiting membranes are to be investigated (238, 251, 252); the current investigation has endeavored to maintain this consistency. A range of methods have been utilised to investigate the autophagy/xenophagy process in host cells, with in situ adherent cell types commonly used. However, many are time- consuming, laborious and technically difficult to replicate without adequate technical expertise and specialised equipment. The goal in sample preparation of in situ adherent cells is to successfully carry out the preparation procedure without disturbing the cell monolayer sheath or damaging it (242, 243). The most common sample procedures used include the agar-peel technique for in situ cells, agar embedment and non-agar embedment procedures (242, 243). The agar-peel method is a technique that is typically used on in situ cells (242), the principle behind this technique being to contain the adherent monolayer sheath within molten agar (242). However, this technique carries the risk of the cell monolayer being lifted off and damaged during ethanol treatment, resulting in disintegration of the cell monolayer. Moreover, this method uses the conventional TEM procedures of fixation, dehydration, and resin polymersiation that requires the use of a Jeweler’s saw to cut out each individual agar piece on blank blocks for sectioning (242), making it more physically demanding and a riskier procedure in relation to the preservation of biological material. 204 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus The agar embedment method has also been utilised for adherent cell monolayers (228, 243). In contrast to the non-agar embedment method, agar embedment has fewer disadvantages and has been commonly used for TEM investigation of host mammalian cells (228, 243). This procedure involves the embedment of the sample in molten agar to cover and protect it from damage or loss throughout the preparation process. However, the use of molten agar and centrifugation may potentially cause heat shock to the already sensitive cells that have been exposed to fixative solutions (228, 243). The non-agar embedment process involves the careful removal of the monolayer sheath off a flask, petri dish or microtiter plate post fixation. While this method has been reported to result in minimal loss of the sample, studies investigating autophagy ultrastructures in mammalian cells have successfully used both agar embedment (228) and the non-agar embedment procedure for these investigations (236, 245, 250). Without the effective use of a resin medium the visual resolution capacity of TEM would not be so sharp. The use of resin embedment is a critical process for the stability and uniformity of biological specimens. Thus, the correct choice of resin medium is critical for the interpretation of the autophagy ultrastructures, as the resins help maintain the lipid density that is required for visualisation of the biological sample. Resin mediums are used to embed the specimen in a medium that can later be polymerised into a solid block for sectioning and visualisation (241, 242). The three main commercially available mediums include epoxy resins, methacrylates and polyester resins (240). Essential qualities of resin embedding media include uniformity in resin components from batch to batch, good stability and solubility in dehydration agents, low viscosity as a monomer, minimal shrinkage and distortion with little change in sample volume, uniform polymerisation, stability under the electron beam, and minimal granularity, although in many cases all of these ideal qualities is not easily attainable 205 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus (241, 242). For instance, resins with low viscosity tend to shrink significantly, which affects the sample proportionality and thus interpretation of the image (241, 242). Epoxy resins such as Epon 812 have the advantage of polymerising uniformly with little change in sample volume (241, 242). The epoxy resins are also relatively stable in the presence of the electron beam and usually avoid floating off while being visualised (241, 242). A primary disadvantage of epoxy resins, however, is their high viscosity and the lengthy infiltration process required as compared with methacrylate resins (241, 242). Finally, methracylate or acrylic resins like London Resin (LR) white, which were used in the current investigation are still widely used as embedding media in electron microscopy (241, 242). These types of resin have a number of advantages, they are less toxic and carcinogenic than epoxy and polyester resins, which makes them more suitable for cyto-chemical studies (241, 242). Moreover, they remain stable in a liquid state without polymerising and unlike epoxy resins are very useful for dehydration and infiltration procedures in cold temperatures, (241, 242). This allows a more optimal resin medium infiltration process, ensuring that the ethanol-dehydrating agent is properly eliminated. Further, rotation of the sample on a slow rotator throughout the duration of infiltration process is recommended to avoid the deterioration of the mammalian cells in the sample (241, 242). Regardless of which resin is utilised all have advantages and disadvantages. Importantly, the general changes which occur post infiltration are minimal when compared with fixation and dehydration processes which cause the majority of cellular sample shrinkage (241, 242). A key factor is achieving uniformity in the resin medium 206 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus components from batch to batch. Importantly, TEM sample preparation methods used for sample resin embedment and polymerisation are focused on eliminating oxygen using a flat embedding process, or a capsule embedding process in gelatin or polyethylene capsules (241, 242). Given that sample preparation is known to be critical to the outcome of the TEM studies, we chose, to compare two different approaches to sample preparation, these being, with and without agar embedment prior to performing the TEM studies. The rationale for this is that previous studies have reported agar embedment to protect samples from damage or loss during the washing, dehydration and resin embedding process. 5.1.1. Aims The aims of this chapter are: (i) To develop and optimise the TEM sample preparation procedure for the visualisation of autophagy ultrastructures and C. concisus infection within Caco-2 cells. (ii) To visualise and examine the process of autophagy within intestinal epithelial Caco-2 cells following infection with C. concisus UNSWCD. 207 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus 5.2. Materials and Methods 5.2.1. Biological sample preparation using the agar embedment method For these studies, Caco-2 cells were seeded and grown as outlined in Chapter 4 section 4.2.3. The Caco-2 cells were then infected with C. concisus at a MOI of 200 and incubated for 6 h as outlined in Chapter 4 section 4.2.3. Following the 6 h infection incubation period the Caco-2 cells were washed three times for 10 min using 0.1 M Sorensen’s Phosphate buffer (SPB) pH 7.2 (0.133M Na2HPO4; 0.133M KH2PO4), after which the monolayers were fixed overnight at 4°C in 2 ml of a fixative solution comprising 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M SPB (ProSciTech; Kirwan, QLD, Australia). Following chemical fixation, the samples were washed three times for 10 min using 0.1 M SPB after which 1% osmium tetroxide (ProSciTech) was added to the cell monolayer. Following this immersion the osmium tetroxide solution was allowed to set over the cell monolayers for 1 h. The cell monolayer was then washed three times for 10 min using 0.1 M SPB after which the monolayer was lifted off the plate using a cell scraper (Nunc) and centrifuged in an eppendorf tube at 232 × g for 5 min (Figure 5.3, Phase 1). Following centrifugation, 1 ml of molten Nutrient Agar # LP0011 (Oxoid; Hampshire, England) at a concentration of 4% was added to the eppendorf and this was placed on ice to allow the agar to set (Figure 5.2). To avoid shock to the cells the agar was left in ice for no longer than 2 min. The agar was then removed from the eppendorf by cutting the base of the tube. The agar encased biological sample was then trimmed to approximately 1 mm in size using a thin steel blade (ProSciTech) as outlined in (Figure 5.3, Phase 2). The agar embedded samples were then collected and transferred to a sterile 2 ml eppendorf tube (Axygen Scientific; Union City, CA, USA), after which they 208 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus were dehydrated using increasing concentrations of ethanol (30%, 50%, 70%, 90%, 95%), each for 10 min (Figure 5.3, Phase 1). The agar embedment method described by Dykstra et al. (243) was used. This method was based on the objective of providing a protective matrix around the biological material so that the loss of biological material throughout the sample preparation process did not occur (243). Throughout the sample preparation process the sample fragments embedded in agar were handled like sliced tissues, thus avoiding shearing artifacts on the cells that can occur due to vigorous pipetting and centrifugation (243). A detailed schematic representation of the agar embedding procedure is outlined in (Figure 5.2, Panel A). This image shows the agar layer providing a protective coating on top and around the Caco-2 cells. To improve resin embedment the samples were trimmed down to smaller fragments using a fine blade (ProSciTech) (Figure 5.2, Panel B). 209 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Figure 5.2. Agar embedment and trimming of the biological samples to produce smaller sample fragments. A: The image on the left shows the sample embedded in agar (yellow) and on the right a schematic representation of the sample after removal from the eppendorf tube, which shows the agar encasing the entire cell pellet (brown colour). B: A schematic representation of the trimming of the biological sample and the agar (yellow colour) coating and protecting the monolayer cell pellet (brown colour). Once dehydrated the samples were infiltrated with LR white resin at 1 h intervals. Initially, a 1:1 mixture of 100% ethanol and LR white resin was used, followed by a 9:1 mixture of ethanol: LR white resin for 1 h. This was then followed by three final infiltrations with 100% LR white resin each for a period of 1 h. For the resin embedment process a roller mixer (BTR5-12V) (Ratek Laboratory Equipment; Victoria, Australia) was used, which allowed the samples to come in contact with either the solvent or resin. After the final 1 h infiltration with 100% LR white resin, the samples were placed in silicon flat embedding molds half filled with 100% LR white resin, and placed as close to the edge as possible before covering them with a rectangular 210 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Thermanox slide (ProSciTech). The material in the mold was then allowed to polymerise for 24 h at 60°C in a drying oven (Thermoline Scientific; Wetherill Park, Sydney, Australia). The resin blocks were then removed from the Thermanox slide casing, released from the silicone mold after which they were stored at room temperature (Figure 5.3, Phase 2) until semi-thin (1 µm) and ultra-thin (70 nm) sectioning. Figure 5.3. An outline of sample preparation using the agar embedment method for TEM. Sample preparation involved three distinct phases. Phase 1 involved Caco-2 cell seeding, bacterial infection, fixation, washing, osmium tetroxide post fixation, agar embedment and ethanol dehydration at concentrations ranging between 30-100%. Phase 2 involved embedding the samples in nutrient agar, trimming the samples to the appropriate size followed by the LR white resin embedment process. Phase 3 involved the cutting of ultra-thin (1 µm) and semi-thin (70 nm) sections using an ultramicrotome, followed by staining of the sections with uranyl acetate (UA) and Reynold’s lead citrate (RLC) on a formvar grid. Samples were then viewed using a TEM JEOL 1400 microscope (JEOL USA, Inc., Peabody MA) at 100 kV. 211 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus 5.2.2. Biological sample preparation without agar embedment To determine if the agar embedment process was essential to maintaining sample integrity, sample preparation without agar embedment was also investigated. The initial steps of this procedure were identical to that outlined in section 5.2.1. However, in this case the monolayer sheaths were not centrifuged inside an eppendorf tube and no agar was added. In this case the samples were directly embedded in 100% LR White (ProSciTech) and then encapsulated in gelatin capsules with 100% LR white and polymerised for 24 h at 60oC in a drying oven (Thermoline Scientific) (Figure 5.4, Phase 1). The sample blocks were then stored at room temperature until semi-thin (1 µm) and ultra-thin (70 nm) sectioning was performed (Figure 5.4, Phase 2). (243). The general principle behind this procedure is that the cell pellets and tissues are trimmed into smaller fragments, before being immersed with freshly prepared fixative. For cellular monolayers, the monolayer is flooded with fixative solution, after which the monolayer is scraped off the plate with a rubber spatula, collected and pelleted into a cellular pellet via centrifugation. 