Engineering Probiotic Bacteria for Use as Antibiotic Alternatives

123 Pages·2017·4.11 MB·English

Engineering Probiotic Bacteria for Use as Antibiotic
Alternatives

A DISSERTATION
SUBMITTED TO THE FACULTY OF THE
UNIVERSITY OF MINNESOTA
BY

Brittany Anne Forkus

IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

Wei-Shou Hu

February 2018
© Brittany A. Forkus 2018
Acknowledgements

This dissertation would not have been completed without the critical help and
support I have received along the way from my advisors, peers, and family.
Firstly, I must thank Professor Wei-Shou Hu for helping me finish strongly with a
dissertation I am proud of. The way you advise students and care about their personal and
professional development really embodies what the University of Minnesota stands for
and I am so happy I got the opportunity to work closely with you.
I would also like to thank my committee members, Professor Hackel, Professor
Zhang, and Professor Johnson, for their continued support and investment in my
education. Professor Hackel is not listed on my transcript as an official advisor, however,
your technical and professional support has largely shaped my development as a
researcher. Professor James Johnson, collaborating with you has been a highlight of my
graduate school experience and has challenged me in areas outside of classical chemical
engineering, I thank you for the opportunity.
I must also extend my gratitude to Professor Paul Dauenhauer who has pushed me
academically since teaching me Kinetics at UMASS Amherst. Thanks to you and your
continued investment in me, I have found a career I am passionate about and have a
strong foundation to continue to grow from. I will always remember how you said that
once you study engineering you see the world differently and now, in this position, I
couldn’t agree more.
I thank the Kaznessis and Hu group members for their critiques and support
throughout my studies. I especially thank Seth Ritter, who has been my co-pilot in this
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journey, you have continued to challenge me, offer comic relief, and broaden my skillset,
and it has been a privilege to work with you.
I thank my peers, Sadie Johnson, Jeff Ting, Larry Stern, Panagiota Kryiakou, and
Koustav Ganguly for making my graduate school experience a memorable and positive
journey. Scott White, I thank you for carrying me through some of the darker times of
grad school and celebrating the highs with me.
Lastly I thank my family. They have supported me throughout my entire
academic career and I know they will continue to support me after. I am lucky to have
such great role models in my life and whose company make my life so rich. Shannon and
Paul, your support and friendship are two of the most valuable assets of my life. Sophia
and Corin, I look forward to seeing what great things you two will accomplish. Renee,
thank you for ignoring the job description of a stepmom and being there for me whenever
I need it. Mom, thank you for being a working female who has inspired me to build my
own career. Dad, thank you for always believing I would make it to this point even when
I doubted it.

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Dedication
To my family

‘The beautiful thing about learning is that no one can take it away from you’ –B.B. King

