ENGINEERING B. SUBTILIS TRANSCRIPTIONAL CONTROL AND PHYSIOLOGY FOR THE ADVANCEMENT OF BACTERIOTHERAPIES By Emily Marilynn Greeson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Microbiology and Molecular Genetics – Doctor of Philosophy 2022 ABSTRACT ENGINEERING B. SUBTILIS TRANSCRIPTIONAL CONTROL AND PHYSIOLOGY FOR THE ADVANCEMENT OF BACTERIOTHERAPIES By Emily Marilynn Greeson This dissertation explores to avenues of improvement for current bacteriotherapy approaches. Cody Madsen and I worked closely to advance engineered B. subtilis as a modular platform technology and Dr. Ashley Makela was instrumental in the in vivo studies (Chapter 2). In Chapter 2, transcriptional control of B. subtilis will demonstrate the ability to use magnetothermal energy generated by superparamagnetic iron oxide nanoparticles (SPIONs) and alternating magnetic fields (AMF) to induce temperature-sensitive repressors. Chapter 3 demonstrates how synthetic biology techniques can allow engineered B. subtilis to invade epithelial cells with the “zipper” mechanism. This was a collaborative effort as it was a multidisciplinary study and the contributions of Cody Madsen, Evran Ural, Dr. Ashley Makela, Dr. Bige Unluturk, and Victoria Toomajian were important and have been specifically noted in author contributions at the end of Chapter 3. Most patients on organ transplant waitlists will need alternative therapeutics due to a deficit of organ donations. Regenerative medicine approaches, including cellular reprogramming are being used to help address the deficit, but there are limitations. Bacteriotherapies aim to better deliver the therapeutics to a variety of targets, however, most approaches do so externally to the target cells. B. subtilis, a generally recognized as safe organism, engineered to express listeriolysin O (LLO) has been shown to replicate in the cytoplasm of macrophages and deliver transcription factors and modulate cell surface markers, cytokines, and chemokines. This mechanism of uptake only works with phagocytic cells creating an opportunity for the engineering of B. subtilis that targets internalization into non-phagocytic cells. When introducing known virulence factors into non-native organisms it is important to consider controlling the gene expression while trying to remain as minimally invasive as possible. Alternating magnetic fields (AMF) cause local temperature increases in regions with Superparamagnetic iron oxide nanoparticles (SPIONs), and we investigated the ability of this magnetic hyperthermia approach to regulate temperature-sensitive repressors (TSRs) in bacteria. Magnetic hyperthermia-based control of bacterial gene expression would advance development of bacteriotherapies and expand options of regulated bacterial transcription. TSRs block transcription in a temperature-dependent manner. B. subtilis was coated with three SPION variations, plain-dextran, amine- or carboxyl-coated and the interactions and AMF responses were characterized and induction of the TSRs was demonstrated using AMF. Murine intramuscular injections revealed continual association of SPIONs with B. subtilis. While there was no induction via AMF in vivo, pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to regional bacterial transcriptional control, a minimally invasive method that could be used with virulence factors and therapeutics. To delivery therapeutics to epithelial cells, B. subtilis llo was engineered to express internalin A (InlA), a protein native to Listeria monocytogenes. Internalin A is an adhesin that binds to the E-cadherin host cell receptor found in epithelial cells and mediates a “zipper” mechanism of invasion. B. subtilis llo inlA demonstrated cytosolic persistence and B. subtilis llo remained extracellular. Ultimately, the engineering of B. subtilis transcriptional control and physiology creates a new modular approach to regenerative medicine, cellular reprogramming, and cancer therapy that can be used in human health applications. Copyright by EMILY MARILYNN GREESON 2022 iv This work is dedicated to the individuals who sparked my curiosity and love of science. Especially my family, friends, and MSU and SVSU mentors and colleagues, without their support I would not be on my current trajectory. v ACKNOWLEDGEMENTS Loving science and the work that you do is not enough on its own to complete a dissertation. A strong support system and dedication are necessary for getting through a graduate program almost as much as the enthusiasm for your field. As I write this acknowledgements section I fully realize and appreciate how strong of a network of mentors, mentees, colleagues, friends, and family have helped me achieve my goals and continue to strive for personal growth. There are far too many supporters to name in this document, you know who you are. I want to thank Dr. Christopher H Contag and the members of the Contag lab that I worked closely with throughout this dissertation. Dr. Ashley Makela and Cody Madsen were instrumental in working on the engineered endosymbiont project and without them this work would not have reached this point. Dr. Bige Unluturk, Dr. Chima Maduka, Victoria Toomajian, and Evran Ural were very supportive lab members that helped me think through studies and provided comradery. Ashley Kimmel was an outstanding mentee I had the opportunity to work with and I appreciate the newfound black bear knowledge. I also want to thank the many collaborators that were associated with this work and especially thank the Hardy lab from Dr. Jonathan Hardy to the lab members (Dr. Kayla Conner, Dr. Jon Kaletka, Dr. Justin Lee) in advancing this project through resource sharing and critical discussions. I would like to thank our other collaborators including Dr. Daniel Portnoy for the B. subtilis llo background strain, Dr. Lee Kroos for B. subtilis plasmids, Dr. Mikhail Shapiro for temperature-sensitive repressor troubleshooting and advice, and Dr. Arash Komeili for magnetotactic bacteria related training and protocols. I would also like to thank my committee members Dr. Michael H Bachmann, Dr. Vic DiRita, Dr. Jonathan W Hardy, and Dr. Bryan Smith that provided invaluable suggestions and guidance throughout the completion of this vi dissertation. Thank you to Brenda Lippincott, Roseann Bills, Katie Conley, Dr. Rob Abramovitch, and Dr. Donna Koslowski for their help and insightful advice while navigating my degree. I would like to thank the staff at MSU Flow Cytometry Core, Center for Advanced Microscopy, and Advance Molecular Imaging Facility for providing crucial support in data acquisition and analysis. Additionally, the staff at these facilities provided training that allowed me to develop and advance my skill sets. Specifically, Dr. Melinda Frame, Dr. Alicia Withrow, Dr. Stanley Flegler, Dr. Carol Flegler, and Dr. Xudong Fan at the Center for Advanced Microscopy for providing training, support, and positive attitudes. Thank you to Dr. Neal Hammer, Dr. Phil Deletka, Dr. Poorna Viswanathan, and Dr. Dennis Arvidson for support during my teaching assistantships with the MSU Microbiology and Molecular Genetics (MMG) department. Dr Tami Sivy at the Saginaw Valley State University (SVSU) was a phenomenal academic advisor, biochemistry instructor, research mentor, and honors thesis advisor during my time at SVSU and the Saginaw Bay Environmental Science Institute. Bruce Hart with the SVSU Independent Testing Laboratory and Steve Erdody with the SVSU Chemistry Stockroom were also critical to my success during my undergraduate career and in turn, graduate school. I would like to thank the following mentors and programs that provided me with additional training throughout my graduate education, but also with countless memories and lasting friendships. Thank you to Dr. Julie Rojewski and Dr. Stephanie Watts from the MSU Broadening Experiences in Scientific Training (BEST) program and the other cohort IV members. Thank you to Dr. Michael O’Rourke and Dr. Stephanie Vasko from the MSU Center for Interdisciplinarity Transdisciplinary Graduate Fellows Program (C4I TGFP) including the other 2020-2021 fellows and our community partners at the MyMichigan Medical Center Alma. Thank you to Dr. Rique Campa from the MSU Future Academic Scholars in Teaching (FAST) fellowship program as well vii as the FAST steering committee members, Vicky Phun, other 2021-2022 fellows, and my faculty teaching mentor Dr. Ashley Shade. Thank you to the students in MMG 425 Fall semester 2021 for participating in my study and expanding my research experience. I also am appreciative of the support and opportunities provided to me through the Graduate Women in Science mid-Michigan chapter and the MSU MMG department graduate student workshop. Funding for this dissertation came from the James and Kathleen Cornelius Endowment Fund, the MSU College of Natural Science Dissertation Continuation Fellowship, MSU College of Natural Science Dissertation Completion Fellowship, and teaching fellowships from the MSU Department of Microbiology and Molecular Genetics. I would like to emphasize my gratitude to my friends and family. My friends have been a supportive and understanding throughout the completion of this degree. I want to thank all my friends for being a part of my network and an important part of my life, I appreciate your dedication to me from trying to figure out what I am studying to working alongside me for accountability. My family have been very supportive for as long as I can remember supporting my academic pursuits. I would like to thank my sister, Dr. Megan Greeson, for being my personal statistics hotline. Thank you to Nanny for bragging about me on Facebook and to anyone who will listen and to Daniel for always knocking me down a peg afterwards with a well-timed burn. A special thank you to Ellie (i.e. Queen Elsa), Ruby, and James for reminding me that no matter how busy you get there is always time to have fun and be silly. Finally, I am especially grateful that I have had so many personal examples of women in science providing me with crucial representation starting at an early age. Specifically, my mother Dr. Linda McAllister, my aunt Maggie McAllister, my sister Dr. Megan Greeson, Sue Bentley at Durand Area High School, and Dr. Tami Sivy at Saginaw Valley State University. viii TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................ xii LIST OF FIGURES ..................................................................................................................... xiii KEY TO ABBREVIATIONS .. ....................................................................................................xiv CHAPTER 1 INTRODUCTION AND BACKGROUND: REGENERATIVE MEDICINE AND BACTERIOTHERAPY, TEMPERATURE-SENSITIVE REPRESSORS, AND SYNTHETIC BIOLOGY ................................................................................................................1 INTRODUCTION ...............................................................................................................2 Significance and innovation .........................................................................................2 Temperature-sensitive bacterial repressors ..................................................................5 Internalin A and E-cadherin interactions ......................................................................6 Regenerative medicine and cellular reprogramming ....................................................7 Innate immune response to cytosolic Gram-positive bacteria ......................................9 Platform technology design and approach .................................................................11 Concluding remarks and dissertation overview .........................................................12 REFERENCES ..................................................................................................................14 CHAPTER 2 MAGNETOTHERMAL CONTROL OF TEMPERATURE-SENSITIVE REPRESSORS IN SUPERPARAMAGNETIC IRON NANOPARTICLE-COATED BACILLUS SUBTILIS ......................................................................................................................................25 PUBLICATION NOTICE .................................................................................................26 ABSTRACT.......................................................................................................................27 INTRODUCTION .............................................................................................................28 RESULTS ..........................................................................................................................31 Temperature-sensitive repressor’s control of transcription in B. subtilis ..................31 SPION coating of B. subtilis ......................................................................................34 B. subtilis viability after coating with SPIONs and AMF application .......................38 Magnetothermal induction of B. subtilis transcription ...............................................39 Reducing thermal energy application to in vivo timeframe .......................................42 Iron association with B. subtilis and magnetic hyperthermia in vivo ........................44 DISCUSSION ....................................................................................................................50 MATERIALS AND METHODS .......................................................................................55 Bacterial growth conditions ........................................................................................55 B. subtilis constructs ...................................................................................................55 Iron coating of B. subtilis ...........................................................................................56 Scanning electron microscopy and elemental analysis ..............................................56 Transmission electron miscroscopy ...........................................................................57 In vitro imaging ..........................................................................................................58 Flow cytometry determination of B. subtilis viability ...............................................58 ix RNA extraction and RT-qPCR ...................................................................................59 In vitro thermocycler inductions ................................................................................60 HYPER theranostic hyperthermia system ..................................................................60 In vivo magnetothermal heating and imaging ............................................................61 Histological analysis ...................................................................................................62 Image analysis ............................................................................................................63 Inductively coupled plasma-mass spectrometry (ICP-MS)........................................63 Statistical analysis and visualization ..........................................................................64 Availability of data and materials...............................................................................64 Competing interests ....................................................................................................64 Acknowledgments ......................................................................................................64 Funding .......................................................................................................................65 Affiliations ..................................................................................................................65 Contributions ..............................................................................................................65 REFERENCES ..................................................................................................................66 CHAPTER 3 ENGINEERED BACILLUS SUBTILIS CAPABLE OF EPITHELIAL CELL INVASION ....................................................................................................................................75 ABSTRACT.......................................................................................................................76 INTRODUCTION .............................................................................................................77 RESULTS ..........................................................................................................................84 Protein expression…... ...............................................................................................84 B. subtilis internalization ............................................................................................87 DISCUSSION ....................................................................................................................92 MATERIALS AND METHODS .......................................................................................96 Data and code availability ..........................................................................................96 Mammalian cell culture ..............................................................................................96 B. subtilis constructs ...................................................................................................96 Bacterial growth conditions ........................................................................................98 Growth curves of B. subtilis .......................................................................................98 Bacterial protein extraction ........................................................................................99 Mammalian cell protein extraction .............................................................................99 Western blot .............................................................................................................100 B. subtilis uptake assay ............................................................................................101 Cell fixation and counter staining ............................................................................102 Confocal microscopy ................................................................................................102 Flow cytometry with cell tracker orange ..................................................................102 Data visualizations ...................................................................................................103 Competing interests ..................................................................................................103 Acknowledgments ....................................................................................................104 Funding .....................................................................................................................104 Affiliations ................................................................................................................104 Contributions ............................................................................................................104 REFERENCES ................................................................................................................106 x CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS ................................................115 REFERENCES ................................................................................................................122 xi LIST OF TABLES Table 2.1 Quantification of iron via ICP-MS..............................................................................37 xii LIST OF FIGURES Figure 2.1. Illustration of magnetothermal control of B. subtilis ............................................30 Figure 2.2. Continuous heating of B. subtilis +TlpA39R and B. subtilis -TlpA39R to induce expression of LuxA-E from PTlpA39 .............................................................................................33 Figure 2.3. Visualization and elemental analysis of B. subtilis and SPION associations.......35 Figure 2.4. Elemental analysis of B. subtilis uncoated control .................................................36 Figure 2.5. Transmission electron microscopy of B. subtilis coated with three SPIONs .......37 Figure 2.6. Flow cytometry determining viability of B. subtilis after coating with the three- particle variations and AMF application ...................................................................................38 Figure 2.7. Magnetic hyperthermia increasing bioluminescent signal (Avg. Radiance) using the HYPER Theranostic Hyperthermia System .......................................................................40 Figure 2.8. Magnetic hyperthermia impact on culture medium temperature with three Synomag-D variations .................................................................................................................41 Figure 2.9 One-hour thermal inductions using continuous heating and magnetic hyperthermia ................................................................................................................................43 Figure 2.10. MPI and histological analysis ................................................................................46 Figure 2.11. MPI of mouse thigh shows no signal from plain-dextran SPION ......................47 Figure 2.12. Magnetic hyperthermia of intramuscular injections ..........................................48 Figure 2.13. Modified Gram stain and hematoxylin and eosin staining of tissue sections ....49 Figure 3.1. Model of engineered B. subtilis invasion into epithelial cells ................................82 Figure 3.2. Western blot showing expression of InlA in B. subtilis .........................................85 Figure 3.3. Growth curves of B. subtilis strains ........................................................................85 Figure 3.4. Western blot showing expression of E-cadherin in epithelial cells ......................86 Figure 3.5. Uptake of B. subtilis into trophoblast stem cells ....................................................89 Figure 3.6. Three-dimensional cross-sections of B. subtilis uptake .........................................90 Figure 3.7. Characterization of B. subtilis interaction with trophoblast stem cells ...............91 xiii KEY TO ABBREVIATIONS EES Engineered endosymbionts MOI Multiplicity of infection RNAP Ribonucleic acid polymerase iPSC Induced pluripotent stem cell GAPDH Glyceraldehyde-3-phosphate dehydrogenase OSKM Oct3/4, Sox2, Klf4, c-Myc (myelocytoma factor