212 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Figure 5.4. An outline of the TEM sample preparation procedure without agar embedment. The process used to prepare the TEM samples without agar embedment involved three distinct phases. Phase 1 involved Caco-2 cell seeding, bacterial infection, fixation, washing, osmium tetroxide post fixation, ethanol dehydration at concentrations ranging between 30-100% and sample embedding into London Resin White directly. Phase 2 involved the preparation of semi- thin (1 µm) and ultra-thin (70 nm) sections using an ultramicrotome. Phase 3 involved staining of the semi-thin section with Toluidine Blue and the ultra-thin samples with UA and RLC on a grid followed by visualisation of the sections using a TEM JEOL 1400 at 100 kV. 213 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus 5.2.3. Ultramicrotomy Ultramicrotomy sectioning was performed using a Leica EM UC6 ultramicrotome (Leica; Wetzlar, Germany). Resin blocks were fixed into an ultramicrotome chuck and trimmed with a fine metal razor (ProSciTech) under the guidance of a Leica dissecting microscope (Leica). This process involved the removal of any excess LR white resin to expose a flat trapezium shaped block face. The sides of the block leading down towards the chuck, and away from the face to be cut, were trimmed at an approximately 45 degree angle to the face. The leading edge and top edges were then trimmed to be parallel and the side edges trimmed at an angle of ~30 degrees, such that the leading edge was slightly narrower than the top edges, which were perpendicular to each other. Glass cutting knives were prepared in a Leica EM KMR2 knife maker (Leica) and a plastic water trough was attached to the glass knife using hot wax to facilitate the capture of the sectioned samples from the water trough. 5.2.4. Semi-thin sectioning and staining Semi-thin sections were cut from the block face using a setting of 1 mm/s, and a 1000 nm section thickness. The semi-thin sections were retrieved onto a glass cover slide (Livingstone; Rosebery, NSW, Australia) and stained with Toluidine Blue for 1 min. Semi-thin sections were then dried on a heat block (Leica) and viewed using a DP-37 light microscope (Olympus; Notting Hill, VIC, Australia) with a DP-37 mounted camera (Olympus). Samples were then viewed to locate areas where cells were present in high abundance, thus narrowing down the areas to be ultra-thin sectioned. 214 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus 5.2.5. Ultra-thin sectioning Glass knives containing attached water baths were prepared as described in Figure 5.4. Ultra-thin sections (70 nm) were cut from the block face using a setting of 1 mm/s. The entire glass knife was changed and re-aligned after every 20 sections to avoid cutting marks within the sections. Sections with a silver to pale gold colour were selected for further analysis, this selection process being applied to both the agar and non-agar methods. Following selection, the sections were placed in close proximity to one another using an eyelash tool (constructed by attaching an eyelash to a wooden toothpick using super glue). Groups of sections (on average 4-5 sections) appearing in a grouped ribbon formation were then picked up from the surface of the water and placed onto 6 mm × 6 mm square formvar coated copper grids. The grids were subsequently dried by capillary action using filter paper and then stored at room temperature prior to staining. 5.2.6. Staining of ultra-thin sections A standard contrasting technique recommended for TEM was used for staining the sections (228, 241-243). This method employs a double contrast approach in which uranyl acetate (UA) and Reynold’s lead citrate (RLC) are employed (228, 241-243). UA enhances the contrast by interaction with lipids and proteins, while RLC enhances the contrast by interacting with proteins and glycogens. Each sample grid containing the collected ultra-thin section was stained using 8% UA and 8% RLC (ProSciTech). For each grid a drop (constant volume) of RLC (~ 26 g/L PbNO3, 35 g/L sodium citrate) was placed onto a square of parafilm within a glass petri dish. The glass petri dish was then humidified to remove CO2 and to prevent the precipitation of the RLC. To establish optimal humidification, one pellet of sodium hydroxide (NaOH) per grid section was 215 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus added to ensure that the conditions within the petri dish remained alkaline. The petri dish was then covered and set aside for later use. In another glass petri dish, one drop (constant volume) of UA (ProSciTech) in 50% ethanol was placed onto a square of parafilm. Initially, the grids containing the collected ultra-thin sections were placed face down into the drops of UA for 2 min, after which the front and back face of the grids were washed in running distilled water for 30s. After washing, the grids were placed face down in the RLC solution for 20 min after which they were thoroughly washed under a steady stream of dH2O. The grids were then dried using filter paper and placed face down in the RLC solution and stained for a further 4 min in a humidified environment, prepared as outlined above. The grids were then washed thoroughly with dH2O on both surfaces and dried with filter paper. The grids were then stored in sample holders at room temperature until the time of TEM examination. 5.2.7. Transmission Electron Microscopy imaging procedure Both agar embedded and non-agar embedded samples were viewed using a JEOL JEM- 1400 TEM microscope (JEOL USA, Inc., Peabody MA) mounted with a Gatan Erlangsheen ES5000W CCD camera model 782. The samples were visualised at an acceleration voltage of 100 kV at 54 µA. 216 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus 5.3. Results 5.3.1. Optimisation of Transmission Electron Microscopy sample preparation To compare the integrity of samples prepared with and without agar embedment, both semi-thin and ultra-thin sections of non-infected Caco-2 cells and those infected with C. concisus UNSWCD were prepared and then examined using TEM. 5.3.2. Visualisation of semi-thin sections prepared using agar embedment, as an assessment for progression to ultra-thin sectioning The semi-thin sections (1 µm) were initially assessed to evaluate the structure and integrity of the Caco-2 cells and ensure that sufficient biological material was present after the chemical fixation, dehydration and resin embedding procedures were completed. Visualisation of the samples that had undergone agar embedment, showed agar encasing the Caco-2 cells with and without C. concisus infection and (Figure 5.5). Moreover, the integrity and cellular structure of the Caco-2 cells was well preserved, and distinctive basolateral and apical membranes were observed (Panels A & B). Additionally, the possible presence of microvilli were observed on the apical membrane surface of the Caco-2 cells (Panels A & B) indicated by (single arrow heads). However, the agar encasing the sample made it difficult to differentiate these microvilli from the surrounding agar. 217 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Figure 5.5. Agar embedded semi-thin sections (1 µm) of uninfected Caco-2 cells and C. concisus UNSWCD infected Caco-2 cells. Semi-thin sections (1 µm) prepared as described above were examined using a DP-37 light microscope. A: Non-infected Caco-2 cells surrounded by agar (*). The Caco-2 cells appear to be structurally well preserved and can be seen surrounded by agar (*). B: Caco-2 cells infected with C. concisus UNSWCD at a MOI of 200. The Caco-2 cells surrounded by agar (*) appear well preserved and are not structurally altered or damaged. The boxed region shows what appear to be C. concisus UNSWCD within the Caco-2 cells. Possible microvilli are indicated by single arrow heads. Scale bars represent 20 µm with the images taken at 40 × magnification. 218 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus 5.3.3. Visualisation of semi-thin sections without agar embedding, as an assessment for progression to ultra-thin sectioning The samples prepared without agar embedment showed very similar results to those prepared with agar embedment. Moreover, the integrity and cellular structure of the Caco-2 cells was well preserved, and distinctive basolateral and apical membranes were observed (Fig 5.6, Panel A) with microvilli projecting outwards from the apical membrane surface (Figure 5.6, Panel A-boxed area). Upon C. concisus UNSWCD infection, no structural differences or loss of integrity of the Caco-2 cells were observed (Figure 5.6, Panel B), when compared with samples prepared using agar embedment. Distinctive basolateral and apical membranes were again observed, with microvilli projecting outwards from the apical membrane surface. Additionally, C. concisus UNSWCD bacterial cells were observed inside Caco-2 cells (Figure 5.6. Panel B - boxed area). 219 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Figure 5.6. Non-agar embedded semi-thin sections (1 µm) showing uninfected Caco-2 cells and C. concisus UNSWCD infected Caco-2 cells. Semi-thin images of sections (1 µm) were prepared as described above and examined using a DP-37 light microscope. A: Shows non-infected Caco-2 cells. The structure and integrity of the Caco-2 cells is well preserved. Microvilli are observed projecting outwards from the apical membrane surface (single arrow heads). B: Shows that the structure and integrity of the Caco-2 cells infected with C. concisus are also well maintained, with microvilli being observed projecting from the apical membrane surface (singular arrow head). Aggregates of C. concisus UNSWCD can be observed inside Caco-2 cells (boxed region). Scale bars on all images are 10 µm with the images taken at 100 × magnification. 220 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Given that visualisation of the semi-thin sections of samples prepared both with and without agar embedment showed sufficient biological sample to be present and the cell structure and integrity to be maintained, ultra-thin sections were prepared to provide a more in depth view of the samples prepared with and without agar embedment. 5.3.4. Visualisation of ultra-thin sections prepared using Caco-2 cells and C. concisus UNSWCD agar embedded samples under Transmission Electron Microscopy Visualisation of the ultra-thin sections prepared from agar embedded samples of Caco-2 cells alone using TEM showed the microvilli on the apical membrane of the cell to appear disjointed and unevenly distributed over the apical membrane surface (Figure 5.7, Panel A). In some cases the microvilli on the apical membrane were completely non-existent (Figure 5.7, Panels B & C). Visualisation of Caco-2 cells infected with C. concisus UNSWCD for 6 hours showed the bacterium inside the Caco-2 cells (Figures 5.7, Panels B & C), thus providing evidence that C. concisus UNSWCD had internalised into the cell, which is in line with our previous results (Chapter 4). Additionally, C. concisus UNSWCD appeared to be in aggregated masses within the Caco-2 cell with a clear layer surrounding each bacterium (Figures 5.7, Panels B & C). It is possible that this layer may represent the formation of a Campylobacter containing vacuole (CCV), a finding that has been reported in other gastrointestinal pathogens such as C. jejuni (129). Furthermore, C. concisus UNSWCD was observed in close proximity to what appears to be the formation of an early or an intermediary phagophore-like ultrastructure (Figure 5.7, Panel D) indicated by the presence of a single membrane phagophore with the mitochondria being in close proximity to the bacterial cell. 221 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Figure 5.7. Transmission Electron Microscopy images of ultra-thin sections obtained from samples prepared using agar embedment. A: An uninfected Caco-2 cell showing the apical membrane surface with microvilli, which appear to have broken away from the apical membrane surface (triangular-headed arrows). A clear nucleus was observed (Nu) (10,000 × magnification, 2 µm scale bar). B: Aggregates of C. concisus UNSWCD bacterial cells (*) can be observed inside the cell. The structure of the Caco-2 cell appears to be damaged on the basolateral surface (#). A lack of uniformity and distribution of the microvilli is also observed on the apical membrane surface (double-headed arrows) (20,000 × magnification, 1 µm scale bar). C: An aggregate of C. concisus UNSWCD bacterial cells (*) can be seen within the Caco-2 cells that appear to be surrounded by a capsule like structure (boxed region) (20,000 × magnification, 1 µm scale bar). D: A mitochondria organelle with clear distinct cristae can be observed in close proximity to what could be a maturing autophagosomal phagophore complex within the boxed region. Additionally, staining artifacts were observed, appearing as crystal precipitates (oval region), (50,000 × magnification, 0.5 µm scale bar). 