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Engineering Antimicrobial Probiotics for the Treatment of Vancomycin-Resistant Enterococcus

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Engineering Antimicrobial Probiotics for the Treatment of Vancomycin-Resistant Enterococcus Engineering Antimicrobial Probiotics for the Treatment of Vancomycin-Resistant Enterococcus A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY KATHRYN GELDART IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ADVISOR: YIANNIS N. KAZNESSIS December, 2016 © Kathryn Geldart 2016 ACKNOWLEDGEMENTS I am deeply grateful to my Ph.D. advisor, Yiannis Kaznessis for his guidance, encouragement, and unwavering faith in my abilities throughout my time as his graduate student. His optimism and enthusiasm towards this project provided a constant fuel of inspiration, both in times of success and frustration. The trust he instilled in me gave me confidence and freedom to explore unfamiliar techniques and topics. We now know more about E. faecium resistance than either one of us originally intended. Once again, thank you Yiannis, for everything. I must also thank our collaborator, Gary Dunny, for his support and advice on bacterial techniques used throughout this project. Gary’s expertise on Enterococcus has played a key role in this project and I am incredibly grateful for the time he has taken for all of our discussions. I thank the members of Gary’s lab, especially PostDocs Dawn Manias, Jennifer Dale, and Yuqing Chen who taught me numerous techniques that have been fundamental to this work. I also thank our other collaborators, Nita Salzman and her PostDoc Sushma Kommineni for their expert advice on Enterococcus in in vivo settings and for the vast efforts they have put into the mouse trials. I thank my current lab mates Brittany Forkus and Seth Ritter for your invaluable input, support, and daily comic relief. I am truly fortunate to have worked with such brilliant researchers and more importantly, friends. Brittany; thank you for this tumultuous path we have taken together starting as roommates at UMass. You have been one of the most influential people in my life and will always be special to me. I also thank the computational half of the group-Michail Vlysidis, Panagiota Kyriakou, Pin-Kuang Lai, and Pedro Constantino for providing the much-needed alternative perspectives on this work and helping to broaden my outlook through fascinating discussions of computational and biological topics. Lastly, I thank the past members of our group- Juan Borrero, Kat Volzing, Patrick Smadbeck, and Kostas Biliouris. In particular, thank you Juan for taking the time to teach me the fundamental techniques I have used throughout my research. I would also like to thank our department as a whole for the incredibly supportive environment. I greatly appreciate Benjamin Hackel’s willingness to help me troubleshoot a myriad of random problems throughout these past four years. I thank Samira Azarin for this i same kindness and openness in assisting me with my qRT-PCR studies. My experience as a graduate student has been enriched by this unique sense of community fostered by CEMS. Finally, I thank my amazing family. Mom- thank you for teaching me to be strong, practical, and self-reliant. Dad- thank you for passing on your love of learning and optimistic personality and for reminding me not to take life so seriously. JR- thanks for providing me with a lifetime of entertainment and reminding me that it’s okay to relax. I’m so proud to be your sister. Lastly Max Kruziki. Thank you for supporting me even on my worst days and for constantly reminding me that the sacrifices I make are my choice. It is because of you that I have a clearer image of what I want from life and who I want to be as a person. I am truly lucky to have you in my life. ii Dedication To my family iii Abstract The rise of antibiotic resistance in bacteria has become an urgent concern in global healthcare. There is now a strong drive for the preservation of our current antibiotics as well as for the rapid development of new antibacterial therapies. Antimicrobial peptides (AMPs) are a vast collection of proteins naturally produced by living organisms as a defense mechanism against invading microbes. Unfortunately, though society has been aware of the therapeutic potential of AMPs for many years, their utility has been limited to topical applications because of toxicity and degradation in the body. Moreover, many bacterial infections originate in the gastrointestinal (GI) tract, which is largely unreachable for most AMPs by oral administration. To overcome this delivery challenge, we are engineering probiotic bacteria that can actively produce and deliver AMPs inside the GI tract. Vancomycin-resistant enterococci (VRE) are among the most difficult to treat pathogens in hospital environments. These bacteria frequently reside in