C) Oct3/4 octamer-binding transcription factor 4 Pou5f1 POU class 5 homeobox 1 Sox2 SRY (sex-determining region Y) – box 2 Cdx2 caudal type homeobox 2 Klf4 Krüppel-like factor 4 Tat Twin-arginine translocation SD Standard deviation PCR Polymerase Chain Reaction RT-qPCR Reverse transcription quantitative Polymerase Chain Reaction DNA Deoxyribonucleic acid LLO Listeriolysin O InlA Internalin A ANOVA Analysis of Variance TSC Trophoblast stem cell IPTG Isopropyl β-D-1-thiogalactopyranoside xiv TSR Temperature-sensitive repressor MPI Magnetic particle imaging MRI Magnetic resonance imaging AMF Alternating magnetic field SPION Superparamagnetic iron oxide nanoparticle xv CHAPTER 1 INTRODUCTION AND BACKGROUND: REGENERATIVE MEDICINE AND BACTERIOTHERAPY, TEMPERATURE-SENSITIVE REPRESSORS, AND SYNTHETIC BIOLOGY 1 INTRODUCTION Significance and innovation In the United States alone, approximately 114,000 people annually need some type of tissue or organ transplant and are on lists waiting for compatible donations from living or deceased donors.1 Despite a record-breaking number of donors (living and deceased) for the last seven years, there were still only 36,500 transplantations performed in 2018.1–3 This deficit leaves the remaining 68% of the transplant waitlist in need of other forms of therapeutic options, possibly including regenerative medicine. Additionally, tissue and organ transplantation often come with complications and side effects due to molecular incompatibilities.4 To address this deficit, regenerative medicine has developed cellular reprogramming to pluripotency as a therapy. This dissertation will layout a new therapy that can be used for a wide variety of applications and address current limitations including, but not limited to, tight control of the system, expanded range of delivery mechanisms, and modular nature for future development. Current approaches of cellular reprogramming to pluripotency include overexpression of Oct3/4, Sox2, Klf4, and c-Myc known as OSKM factors (i.e. Yamanaka factors).5 As a therapy this involves collecting healthy or diseased donor skin cells and delivering OSKM factors to the nucleus and culturing the cells in vitro until they reach an induced-pluripotent stem cell (iPSC) state.6 From pluripotency, the cells can then be differentiated into any cell type needed for therapy and implanted into the patient.6 By using donor cells to create the needed cell type molecular incompatibilities can be reduced.5,6 Somatic cells can be directly reprogrammed by the following methods: retro- or lenti-viral transduction, protein and microRNA transduction, or chemical/small molecule-based reprogramming.6 One of the risks of using induced pluripotent 2 stem cells (iPSCs) as a therapeutic is the potential to create teratomas in vivo6 which highlights the need for precise methods of control when building upon these approaches. In addition to cellular reprogramming to address various regenerative medicine and diseases bacteriotherapies are being investigated for a variety of treatment models. Bacteriotherapies are being studied for a variety of targets currently including treatment of hyperammonemia,7 phenylketonuria,8 in situ microbiome engineering,9 recurrent streptococcal pharyngotonsillitis,10 and adenoidectomy prevention. 11 These studies demonstrate the efficacy of bacteria as treatment options for diverse illnesses and disease states creating a body of work that can be expanded upon into the field of regenerative medicine. To exhibit tight control of a bacterial therapy system, cancer therapy applications were investigated as a target and a new method of control was discovered. By coupling existing systems of cancer therapy with genetic engineering in bacteria a new niche was explored. Magnetic nanoparticles have been applied to a variety of methodologies including imaging, drug delivery, theranostics and therapeutic hyperthermia.12–14 Superparamagnetic iron oxide nanoparticles (SPIONs) can be used as contrast agents for imaging and can be coupled with the application of electromagnetic energy generated by alternating magnetic fields (AMF). 15–20 This AMF application to SPIONs can cause a local temperature increase referred to as magnetic hyperthermia21–23 to provide precise, local heating.24 Magnetic hyperthermia has been proposed to be used for tumor microenvironment, but with limitations.14,18,21,22,24 This localized heating can be coupled with temperature responsive systems in bacteria to exhibit the level of control needed for in vitro and in vivo bacteriotherapies and will be explored further in chapter 2. Further expansion of host cell type is necessary for a tightly controlled, modular bacteriotherapy. To bridge the gap between magnetic hyperthermia and cancer therapy 3 approaches epithelial cells were investigated as potential host cells which is explored in chapter 3. Epithelial cancers, categorized as carcinomas by the International Classification of Diseases for Oncology, Third Edition, is malignant growth in the internal and external lining of the body.25 According to the National Cancer Institute these epithelial tissue malignancies make up 80-90% of all cancer cases.26 Epithelial tissue is present in skin and the linings and coverings of internal passageways and organs.25,26 This makes epithelial tissues and more specifically, carcinomas, an important target for bacterial cancer therapies. Current approaches to carcinoma treatments include surgery, chemotherapy, radiation therapy, bone marrow transplant, immunotherapy, hormone therapy, targeted drug therapy, cryoablation, radiofrequency ablation, and clinical trials of new treatments such as cytokine-based27 and bacterial-based therapy.28–33 Cell cycle regulators and tumor suppressors are often targets of these clinical trials; however, delivery methods remain a limitation.34–40 Bacterial cancer therapy with Bacille Calmette-Guerin (BCG, the Mycobacterium bovis strain used as a tuberculosis vaccine) is the standard of care for early-stage bladder cancer41 and there are many other approaches under investigation and FDA review for various human cancers.29,31–33,42 This demonstrates the need for targeting of epithelial cells to gain access to carcinoma applications from within the cytoplasm of the cells. This dissertation will outline the efforts to create a modular technology that can be used to advance the field of bacteriotherapies. To accomplish this the following systems were engineered to address the limitations of current approaches such as control of the therapeutics and delivery to the nucleus of new types of host cells. First, temperature-sensitive repressors were coupled with magnetic hyperthermia and tested in vivo and in vitro with a reporter gene as a proof-of-concept. Second, epithelial cells were targeted for applications by engineering an epithelial-specific ligand into B. subtilis to demonstrate invasion into trophoblast stem cells with 4 the ultimate goal of cellular reprogramming to pluripotency. The next sections of this chapter will go more in depth into the underlying research that has allowed for the creation of these new systems. Temperature-sensitive bacterial repressors Temperature-sensitive repressors (TSRs) are a class of repressors that bind an operator- promoter region in a low-temperature, often dimeric, state blocking sigma factors and RNAP from binding, therefore repressing transcription. With the addition of heat to the system, there is a structural change to a high-temperature, often uncoiled or monomeric, state where the DNA in the operator-promoter region is unbound and free for the RNAP holoenzyme. TSRs are different from heat shock promoters in that they rely on sigma factor 70 (i.e. σA) the normal, housekeeping sigma factor that enables specific binding of RNAP to gene promoters instead of σ H, the heat shock sigma factor, which is upregulated in higher temperatures associated with stress responses.43 This allows for tighter transcriptional control via a switch-like mechanism of repression with well-defined “on” and “off” states.43 TSRs work in E. coli by relying on a highly conserved bacterial protein, sigma factor 70, to initiate bacterial transcription.44 Sigma 70 in E. coli has been shown to be structurally and functionally similar to sigma A in B. subtilis.45 Furthermore, previous studies show in vitro transcription can be carried out successfully by either E. coli or B. subtilis core RNAP and the other’s sigma factor.46 One well studied TSR is the TlpA repressor system which is a transcriptional auto- repressor from the virulence plasmid of Salmonella typhimurium. At the C-terminus this protein contains a 300-residue coiled-coil domain that allows for abrupt, temperature-dependent uncoiling between 37C and 45C. At the N-terminus there is a DNA-binding domain that blocks transcription by binding the 52-base pair TlpA operator-promoter in the lower temperature 5 dimeric form.47,48 TlpA thermal switches have been previously described in Escherichia coli (E. coli) DH5α.44 The TlpA construct in E. coli shows a 355-fold change in fluorescence which is eleven times greater than the best heat shock promoter construct tested (32-fold, HtpG).44 Piraner et al. created TlpA temperature mutants in E. coli to encompass a greater range of physiologically relevant switch temperatures.44 There is precedent for thermal control of B. subtilis in both native and recombinant systems.49–54 The B. subtilis heat shock regulon has been well studied and has shown activation of over 100 different genes. Some of these genes are a part of the general stress response regulon and are controlled by sigma B and others are controlled by HrcA or CtsR which are heat shock regulators.49 Two temperature-inducible promoters were isolated from B. subtilis, P2 and P7, with higher expression at 45C compared to 37C.50 The CI857-encoded repressor is from a temperature-inducible mutant of the well characterized E. coli bacteriophage CI. The λc1857 repressor-PR promoter system was cloned into B. subtilis and was insufficient to produce the repressor. To overcome this, the repressor gene was fused to sak42D transcription and translation signals and subsequently demonstrated temperature-inducible expression at 37C and 42C.52 Multiple cold-inducible systems have been discovered with higher gene expression at 25C and 15C compared to 37C.53,54 Internalin A and E-cadherin interactions Bielecki et al. transferred the hlyA gene (LLO protein) from Listeria monocytogenes into B. subtilis and studied bacterial invasion of phagocytic J774A.1 cells.55–57 Previous work has shown that this chassis organism can be coupled with different proteins to function as a modular tool and potential bacteriotherapy.58 One mechanism of invasion into non-phagocytic host cells that L. monocytogenes utilizes is the “zipper” mechanism between the bacterial ligand internalin 6 A (inlA) and the host cell receptor E-cadherin.59 The “zipper” mechanism is broadly described with four steps in L. monocytogenes, infection, receptor binding, membrane engulfment, and vacuole disruption. Infection is a precursor to binding that requires the bacteria and host cells to be in the same space.60 Receptor binding for L. monocytogenes in epithelial cells specifically is between the adhesin molecule internalin A (InlA; inlA) and the receptor E-cadherin.59,61,62 At this stage the cytoskeleton (i.e. F-actin, G-actin) inside the host cell starts to reorganize to start the membrane engulfment.60 Once fully engulfed in the vacuole, L. monocytogenes uses listeriolysin O (LLO, hlyA) to disrupt the vacuole membrane and allow the bacteria to escape into the cytoplasm of the epithelial cell.59 Additionally, L. monocytogenes and other pathogens have evolved many different mechanisms of invasion such as L. monocytogenes protein internalin B mediating host uptake via a “zipper” mechanism, Yersinia pseudotuberculosis invasion proteins binding host integrin proteins in a “zipper” mechanism, and Shigella or Salmonella spp. using type-III secretion systems with a “trigger” mechanism.59,60 Pathogenicity, host cell toxicity, and an excess of virulence factors are limitations to using known bacterial pathogens as chassis organisms for therapies.63,64 Synthetic biology techniques create an opportunity to selectively choose one or more virulence factors and move them into bacteria more amenable to treatments.65,66 B. subtilis physiology is ideal for a bacteriotherapy chassis as it is a GRAS, non- pathogenic, Gram-positive, soil bacterium that respires as a facultative anaerobe67–69 and does not have a lipopolysaccharide- (LPS) mediated immune response.70 Regenerative medicine and cellular reprogramming Regenerative medicine is a branch of translational research in tissue engineering and molecular biology which deals with the process of replacing, engineering or regenerating human or animal cells, tissues or organs to restore or establish normal function.71 Cell therapies include 7 delivery of cells, often from a donor, to the patient, as a therapeutic approach.72 Specifically, cellular reprogramming is important to the field because iPSCs and their derivatives allow for autologous cell replacement of immune-compatible cells.6 However, large-scale productions are difficult with the current systems.6 An EES can help make these types of therapies scalable by providing a means of studying and controlling differentiation conditions to inform defined, reproducible conditions. Cellular reprogramming is the reconfiguration of a cellular gene expression program to drive the cell to a different state in a more or less deterministic fashion, including modification of the epigenetic marks of mammalian germ cell development.73 The field of cell biology was dramatically changed when Gurdon described nuclear reprogramming74 and led to Takahashi and Yamanaka publishing four transcription factors that are necessary and sufficient to drive a terminally differentiated cell, such as a fibroblast, to an iPSC.5 These four factors known now as OSKM factors (i.e. Yamanaka factors) are Oct3/4, Sox2, Klf4, and c-Myc.5 Currently, systems of reprogramming include viral transduction (e.g. lentivirus, adeno-associated virus, etc.), DNA transfection (e.g. plasmid, minicircle, transposon), OSKM protein transduction, synthetically modified RNA transfection (e.g. microRNA, in vitro transcribed-RNA, etc.), and chemical/small molecule-mediated reprogramming.6 Most of these systems have a low efficiency, on average 1%, and require 4-6 weeks to complete reprogramming.75 Once pluripotent, the cells can be differentiated into desired cell types, and transplanted, however, if the cells are not differentiated enough, teratomas can form in vivo.6 Additionally, a recent study investigated using Pseudomonas aeruginosa to deliver transcription factors to iPSCs via type III secretion system to drive differentiation to cardiomyocytes.76 This study demonstrates that a pathogenic, extracellular bacteria can deliver 8 transcription factors to drive mammalian cell fate. However, previous studies reveal an opportunity to create a non-pathogenic EES delivering proteins in a controlled fashion to mammalian cells to alter cell fate. Innate immune response to cytosolic Gram-positive bacteria When engineering a bacterial system to exist in a host cell it is vital to consider the host responses that will be triggered by the presence of the bacteria. Innate immunity is the immune system’s first response to foreign substances such as invading bacteria or viruses. 77,78 Both hematopoietic and non-hematopoietic cell types are involved in innate immunity such as macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, natural killer cells, T-cells, and epithelial cells and the response of this system is immediate although there is no immunologic memory after subsequent exposure.78 By contrast, adaptive immunity takes place in only hematopoietic T-cells and B-cells and takes hours to days to respond to stimuli, however, the response is enhanced by repeated exposure to the antigen.78 Pathogens in the cytoplasm of mammalian cells trigger innate immune responses. These responses vary depending on the type of pathogen. Toll-like receptors (TLRs) recognize conserved molecules from microbes and are expressed on the membranes of leukocytes including dendritic cells, macrophages, natural killer cells, T-cells, B-cells, and non-immune cells (i.e. epithelial cells, endothelial cells, fibroblasts).77,79 TLR-3 is expressed in endosomes of cells in the placenta, pancreas, and dendritic leukocytes and recognizes double-stranded RNA which often marks a viral infection.79 TLR-4 is expressed in the membrane of myeloid cells and responds to endotoxin, including lipopolysaccharide (LPS) an important component of the cell wall in Gram-negative bacteria.79 Gram-positive bacteria can trigger an innate immune response via TLR-2 which responds to lipoprotein in cell walls and TLR-5 which reacts to flagellin the main protein component of 9 bacterial flagella.81 TLR-2 stimulates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in peripheral blood and leukocytes which plays a key role in regulating immune responses to infection.78–80 In addition to detecting structures on bacterial surfaces, the innate immune system can also detect DNA. The best understood of these systems are TLR-9, a membrane-associated sensor of bacterial CpG DNA,82 the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway,83 and the inflammasome using the AIM2 sensor.84–86 cGAS-STING pathway and AIM2 sense double-stranded DNA in the host cell cytoplasm and induce an innate immune defense pathway.83,85 Whether microbial or host-derived, DNA in the host cell cytoplasm is a danger-associated molecule and indicates a state of infection, cellular stress, and/or tissue damage for the host cell.77,78 The innate immune system has been studied in numerous cell types including epithelial cells,87 macrophages,84,86 and embryonic and trophoblast stem cells88,89 highlighting differences in responses. A review of the immunity in epithelial cells emphasizes the epithelium as an important niche for both innate and adaptive immunity.87 Macrophage studies often investigate phagosome evasion and mechanisms specific to macrophage behavior.84,86 Listeria monocytogenes triggers AIM-2 dependent inflammasome activation, and in some cases pyroptosis, or host cell death.86 To understand how early in embryogenesis innate immunity is developed studies have utilized embryonic stem cells 88 as well as blastocyst models (i.e. embryonic and trophoblast stem cells).89 A 2013 study using an in vitro murie blastocyst model confirmed the expression of TLR-2, -3, and -5 in both embryonic stem cells (ESCs) and trophoblast stem cells (TSCs).89 And a 2019 study concluded that innate immunity is underdeveloped in ESCs and matures further along in development despite expression of pattern 10 recognition receptors.88 Investigations of first trimester human trophoblasts have identified TLR- 3 and TLR-4 as mediating antiviral and antibacterial responses, respectively, however the immunological properties of TSCs are not as well characterized.88 Platform technology design and approach For this dissertation, an EES will be defined as a normally extracellular, non-pathogenic bacterium that can be genetically and physically manipulated to exist viably in the cytoplasm of a mammalian cell.58 With the EES system, both applied and basic science questions can be examined. These topics include, virulence and pathogenicity factors of intracellular bacterial pathogens, directing cellular fates and functions in vitro and in vivo, and creating a tool for non- invasive tissue regeneration in vivo.90 This tool can be applied not only to cellular reprogramming and tissue regeneration, but also more broadly to the field of regenerative medicine (e.g. immunomodulation therapy, delivery of cancer therapeutics, etc.).58,90 B. subtilis LLO within J774A.1 cells was first studied to understand the behavior of LLO and its role in evading phagosome destruction.55–57 The current mammalian cell type being explored as an EES host cell is trophoblast stem cells (TSCs) derived from a female C57B6 mouse. TSCs were chosen for their ability to be reprogrammed in half the time of terminally differentiated cells91,92 (i.e. macrophages) as well as their tendency to take up extracellular bacteria.93–95 Previous work has shown that β-gal can be effectively produced, secreted, and trafficked from intracellular B. subtilis LLO to the J774A.1 nucleus.58 Our basic EES system involves a co-incubation of bacteria and mammalian cells to promote an interaction allowing the bacteria entry into the host mammalian cell. 58,90 The main mechanisms of invasion utilized in this setup are first, an E-cadherin-mediated entry into murine epithelial cells facilitated by the inlA gene encoding internalin A (InlA) followed by vacuole 11 disruption with the hlyA gene encoding listeriolysin O (LLO).61 Both InlA and LLO are originally Listeria monocytogenes virulence factors and were engineered into B. subtilis.55 A secondary mechanism of invasion which may contribute to host cell entry is phagocytosis and vacuole disruption with LLO.55 This requires the host cell type to exhibit enough phagocytic activity to engulf B. subtilis which is a limitation mitigated by the inclusion of InlA. Once inside the host cell, an engineered bacterium can be induced to secrete folded proteins to the nucleus which allows for control of host cellular functions.58 The bacterium is described as a chassis organism, emphasizing the modular nature of the prokaryotic components. The chassis organism can be genetically modified using synthetic biology techniques to include operons encoding mammalian transcription factors with a nuclear localization signal and bacterial secretion peptide tag.58 Additionally, transcriptional control will be incorporated into the approach to ensure precise, inducible timing for protein production. B. subtilis has several options for alternative inducible systems because it is an environmental organism that maintains fitness by responding to dynamic stimuli in the soil environment.96 Some well-defined inducible systems for B. subtilis expression include D-xylose,97 L-rhamnose,98 D-mannose,99 and IPTG100 for chemical control. In addition to chemical control this dissertation demonstrates the ability to utilize magnetothermal induction of a temperature-sensitive repressor (TSR) construct, to control the chassis organism operon at a transcriptional level. Concluding remarks and dissertation overview An EES could be used in the field of regenerative medicine for cellular reprogramming, tissue regeneration, and organogenesis. The modular nature of the EES and the polycistronic