222 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus 5.3.5. Visualisation of C. concisus using Transmission Electron Microscopy without agar embedment In order to better understand the interaction between C. concisus and autophagosomes within Caco-2 cells, it was important to visually confirm the morphology of C. concisus under TEM conditions and establish its presence inside Caco-2 cells (Figure 5.8, Panels A & B). The association between C. concisus infection and the autophagy process was also investigated using samples prepared without the agar embedment procedure. Visualisation of C. concisus using TEM revealed a curved rod, 4 µm long × 0.5 µm wide, which is in line with its reported morphological appearance (Figure 5.8 A & B). Figure 5.8. Transmission Electron Microscopy ultra-thin images of Campylobacter concisus UNSWCD without agar embedment. A: The bacterium was densely stained, showing a spiral shaped morphology. B: The dimensions of C. concisus UNSWCD appeared to be 4 µm long × 0.5 µm wide. An outer layer appeared to be forming around the bacterium (indicated by arrows). Both images shown in (Panels A & B) were taken at 20,000 × magnification. 223 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus 5.3.6. Identification of autophagosomes using Transmission Electron Microscopy following C. concisus infection of Caco-2 cells samples without agar embedment TEM examination of the Caco-2 cells alone prepared without agar embedment showed these cells to be structurally normal, the nucleus being apparent within the cells with the microvilli on the apical surface being well preserved (Figure 5.9). TEM examination of Caco-2 cells infected with C. concisus UNSWCD at MOI 200 revealed that C. concisus UNSWCD was able to invade these cells (Figure 5.10). Moreover, C. concisus UNSWCD was shown to interact with the autophagy process in Caco-2 cells via autophagosomes (Figure 5.10). 224 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Figure 5.9. Transmission Electron Microscopy ultra-thin images of Caco-2 cells without agar embedment, showing the initial stages of Campylobacter concisus UNSWCD infection. A: An image of the characteristics of a Caco-2 cell. The microvilli are observed on the apical membrane surface, vacuolar compartments with no cellular material observed between the apical membrane surface and the nucleus (Nu) (15,000 × magnification). B: An image of a Caco-2 cell infected by C. concisus UNSWCD. Notable characteristics include lysosomal compartments, vacuoles and a Campylobacter-containing vacuole (10,000 × magnification). C: A high magnification image of a Caco-2 cell vacuole showing a granular compartment inside the vacuole (30,000 × magnification). D: A high magnification image of two lysosomes showing a one-layered membrane encapsulating dense granular material (30,000 × magnification). E: A high magnification image of Caco-2 cell mitochondria and a densely stained bacterium. The mitochondria show a clear cristae lining and appear close to a bacterial cell, which may suggest a possible association between the mitochondria and C. concisus UNSWCD in the formation and maturation of autophagosomes (30,000 × magnification). F: C. concisus UNSWCD within Caco-2 cells inside a Campylobacter containing vacuole (30,000 × magnification). 225 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus Figure 5.10. Visualisation of the internalisation of C. concisus into Caco-2 cells without agar embedment using Transmission Electron Microscopy. A: C. concisus UNSWCD invading Caco-2 cells shown in cross section measuring 0.5 µm wide. The bacterial cell appears to be in close proximity to a mitochondrion with a fine layer surrounding them. (30,000 × magnification). B: A membrane forms around internalised C. concisus UNSWCD. A fine layer surrounds the bacterium alongside mitochondria and could be indicative of early stage phagosome formation in Caco-2 cells (50,000 × magnification). C: The appearance of fine filament arrangements close to the bacterium (30,000 × magnification). D: C. concisus UNSWCD associated with an autophagosome showing a double membrane (50,000 × magnification). E: C. concisus UNSWCD shown in cross section (0.5 µm wide) in close proximity to an intermediary phagosome. The bacterium appears to be within a vacuole (40,000 × magnification). F: C. concisus UNSWCD shown in cross section (0.5 µm wide). The bacterium is shown in close proximity with a vacuole compartment forming one 226 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus large phagosome (30,000 × magnification). G: High magnification image of the bacterium fusing with a vacuole compartment (50,000 × magnification). H: C. concisus UNSWCD (0.5 µm wide) observed inside an autophagolysosome containing dense granular material (50,000 × magnification). I: A Caco-2 cell showing a clear apical membrane surface with microvilli. C. concisus UNSWCD, which has maintained its spiral morphology, is visualised inside an autophagolysosome (20,000 × magnification). 227 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus 5.4. Discussion To determine the optimal method of sample preservation for TEM analysis, two approaches were compared. Firstly, an agar embedment technique, which had previously been reported to protect biological samples throughout the sample preparation process (243) was examined. In addition, given the extremely time consuming nature of the agar embedment process and the fact that a number of other studies had reported successful visualisation of autophagy without agar embedment (236, 245, 250), this latter approach was also investigated. Comparison of the two methodologies based on visualisation of semi-thin sections of non-infected and infected Caco-2 cells demonstrated that both approaches resulted in samples that were well preserved and showed no evidence of damage or loss of biological sample as observed in (Figures 5.5, Panels A & B and Figure 5.6, Panels A & B). Given that no significant changes in cell structure or integrity were observed between the two sample preparation approaches, and that both approaches retained sufficient biological material, ultra-thin sections were prepared from both agar embedded and non-agar embedded samples. These were examined using TEM as this not only provides a higher magnification of the cells but also has increased resolution, as compared with light microscopy, thus allowing a more in depth view of the autophagy process following C. concisus infection of Caco-2 cells. Upon inspection of the ultra-thin images prepared using the agar embedment approach using TEM (Figure 5.7), the shape of the non-infected Caco-2 cells was normal, with a distinct apical membrane surface and a nuclear region being observed (Nu). However, the microvilli on the apical membrane surface appeared to lack an even homogenous distribution across the membrane surface (Figure 5.7, Panel A). This irregularity in the 228 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus microvilli distribution could have resulted from the ultramicrotome sectioning, with the resulting sections either being cross-sectional or longitudinal sections. However, the fact that some of the microvilli extending from this membrane surface appeared to have split off from the apical membrane surface raised the possibility that this may relate to the agar embedment procedure. Examination of Caco-2 cells infected with C. concisus UNSWCD prepared using the agar embedment procedure also showed that the microvilli on the apical membrane were not homogenous in their distribution (Figure 5.7, Panels B & C). Moreover, the C. concisus infected cells appeared structurally less rigid, (Figure 5.7, Panel B), particularly the basolateral surface of the cell. Whether this damaged appearance was due to infection with C. concisus is unclear. However, given that the agar embedment process is meant to protect the biological specimen from potential damage during the dehydration, fixation and resin embedment process (242, 243), The presence of cellular damage observed using the agar embedment process raised concerns regarding the appropriateness of this embedding technique in this particular instance. TEM examination of the uninfected and infected Caco-2 cells prepared without agar embedment showed no evidence of fixative artifacts such as unusually shaped or swollen mitochondria, that may occur due to hypo-osmotic fixative solutions (253). Indeed, the mitochondria appeared normal and showed distinct cristae and a dense cellular matrix, which are the key criterion for the visualisation of a mitochondrial organelle using TEM (244). Although post-fixation was undertaken using osmium tetroxide which has been reported to result in high quality TEM images and to prevent fixative artifacts (244, 253), crystal precipitates were observed (Figure 5.7, Panel D) were observed, which are a known indication of staining artifacts (253). The build-up of 229 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus crystal precipitates in TEM sections is reported to be caused by lead citrate and may have occurred as a result of inadequate washing of the sample grid with distilled water (253). While in Figure (5.7, Panel D) what appeared to be a maturing autophagosomal phagophore was observed, this compartment did not exhibit a double-layered membrane, which is the hallmark of an autophagosome compartment (238), raising some concern as to whether this is truly an autophagosome. Examination of ultra-thin images of the non-infected Caco2 cells prepared using the non-agar embedment method showed the microvilli to be homogenously distributed across the apical membrane surface (Figure 5.9, Panel A). Moreover, the integrity and cellular structure of the Caco-2 cells was well preserved, and distinctive basolateral and apical membranes were observed (Figure 5.9, Panel A & B). Examination of the C. concisus UNSWCD infected Caco-2 cells showed that C. concisus UNSWCD was able to invade and infect the Caco-2 cells (Figure 5.10, Panels A-I), a finding that confirmed the results of previous gentamicin assays conducted by our group (108, 109), as well as the results of our examination of semi-thin sections, which showed what appeared to be C. concisus inside Caco-2 cells (Figure 5.5, Panel B and Figure 5.6, Panel B). Determination of the morphological characteristics of C. concisus UNSWCD using TEM not only showed C. concisus UNSWCD morphology (spiral shaped and 4 µm length and 0.5 µm wide) (Figure 5.8, Panels A & B) to be consistent with the morphological description of C. concisus (1, 232, 254) but also identified an outer layer to be present around the bacterium (Figure 5.8, Panels A & B). While the structure of the outer membrane layer of C. concisus has not been characterised it is possible that this outer layer may correspond to a polysaccharide capsule given that some strains of 230 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus C. jejuni have been reported to have a polysaccharide capsule (255). In C. jejuni this capsule has been shown to contribute to serum resistance, invasion of intestinal epithelial cells in vitro, as well as virulence in ferret models of disease (255, 256). Alternatively, the outer membrane may correspond to the surface (S)-layer that has been reported in both C. fetus and C. rectus (257). This layer, which coats the outer membrane, has been reported to be a proteinaceous paracrystalline lattice and has been reported to play a key role in immune evasion by conferring resistance to complement mediated death, by preventing the binding of complement factor C3b to the C. fetus cell surface (257-259). Characterisation of the S-layer of a C. fetus sub spp fetus strain isolated from a patient with a C. fetus infection by Fujimoto et al. TEM has shown based on TEM that its S-layer comprises two formations, one hexagonal and the other a tetragonal (260, 261). Fujimoto