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Abstract

Decades of overuse of antibiotics has led to the emergence of resistant infections
across the globe. Healthcare professionals are running out of viable options, as clinical
isolates have begun resisting treatment to even last resort therapies. The emergence of
these ‘superbugs’, coupled with the lack of new drugs in the discovery pipeline, has led
to the possibility of a ‘post-antibiotic’ era.
With the primary driving force for resistance development being the overuse of
antibiotics, technologies are being sought to limit their injudicious application within the
clinical and agricultural sectors. An attractive contender in the fight against microbial
resistance are antimicrobial peptides (AMPs). AMPs are small peptides that are produced
natively from organisms across all domains of life as a first line of defense against
microbial challenge. However, despite decades of research on their therapeutic potential,
AMPs have widely failed in translational success due to delivery and synthesis
challenges.
Many AMPs are unable to survive passage to the gastrointestinal (GI) tract, the
residence of many bacterial pathogens, limiting their utility to topical applications. In this
work, we propose engineering probiotic bacteria as AMP-delivery vehicles to overcome
their inherent transport barriers and localize their production at the site of infection. We
focus on modifying the probiotic, E. coli Nissle 1917 (EcN), which has shown promise in
both human and animal health.
This work takes a synthetic biology approach to iteratively improve and redesign
AMP biosynthetic gene clusters. Through strong collaborations with the Veterans’
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Affairs Medical Center and Department of Veterinary Sciences, the work within takes a
highly translational approach incorporating professional veterinary and clinical oversight
at the design stages to develop systems with meaningful downstream applications.
We describe our development of an engineered EcN derivative, EcN(J25), that
has antagonistic activity against foodborne Salmonella. With EcN(J25), we demonstrated
the first in vivo success of AMP-producing probiotics. EcN(J25) was capable of reducing
Salmonella carriage in poultry by 97% after a two-week period with no detectable impact
on the native microbiome. This proof-of-concept opens an alternative method to reduce
pathogen counts in livestock without incorporating medically important antibiotics in the
feed supply.
We then developed an alternative EcN derivative, EcN(C7), which targets the
rising multi-drug resistant E. coli strain, sequence Type ST131. The goal was to develop
an engineered probiotic that could be used as a decolonization measure for ST131 in the
clinic. We further explored mechanisms of resistance of ST131 to aid in the development
of future combination therapies.
We also describe our development of two new synthetic biology tools, ProTeOn+
and pMPES. ProTeOn+ is a synthetic-hybrid promoter that enables robust protein
expression without exogenous induction. ProTeOn+ has demonstrated functional utility
in many of the described engineered AMP networks. pMPES serves as a modular peptide
expression system that allows heterologous secretion of a variety of AMPs from EcN.
This work take a synthetic biology perspective to describe many of the challenges
and potential of engineered probiotics, laying a foundation for future work in this field.
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Table of Contents