capabilities of a bacterial chassis organism make it possible to achieve multistep cellular manipulation in tandem or concurrently, as needed. The use of thermal control allows for the 12 EES system to be activated in vivo, noninvasively through technologies that already exist, such as focused ultrasound,101 infrared light,102 and magnetic particle hyperthermia.103,104 The scope of this dissertation is to investigate engineering transcriptional control and physiology of B. subtilis to develop an in vitro and in vivo tool for cellular reprogramming and, more broadly, as a tool for a variety of medical therapeutics. The field of regenerative medicine has advanced greatly since the discovery of OSKM factors. Unfortunately, the need for tissue and organ donations in the United States is ever present and despite the increase in transplants occurring annually, there is still a deficit. Organogenesis, tissue regeneration, and cellular reprogramming could all be performed via an EES. Our EES model involves B. subtilis llo inlA with a Tat secretion system and SV40 nuclear localization signal to deliver proteins of interest to the host cell (e.g. TSCs, 4T1s) nuclei to alter cell fate. While developing such an EES system, it is necessary to consider how to tightly control the delivery of the proteins (e.g. pro-drug converting enzyme, transcription factors, tumor suppressors, cell cycle regulators). This dissertation will cover the characterization of the TlpA39 TSR system in B. subtilis demonstrating magnetothermal control of bioluminescence in vitro and exploration of the system in vivo. Additionally, in vitro chemical control (e.g. IPTG, D-xylose) of engineered B. subtilis llo inlA to promote cellular uptake via E-cadherin receptors on TSCs will be explored. 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Appl. 2019, 265–286. https://doi.org/10.1016/B978-0-12-813928-8.00010-7. 24 CHAPTER 2 MAGNETOTHERMAL CONTROL OF TEMPERATURE-SENSITIVE REPRESSORS IN SUPERPARAMAGNETIC IRON NANOPARTICLE-COATED BACILLUS SUBTILIS 25 PUBLICATION NOTICE The following dissertation chapter describes coating B. subtilis engineered with thermally responsive genetic switches with superparamagnetic iron as a way of non-invasive transcriptional control of bacteria using alternating magnetic fields. I constructed new strains of B. subtilis with genetic switches (temperature sensitive repressors) that respond to thermal energy and characterized the response of these switches to thermal energy. Cody Madsen and I worked jointly on this chapter, he focused on coating B. subtilis with superparamagnetic iron and testing alternating magnetic field parameters to generate thermal energy (magnetothermal energy) to regulate transcription in the strains I constructed. I focused on imaging, and in vitro characterization of the strains. Dr. Ashley Makela supported the characterization of the magnetothermal energy effect on B. subtilis. Dr. Christopher Contag supervised and aided in the conception and development of controlling B. subtilis with magnetothermal energy. This chapter was published as a preprint in “Greeson, E. M., Madsen, C. S., Makela, A. V & Contag, C. H. Magnetothermal control of temperature-sensitive repressors in superparamagnetic iron nanoparticle-coated Bacillus subtilis. bioRxiv 2022.06.18.496685 (2022).” and reprint permission is reserved to the authors for use in this dissertation. The following chapter has been submitted to “Greeson, E. M., Madsen, C. S., Makela, A. V & Contag, C. H. Magnetothermal control of temperature-sensitive repressors in superparamagnetic iron nanoparticle-coated Bacillus subtilis. ACS Nano. (2022).” and reprint permission was granted for use in this dissertation. 26 ABSTRACT Superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) and resulting images can be used to guide magnetothermal heating. Alternating magnetic fields (AMF) cause local temperature increases in regions with SPIONs, and we investigated the ability of magnetic hyperthermia to regulate temperature-sensitive repressors (TSRs) of bacterial transcription. The TSR, TlpA39, was derived from a Gram-negative bacterium, and used here for thermal control of reporter gene expression in Gram-positive, Bacillus subtilis. In vitro heating of B. subtilis with TlpA39 controlling bacterial luciferase expression, resulted in a 14.6-fold (12 hour; h) and 1.8- fold (1 h) increase in reporter transcripts with a 10-fold (12 h) and 12.1-fold (1 h) increase in bioluminescence. To develop magnetothermal control, B. subtilis cells were coated with three SPION variations. Electron microscopy coupled with energy dispersive X-ray spectroscopy revealed an external association with, and retention of, SPIONs on B. subtilis. Furthermore, using long duration AMF we demonstrated magnetothermal induction of the TSRs in SPION- coated B. subtilis with a maximum of 5.6-fold increases in bioluminescence. After intramuscular injections of SPION-coated B. subtilis, histology revealed that SPIONs remained in the same locations as the bacteria. For in vivo studies, 1 h of AMF is the maximum exposure due to anesthesia constraints. Both in vitro and in vivo, there was no change in bioluminescence after 1 h of AMF treatment. Pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to transcriptional control that expands options for targeted bacteriotherapies. 27 INTRODUCTION Magnetic nanoparticles have broad applications in biomedicine including imaging, drug delivery, theranostics and therapeutic hyperthermia.1–3 Nanoparticles have also been used to study and treat bacterial infections through the coating of bacterial membranes for imaging and as anti-microbial agents.4–10 Superparamagnetic iron oxide nanoparticles (SPIONs) are useful imaging contrast agents for magnetic resonance imaging (MRI) and more recently in magnetic particle imaging (MPI).11–16 MPI detects SPIONs directly, providing a readout of both iron content and location with high specificity and sensitivity.11,17–19 Further, MPI can guide the application of electromagnetic energy generated by alternating magnetic fields (AMF) to cause local temperature increase known as magnetic hyperthermia20–22 to precisely heat the iron- containing area.23 Bacillus subtilis is a model Gram-positive organism24 with numerous synthetic biology strategies for manipulating gene expression,25,26 global metabolic networks27 and the entire genome28–30 making it well-suited for engineering systems for spatial and temporal regulation.27 B. subtilis is a generally recognized as safe organism that is used for industrial protein production and is highly resistant to environmental stressors such as heat with a heat shock response at 48ºC.31,32 High heat resistance and well characterized protein production pathways may make B. subtilis an ideal chassis organism for thermal energy controlled protein production that could act as therapeutics.33–35 B. subtilis also has multiple characterized inducible systems including several sugar-regulated inducible systems.36 B. subtilis has been well studied for a variety of in vitro industry applications in areas such as pharmaceutical/nutraceutical production, recombinant protein production and secretion, and production of functional peptides and oligopeptides.37–40 28 However, these inducible systems have limited control for both in vitro and in vivo applications due to potential host toxicity, cost and carbon-source dependence.41 Temperature-sensitive repressors (TSRs) are a class of repressors that bind an operator- promoter region with temperature dependence, and show promise for in vivo control with local heating for localized delivery.42 With the addition of thermal energy to the system, a structural change occurs that releases the repressor from DNA resulting in transcription.43 Thus, TSRs are different from heat shock promoters (HSP) and rely on housekeeping sigma factors such as σA in B. subtilis.44,45 TSRs offer a greater dynamic range than HSP and do not necessitate stress conditions for induction.42,43 There is precedent for thermal control of B. subtilis with induction of gene expression at low and high temperatures in both native and recombinant systems.45–50 Additionally, TSRs have been shown to be controlled previously in Gram-negative organisms with ultrasound to create localized thermal energy for transcriptional control.27 The configuration, size and composition of SPIONs have a large effect on MPI performance53–55 and magnetothermal heating.56 Synomag-D is a commercially available multi- core “nanoflower” particle57 and has demonstrated improved MPI performance58,59 as well as high intrinsic power loss under magnetic hyperthermia.60,61 Pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to regional transcriptional control as guided by MPI or MRI for new approaches to bacteriotherapy. There are many bacteriotherapy approaches under investigation, and FDA review, for a variety of human cancers.62–67 We engineered TSRs42,43 into the model organism, B subtilis, towards the development of noninvasive genetic control of a minimally invasive biological therapy (Fig. 2.1). 29 Figure 2.1. Illustration of magnetothermal control of B. subtilis B. subtilis coated with Synomag-D SPIONs can be regulated by thermal energy generated from the SPIONs upon application of AMF which initiates transcription of luxA-E operon from PTlpA39. 30 RESULTS Temperature-sensitive repressor’s control of transcription in B. subtilis Magnetic nanoparticles have been used for several biomedical applications and can be further expanded into a measure of control through non-invasive stimuli. Magnetic hyperthermia has been proposed to be used for tumor microenvironment disruption by combining synthetic and biological magnetic nanoparticles with AMF but with limitations.3,14,20,21,23 We investigated the concept of using magnetothermal energy to control a genetic switch in Gram-positive bacteria. This would comprise a modular platform as the basis for developing a variety of potential therapeutics. Directed delivery and targeted activation can improve therapeutic effects and reduce toxicity of bacteriotherapies while imaging can guide development of novel bacteriotherapies by assessing delivery, retention and activation within the target tissue.68 Here we use magnetic hyperthermia and imaging to characterize the use of superparamagnetic nanoparticle-coated B. subtilis as a new approach for controlling Gram-positive bacterial gene expression with potential use in bacteriotherapies. A TSR (TlpA39)42,43,69 was used to control transcription of the luxA-E operon such that luciferase activity (bioluminescence) could be used as a rapid readout for regulation.70 This construct demonstrated thermal transcriptional control in B. subtilis in response to continuous heating. B. subtilis PTlpA39 luxA-E +tlpa39R (+TlpA39R) and B. subtilis PTlpA39 luxA-E -tlpa39R (-TlpA39R) were heated continuously in a thermocycler for 12 h at 25°C, 37°C, 39°C or 42°C to test induction of PTlpA39. B. subtilis +TlpA39R showed a 10.0-fold increase (p<0.0001) in luciferase activity when normalized to OD600 from 25°C to 37°C while B. subtilis -TlpA39R showed a 1.5-fold increase (p<0.0001; Fig. 2.2A). Bioluminescence did not significantly increase when cells were induced at temperatures above 37°C while mRNA levels, as measured by real- 31 time quantitative PCR (RT-qPCR), showed continual increase