et al. have hypothesised that the pattern and antigenicity of the C. fetus S-layer relates to the specific type of S-protein (260). The C. rectus S-layer has been reported to have a similar complement evasion system to C. fetus, however in C. rectus this layer has been reported to down-regulate pro-inflammatory cytokines (257). The presence of S-layers in C. fetus and C. rectus raises the possibility that the outer layer observed in C. concisus UNSWCD (Figure 5.7, Panels A & B) may be an S-layer. TEM images prepared using non-agar embedded samples of Caco-2 cells showed typical characteristics of this type of cell, including the presence of microvilli on the apical membrane surface and vacuolar compartments between the apical membrane surface and the nucleus (Figure 5.9, Panel A). In comparison to non-infected cells, C. concisus UNSWCD infected Caco-2 cells appeared more rounded (Figure 5.9, Panel A) and contained a higher number of vacuoles (Figure 5.9, Panels B & C). Lysosomes with a single-layered membrane were observed, which contained encapsulated dense 231 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus granular material (Figure 5.9, Panel D). Further, densely stained bacteria were present, which appeared to be in close association with mitochondria (Figure 5.9, Panel E). Moreover, internalised C. concisus UNSWCD cells were found to be inside a vacuole (Figure 5.9, Panel F), most likely to be a Campylobacter-containing vacuole (177). The use of such a defence mechanism, to evade lysosomal degradation has been previously identified in C. jejuni (129). More recently, Bouwman et al. reported that intracellular C. jejuni can reside within membrane-bound CD63-positive cellular compartments (262). Thus, it would be of interest to determine if compartments containing C. concisus are also CD63-positive in order to extrapolate whether a similar evasive strategy exists for C. concisus. C. concisus UNSWCD cells were found in close proximity to mitochondria with both the bacterium and mitochondria being encapsulated in a fine layer (Figure 5.10, Panels A & B), which could be indicative of early stage phagosome formation in Caco-2 cells. The identification of autophagosomes in this study (5.10, Panel D) are in line with the visual markers outlined in the guidelines for the correct interpretation of autophagosomes (238). The visualisation of a clear double membrane layer (5.10, Panel D) is considered to be pivotal to the characterisation of an autophagosome within the host cell as outlined by Eskelinen (227, 238, 239). Additionally, ultrastructures (Figure 5.10, Panel H) showing the presence of denser cellular granular material were observed, a factor that is reported to be essential for the characterisation of autophagic compartments (227, 238, 239). Moreover, C. concisus UNSWCD was observed inside autophagolysosomes containing dense cellular granular material (Figure 5.10, Panels H & I), in some cases its spiral morphology being still detectable (Figure 5.10, Panel I). These results show that C. concisus UNSWCD was 232 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus able to persist within the autophagolysosomes without been degraded, thus avoiding the autophagy process. Interestingly, Coxiella burnetii is able to internalise into host cells where it interacts with phagosomes, facilitated by the fusion of the bacterium with vesicles termed parasitophorous vacuoles (PV) (263, 264). These PVs allow the bacterium to replicate within the host cell (263, 264). Moreover, studies have shown that this interaction between the PVs and the autophagic machinery is caused by a delay in the lysosomal fusion procedure, which enables C. burnetii to continue to replicate within the lysosome (265, 266). The appearance of C. concisus in autophagolysosomes might also suggest a delay in the lysosomal fusion procedure similar to that occurring in C. burnetii. This fusion delay could facilitate the persistence and replication of C. concisus in the host cell utilising autophagic compartments. Thus, based on both confocal microscopy and TEM studies it would appear that C. concisus internalisation into host cells is associated with the autophagy process (81), suggesting that C. concisus may manipulate the autophagy process to persist within epithelial cells. Many other intracellular bacteria have evolved mechanisms to redirect or manipulate the autophagic pathway, including: Listeria monocytogenes, Shigella flexneri, Yersinia pestis, Yersinia pseudotuberculosis and Porphyromonas gingivalis (263, 264, 267, 268). Interestingly, much like C. concisus UNSWCD the Gram-negative pathogen Brucella abortus has been shown to be able to manipulate the autophagy process to allow it to replicate and survive within the host (269), and with this knowledge its infectivity cycle within host cells is becoming better understood. Initially, a study by Starr et al. noted that these Brucella capsular vesicles (BCVs) relied on endoplasmic reticulum ER trafficking for their formation (270). However, in a subsequent study Starr et al. 233 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus examined the later stages in infection and reported the observation of modified endoplasmic reticulum derived Brucella containing vacuoles (BCVs) in both macrophages and epithelial cells (269). Using TEM and confocal laser scanning microscopy (CLSM), Starr et al. observed that the intracellular infection cycle of B. abortus into BCVs required the use of autophagy-related protein markers: Beclin-1, ULK-1, and ATG14L (269). Subsequently, Starr et al. showed that BCV conversion to autophagosomal compartments termed an autophagic BCV (aBCV) completes the intracellular cycle of B. abortus and promotes cell-to-cell spread (269). Interestingly, these autophagic compartments were independent from key markers of autophagosomes, which include: ATG5, ATG4B, ATG16L1, ATG7, and LC3B (269). Given this, Starr et al. hypothesised that B. abortus engaged in autophagosome-like rearrangements that are dependent upon a subset of autophagy-associated proteins that may contribute to an unconventional autophagic response (269). It has been suggested by Starr et al. that these autophagosome-like compartments may contribute to the release and spread of B. abortus in a process aimed at hijacking the autophagy framework to subvert its degradation (269). Thus, the autophagy process is manipulated in a manner benefiting B. abortus infection and not the host’s lysosomal bacterial clearance. Based on the findings of the current study, and comparisons to other pathogens that that can evade autophagy, it is possible that C. concisus could potentially impair phagosome-lysosome fusion. This view is reinforced by the finding that important genes functioning within the autophagolysosome such as LAMP1, CTSD and CTSS were all down-regulated upon infection with C. concisus UNSWCD (81). The down- regulation of key autophagolysosome genes by C. concisus could explain how C. concisus UNSWCD can survive inside autophagolysosomes. Evidence supporting 234 Chapter 5: TEM procedure to investigate the modulation of autophagy by C. concisus this view is observed in (Figure 5.10, Panels H & I), which show that the spiral morphology of C. concisus is still detectable within these autophagic compartments (Figure 5.10, Panel I). Given that in C. jejuni infection, CCVs have been proven to be a key element in hijacking the autophagy machinery required to evade lysosomal degradation at low pH (129), it is possible that formation of a C. concisus CCV could potentially function in a similar manner to the CCV of C. jejuni. 5.5. Conclusion The studies presented in Chapter 4 and the above TEM studies presented in this Chapter demonstrate, for the first time, that autophagy plays an important role in modulating the intracellular levels of C. concisus UNSWCD. Moreover, these results would suggest that differences in the intracellular levels of C. concisus strains are associated with the ability of these strains to interact with the autophagy process, with some strains being able to evade destruction by autophagy more efficiently than others. Previous studies would suggest that the increased ability of C. concisus to survive intracellularly might be due to four conserved genes on a plasmid within some strains (109, 126). Thus, it is plausible that these C. concisus genes may interact with the host autophagy process to enhance the intracellular survival of this bacterium. Interestingly, C. concisus has been associated with Crohn’s disease (CD) (41, 105, 136). Given that polymorphisms in the autophagy genes ATG16L1 and IRGM are known to confer susceptibility to CD (145, 146) and that an increased number of autophagosomes are observed in CD patients (208), further investigation of the role of C. concisus as an initiator of this disease should be the focus of future studies. 235 CHAPTER 6 6. GENERAL DISCUSSION AND FUTURE DIRECTIONS 6.1. General Discussion The overall aim of this thesis was to investigate the pathogenic potential of two emerging pathogens, C. ureolyticus and C. concisus. 6.1.1. C. ureolyticus At the commencement of this thesis, limited information existed regarding the pathogenic potential of C. ureolyticus. A study by Man et al., which investigated the ability of a number of non-jejuni Campylobacter spp to attach to and invade host intestinal cells, had shown using Scanning Electron microscopy (ScEM) that C. ureolyticus was capable of adhering to Caco-2 and HT-29 cells (108). The aims of the studies undertaken in Chapter 3 were to build upon the work established by Man et al. (108), as well as to undertake an in depth investigation of the pathogenic mechanisms used by C. ureolyticus. 6.2. Summary of major findings on the pathogenesis of C. ureolyticus The initial study conducted in Chapter 3 was to confirm the findings of Man et al. (108) using adherence and gentamicin protection assays to quantify the level of adherence to and invasion of C. ureolyticus into host cells. The results of these assays showed that C. ureolyticus could adhere to, but not invade either Caco-2 cells or HT-29 cells at a MOI of 200, a result that was in line with findings of Man et al. (108). Investigation into 236 Chapter 6: General Discussion and Future Directions whether the presence of inflammation, as might be found in the intestinal tract altered the ability of C. ureolyticus to attach to and invade Caco-2 and HT-29 cell lines, showed that the presence of inflammation did not significantly affect the level of attachment to these intestinal cell lines, (Caco-2 cell line treated with TNF-α: P = 0.270 and IFN-γ: P = 0.235; HT-29 cell line treated with TNF-α: P = 0.519 and IFN-γ: P = 0.595) or induce invasion. Confirmatory studies to determine whether addition of TNF-α and IFN-γ actually induced inflammation in host cells showed that intestinal cell lines exposed to TNF-α produced significantly higher levels of IL-8 (687.5 ± 3.1 pg/ml; P < 0.0001) and IFN-γ (58.2 ± 2.0 pg/ml; P < 0.01) than the negative control (33.8 ± 1.5 pg/ml), indicating that addition of these cytokines did indeed result in an inflamed state. Subsequent examination of the adherence potential of two further C. ureolyticus strains, designated UNSWE and UNSWR, that had been isolated from the faeces of patients with no pathology showed that C. ureolyticus UNSWE and UNSWR adhered to both Caco-2 and HT-29 cells in vitro, with both these strains showing similar levels of attachment to that observed for C. ureolyticus UNSWCD (UNSWE: P = 0.977 and P = 0.322; UNSWR: P = 0.471 and P = 0.404) for Caco-2 and HT-29 cells respectively. The finding that C. ureolyticus did not invade host intestinal cells led us to investigate whether C. ureolyticus used an alternative route of entry into host cells. Given that C. concisus had been shown to undergo transcellular infection by translocating through tight junction (108, 133), we investigated whether C. ureolyticus was able to use this alternate method of entry into host cells. Based on the use of in vitro transwell assays this study showed, for the first time that C. ureolyticus UNSWCD was able to 237 Chapter 6: General Discussion and Future Directions translocate across the HT-29 cell monolayer (0.018 ± 0.002%), suggesting that C. ureolyticus UNSWCD is able to invade paracellularly through cell tight junctions (156). To provide a more in depth understanding of the interaction and adherence of C. ureolyticus UNSWCD with intestinal cells ScEM studies were undertaken. This showed that C. ureolyticus UNSWCD used a ‘sticky-end’ flagellum independent mode of adhesion to adhere to the microvilli of Caco-2 cells, which not only confirmed that C. ureolyticus UNSWCD could adhere to intestinal