Acknowledgements .............................................................................................................. i
Abstract .............................................................................................................................. iv
Table of Figures ................................................................................................................. ix
List of Tables ..................................................................................................................... xi
Chapter 1 Introduction ........................................................................................................ 1
1.1 The Rise of Antibiotic Resistant Bacteria ................................................................. 1
1.2 Antimicrobial Peptides (AMPs): A Potential Antibiotic Alternative........................ 3
1.3 Recruiting Probiotics to Treat Enteric Infections ...................................................... 5
1.3.1 Enteric Infections and Foodborne Illness ........................................................... 5
1.3.2 The Role of Probiotics in Gastrointestinal (GI) Health ...................................... 6
1.3.3 Engineered Probiotics as AMP-delivery vehicles .............................................. 7
1.4 Scope and Organization of Thesis ............................................................................. 8
Chapter 2 ProTeOn+: A Synthetic Promoter for the Delivery of Antimicrobial Peptides 11
2.1 Scope ....................................................................................................................... 11
2.2 Introduction ............................................................................................................. 11
2.3 Results ..................................................................................................................... 14
2.3.1 ProTeOn+ Network Mechanics ........................................................................ 14
2.3.2 Stochastic-Kinetic Simulations of ProTeOn+ .................................................. 18
2.3.3 Incorporate ProTeOn+ into MccV-gene cluster ............................................... 21
2.4 Discussion ............................................................................................................... 26
2.5 Materials and Methods ............................................................................................ 27
2.5.1 Bacterial Strains and Plasmid Construction ..................................................... 27
2.5.2 Spectrophotometer Fluorescence Assays ......................................................... 28
2.5.3 GFP Quantification via Flow cytometry .......................................................... 29
2.5.4 Stochastic chemical reaction simulations ......................................................... 30
2.5.5 Liquid Supernatant Activity Assays ................................................................. 30
2.5.6 Agar Diffusion Assay ....................................................................................... 31
Chapter 3 Antimicrobial Probiotics Reduce Salmonella enterica in Turkey
Gastrointestinal Tracts ...................................................................................................... 32
3.1 Scope ....................................................................................................................... 32
3.2 Introduction ............................................................................................................. 33
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3.3 Design of System for AMP Production and Secretion ............................................ 35
3.4 Results ..................................................................................................................... 37
3.4.1 In Vitro SE Growth Inhibition .......................................................................... 37
3.4.2 SE reduction in turkey ceca following EcN(J25) treatment ............................. 39
3.4.3 Microbiome Analysis ....................................................................................... 42
3.5 Discussion ............................................................................................................... 44
3.6. Materials and Methods ........................................................................................... 46
3.6.1 Bacterial strains and plasmid construction ....................................................... 46
3.6.2 Zone of Inhibition Activity Assay .................................................................... 46
3.6.3 In vitro supernatant activity assay .................................................................... 47
3.6.4 Bacterial challenge/treatment of turkey poults ................................................. 47
3.6.5 Bacterial Challenge Strains .............................................................................. 48
3.6.6 Animal trial 1 .................................................................................................... 48
3.6.7 Animal trial 2 .................................................................................................... 49
3.6.8 Enumeration of Salmonella and Nissle in cecal contents ................................. 50
3.6.9 Statistical analysis of cecal counts.................................................................... 50
3.6.9 Microbiome Extraction and Analysis ............................................................... 52
3.6.10 Vertebrate Animal Experiments ..................................................................... 52
Chapter 4 Engineered Probiotics to Target Multidrug-Resistant E. coli .......................... 53
4.1 A Rising Superbug: E. coli Sequence Type ST131 ................................................ 53
4.2 Microcin C7: A Trojan Horse Antimicrobial Peptide ............................................. 55
4.3 Development of a Mcc7-Producing Probiotic ......................................................... 59
4.3.1 Analysis of the Mcc7 Promoter ........................................................................ 59
4.3.2 In Vitro activity of Mcc7 producing E. coli ...................................................... 62
4.4 Mechanism of Resistance in JJ1886........................................................................ 65
4.5 Future Directions ..................................................................................................... 68
4.5.1 Test EcN(C7) in mouse decolonization models ............................................... 68
4.5.2 Peptide Engineering of Mcc7 ........................................................................... 68
4.6 Materials and Methods ............................................................................................ 70
4.6.1 Promoter assays with GFP ................................................................................ 70
4.6.2 Kinetic supernatant inhibition assays ............................................................... 70
4.6.3 Isolation of JJ1886 mutants stably resistant to Mcc7. ..................................... 71
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4.6.4 qRT-PCR analysis of yejABEF ......................................................................... 71
Chapter 5 Microcin V: Foundation for modular AMP-expression systems ..................... 73
5.1 Introduction ............................................................................................................. 73
5.1.1 Heterologous Secretion of Antimicrobial Peptides .......................................... 74
5.2 Genetic Organization of Microcin V ....................................................................... 75
5.3 Results ..................................................................................................................... 77
5.3.1 Development of pMPES 1.0 ............................................................................. 77
5.3.2 Bacteriocins tested for secretion ....................................................................... 78
5.3.3 Development of pMPES 3.0 ............................................................................. 80
5.4 Secretion of peptides from EcN using MccH47 Export Machinery ....................... 84
5.5 Conclusions ............................................................................................................. 86
5.6 Materials and Methods ............................................................................................ 87
5.6.1 Development and testing of pMPES 1.0 .......................................................... 87
5.6.2 Construction of pMPES 3.0 .............................................................................. 87
Chapter 6 Conclusions and Future Perspectives ............................................................... 89
Chapter 7 Bibliography ..................................................................................................... 93
Chapter 8 Appendix ........................................................................................................ 101
Appendix A: ProTeOn+: A Synthetic Hybrid Promoter for the Delivery of
Antimicrobial Peptides ................................................................................................ 101
Appendix B: Antimicrobial Probiotics Reduce Salmonella enterica in Turkey
Gastrointestinal Tracts................................................................................................. 105
Appendix C: Engineered Probiotics to Target Multidrug Resistant E.coli ................. 110

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