in PTlpA39 activity up to 42°C in B. subtilis +TlpA39R (Fig. 2.2B). The second gene in the luxA-E operon engineered for expression in Gram-positive organisms,69 luxB, was chosen as the target for RT-qPCR analysis since it encodes for the β subunit of the alkanal monooxygenase enzyme that provides structure for the active conformation of the α subunit of the heterodimeric luciferase.71 In the +TlpA39R strain luxB levels increased by 1.9-, 3.6-, and 14.6-fold change at 37°C, 39°C and 42°C, respectively. The tlpa39R transcript fold change was 1.6, 2.9, and 7.4 at 37°C, 39°C and 42°C, respectively in B. subtilis +TlpA39R. B. subtilis -TlpA39R showed no significant change in bioluminescence signal and minimal change in PTlpA39 activity from mRNA levels as expected from the unregulated promoter (Fig. 2.2C). In the -TlpA39R strain luxB levels increased by 2.1-, 1.8-, and 1.6-fold change at 37°C, 39°C and 42°C, respectively. The increase in bioluminescence in the - TlpA39R strain from 25°C to 37°C can be attributed to increased activity of the Lux enzymes over those temperatures and more so to a shift in B. subtilis metabolism which is consistent throughout the study.50,70,77 32 Figure 2.2. Continuous heating of B. subtilis +TlpA39R and B. subtilis -TlpA39R to induce expression of LuxA-E from PTlpA39 Error bars are mean ± standard error mean. (A). Transcript levels determined by RT-qPCR for luxB and tlpa39R from +TlpA39R strain (B) along with luxB from -TlpA39R strain (C) at induction temperatures compared to 25°C. RT-qPCR shown as mean with error bars as 95% confidence intervals. Statistics were displayed when comparing to 25°C for both +/- TlpA39R strains and between each increasing temperature in +TlpA39R strain. ****p<0.0001. 33 SPION coating of B. subtilis To test magnetothermal activation, B. subtilis ZB30772 (derivative of B. subtilis strain 168) was coated SPIONs using plain-dextran, carboxyl or amine-coated Synomag-D;58,59 each were assessed for coating efficiency, interactions between SPION and bacteria and magnetothermal heating. Scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDS) was performed and displayed associations with, and retention of, the nanoparticles and B. subtilis. All three variations were found surrounding and associating with B. subtilis as confirmed by Fe signal from EDS, but with varied consistency of coating observed (Fig. 2.3). The plain-dextran and amine-coated evenly covered and associated with B. subtilis while the carboxyl-coated appeared to heterogeneously associate with B. subtilis in large aggregates (Fig. 2.3). Iron signal was absent from a B. subtilis sample without SPIONs (Fig. 2.4). SPIONs were not found in the cytoplasm of the B. subtilis after coating as shown by transmission electron microscopy (TEM) cross-sections (Fig. 2.5). To further investigate the three B. subtilis coatings, inductively coupled plasma mass spectrometry (ICP-MS) was performed to measure iron content. There was more iron in the carboxyl-coated samples compared to the plain-dextran and amine-coated, 464.8 and 294.7 times, respectively (Table 2.1). This highlights the disparity between the way the three SPION variations associate with B. subtilis. 34 Figure 2.3. Visualization and elemental analysis of B. subtilis and SPION associations Plain-dextran SPIONs; mag. = 27,000X (A), carboxyl SPIONS; mag. = 23,000X (B) and amine SPIONS; mag. = 33,000X (C) show various associations with B. subtilis as observed by scanning electron microscopy. Elemental analysis was performed on each of the samples (D-F) in the regions indicated with white borders to show iron (Fe) signal to identify the SPIONs. Scale bars = 1 µm. 35 Figure 2.4. Elemental analysis of B. subtilis uncoated control Sample analyzed with SEM-EDS in two regions (A) showing no iron (Fe) signal from the extracellular material (B) or from the bacterium (C). Magnification = 12,000X; scale bar = 1 µm. 36 Figure 2.5. Transmission electron microscopy of B. subtilis coated with three SPIONs Embedded and cross-section of each B. subtilis-SPION coating variation: plain-dextran (A, D), carboxyl (B, E), and amine (C, F) showing nanoparticles located outside of the bacterial cells. Mag. = 8,000X; scale bars = 1 µm (A-C). Mag. = 20,000X; scale bars = 200 nm (D-F). Table 2.1. Quantification of iron via ICP-MS ICP-MS Iron Quantification Sample Adjusted Total Iron (ng in 25 µl) Adjusted Iron Concentration (ppm) Plain 19.80 0.792 COOH 9203.15 368.126 Amine 31.23 1.249 Each of the three SPION variations and an untreated control sample were analyzed via ICP-MS. Values for the three experimental samples were adjusted by subtracting the background iron present from the untreated sample. n=1 for all samples. 37 B. subtilis viability after coating with SPIONs and AMF application B. subtilis viability was assessed by flow cytometry after coating with each of the SPIONs. Two bacterial concentrations, equal to OD600 = 1 or 2, were tested while maintaining the same concentration of the Synomag-D variations to determine if a high ratio of iron to B. subtilis would cause toxicity. After 2 h of coating, none of the Synomag-D variations at either B. subtilis concentrations demonstrated reduction in viability compared to the untreated control and all treatments were significant when compared to the 98°C control for cell death (Fig. 2.6A, B). Furthermore, viability was assessed after 12 h of AMF using the plain-dextran particle as it produced the most reproducible heating response from B. subtilis +TlpA39R at an OD600 = 2 with a 16.0 mT radio frequency (RF) amplitude (data not shown). The B. subtilis +TlpA39R strain had slight, but not significant differences in viability compared to the -TlpA39R strain with or without AMF treatment (Fig. 2.6C). Figure 2.6. Flow cytometry determining viability of B. subtilis after coating with the three- particle variations and AMF application Viability of B. subtilis was determined after coating with the three SPION variations at two bacterial concentrations (A, B). B. subtilis +TlpA39R and B. subtilis -TlpA39R were compared in and outside the AMF after 12 h of heating when coated with the plain-dextran particle (C). Error bars are mean ± standard deviation. ****p<0.0001. 38 Magnetothermal induction of B. subtilis transcription To illustrate transcriptional control of potential bacteriotherapies, magnetic hyperthermia using the HYPER system, was applied to B. subtilis coated with each of the three variations of the SPION. The growth temperature of the B. subtilis for the HYPER experiments was 37°C as opposed to the thermocycler experiments where the growth temperature is 25°C. This was intended to support in vivo studies as the core body temperature of mice and humans is approximately 37°C.73 Magnetic hyperthermia increased bioluminescent signals in bacteria coated with all particle variations with plain-dextran producing the most reproducible and significant result in the higher bacterial concentration at the max RF amplitude (16.0 mT). The carboxyl-coated SPION caused the highest fold changes in signal compared to the -AMF condition but with the most variability between replicates. The plain-dextran coated B. subtilis +TlpA39R showed only a 1.2-fold change (p=0.0456) at the lower concentration when exposed to AMF while at the higher bacterial concentration with a 16.0 mT RF amplitude showed a reproducible 2.4-fold change (p<0.0001; Fig. 2.7A, D). Carboxyl-coated B. subtilis +TlpA39R showed a 5.6-fold change (p=0.0214) and a 4.4-fold change (p=0.014) in bioluminescence when exposed to AMF at the lower and higher bacterial concentrations respectively but with variability (Fig. 2.7B, E). Additionally, the -TlpA39R strain showed a 2.9-fold change (p=0.1689) and a 2.1-fold change (p=0.014) in bioluminescence when exposed to AMF at the lower and higher bacterial concentrations respectively and with high variability. Finally, the amine-coating produced a 1.6-fold increase (p=0.0026) at the lower bacterial concentration in the +TlpA39R strain but showed a small decrease in signal at the higher bacterial concentration when exposed to AMF (p=0.0217; Fig. 2.7C, F). Due to the plain-dextran coating producing the most significant and reproducible result at the higher bacterial concentration, this condition was 39 chosen for transcript measurements. There was a 1.2-fold increase in luxB levels even with increasing tlpa39R levels (1.7-fold change) in the +TlpA39R strain (Fig. 2.7G) and 1.4-fold increase in luxB in the -TlpA39R strain after AMF exposure (Fig. 2.7H). Thermal probes indicated that only the carboxyl-coated SPION increased the culture medium by +3°C (Fig. 2.8). Figure 2.7. Magnetic hyperthermia increasing bioluminescent signal (Avg. Radiance) using the HYPER Theranostic Hyperthermia System B. subtilis +TlpA39R and B. subtilis -TlpA39R were compared in and outside the AMF with the three Synomag-D coating variations at OD600 = 1 (A-C) or 2 (D-F). Error bars are mean ± standard error mean. RT-qPCR was used to determine transcript levels of the two strains and compare AMF to -AMF(G-H). RT-qPCR shown as mean with error bars as 95% confidence intervals. *p<0.05, **p<0.01, ****p<0.0001. 40 Figure 2.8. Magnetic hyperthermia impact on culture medium temperature with three Synomag-D variations Points indicate temperature every 0.5 h over 12 h with 60 temperature reads taken every 1 min cycle considered as technical replicates. Error bars are mean ± standard deviation. 41 Reducing thermal energy application to in vivo timeframe For small animal in vivo applications, anesthesia for times >1 h can cause negative impacts on animal health75,76. Therefore, reducing AMF application time to around 1 h was necessary for demonstration of translatability of this approach. Increases in bioluminescent signals and luxB levels were seen after 1 h of continuous heating in a thermocycler (Fig. 2.9A, B). B. subtilis +TlpA39R had a 12.1-fold increase (p<0.0001) in luciferase activity when normalized to OD600 from 25°C to 37°C while B. subtilis -TlpA39R showed a 2.3-fold increase (p<0.0001). When increasing the temperature from 37°C to 39°C and from 39°C to 42°C in the regulated strain (+TlpA39R) there was a 1.5-fold (p<0.0001) and 1.1-fold change (p<0.0001) between each step up, respectively, whereas the –TlpA39R strain had a negative fold change when comparing bioluminescent signal between 37°C to 39°C (-1.02-fold;p=0.9061) and 39°C to 42° (-1.3-fold;p<0.0001). In the +TlpA39R strain luxB transcript levels increased by 0.9-, 0.9- , and 1.8-fold change at 37°C, 39°C and 42°C, respectively, indicating induction between 39°C and 42°C. The tlpa39R transcript fold change was 0.8, 0.9, and 0.9 at 37°C, 39°C and 42°C, respectively in B. subtilis +TlpA39R showing consistent levels as temperature increased. The B. subtilis -TlpA39R showed similar changes to +TlpA39R in luxB levels at 37°C and 39°C (0.9- and 0.9-fold, respectively), but showed a lesser fold change of 0.4 at 42°C compared to the regulated strain (Fig. 2.9C). This indicates that there is some temperature dependent induction of luxB in the +TlpA39R strain after 1 h of continuous heating. However, AMF application for 1 h only increased bioluminescent signal by 1.02-fold in the +TlpA39R strain and a minimal 1.0-fold increase (doubling) in luxB transcripts which was similar to the –TlpA39R luxB mean increase of 1.5 (Fig. 2.9D-F). 