cell lines, but also showed that attachment to the host cell led to cellular damage, with degradation of the microvilli on the apical membrane surface of Caco-2 cells (156). Further, ScEM revealed that in the presence of pre-existing inflammation (induced by TNF-α or IFN-γ) the sticky-end mode of adhesion and cellular degradation also occurred. Determination of whether C. ureolyticus UNSWCD infection per se resulted in an inflammatory response, showed that following infection of HT-29 cells C. ureolyticus UNSWCD induced IL-8 levels (58.2 ± 2.8 pg/ml) significantly higher (P < 0.01) than that in the uninfected negative control (33.8 ± 1.5 pg/ml). Interestingly, addition of TNF-α, prior to the infection of HT-29 cells with C. ureolyticus UNSWCD resulted in IL-8 levels (681.7 ± 6.2 pg/ml; P = 0.12) very similar to that observed in non-infected cells exposed to TNF-α (687.5 ± 3.1 pg/ml). In contrast, addition of IFN-γ prior to infection with C. ureolyticus UNSWCD led to a significantly higher level of IL-8 (249.0 ± 9.1 pg/ml; P = 0.002) than that observed in non-infected cells exposed to IFN- γ (58.2 ± 2.0 pg/ml). Investigation of the ability of heat-killed C. ureolyticus UNSWCD to induce IL-8 showed IL-8 levels to be very similar to that produced by viable bacteria (62.3 ± 3.5 pg/ml), suggesting that endotoxin released from C. ureolyticus upon cell 238 Chapter 6: General Discussion and Future Directions lysis may play a role in IL-8 induction (156). Overall, the above results suggest that C. ureolyticus UNSWCD could potentially induce a mild inflammatory response from intestinal epithelial cells and that upon exposure to IFN-γ this response is considerably increased, as opposed to stimulation by TNF-α (156). Given our observation that cellular degradation and damage to the microvilli occurred following infection with C. concisus, we investigated whether C. ureolyticus secreted virulence factors that could induce the observed cellular degradation. To do this we characterised for the first time the secretome of C. ureolyticus UNSWCD using mass spectrometry based proteomics (156). Based on bioinformatic analysis 29 proteins were predicted to be secreted proteins. Functional classification of these secreted proteins showed that three were putative, virulence or colonisation factors. The first of these, the surface antigen CjaA, is reported to be homologous to ABC transport proteins, and has previously been shown be highly immunodominant in C. jejuni. An outer membrane fibronectin binding protein (a CadF homolog) was also identified as a secreted protein. Interestingly, this protein has been shown in C. jejuni to mediate adhesion to host cells, the OMP CadF being shown to promote the binding of C. jejuni to fibronectin on host cells (271), for maximal adherence and invasion of INT407 cells, and for colonisation of the chicken cecum (272, 273). While this study did not specifically investigate the role of CadF in the binding of C. ureolyticus UNSWCD to the intestinal cell lines, the identification of CadF in the secretome suggests a role for this protein in binding C. ureolyticus to host intestinal cells. Further, an S-layer RTX toxin was secreted by C. ureolyticus. Given that RTX proteins are reported to be pore- forming toxins and are synthesised by a diverse group of Gram-negative pathogens, it is possible that this S-layer RTX toxin may be responsible for the cellular damage and 239 Chapter 6: General Discussion and Future Directions microvilli degradation observed using ScEM. To examine this possibility the effect of the C. ureolyticus secretome (2.6 µg, 13 µg, 26 µg, and 130 µg of purified secretome) on the viability of HT-29 cells was investigated. This showed that cell viability was significantly reduced as compared with untreated cells (2.6 µg, P < 0.037; 13 µg, P < 0.007; 26 µg, P < 0.026; and 130 µg, P < 0.0003), suggesting that the secretome of C. ureolyticus is toxic to host cells. Investigation of IL-8 production by HT-29 cells following exposure to the same concentrations of the C. ureolyticus secretome showed that similar levels of IL-8 (102.7 ± 12.9 pg/ml; 88.0 ± 5.8 pg/ml; 96.1 ± 5.9 pg/ml and 90.5 ± 2.4 pg/ml) were produced across all secretome concentrations, which were significantly higher (P < 0.0001) than that of the negative control (33.8 ± 1.5 pg/ml) and cells exposed to C. ureolyticus alone (58.2 ± 2.8 pg/ml/). Based on these findings it would appear that components of the purified C. ureolyticus secretome lead to IL-8 production. 6.2.1. The putative pathogenic mechanism of infection used by C. ureolyticus Overall, the current study has provided novel information regarding the pathogenesis of C. ureolyticus and its interaction with host cells. Based on the results of this thesis, the possible pathogenic mechanisms used by C. ureolyticus UNSWCD to cause infection of the intestinal tract are outlined below and can be visualised in Figure 6.1. C. ureolyticus UNSWCD potentially makes its way through the normal microflora in the luminal space [1] to the mucus layer, where it potentially uses its twitching motility to reach the intestinal epithelial cells [2]. C. ureolyticus UNSWCD is then attracted to the microvilli on the apical membrane surface of the intestinal cell, possibly through recognition of surface antigens such as the surface antigen CjaA [2]. The bacterium then 240 Chapter 6: General Discussion and Future Directions adheres to the microvilli of the intestinal epithelial cells, which may facilitate contact with the extracellular matrix of the intestinal cell, possibly mediated by adhesins such as the outer membrane fibronectin binding protein CadF [3]. C. ureolyticus UNSWCD may then colonise the intestinal cell surface using its ‘sticky end’ flagellum-independent mechanism of adhesion [4]. Further, C. ureolyticus UNSWCD may also translocate through the intestinal epithelial cell tight junctions via the paracellular route. Moreover, C. ureolyticus carries out this process by the secretion of the Zot toxin, which could facilitate the breakdown of the cell tight junctions allowing C. ureolyticus to translocate paracellularly [5]. Further, exposure of epithelial cells to C. ureolyticus UNSWCD secreted proteins may result in degradation of microvilli, causing the intestinal cells to become porous, resulting in leakage of its cellular contents, thereby providing C. ureolyticus UNSWCD with nutrients required for its survival in the human gastrointestinal tract [6-7]. Additionally, upon passing through the basolateral surface and into the submucosa, C. ureolyticus UNSWCD is detected by the innate immune system, which mounts an immune response to destroy C. ureolyticus. As a result of C. ureolyticus infection, the intestinal cells produce IL-8, instigating the recruitment of resident macrophages, monocytes and polymorphonuclear (PMN) cells, such as neutrophils, from the bloodstream into the submucosa [8]. C. ureolyticus also encounters resident macrophages in the area, which may lead to phagocytosis of C. ureolyticus that stimulates the release of IL-8 and TNF-α could then propagate an inflammatory response at the infection site [9]. The stimulation of IL-8 by C. ureolyticus throughout the submucosa activates macrophage secretion of further IL-8, which leads to the influx of PMNs, which in turn produces more inflammation in the area [10] and possibly the formation of granulomas observed in patients with CD. 241 Chapter 6: General Discussion and Future Directions Figure 6.1. The putative pathogenic mechanisms used by C. ureolyticus to interact with human intestinal epithelial cells in the gastrointestinal system. The numbers (1-10) in the figure above relate to the postulated sequence of events occurring following infection of intestinal epithelial cells with C. ureolyticus as outlined in (section 6.2.1). 242 Chapter 6: General Discussion and Future Directions 6.3. C. concisus The emergent pathogen C. concisus is considered to be a highly fastidious member of the Campylobacter genus (1) which has been associated with a range of gastrointestinal infections including Barrett’s oesophagus, gastroenteritis and inflammatory bowel diseases (80). However, the presence of C. concisus in healthy patients and the failure of some studies to demonstrate a significant difference in the prevalence of C. concisus in diseased patients as opposed to healthy patients has led to controversy as to its role as a pathogen in human disease. Recent studies have indicated that C. concisus possesses a diverse array of pathogenic mechanisms as well as unique genetic and functional virulence repertoires. Studies have shown that in vitro the invasive potential of C. concisus strains isolated from chronic intestinal diseases (including IBD), are > 500-fold higher than that of C. concisus strains isolated from acute intestinal diseases and a healthy subject (108, 109). Given that many intestinal pathogens including C. jejuni have been shown to be able to manipulate the autophagy process to facilitate their survival in host cells, Chapters 4 and 5 investigated the ability of C. concisus to survive intracellularly in intestinal cells through the manipulation of the autophagic system. 6.4. Summary of the major findings on the pathogenesis of C. concisus Considering that numerous studies have highlighted the importance of autophagy for the Considering that numerous studies have highlighted the importance of autophagy for the elimination of intracellular bacteria and the subversion of this process by pathogenic bacteria (137, 140, 141, 222), the role of autophagy in the intracellular survival of C. concisus was investigated. Gentamicin protection assays were employed to assess intracellular levels of the highly invasive strain C. concisus UNSWCD and three C. concisus strains with no or low invasive ability (C. concisus BAA-1457, 243 Chapter 6: General Discussion and Future Directions UNSWCS and ATCC 51562) within Caco-2 cells, following autophagy induction and inhibition. Autophagy inhibition resulted in two to four fold increases in intracellular levels of C. concisus UNSWCD within Caco-2 cells, while autophagy induction resulted in a significant reduction in intracellular levels or bacterial clearance. Interestingly, C. concisus strains BAA-1457, UNSWCS and ATCC 51562, which were previously shown to have no or low intracellular invasion levels, showed a dramatic increase in their levels upon autophagy inhibition, suggesting that differences in the intracellular levels of C. concisus strains are associated with the ability of strains to interact with the autophagy process, with some strains being able to evade destruction by autophagy more efficiently than others. To assess the interaction between C. concisus and autophagosomes in Caco-2 cells, confocal microscopy and ScEM were employed. The use of confocal microscopy and fluorescence staining revealed co-localisation between C. concisus UNSWCD and autophagosomes, the strength of this co-localisation, based on the Pearson’s coefficient test, showing a positive correlation to exist between the bacterium and LCB3 (274) Importantly, this finding indicated for the first time that C. concisus UNSWCD could interact with autophagosomes, suggesting that C. concisus UNSWCD may be able to subvert the autophagy process. To investigate the effect of C. concisus infection on autophagy gene expression levels in Caco-2 cells, the expression levels of 84 genes involved in the autophagy process were measured using RT-qPCR. Following infection, 13 genes involved in the autophagy process were found to be significantly regulated, and a further 6 genes showed borderline results, with the overall indication being that a dampening effect was exerted by the bacterium on the autophagy process (275). Interestingly, C. concisus UNSWCD 244 Chapter 6: General Discussion and Future Directions appeared to have a direct effect on early stage phagophore formation, as well as autophagosome maturity, as shown by the up-regulation of MAPLC3B and the down regulating of the key genes ATG4B and ATG7. Both these genes are required in the cycling of LC3-II back to LC3-I, which is an essential part of the autophagosome replenishment and maturation process (275). Moreover, phagosome-lysosome fusion appeared to be somewhat impaired as witnessed by the down-regulation of LAMP-1 (275). Additionally, Cathepsins S and D were down-regulated, which could suggest that lysosomal enzymes responsible for degradation may have also been impaired (275). Overall the above findings provide evidence that C. concisus UNWSCD is capable of manipulating the autophagy system through the utilisation of autophagy evasion strategies for its own intracellular survival within Caco-2 cells. Importantly, these autophagy evasion strategies utilised by C. concisus UNSWCD, are to our knowledge, the first report of the manipulation of the autophagy process by a Campylobacter species. Moreover, the results of this study add significantly to our understanding of the pathogenic potential of C. concisus. 