the GI tract, often in low counts because of competition from the surrounding microbiota. When patients are given broad- spectrum antibiotics, the competition is reduced and VRE dominate in numbers. The pathogen can then spread to other host organs or to the surrounding hospital environment. Delivery of AMPs targeting VRE by probiotics may provide an alternative treatment or prevention option against these deadly pathogens. Importantly, the elimination of VRE from the GI tract using VRE-specific AMPs may allow removal of VRE while minimizing disruption of the surrounding bacteria. In this thesis, we describe the development of two different probiotic delivery systems for the reduction of the two major causative species of VRE infections, Enterococcus faecium and Enterococcus faecalis. The first probiotic platform employs the Gram-positive species, Lactococcus lactis, for the production of three class IIa bacteriocins, AMPs endogenously produced by bacteria. We have developed a chloride-inducible expression vector for AMP delivery from L. lactis, which we show to be activated by physiologically-relevant chloride concentrations. Herein, we demonstrate the ability of this system to inhibit VRE, first in in vitro cultures. VRE intestinal colonization models in mice were then developed and used to test the iv efficacy of our engineered L. lactis in the GI tract. Multiple trials showed statistically significant reduction of Enterococcus faecium in L. lactis treated mice compared to untreated mice. The second probiotic delivery system uses probiotic Escherichia coli Nissle 1917 (EcN). Currently, no anti-enterococcal peptides are known to be naturally produced from E. coli. In this project, we developed a modular AMP expression system that can be used in E. coli to express and secrete a variety of AMPs derived from a wide range of producer strains. With this system, we are able to produce AMPs targeting not only Gram-positive pathogens like VRE, but also Gram-negative pathogens including Salmonella and diarrheagenic E. coli. We show this system can be used to simultaneously express multiple anti-enterococcal peptides in vitro. Lastly, we demonstrate the efficacy of Nissle producing Enterocin B, Enterocin A, and Hiracin JM79 in reducing VRE colonization in mice. The final section of this thesis addresses the concern of bacterial resistance development to our antimicrobial probiotics. Class IIa bacteriocins are currently the most thoroughly-studied class of AMPs targeting enterococci. Though the mechanism of resistance to these peptides has been studied in E. faecalis, it has never been examined in E. faecium. In this project, we identified a mannose phosphotransferase in E. faecium that appears to be directly involved in E. faecium susceptibility to class IIa bacteriocins. We show that resistant mutants exhibit either downregulation or direct mutation of this transporter and that heterologous expression of this transporter in L. lactis confers susceptibility to the otherwise unsusceptible strain. We then include a brief discussion of the implications of this mode of resistance and potential methods for preventing resistance development in the future. v Table of Contents List of Tables ............................................................................................................................ ix List of Figures .......................................................................................................................... ix Chapter 1: Introduction ........................................................................................................... 1 1.1 The Rise in Antibiotic Resistance ........................................................................................... 1 1.2 Antimicrobial Peptides: An Untapped Reservoir of Antimicrobial Activity ........................... 3 Chapter 2: Delivery of AMPs Targeting Vancomycin-Resistant Enterococci .............................. 9 2.1 Vancomycin-Resistant Enterococcus: A Pathogen of Interest ............................................... 9 2.2 Antimicrobial Peptides Targeting VRE ................................................................................. 10 2.3 Natural and Heterologous Bacteriocin Production from Gram-Positive Species ................ 13 Chapter 3: Probiotic Platform 1 - Lactococcus lactis ............................................................... 16 3.1 Lactococcus lactis as a Delivery Organism ........................................................................... 16 3.1.1 Lactococcus lactis .......................................................................................................... 16 3.1.2 Lactococcal Gene Expression Systems .......................................................................... 17 3.2 A Chloride-Inducible Expression Vector for AMP Delivery from L.
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