42 Figure 2.9. One-hour thermal inductions using continuous heating and magnetic hyperthermia Reporter gene activity (Avg. Radiance) and transcript levels were measured for continuous and magnetic hyperthermia (A-C). B. subtilis +TlpA39R and B. subtilis -TlpA39R were compared in and outside the AMF (D-F). Error bars are mean ± standard error mean. RT-qPCR shown as mean with 95% confidence intervals. Statistics were displayed when comparing to 25°C for both +/- TlpA39R strains and between each increasing temperature in +TlpA39R strain for continuous heating. 43 Iron association with B. subtilis and magnetic hyperthermia in vivo MPI was performed to quantify iron content in each sample, and these values were compared to those identified by ICP-MS (Table 2.1). Samples containing 1x108 B. subtilis coated with the three Synomag-D coatings were resuspended in a volume relevant to intramuscular (IM) injections (25 µL). Only the carboxyl-coated B. subtilis could be detected in these conditions (Fig. 2.10B), with iron concentration at 384.8 ppm. The plain-dextran could not be detected when the 25 µL samples were imaged using MPI neither in vitro (Fig. 2.10A) nor in vivo (Fig. 2.11). When the plain-dextran samples were pooled to a total volume of 100 µL, MPI signals were detected (Fig. 2.10C inset) and iron quantified was 0.5 ppm, or 13.6 ng per 25 µL sample injected in vivo (Fig. 2.10D). The amine-coated sample was not detectable in a 25 µL sample volume (Fig. 2.10C) and was not pursued further due to the poor AMF response observed previously (Fig. 2.7C, F). MPI quantification showed that the carboxyl-coated SPION sample was 707.3 times that of the plain-dextran SPION (Fig. 2.10D). A murine model of IM thigh injections was paired with the HYPER system and histology to assess iron association with the bacteria and potential changes in bioluminescence. Bacteria coated with SPIONs were prepared and imaged for bioluminescence quantification pre-injection, immediately post-injection, and 1 h post-treatment (+/-AMF). The bioluminescence levels decreased 5.1-fold and 9.0-fold from pre-injection to post-injection in +AMF and -AMF treatments, respectively, but the variance was high between replicates so there was no significance (p=0.4841; p=0.3446; Fig. 2.12). The change in bioluminescence before and after treatment was negligible with a 1.43-fold decrease and a 1.56-fold increase in the +AMF and – AMF conditions, respectively (p=0.4813; p=0.4760; Fig. 2.12). Histology using a modified Gram stain confirmed presence of B. subtilis within sectioned intramuscular tissue after 1 h 44 treatments. Further, consecutive staining with a Perls’ Prussian Blue protocol revealed B. subtilis and iron staining in the same location within the tissue (Fig. 2.10E-G). The white arrows (Fig. 2.10F-G) indicate the presence of iron due to the insoluble Prussian blue pigment which is formed after the potassium ferrocyanide reagent reacts with ferric iron in the sample.77 The use of the consecutive staining scheme which included multiple counterstains and decolorizing steps led to an atypical Gram stain result for B. subtilis. A sequential tissue section was stained using only the modified Gram stain and the standard purple rods of B. subtilis were observed adjacent to the muscle tissue stained yellow from the alcoholic saffron (Fig. 2.13A-C). 45 A Plain C Amine 3.52 1.99 MPI signal (a.u.) MPI signal (a.u.) 1.88 0 0 B COOH 74 D MPI signal (a.u.) 0 E F G Figure 2.10. MPI and histological analysis B. subtilis +TlpA39R coated with the three SPION variations were analyzed via MPI in triplicate. MPI signals could not be detected in the plain-dextran sample in a 25 µL volume (A). Inset shows a pooled volume of 100 µL plain-dextran sample, adjusted to visualize MPI signals. Carboxyl-coated samples showed signal (B) while the amine-coated samples (C) were not 46 detected in 25 µL volumes. MPI scale bars are individual for each condition and represent the full dynamic range of the image. Iron content was quantified using MPI data (D). *Quantification of the plain-dextran sample was performed on a 100 µL pellet and then calculated for a 25 µL volume. Sectioned tissue stained with a modified Gram stain followed Perls’ Prussian Blue (PPB); magnification = 100X (E) and zoomed in regions indicated by magenta (F) and purple (G) boxes with magnification = 400X. White arrows indicate PPB- stained iron. a.u. = arbitrary units; ND = not detected. Scale bars = 50 µm. Figure 2.11. MPI of mouse thigh shows no signal from plain-dextran SPION After injection of plain-dextran coated B. subtilis the -AMF control mouse was imaged via MPI and no discernible signal above background was found. Because of the injections of SPION- coated bacteria being below the limit of detection no additional mice were imaged (n=1). a.u. = arbitrary units. 47 Figure 2.12. Magnetic hyperthermia of intramuscular injections Magnetic hyperthermia did not significantly change bioluminescent signal (Avg. Radiance) when using the plain-dextran SPION. B. subtilis +TlpA39R was compared in and outside the AMF after injection and after 1 h of magnetic hyperthermia. Error bars are mean ± standard error mean; n=3; two-way repeated measures ANOVA with Tukey’s post hoc showed no significance for any comparisons. 48 Figure 2.13. Modified Gram stain and hematoxylin and eosin staining of tissue sections Mouse thigh muscles were sectioned and subsequently stained with a modified Gram stain method. Samples were imaged at various magnifications: 100X (A), 400X (B), and 1000X (C) with scale bars of 50 µm, 25 µm, and 10 µm, respectively. Muscle tissue is yellow from the alcoholic saffron counterstain and the purple rods are Gram positive from crystal violet which supports identification as B. subtilis. Hematoxylin and eosin staining was performed on mouse thigh controls with no samples injected. Imaging showed pink, eosin-stained longitudinal view of quadriceps muscle fibers; Magnification = 400X; scale bar = 25 µm (D). 49 DISCUSSION The introduction of the TlpA39 regulatory system into B. subtilis demonstrated that a temperature-sensitive repressor optimized in a Gram-negative organism can be utilized in a Gram-positive organism to drive controlled transcription of the luxA-E operon by continuous or magnetothermal heating. The results indicate that the TlpA39 promoter and regulator system is functional in B. subtilis and able to regulate an operon with a slight temperature shift from what was observed in Escherichia coli previously (Fig. 2.2).42 This is further demonstrated by the increased levels of luxB transcription at increasing temperatures despite the increased levels of tlpa39R transcripts indicating more regulator protein available to bind the PTlpA39 operator- promoter region as indicated by RT-qPCR. The TlpA39 promoter and regulator system could be further optimized in B. subtilis as was done previously in E. coli42 and B. subtilis.48 Further optimization by directed mutagenesis42,48 or other measures could improve the PTlpA39 genetic switch to have a more stringent on/off state which would be more ideal for in vivo studies. After coating the bacteria with three SPION variations (plain-dextran, carboxyl-coated, amine-coated), SEM-EDS confirmed that the plain-dextran and amine-coated SPIONs covered the B. subtilis in an even, thin coating compared to the carboxyl-coated particle that formed large aggregates that heterogeneously associated with B. subtilis (Fig. 2.3). The variations in association and retention of the three types of SPIONs with B. subtilis are primarily influenced by electrostatic and dispersive forces between the bacteria and the SPION coatings.78 B. subtilis has a net negative electrostatic charge and a zeta-potential of – 41 mV when grown at a physiological pH.79,80 Previous studies have demonstrated that with increasing negative zeta- potential, the higher the adhesion potential extends from the bacteria.80 Additionally, the DLVO (Derjaguin–Landau–Verwey–Overbeek) theory can be used to explain the potential interaction 50 between a given nanoparticle and bacteria.78,80 The SPIONs used in this study have a net electrostatic charge of negative (plain-dextran),58,59 low negative to neutral charge (carboxyl- coated), or a positive charge (amine-coated) when at physiological pH or pH 6.5 for the amine- coated (MicroMod). Even though the plain-dextran is negatively charged, the difference in zeta potential between B. subtilis and the particle was enough to allow for coating similar to previous coatings of B. subtilis with gold nanoparticles.80 The carboxyl-coated SPION has a high potential for Van der Waals interactions due to its hydroxyl functional groups which contributes to the DLVO theory and increases the aggregation and agglomeration of the nanoparticle in suspension and around B. subtilis.81 The positive charge of the amine-coated SPION at pH 6.5 promoted association with B. subtilis but the pH requirement is a limiting factor for this particle type. Ultimately, the variations in coating between the promising plain-dextran and carboxyl-coated SPIONs at physiological pH were more well-suited for downstream applications. Additionally, none of the SPION variations reduced B. subtilis viability after coating (Fig. 2.6). The SEM-EDS and TEM (Fig. 2.3, 2.5) provide some explanation for the results seen following magnetic hyperthermia. The plain-dextran and amine-coated SPIONs evenly coated B. subtilis while the carboxyl-coated SPIONs formed large aggregates that indicated potentially more iron around B. subtilis but with differences between bacteria in the sample. Therefore, AMF could result in greater thermal energy being delivered to B. subtilis through the carboxyl- coated SPION than with the plain-dextran or amine-coated SPION, but with higher variability due to less reproducible associations with the bacteria. The HYPER parameters were chosen based on several preliminary experiments that optimized RF amplitude for each particle at each bacterial concentration then the best conditions for each particle were performed with maximum biological replicates that could be placed inside the HYPER system (Fig. 2.7). Thermal probes 51 measuring the temperature of the culture medium showed that the carboxyl-coated SPION was the only particle that increased culture medium temperature (+3°C) when exposed to AMF (Fig. 2.8). This was supported by the electron microscopy indicating more free iron throughout the media in addition to the aggregates associated with the bacteria (Fig. 2.3B, 2.5E). The plain- dextran and amine-coated SPIONs did not increase temperature but still induced PTlpA39 indicating potential direct thermal energy transfer to B. subtilis. Classical heat transfer theory based on Fourier’s law of thermal conduction could explain this phenomenon at the micrometer scale taken together with coating observed under SEM-EDS but thermal confinement to B. subtilis is unlikely.82 Explaining the observed thermal energy transfer phenomenon by Fourier’s law is also supported by the observed differences in heating between the three particle variations. The carboxyl-coating caused the largest fold change, though variable, and also increased the culture medium temperature which would be consistent with the law of thermal conduction.82,83 Accordingly, the other two particle variations were diffusing thermal energy that did not cause a detectable