245 Chapter 6: General Discussion and Future Directions Figure 6.2. The schematic representation of the dampening effect exerted on the autophagy pathway by C. concisus UNSWCD infection. Image adapted from Tanida et al. (130) to reflect our findings within the autophagy pathway. Down-regulated genes are shown in blue while up-regulated genes are shown in red. Further, the findings presented in Chapter 4 showing the co-localisation between autophagosomes and C. concisus UNSWCD in Caco-2 cells, led us to further characterise the autophagic ultrastructures mediating the interaction between C. concisus and intestinal host cells in more depth, using transmission electron microscopy (TEM) (Chapter 5). Prior to this examination, development of an optimised protocol for sample preparation for use in these TEM studies was undertaken, which showed that the sample preparation without agar embedment was optimal. Examination of Caco-2 cells infected with C. concisus UNSWCD using TEM showed the presence of C. concisus UNSWCD inside Caco-2 cells. Moreover, C. concisus 246 Chapter 6: General Discussion and Future Directions UNSWCD was shown to interact with the autophagy process via autophagosomes. Additionally, intracellular bacteria were observed to persist within autophagic vesicles (275). Primarily, C. concisus UNSWCD was shown to reside within what appeared to be Campylobacter containing vacuoles (CCV), similar to the intracellular protective mechanism observed in C. jejuni (129), where the CCV allows C. jejuni to deviate the endocyctic pathway of lysosomal degradation (129). The identification of this putative CCV in C. concisus UNSWCD is a novel finding and adds considerably to our understanding of the mechanisms by which invasive strains of C. concisus may mediate their intracellular survival within host cells (81). Importantly, the studies conducted in Chapters 4 and 5 established that some strains of C. concisus have the ability to evade the autophagy process, allowing them to survive inside the host cell. Further, these studies suggest that C. concisus UNSWCD forms a Campylobacter containing vacuole (CCV), which allows it to block autophagosome- lysosomal fusion, avoid degradation and survive intracellularly within host cells. Based on these findings, it was postulated that highly invasive strains like C. concisus UNSWCD may in fact have virulence factors that inhibit the autophagy pathway, which sets them apart from other C. concisus strains (81). Furthermore, this thesis provides novel insight into the processes that govern the intracellular survival of C. concisus within host intestinal cells, post invasion through the manipulation of the autophagy process. The putative pathogenic mechanisms used by C. concisus are discussed below and shown in Figure 6.3. 247 Chapter 6: General Discussion and Future Directions 6.5. The putative pathogenic mechanisms used by C. concisus The investigations conducted in this thesis have shown for the first time that some strains of C. concisus have the ability to evade removal by the host through manipulation of the autophagy process. The putative steps involved in pathogenesis of C. concisus infection are outlined in (Figure 6.3). Following infection of a human host C. concisus enters the lumen of the gastrointestinal tract [1]. Once in the intestine it may be chemo-attracted to the mucus layer after which it uses its polar flagellum to swim though the mucus layer to the epithelial cell surface [2]. Upon reaching the epithelial cell surface C. concisus initially attaches via its flagellum, which wraps itself around the microvilli on the apical membrane surface of the epithelial cell [3.1]. Following this, further attachment of C. concisus may be mediated by the C. concisus adhesive factor, Cad-F that binds to fibronectin, a component of the extracellular matrix of the epithelial cell, thus facilitating better adherence. Following adherence, C. concisus could potentially secrete an S-layer RTX-toxin, which may damage the host cell [3.2]. This S-layer RTX-toxin can potentially cause a degradative effect on the host cell that may result in cell lysis [3.2.1]. C. concisus AToCC pathotype strains adhere to host cell microvilli, do not invade, but are able to translocate through host cell tight junctions [3.3]. This paracellular translocation involves the Zot toxin, which targets the ZO-1 host cell tight junction protein leading to barrier dysfunction, thus allowing C. concisus to translocate through host cell tight junctions [3.3.1]. The adherence of C. concisus AToCC strains to the host cell microvilli induces the production of IL-12 and IL-8. Additionally, following passage of C. concisus through the host cell tight junctions to the submucosa IL-8 production may cause stimulation of macrophages and PMN cells aimed at eliminating the infection of C. concisus AToCC strains [3.3.2]. 248 Chapter 6: General Discussion and Future Directions Moreover, highly invasive C. concisus AICC pathotype strains could potentially become compartmentalised into a Campylobacter containing vacuole (CCV), which forms around the bacterium and eventually protects it from lysosomal degradation [4]. Additionally, these highly invasive C. concisus AICC strains associate with autophagosomes in an interactive process [5] where the bacterium resides within the autophagosome, and upon lysosomal fusion associates with this complex to form an autophagolysosome [6]. C. concisus AICC strains however, are able to block lysosomal degradation within the autophagolysosome through disruption of the cathepsins (lysosomal enzymes) [7]. This evasion strategy allows C. concisus AICC strains to evade the autophagy process and reside within the autophagolysosome [8] where it can replicate and persist within the host cell [9]. The adherence of C. concisus AICC pathotype strains to the host cells produces IL-12 and IL-8, with IL-8 production potentially inducing macrophages and PMN cells to the site [11]. The invasion of highly invasive C. concisus AICC pathotype strains into the host cell [12] results in IFN-γ production [12], which in turn activates NF-κB resulting in increasing inflammation [13]. Mechanistically, the production of IFN-γ by AICC infected host cells, stimulates the immunoproteosome to ubiquitinate and subsequently degrade NF-κB inhibitors such as IκBα, which would normally repress NF-κB production [13]. Thus, this process activates NF-κB leading to the production of pro- inflammatory cytokines such as TNF-α [13] as shown by Kaakoush et al. (109). Moreover, the stimulation of pro-inflammatory cytokines such as IL-8 and TNF-α by C. concisus AICC infected host cells, drives the stimulation of macrophages and PMNs as an innate immune response to combat the infection of C. concisus AICC strains, but as a consequence produces more inflammation in the area [14]. 249 Chapter 6: General Discussion and Future Directions Figure 6.3. The putative pathogenic mechanisms used by C. concisus to attach to human intestinal cells and cause disease. The numbers in this diagram (1-14) relate to the possible sequence of events occurring following infection of intestinal epithelial cells with C. ureolyticus as detailed in (section 6.5). 250 Chapter 6: General Discussion and Future Directions 6.6. Future Directions 6.6.1. Future directions for C. ureolyticus investigations In relation to the findings of Chapter 3 there are a number of avenues that would be important foci for future studies examining the pathogenic mechanisms used by C. ureolyticus. Further in vitro studies, in which the effect of decreasing doses of the C. ureolyticus secretome on the cellular release of IL-8 would allow the identification of the minimal concentration of secretome required to generate cellular release of IL-8. Furthermore, the observation that C. ureolyticus UNSWCD forms aggregates on an inert surface (Poly-L-Lysine cover slip), as well as on the apical membrane surface of Caco-2 cells suggests that C. ureolyticus UNSWCD has the ability to form biofilms. Given, that within the gastrointestinal tract C. ureolyticus is continually subjected to peristalsis and mucus turn over (159) the ability of pathogens to adhere to the gastrointestinal tract is of paramount importance. Further, the ability of bacterial pathogens to aggregate and form biofilms assists in the survival of pathogens in hostile environments by protecting them from the host immune response and antibiotic therapy, thus allowing them to continue to multiply within the host (276-278). Given this, further research into the ability of C. ureolyticus UNSWCD to form biofilms in different environments using biofilm assays (27, 28) has the potential to lead to a more in-depth understanding of biofilm formation by C. ureolyticus. For example, studies in other pathogens have reported that LuxS signalling homologues play a role in mediating the autoinducer-2 (AI-2) Quorum sensing (QS) system, which drives the formation of biofilms in bacteria (279). Importantly, an investigation by Elver and Park, examining the induction of biofilm formation by C. jejuni, has revealed that AI-2 QS signalling via the LuxS signalling molecule augmented the formation of C. jejuni biofilms (280). Given this, in vitro assays focussing on the possible production of signalling molecules 251 Chapter 6: General Discussion and Future Directions by C. ureolyticus UNSWCD would be of significant interest. Further, C. ureolyticus biofilm production could also be investigated using environmental scanning electron microscopy (ESEM), a technique that favours the visualisation of biofilm formation on various materials (281). In general ESEM is preferred over ScEM for investigation of biofilm formation as the ScEM sample preparation procedure which involves both fixation and dehydration methods (282), can alter and deteriorate the exopolysaccharide structure of the biofilm and introduce artefacts into the biological sample (282). In contrast, ESEM has a distinct advantage over ScEM in that this technique facilitates the visualisation of the biofilm in its natural hydrated state without the requirement to fix or dehydrate the sample (282), thus reducing artefact build up and alteration of the biofilms crystalline structure. Thus, the use of ESEM to examine the development of the C. ureolyticus biofilm structure would appear to be the best approach (282). While the intestinal epithelial cell line, Caco-2 cells are commonly used as an in vitro cell line model to mimic the human gastrointestinal epithelium, its cellular characteristics do not include the expression of a mucus layer (211). Given that the human gastrointestinal tract has a mucosal lining, further studies could be undertaken using the LS174T mucus producing cell line, which may more closely mimic the mucus environment seen in the human gastrointestinal tract (283). In particular, in vitro adherence / invasion assays and ScEM could be performed to further elucidate the interaction between C. ureolyticus UNSWCD and the LS174T mucus producing cell line. Interestingly, a study by Kaakoush et al. which used attachment and invasion assays as well as ScEM to investigate the interaction between four strains of C. concisus isolated from patients with chronic gastrointestinal disease and the LS174T cell line, demonstrated that not only could all four strains attach to and invade the LS174T cell line, but that these strains were chemo-attracted to the mucus layer (109). Further, a 252 Chapter 6: General Discussion and Future Directions study by Lavrencic et al. has shown that upon accumulation of mucus, the adherence levels of C. concisus UNSWCD increased (159). Such in vitro studies could be undertaken using C. ureolyticus UNSWCD, UNSWE and UNSWR, to determine whether the adherence of C. ureolyticus to LS174T also increases with mucus accumulation and whether C. ureolyticus strains are chemo-attracted to mucin. Unlike the majority of Campylobacter species, C. ureolyticus may employ twitching motility (156), a flagella-independent type surface-associated movement that is mediated by type IV pili (284). In vitro studies could also be undertaken to investigate twitching motility in C. ureolyticus strains. Such studies could involve examination of the movement of C. ureolyticus through agar following subsurface agar inoculation. This particular technique was used in a study by Lavrencic et al. to examine C. concisus motility (159). Further, the use of