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Pathogenesis of Enterococcal‑Associated Wound Infections

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Pathogenesis of Enterococcal‑Associated Wound Infections This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Inside‑out : pathogenesis of enterococcal‑associated wound infections Tay, Wei Hong 2018 Tay, W. H. (2018). Inside‑out : pathogenesis of enterococcal‑associated wound infections. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/87455 https://doi.org/10.32657/10220/46716 Downloaded on 30 Sep 2021 23:08:00 SGT INSIDE-OUT: PATHOGENESIS OF ENTEROCOCCAL- ASSOCIATED WOUND INFECTIONS TAY WEI HONG B.Sc. Biological Sciences (Hons) Nanyang Technological University Interdisciplinary Graduate School Singapore Centre for Environmental Life Sciences Engineering A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2018 ACKNOWLEDGEMENTS This is, without doubt, the most difficult section to write. Nevertheless, I will try my best. First and foremost, I would like to express my gratitude and appreciation to my supervisor, Kimberly Kline. I must say, I will be hard-pressed to find another supervisor that is exceedingly kind, patient, supportive and understanding than Kim. Thank you, for never doubting me, giving me the freedom to develop my own research and for guiding me along in asking the right questions. Without your unwavering guidance and support, I would have not grown to be the scientist that I am today. I am thankful to my co-supervisor Swaine Chen and my mentor John Common for their guidance, supervision and technical expertise. I would not have made it thus far, without the critical inputs and suggestions given. Special thanks to all members of the Kline lab, both past and present. Everyone’s input during the Monday, pathogenesis and BUG meetings allowed us to drive this project forward. I am grateful to my partner in crime, Kelvin Chong. Thank you, for the boisterous discussions that we had. We will always make the best tag-team. I am especially thankful to have Pei Yi around, thank you, for all the scientific and non-scientific help given and for being there, when I needed a listening ear. Life in the Kline lab will never be the same, without your infectious laughter and your cheerful personality. I am also thankful to Sharon Goh, Rafi Rashid, Eliza Soh, Artur Matysik, Irina Afonina and Sumitra Mitra for their scientific advises and friendship. I am also incredibly fortunate to have worked with Rayson Ho, Hui Yi, Joyce Pek, Jun Jie, Eunice and Wan Xin. Thank you for being such wonderful students and I hope that all of you will be successful in all your future endeavors. I have learnt a lot from all of you! I am indebted to Brenda Tien, Foo Kiong, Anu, Wooi Keong, Song Lin and Sarah Kay for helping me with the maintenance and upkeep of the Tissue Culture Facility. I am also grateful to the following laboratory support personnel: Krager, Wey Yeeng, Kartini, Deborah, Ley Byan, Wahyuna and Lee Chin Weng for assisting me with purchasing, facility management as well as consumables collections. All of you have always been under- appreciated by the rest of the researchers. Without all of you, how could we carry on with our research in SCELSE? I would also like to extend my appreciation to my fellow PhD course mates Hafiz, Jia Wang, Milton Kwek, Erica Teo, Lian Yilong, Wendy Phua, Penny Oh, Jeremy Lim and Mark Tan for their friendship and help throughout this journey. Thank you for your support throughout these 4 long years. I must also thank Evelyn Wong, Serene Tan, Melissa Tan and Lee Jing for supporting and helping me. Purchasing equipment and consumables would have been a lot more expensive and troublesome without your assistance. Finally, I am immensely fortunate to have the full support of my wife, Yi Qi and my family members. This journey would not have been possible without your understanding and accommodation to my schedule for family events. Thank you for always believing in me and I dedicate this thesis to all of you. I do not usually say this out loud, or put it in writing, but I love you all! This section took an unbelievable two weeks to craft. I hope that I did not miss anyone out. I would like to end off, with a quote from the 1997 science fiction film, Gatacca: “There is no gene for the human spirit” TABLE OF CONTENTS Sections Page SUMMARY i LIST OF TABLES iii LIST OF FIGURES iv LIST OF ABBREVIATIONS vii PUBLICATIONS x PRESENTATIONS xi CHAPTER 1: BACKGROUND AND SIGNIFICANCE 1 1.1 Research motivation 1 1.2 Epidemiology of wound infections and factors involved 2 1.2.1 Microbiology of wound infections 3 1.2.2 Biofilms in wound infections and the host response 4 1.2.3 Polymicrobial wound infections 7 1.3 Enterococci 8 1.3.1 Epidemiology of Enterococcal infections 9 1.3.2 Enterococcal wound infections 10 1.3.3 Pathophysiology and virulence factors involved in 11 infection 1.3.4 