subsurface twitch motility assays in conjunction with commassie blue staining (285) and video microscopic analysis have the potential to increase our understanding of the colony expansion of C. ureolyticus by twitching motility, as has previously been reported for P. aeruginosa with the use of similar assays and procedures (285). Given that the genome of C. ureolyticus CIT007, an isolate cultured from a patient with diarrhoea has now been sequenced (77) a further important focus of research would be to sequence the genomes of C. ureolyticus UNSWCD, isolated from a child with CD as well as C. ureolyticus UNSWE and UNSWR that were isolated from the faeces of children with no pathology and to compare their genomes with C. ureolyticus CIT007. Using this approach novel putative virulence and colonisation factors are likely to be identified. Given the heterogeneity observed amongst C. ureolyticus spp (60), sequencing of further genomes of C. ureolyticus isolates and identification of 253 Chapter 6: General Discussion and Future Directions interspecies differences would aid further characterisation of its pathogenic repertoire. Importantly, the use of genomic sequencing aided in the distinction of different pathotypes (AICC and AToCC) in C. concisus (81). Similarly, the same application could also help determine whether C. ureolyticus isolates can also be broken down into pathotypes based on their pathogenic potential. Given that the functional role of the S-layer RTX toxin in C. ureolyticus remains unknown, it would also be worthwhile extracting and purifying the S-layer RTX toxin produced by the secretome of C. ureolyticus UNSWCD to determine whether it has cytotoxic effects on host cells. To conduct these studies a tandem approach using flow cytometry and a tetrazolium salt-based XTT assay measured spectrometrically could also be used. The application of flow cytometry via FACS analysis could be used to determine the viability of host intestinal cells and immune cells exposed to the purified C. ureolyticus S-layer RTX toxin. Additionally, the effects of toxicity on host intestinal cell viability could be examined using in vitro XTT assays, an approach that has been used to examine the cytotoxicity of the Shiga toxin produced by Enterohemorrhagic E. coli (286). Moreover, expression levels of the S-layer RTX toxin gene could be evaluated using qRT-PCR from cDNA obtained from biopsies or faecal samples of a large and statistically strong population of paediatric or adult patients suffering from CD or gastroenteritis. Moreover, healthy control paediatric or adult patients could be used as a comparative sample population to better interpret the results of this investigation. Similar studies have been carried out on C. concisus, with Kalischuk et al. using qPCR to determine the prevalence of the RTX toxin in faecal samples obtained from patients with C. concisus induced diarrhoea (118). Understanding the function of the C. ureolyticus S-layer RTX toxin could shed greater insight into its pathogenesis 254 Chapter 6: General Discussion and Future Directions against the host and also determine whether the plausible cytotoxicity effect of the RTX toxin is strain specific. 6.6.2. Future directions for C. concisus investigations Since autophagy is such a dynamic and intricate process within host cells (134, 194), future studies could shed additional insight into key C. concisus autophagy evasive strategies, which would help in the interpretation of its infectivity cycle within host cells. For example future studies aimed at identifying key proteins and mediators involved in the process governing the activation of certain autophagy genes could be undertaken using siRNA knock down experiments. Moreover, cell lines in which different autophagy-related genes have been knocked out could be used to investigate the ability of C. concisus chronic and acute strains to invade and survive intracellularly within host cells. Key genes that could be investigated include ATG5, ATG9, BECLIN-1 and LAMP-1. This would help determine how strains such as C. concisus UNSWCD manipulate the autophagy system in host intestinal cells. Other studies have successfully used knock out cell lines for the investigation of the role of xenophagy in the intracellular survival of pathogens (223, 287-289). For example, a study by Kuballa et al. utilised RNAi to knock down ATG16L1 and investigate the role of ATG16L1 variants in autophagy following S. Typhimurium infection (223). A similar system could be utilised in which Caco-2 cell line knockouts of ATG16L1 are infected with AToCC and AICC C. concisus pathotypes. Characterisation of the Campylobacter containing like vacuole (CCV) of C. concisus UNSWCD could potentially aid in determining how this CCV is involved in the 255 Chapter 6: General Discussion and Future Directions autophagy process. Given the observation in the TEM images of a clear structure around C. concisus UNSWCD, further studies investigating this structure are clearly warranted. Further, investigations concerning the C. concisus CCV could centre on extracting and purifying the CCV itself and the surface layer proteins coating the CCV, through the characterisation of protein profiles. Importantly, this would allow greater interpretations of the function behind the CCV in mediating interactions with processes like autophagy and the canonical endocytic pathway. Moreover, determination of how the C. concisus CCV is involved in lysosomal evasion is also be an interesting area to follow up, given that the C. jejuni CCV endosomal markers mediate the lysosomal evasion by C. jejuni from the canonical endocytic pathway of bacterial degradation (129). In particular the interaction between the C. concisus CCV with endosomal markers Rab 5 or Rab 7 as well as the late endosomal marker LAMP-1, could be examined to gain greater understanding into the CCVs role in evading lysosomal degradation by bypassing the canonical endocytic pathway. For example, CLSM or live cell imaging could be used to visualise the interaction between the C. concisus CCV and the stained endosomal markers mentioned above, to determine how the C. concisus CCV modulates intracellular trafficking and intracellular survival. A further step in assessing the mechanistic action of C. concisus invasion would be the development of a suitable animal model for in vivo investigations. Although a number of research groups have attempted to develop a mouse model of C. concisus, to date this has been unsuccessful. For example, Aabenhaus et al. used BALB/cA mice as an in vivo mouse model for studies of C. concisus. In this study 5 clinical C. concisus strains were used to infect immune-competent BALB/cA mice (290). However, this attempt at establishing an effective C. concisus mouse model failed, as C. concisus was found to only transiently colonise the gastrointestinal tract (290). Moreover, this transient 256 Chapter 6: General Discussion and Future Directions colonisation produced no pathology, histological examination revealing no signs of inflammation along the gastrointestinal tract (290). Ultimately, if an animal model of C. concisus could be developed that allowed effective colonisation by C. concisus, this would significantly impact on our understanding of the pathogenesis of C. concisus. For example, future investigations could be conducted in which transgenic mouse models such as C57BL/6 mice with ATG5 -/- for autophagy deficiency, and ATG16L1 -/- mice for CD association could be used as have been reported previously (236, 268, 291-294). These mouse models can be used and adapted for C. concisus colonisation, as has been done in other studies using both ATG5 and ATG16L1 knock out models (236, 268, 291-294). Another useful mouse model for the determination of autophagosomes is the green fluorescent protein (GFP)-LC3 transgenic mouse model, which facilitates the quantification of autophagosome puncta through CLSM (295). The effective generation of a mouse model will significantly aid in understanding not only the virulence mechanisms of C. concisus and its interaction with the host microbiota, but also host immune recognition and response to C. concisus. Apart from the utilisation of mouse-model studies, the Zebra fish is becoming an increasingly used animal model for investigating the role of autophagy during bacterial infection (296, 297). An important tool is the GFP-LC3 transgenic Zebra fish line, which enables the visualisation of autophagosomal structures and has been used extensively to show the autophagy response to infection by Shigella Flexneri and Mycobacterium marinum (296-299). Using a different live animal model besides transgenic mice enables the study of bacterial infection across various cell types, which could facilitate the development of novel therapies against autophagy-related disorders (296, 300, 301). Finally, given that autophagy is such a dynamic process (134, 194), 257 Chapter 6: General Discussion and Future Directions future in vivo studies could apply live cell light microscopy imaging techniques to view the process of autophagy in intestinal cells following C. concisus infection. Live cell imaging has been used as a super resolution imaging application to investigate and assess the progression of the autophagy process such as autophagosome formation, following bacterial infection of other pathogens such as S. Typhimurium and S. Flexneri (134, 289, 296, 297). Conventional microscopy imaging techniques can occasionally cause confusion in the interpretation of autophagy events due to the biochemical complexity associated with uncharacterised bacterial components, and the inability to predict the location and timing of autophagosome formation. Thus, the use of live cell imaging could be implemented to overcome such limitations caused by other microscopy techniques. Moreover, live cell imaging could be applied to C. concisus infected in vivo models to obtain a better resolution, as well as an improved prediction and location of autophagosome formation. Importantly, this would facilitate a better judgement of the interplay occurring between C. concisus and the autophagy process, live in real time over an extended period of time. In conclusion while the findings of this thesis represent a significant advancement in our understanding of the pathogenesis of the emergent pathogens C. concisus and C. ureolyticus, further studies examining a range of both C. concisus and C. ureolyticus strains isolated from healthy subjects and subjects with different disease outcomes will be required to verify these findings. Nevertheless, the findings of this thesis represent a significant advancement in our understanding of the pathogenesis of the emergent pathogens C. concisus and C. ureolyticus. Given these findings, continuation and expansion of research into these two emergent gastrointestinal pathogens is clearly warranted. 258 REFERENCES 1. Vandamme P, Dewhirst FE, Paster BJ, On SLW. 2005. Genus I Campylobacter, p. 1147-1160. 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Zebrafish as a model to understand autophagy and its role in neurological disease. Biochim Biophys Acta 1812:520- 526. 