Enterococcal biofilms and infection 13 1.3.5 Intracellular survival and dissemination of E. faecalis 14 1.4 Keratinocytes and the innate immune response 16 1.5 Thesis outlines and specific aims 18 1.6 References for Chapter 1 21 CHAPTER 2: ENTEROCOCCAL METABOLITE CUES 31 FACILITATE INTERSPECIES NICHE MODULATION AND POLYMICROBIAL INFECTION 2.1 Introduction and motivation 31 2.2 Results 38 2.2.1 Enhanced E. coli growth by E. faecalis is dependent on 38 enterobactin siderophore production 2.2.2 E. coli ferrous uptake systems are not important for E. 39 faecalis-driven E. coli growth promotion 2.2.3 Co-culture of E. faecalis and E. coli results in enhanced 41 biofilm/biomass under iron deplete conditions 2.2.4 E. faecalis Transposon library screen and validation 42 2.2.5 E. faecalis Transposon library single species secondary 46 validation 2.2.6 L-ornithine, but not branched-chain amino acids, 48 augments E. coli biofilm formation under iron limitation 2.3 Discussions 51 2.4 Materials and methods 56 2.5 Contributions 62 2.6 References for Chapter 2 64 CHAPTER 3: CHARACTERIZATION OF THE 69 INTRACELLULAR LIFESTYLE OF ENTEROCOCCUS FAECALIS 3.1 Introduction and motivation 69 3.2 Results 74 3.2.1 E. faecalis forms microcolonies on the wound surface and 74 under the wound bed 3.2.2 Intracellular E. faecalis can be recovered from CD45+ and 76 CD45- cells 3.2.3 E. faecalis adheres to and enters keratinocytes 77 3.2.4 E. faecalis entry is not cell line specific 79 3.2.5 E. faecalis survives up to 72 hpi within keratinocytes 80 3.2.6 E. faecalis is able to multiply within keratinocytes and 82 escape 3.2.7 E. faecalis escapes from infected keratinocytes and 83 subsequently infects other keratinocytes when conditions are favourable 3.2.8 E. faecalis exists within early and late endosomes 84 3.2.9 Lamp1+ vacuoles that contain E. faecalis lack Cathepsin D 87 3.2.10 E. faecalis is present in compartments lacking Lamp1 at 88 late timepoints in the cell periphery 3.3 Discussions 93 3.4 Materials and methods 98 3.5 Contributions 107 3.6 References for Chapter 3 108 CHAPTER 4: ENTRY OF ENTEROCOCCUS FAECALIS INTO 112 KERATINOCYTES AND IMMUNE-MODULATION 4.1 Introduction and motivation 112 4.2 Results 117 4.2.1 E. faecalis entry into keratinocytes is dependent on 117 microfilament polymerization but independent of microtubule polymerization 4.2.2 E. faecalis entry into keratinocytes is clathrin and 119 caveolae independent 4.2.3 E. faecalis entry into keratinocytes is dependent on PI3K 121 signalling 4.2.4 Classical E. faecalis virulence factors are dispensable for 122 attachment and uptake into keratinocytes 4.2.5 Intracellular E. faecalis can modulate the immune 124 response in keratinocytes 4.3 Discussions 127 4.4 Materials and methods 131 4.5 Contributions 134 4.6 References for Chapter 4 135 CHAPTER 5: SUMMARY AND PERSPECTIVES 140 5.1 Summary of findings 142 5.2 Future perspectives 145 5.3 References for Chapter 5 152 Inside-Out: Pathogenesis of Enterococcal-associated wound infections by Wei Hong TAY Interdisciplinary Graduate School Singapore Centre for Environmental Life Sciences Engineering Nanyang Technological University Summary Wound infections represent a significant health problem, due to their difficulty in management and the cost associated with their treatment. Surgical wounds are often infected with Enterococci, which is one of the top 3 most frequently isolated bacterial species from this site. However, mechanisms that govern and contribute to Enterococcal pathogenesis in wounds have not been elucidated. The following thesis hypothesized that polymicrobial wounds with Enterococci will result in a poorer disease outcome, due to synergistic effects with other microbial species. We also proposed that persistent and recurring wound infections are due to Enterococci having a dual extracellular and intracellular lifestyle. We used an unbiased screening approach mimicking in vivo wound conditions to screen for Enterococcal factors that are responsible for enhanced E. coli biofilm growth in wounds. We demonstrated that the Enterococcal metabolite, L-ornithine can induce siderophore production in E. coli, resulting in enhanced virulence under iron limiting conditions. Using both in vitro and in vivo models, we also showed that Enterococci have a dual extracellular and intracellular infective lifestyle. Together, these studies demonstrate the contributions of Enterococci in wound infections, both outside of the host cells and inside of the host cells. Our work provides evidence of cooperative extracellular interactions between Enterococci and other co-infecting microbial species in wounds, and demonstrates that it has a dual infective lifestyle. Our findings will be useful to clinicians in managing persistent wound infections and could potentially result in the development of improved treatment strategies or therapeutics.
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