288 APPENDICES APPENDIX Table 1. Proteins identified within the secretome fraction of Campylobacter ureolytcius UNSWCD (n = 111). GI MASCOT SIGNALP PROTEIN NAME SECRETED NUMBER SCORE POSITION 218129864 hypothetical protein BACEGG_01445 61 YES* POSITION 19 & 20 157164945 DNA-binding protein HU 1 DNA-binding protein II 68 YES# 157165109 L-asparaginase 110 YES# 157163999 3-isopropylmalate dehydrogenase 153 NO 157165445 dihydrodipicolinate synthase 122 NO 157163926 7-alpha-hydroxysteroid dehydrogenase 147 NO 157164709 peptidoglycan associated lipoprotein 43 YES* POSITION 28 & 29 157165429 phosphoribosylaminoimidazole-succinocarboxamide 116 NO synthase 157164629 elongation factor Tu 217 NO 157165039 transcription antitermination protein NusG 108 NO 157165495 50S ribosomal protein L11 151 YES# 157164541 DNA-directed RNA polymerase, beta subunit 138 NO 189027839 RecName Full Elongation factor G Short EF-G 85 NO 157165207 fumarate hydratase 105 YES# 157163909 serine hydroxymethyltransferase 101 NO 157164022 long-chain-fatty-acid-CoA ligase 56 NO 157164305 ribonucleotide-diphosphate reductase subunit beta 51 NO 157165314 thiamine biosynthesis protein ThiC 195 NO 289 Appendices 157165747 fumarate reductase flavoprotein subunit 62 YES* POSITION 23 & 24 157164734 UTP-glucose-1-phosphate uridylyltransferase 63 NO 157165165 enoyl-acyl carrier protein reductase 213 NO 157164277 glyceraldehyde-3-phosphate dehydrogenase, type I 69 YES# 157164319 elongation factor Ts 85 NO 157164063 3-oxoacyl-acyl carrier protein synthase II 136 YES# 157164484 3-ketoacyl-acyl-carrier-protein reductase 78 YES# 157164747 ADP-glyceromanno-heptose 6-epimerase 58 YES* POSITION 22 & 23 157165083 transcription termination factor Rho 53 NO 157164816 surface antigen, CjaA 98* YES* POSITION 21 & 22 157165097 chaperonin GroEL 224 NO 157165660 co-chaperonin GroES 42 NO 157164953 NAD FAD-utilising dehydrogenase 71 YES# 157165604 50S ribosomal protein L13 58 NO 157164740 outer membrane fibronectin-binding protein 72 YES* POSITION 19 & 20 157165691 C4-dicarboxylate-binding periplasmic protein 47 YES* POSITION 20 & 21 157164791 tryptophanyl-tRNA synthetase 49 NO 157165081 aminobenzoyl-glutamate utilisation protein A 100 NO 157164766 peptidase E 60 NO 157164875 glutamate-1-semialdehyde aminotransferase 133 NO 157164331 DNA gyrase subunit A 71 NO 158604943 aspartate-semialdehyde dehydrogenase 149 NO 157165732 nitrate reductase 277 YES* POSITION 29 & 30 157164647 F0F1 ATP synthase subunit beta 167 NO 157164479 malonyl CoA-acyl carrier protein transacylase 55 NO 290 Appendices 158604925 acetyl-CoA carboxylase, carboxyl transferase, 50 NO beta subunit 157164006 pyruvate kinase 45 NO 157165656 His Glu Gln Arg opine amino acid ABC transporter 139 NO permease 157164360 tryptophan synthase subunit beta 55 NO 157164291 response regulator receiver domain-containing protein 88 NO 157164012 2-cys peroxiredoxin BAS1, Thiol-specific antioxidant 198 NO protein 157163948 nucleoside diphosphate kinase NDK NDP kinase 140 NO nucleoside-2-P kinase 157164147 ribose-phosphate pyrophosphokinase 233 NO 157165645 hydrogenase expression formation protein HypE 46 NO 157165110 hydrogenase accessory protein HypB 51 NO 157165742 N-terminal methylation domain-containing protein 48 YES* POSITION 24 & 25 157165061 GTP-binding protein TypA BipA 76* NO 157165356 dihydrodipicolinate reductase 54 NO 157165021 Thioredoxin 75 NO 157164931 acetolactate synthase small subunit 202 NO 157165398 argininosuccinate synthase 75 NO 157164513 ATP-dependent protease peptidase subunit 190 NO 157165545 hypothetical protein 142 NO 158604975 D-methionine-binding lipoprotein MetQ 63 YES* POSITION 20 & 21 157164498 valyl-tRNA synthetase 43 NO 157164956 aspartate aminotransferase 139 NO 291 Appendices 157164864 2-oxoglutarate-acceptor oxidoreductase subunit OorC 64 NO 157165518 elongation factor P EF-P 78 NO 157164710 molybdenum cofactor synthesis domain-containing 92 YES* POSITION 25 & 26 protein 157165471 radical SAM domain-containing protein 71 YES* POSITION 32 & 33 157163991 molybdenum cofactor biosynthesis protein A 98 NO 157164582 threonine dehydratase 54 NO 157164502 bifunctional aconitate hydratase 2 2- 43 NO methylisocitrate dehydratase 157165235 RNA pseudouridine synthase family protein 52 NO 157164054 cytochrome c oxidase, heme b and copper-binding 67 NO subunit, membrane-bound 157165713 glyceraldehyde-3-phosphate dehydrogenase 2 69 NO 157165286 phosphoenolpyruvate carboxykinase 103 YES# 157165379 peptidyl-prolyl cis-trans isomerase B PPIase B 65 NO rotamase B 157164874 bifunctional GMP synthase glutamine 54 NO amidotransferase protein 157164695 elongation factor P 78 NO 157164116 Transaldolase 71 NO 157163952 50S ribosomal protein L25 general stress protein Ctc 57 NO 157165496 phosphoserine aminotransferase 55 NO 157164621 translation-associated GTPase 124 NO 157164548 leucyl aminopeptidase 54 NO 157165725 adenine phosphoribosyltransferase 137 NO 292 Appendices 157165566 amino acid ABC transporter, periplasmic binding 56 YES# protein 157165509 DNA-directed RNA polymerase subunit alpha 82 NO 157164159 30S ribosomal protein S5 116 NO 157164234 GTP cyclohydrolase I 100 NO 157165243 S-layer-RTX protein 123 YES* POSITION 22 & 23 157164168 phosphoribosylaminoimidazole synthetase 77 NO 157164122 DNA polymerase III subunit beta 44 NO 157165476 adenylosuccinate lyase 89 NO 157164373 pyruvate ferredoxin flavodoxin oxidoreductase 138 NO 157165597 CRISPR-associated protein Cas2 51 NO 157164858 inorganic diphosphatase 96 NO 157165454 amino acid carrier protein AlsT 51 YES# 157165390 30S ribosomal protein S6 116 NO 157164553 molecular chaperone DnaK 120 NO 157165649 DNA-binding response regulator 111 YES* POSITION 25 & 26 157164251 holo-acyl-carrier-protein synthase 72 YES* POSITION 20 & 21 157165707 fructose-bisphosphate aldolase 123 YES# 157164266 recombinase A 101 NO 157164344 biotin carboxylase 60 NO 157165299 ATP-dependent protease ATP-binding subunit 69 NO 157164628 carbamoyl phosphate synthase large subunit 144 NO 157165191 adenylosuccinate synthetase 89 NO 157165072 malate dehydrogenase 60 NO 157165301 deoxycytidine triphosphate deaminase 63 NO 293 Appendices 157165134 2-acylglycerophosphoethanolamine acyltransferase 248 YES# 154149442 hypothetical protein 142 YES* POSITION 6 & 7 154149060 bifunctional ornithine acetyltransferase/N- 72 NO acetylglutamate synthase protein * classically secreted protein # non classically secreted protein The mascot score conveys the protein identification blasted (NCBInr) against a reference genome. In this case the genome of C. concisus 13826 was used, as the genome of C. ureolyticus was not available. The results of this study were filtered so that proteins with Mascot scores < 40 were excluded as were single peptide identifications. 294 Appendices APPENDIX Figure 1. Pie chart outlining the functional classification of C. ureolyticus UNSWCD bioinformatically analysed and predicted to be non- secreted proteins (n = 82), with values expressed as percentages 295 Appendices APPENDIX Table 2. Regulation of (n = 84) genes within the autophagy pathway upon infection with C. concisus UNSWCD. Three biological replicates from each of the non-infected and infected cells were analysed. Fold Gene Gene name Refseq P -value 95% CI change AKT1 V-akt murine thymoma NM_005163 0.8357 0.109772 (0.70, 0.97) viral oncogene homolog 1 AMBRA1 Autophagy/beclin-1 NM_017749 0.7142 0.016645 (0.59, 0.84) regulator 1 APP Amyloid beta (A4) NM_000484 0.9082 0.20646 (0.79, 1.02) precursor protein ATG10 ATG10 autophagy related NM_031482 0.6788 0.424476 (0.12, 1.24) 10 homolog ATG12 ATG12 autophagy related NM_004707 1.0776 0.511287 (0.86, 1.30) 12 homolog ATG16L1 ATG16 autophagy related NM_017974 0.8712 0.265105 (0.70, 1.04) 16-like 1 ATG16L2 ATG16 autophagy related NM_033388 0.8533 0.618848 (0.36, 1.35) 16-like 2 ATG3 ATG3 autophagy related 3 NM_022488 1.053 0.667633 (0.74, 1.36) homolog ATG4A ATG4 autophagy related 4 NM_052936 1.0289 0.802141 (0.81, 1.25) homolog A ATG4B ATG4 autophagy related 4 NM_178326 0.7888 0.0155 (0.69, 0.88) homolog B ATG4C ATG4 autophagy related 4 NM_178221 0.9824 0.926307 (0.76, 1.20) 296 Appendices homolog C ATG4D ATG4 autophagy related 4 NM_032885 0.7744 0.14835 (0.57, 0.98) homolog D ATG5 ATG5 autophagy related 5 NM_004849 1.0457 0.719012 (0.69, 1.40) homolog ATG7 ATG7 autophagy related 7 NM_006395 0.8017 0.012916 (0.72, 0.88) homolog ATG9A ATG9 autophagy related 9 NM_024085 0.8793 0.42937 (0.65, 1.11) homolog A ATG9B ATG9 autophagy related 9 NM_173681 0.7497 0.034192 (0.60, 0.90) homolog B BAD BCL2-associated agonist NM_004322 0.8533 0.157963 (0.70, 1.01) of cell death BAK1 BCL2-antagonist/killer 1 NM_001188 0.9893 0.882286 (0.84, 1.14) BAX BCL2-associated X NM_004324 0.8035 0.030812 (0.70, 0.91) protein BCL2 B-cell CLL/lymphoma 2 NM_000633 1.2874 0.431115 (0.60, 1.98) BCL2L1 BCL2-like 1 NM_138578 0.8692 0.298205 (0.67, 1.07) BECN1 Beclin 1, autophagy NM_003766 1.0077 0.848852 (0.91, 1.10) related BID BH3 interacting domain NM_001196 1.0876 0.025435 (1.04, 1.14) death agonist BNIP3 BCL2/adenovirus E1B NM_004052 1.1364 0.116306 (0.99, 1.28) 19kDa interacting protein 3 CASP3 Caspase 3, apoptosis- NM_004346 0.8999 0.301327 (0.74, 1.06) related cysteine peptidase CASP8 Caspase 8, apoptosis- NM_001228 1.0147 0.899799 (0.83, 1.19) 297 Appendices related cysteine peptidase CDKN1B Cyclin-dependent kinase NM_004064 0.8895 0.444947 (0.64, 1.13) inhibitor 1B (p27, Kip1) CDKN2A Cyclin-dependent kinase NM_000077 1.0926 0.025666 (1.04, 1.15) inhibitor 2A (melanoma, p16, inhibits CDK4) CLN3 Ceroid-lipofuscinosis, NM_000086 0.8632 0.248869 (0.68, 1.05) neuronal 3 CTSB Cathepsin B NM_001908 0.8875 0.196447 (0.76, 1.02) CTSD Cathepsin D NM_001909 0.8572 0.011508 (0.80, 0.91) CTSS Cathepsin S NM_004079 0.6649 0.059751 (0.45, 0.88) CXCR4 Chemokine (C-X-C motif) NM_003467 1.1737 0.514104 (0.06, 2.29) receptor 4 DAPK1 Death-associated protein NM_004938 0.8454 0.468832 (0.46, 1.23) kinase 1 DRAM1 DNA-damage regulated NM_018370 1.0801 0.188463 (0.98, 1.18) autophagy modulator 1 DRAM2 DNA-damage regulated NM_178454 1.0481 0.547052 (0.89, 1.21) autophagy modulator 2 EIF2AK3 Eukaryotic translation NM_004836 0.9712 0.74008 (0.72, 1.22) initiation factor 2-alpha kinase 3 EIF4G1 Eukaryotic translation NM_182917 0.9145 0.371166 (0.76, 1.07) initiation factor 4 gamma, 1 ESR1 Estrogen receptor 1 NM_000125 1.1416 0.282181 (0.89, 1.39) FADD Fas (TNFRSF6)- NM_003824 0.6378 0.051529 (0.46, 0.82) associated via death 298 Appendices domain FAS Fas (TNF receptor NM_000043 0.904 0.760475 (0.34, 1.47) superfamily, member 6) GAA Glucosidase, alpha; acid NM_000152 1.0077 0.944552 (0.83, 1.18) GABARAP GABA(A) receptor- NM_007278 0.8377 0.105573 (0.70, 0.98) associated protein GABARAPL GABA(A) receptor- NM_031412 0.9124 0.021973 (0.87, 0.96) 1 associated protein like 1 GABARAPL GABA(A) receptor- NM_007285 1.0751 0.252664 (0.96, 1.19) 2 associated protein-like 2 HDAC1 Histone deacetylase 1 NM_004964 0.8204 0.002512 (0.78, 0.86) HDAC6 Histone deacetylase 6 NM_006044 0.9273 0.711266 (0.67, 1.19) HGS Hepatocyte growth factor- NM_004712 1.113 0.634149 (0.71, 1.51) regulated tyrosine kinase substrate HSP90AA1 Heat shock protein 90kDa NM_0010179 1.0481 0.399394 (0.94, 1.15) alpha (cytosolic), class A 63 member 1 HSPA8 Heat shock 70kDa protein NM_006597 0.798 0.019944 (0.70, 0.89) 8 HTT Huntingtin NM_002111 0.9622 0.825739 (0.56, 1.37) IFNG Interferon, gamma NM_000619 3.3662 0.371358 (0.00001, 11.01) IGF1 Insulin-like growth factor NM_000618 0.4878 0.088337 (0.15, 0.83) 1 (somatomedin C) INS Insulin NM_000207 0.1154 0.142812 (0.00001, 0.37) IRGM Immunity-related GTPase NM_0011458 0.4036 0.858062 (0.00001, 299 Appendices family, M 05 1.42) LAMP1 Lysosomal-associated NM_005561 0.8166 0.067864 (0.70, 0.93) membrane protein 1 MAP1LC3A Microtubule-associated NM_181509 1.9156 0.198362 (0.33, 3.50) protein 1 light chain 3 alpha MAP1LC3B Microtubule-associated NM_022818 1.2697 0.018016 (1.12, 1.42) protein 1 light chain 3 beta MAPK14 Mitogen-activated protein NM_001315 0.8875 0.064372 (0.81, 0.97) kinase 14 MAPK8 Mitogen-activated protein NM_002750 0.9468 0.548737 (0.79, 1.10) kinase 8 MTOR Mechanistic target of NM_004958 1.0147 0.863695 (0.86, 1.17) rapamycin (serine/threonine kinase) NFKB1 Nuclear factor of kappa NM_003998 0.9209 0.273982 (0.80, 1.04) light polypeptide gene enhancer in B-cells 1 NPC1 Niemann-Pick disease, NM_000271 0.9338 0.589012 (0.73, 1.14) type C1 PIK3C3 Phosphoinositide-3- NM_002647 1.0726 0.450558 (0.90, 1.25) kinase, class 3 PIK3CG Phosphoinositide-3- NM_002649 0.1156 0.119203 (0.00001, kinase, catalytic, gamma 0.39) polypeptide PIK3R4 Phosphoinositide-3- NM_014602 0.9802 0.857482 (0.80, 1.16) kinase, regulatory subunit 300 Appendices 4 PRKAA1 Protein kinase, AMP- NM_006251 1.0147 0.927775 (0.82, 1.21) activated, alpha 1 catalytic subunit PTEN Phosphatase and tensin NM_000314 1.0361 0.775847 (0.76, 1.32) homolog RAB24 RAB24, member RAS NM_130781 1.2667 0.133708 (0.98, 1.55) oncogene family RB1 Retinoblastoma 1 NM_000321 0.8338 0.21895 (0.63, 1.03) RGS19 Regulator of G-protein NM_005873 0.8652 0.350359 (0.64, 1.09) signaling 19 RPS6KB1 Ribosomal protein S6 NM_003161 0.9145 0.323422 (0.78, 1.05) kinase, 70kDa, polypeptide 1 SNCA Synuclein, alpha (non A4 NM_000345 0.9712 0.78983 (0.73, 1.22) component of amyloid precursor) SQSTM1 Sequestosome 1 NM_003900 0.9359 0.539094 (0.73, 1.14) TGFB1 Transforming growth NM_000660 0.7209 0.029435 (0.57, 0.88) factor, beta 1 TGM2 Transglutaminase 2 NM_004613 0.9689 0.759672 (0.80, 1.14) (C polypeptide, protein- glutamine-gamma- glutamyltransferase) TMEM74 Transmembrane protein NM_153015 1.2067 0.309877 (0.79, 1.62) 74 TNF Tumor necrosis factor NM_000594 0.3442 0.461194 (0.00001, 1.12) 301 Appendices TNFSF10 Tumor necrosis factor NM_003810 0.6726 0.207533 (0.33, 1.02) (ligand) superfamily, member 10 TP53 Tumor protein p53 NM_000546 0.7708 0.162883 (0.54, 1.00) ULK1 Unc-51-like kinase 1 NM_003565 0.8091 0.137891 (0.62, 1.00) (C. elegans) ULK2 Unc-51-like kinase 2 NM_014683 1.6145 0.342779 (0.17, 3.06) (C. elegans) UVRAG UV radiation resistance NM_003369 0.904 0.287198 (0.75, 1.05) associated gene WIPI1 WD repeat domain, NM_017983 0.8357 0.057777 (0.72, 0.95) phosphoinositide interacting 1 Three biological replicates from each of the non-infected and infected cells were analysed. 302