culture medium temperature change but could have still caused the biological response from B. subtilis especially when comparing the +/- TlpA39R strains. We chose the plain-dextran SPION for 1 h thermal induction and in vivo studies because of the reproducibility of heating, even coating of B. subtilis to maximize retention, minimal impact on viability after AMF treatment and less thermal energy transfer throughout the culture medium which could translate to less damage to surrounding tissue in vivo. In future studies, the SPION of choice should be determined based on desired effects as the varied particle characteristics could have different advantages in other scenarios. Both thermocycler heating and magnetic hyperthermia by the HYPER system created significant increase in bioluminescent output over 12 h. Yet, the comparison of 12 h of 52 continuous heating to AMF (Fig. 2.2, 2.7) demonstrated that magnetic hyperthermia does not induce the TSRs to the same degree as continuous, direct heating. Accordingly, results obtained after 1 h of heating indicate that thermocycler heating over this limited time can significantly increase the output of the reporter, which demonstrates the potential for in vivo use. However, it is likely that the pulse sequence of magnetic hyperthermia used here would need to be improved to maximize potential for in vivo applications. An immediate change to the current process, that could enhance the magnetic hyperthermia, is increasing the RF amplitude beyond the limitations of the HYPER system (>16.0 mT). However, as an increase in RF amplitude will result in an increase in specific absorption rate (SAR),84 this would have to be further studied to prevent any biological effects. The plain-dextran particle used here to coat B. subtilis is promising and shows potential for enhanced thermal energy transfer from a stronger AMF. Alternatively, other SPIONs could be investigated to further enhance magnetic hyperthermia response in B. subtilis. Various SPIONs have been modified to improve magnetic hyperthermia properties85–88 and these variations should be investigated for efficient coating of B. subtilis and improved magnetic hyperthermia after exposure to AMF. The histology suggests there is association and retention of the SPION with the bacteria after injection in vivo but could not be confirmed with the optical microscopy technique utilized (Fig. 2.10E-G). Yet, the modified Gram stain further supported the finding of the association of the B. subtilis and plain-dextran SPION in vivo by showing a typical Gram stain result for B. subtilis in comparison to the consecutive staining (Fig. 2.10E-G). Hematoxylin and eosin staining performed on adjacent tissue sections showed eosin-stained (pink), longitudinal quadriceps muscle fibers (Fig. 2.13D) confirming samples were injected intramuscularly. 53 Perls’ Prussian Blue staining89 and modified Gram stain90 demonstrated the presence of iron and B. subtilis at the same location, which provides the opportunity to utilize magnetic hyperthermia to control B. subtilis transcription in vivo. Further tuning of the genetic elements to the B. subtilis and characterizing the interaction of improved particles for magnetic hyperthermia with B. subtilis would enhance further in vivo studies. SPIONs can be coated with polymers, small molecules, lipids and composites to increase stability, water solubility and biocompatibility.86 For example, Fe3O4-oleic acid-Na-oleate nanoparticles88 increased stability in a transplanted carcinoma model and polycaprolactone-coated superparamagnetic iron oxide nanoparticles synthesized with a micellular conformation were used to increase cytocompatibility and thermosensitivity as a cancer therapy.86 Additionally, increasing RF amplitude and amount of iron associating with the bacteria could improve heating along with imaging properties in vivo. Yet, increases in bioluminescence were observed after AMF treatment with only ~1 ppm of Fe in the plain-dextran coated condition in vitro. This reduced the amount of Fe that is delivered compared to other magnetic hyperthermia applications, such as for tumor ablation,91 from 1 mg/cm3 to 13.6 ng/cm3. Accordingly, the bacteria can be used as a carrying mechanism for and a responsive mechanism to SPIONs where minimal SPIONs are needed to produce a desired therapeutic outcome through controlling bacteriotherapies. Alternatively, manganese-doped magnetic nanoclusters have been studied for glioblastoma therapy as a nanoparticle that has complementary functionalities and can utilize photothermal and magnetic hyperthermia treatments.87 Additionally, other heating mechanisms could be used for magnetic hyperthermia such as ultrasound which was been shown previously.42,52,92 54 MATERIALS AND METHODS Bacterial growth conditions B. subtilis constructs were grown in Luria-Bertani Miller broth (LB) with spectinomycin (100 µg/mL). The overnight cultures were grown for 16 h at 37°C and 250 RPM unless otherwise specified. B. subtilis constructs The thermal response elements originated from pTlpA39-Wasabi (Addgene plasmid # 86116; http://n2t.net/addgene:86116; RRID:Addgene_86116).42 The TlpA39 promoter and regulator (driven by the LacI promoter) were cloned into the pDR111 plasmid to replace the Phyper-spank promoter and LacI regulator using Gibson assembly.93 Accordingly, the luxA-E operon was inserted into in the NheI restriction site of the pDR111 backbone by the seamless ligation cloning extract (SLiCE) method94 to create the new pDR111 PLacI tlpa39R PTlpA39 luxA- E. coli construct. Three strains were created: empty vector (pDR111 backbone only), experimental PTlpA39 repressed strain (pDR111 PLacI tlpa39R PTlpA39 luxA-E), and PTlpA constitutive strain without the repressor (pDR111 PTlpA39 luxA-E). Constructs were inserted into the genome of B. subtilis at the amyE locus using a homologous recombination plasmid (pDR111,95 a gift from Dr. Lee Kroos). The pDR111 plasmid was transformed into B. subtilis using a natural competence protocol and constructs were selected for by spectinomycin then confirmed by PCR amplification out of the genome.96 Three B. subtilis strains were created: containing the empty vector, the vector with the experimental PTlpA39 repressed strain (PTlpA39 luxA-E + tlpa39R), and the PTlpA constitutive strain without the repressor (PTlpA39 luxA-E - tlpa39R). All constructs were confirmed by PCR, restriction enzyme digestion, functional assays (when applicable), and Sanger sequencing (Azenta Life Sciences). 55 Iron coating of B. subtilis Synomag-D particles possess a maghemite (γ-Fe2O3) core of nanoflower-shaped nanocrystallites with a dextran shell and a hydrodynamic particle diameter of 50 nm.59 We utilized the plain dextran shell nanoparticle, a variation coated with carboxyl groups (carboxyl- coated) and a variation coated with amine groups (amine-coated) (MicroMod; #104-00-501, #103-02-501, #104-01-501). B. subtilis was incubated with plain-dextran or carboxyl-coated Synomag-D (200 µg/mL) in LB broth (pH = 7) or in LB broth (pH = 6.5) for the amine-coated Synomag-D (200 µg/mL) for 2 h at 37°C and 250 RPM after being normalized to OD600 = 1 or 2 in 1 mL. Coated B. subtilis was spun down at 10,000 x g for 2 min and washed with PBS (pH = 7.4) for plain-dextran and carboxyl-coated Synomag-D or with PBS (pH = 6.5) for amine-coated Synomag-D. The cultures were then resuspended in 100 µL of LB broth with appropriate pH mentioned above for use in HYPER or 250 µL of PBS (pH appropriate) for MPI and in vivo experiments. Scanning electron microscopy and elemental analysis Five hundred microliters of coated B. subtilis suspended in growth media was mixed with an equal volume of 4% glutaraldehyde in 0.1M sodium phosphate buffer, pH 7.4. Fixation was allowed to proceed for 30 min at room temperature. Twelve-millimeter round glass coverslips were floated on one drop of 1% poly-L-lysine (Sigma Aldrich P1399) each and allowed to stand for 10 min. The coverslips were removed and gently washed with HPLC-grade water. One drop of fixed sample was placed on the now coated side of the coverslip and allowed to settle for 10 m. After sample addition the coverslip was gently washed with HPLC-grade water and placed in a graded ethanol series (25%, 50%, 75%, 95%) for 10m each with three 10m changes in 100% ethanol.97 56 Coverslips with samples were then critical point dried in a Leica Microsystems model EM CPD300 critical point dryer (Leica Microsystems, Vienna, Austria) using carbon dioxide as the transitional fluid. Coverslips were then mounted on aluminum stubs using System Three Quick Cure 5 epoxy glue (System Three Resins, Inc., Aubur, WA) and carbon conductive paint (Structure Probe, Inc. 05006-AB) was added in a thin line for grounding. Samples were coated with iridium (2.7 - 5.5 nm thickness) in a Quorum Technologies/Electron Microscopy Sciences Q150T turbo pumped sputter coater (Quorum Technologies, Laughton, East Sussex, England BN8 6BN) purged with argon gas. Samples were examined in a JEOL 7500F (field emission emitter) scanning electron microscope (JEOL Ltd., Tokyo, Japan) and energy dispersive X-ray spectroscopy (elemental analysis) was performed using an Oxford Instruments AZtec system (Oxford Instruments, High Wycomb, Bucks, England), software version 3.1 using a 150mm2 Silicon Drift Detector (JEOL 7500F SEM) and an ultra-thin window. Images were analyzed using Fiji (ImageJ, version 2.0.0- rc-69/1.52i). Transmission electron microscopy Transmission Electron Microscopy (TEM; JEM-1400Flash, JEOL, MA USA) was used to confirm external associations of SPIONs with B. subtilis. Pelleted samples were fixed in 2.5% EM-grade glutaraldehyde for 5 min, washed with 0.1M phosphate buffer, and post-fixed with 1% osmium tetroxide in 0.1M phosphate buffer. After fixation, samples were dehydrated in a gradient series of acetone and infiltrated and embedded in Spurr’s resin. Seventy nanometer thin sections were obtained with a Power Tome Ultramicrotome (RMC, Boeckeler Instruments. Tucson, AZ), floated onto 200-mesh, carbon-coated formvar copper grids. Images were taken 57 with JEOL 1400-Flash Transmission Electron Microscope (Japan Electron Optics Laboratory, Japan). Images were analyzed using Fiji (ImageJ, version 2.0.0-rc-69/1.52i). In vitro imaging Plain, carboxyl or amine Synomag-D coated B. subtilis were imaged in triplicates (1x108 cells per sample in 25 µL PBS) using the Momentum MPI scanner (Magnetic Insight Inc, CA, USA). Plain Synomag-D coated B. subtilis were combined to a total of 4x108 cells in 100 µL PBS for detection. Images were acquired using a 2D projection scan with default (5.7 T/m gradient) or high sensitivity (3 T/m gradient) settings, rf amplitude (16.5 mT x-channel, 17 mT z- channel) and 45 kHz excitation with a field of view (FOV) = 12 x 6 cm, 1 average and acquisition time of ~1 minute. Bioluminescence was measured on the in vivo imaging system (IVIS, PerkinElmer) with auto-exposure settings (time = 2-40 sec, binning = medium, f/stop = 1, emission filter = open). Average radiance (p/sec/cm2/sr) was normalized to bacterial growth using optical density measured as absorbance at 600 nm (OD600) on a plate reader (Spectra Max 3, Molecular Devices, San Jose, CA, USA). Bioluminescent signals were quantified using the 8x12 grid ROI for all wells (in vitro thermocycler induction) and ellipse ROIs with standardized area for all tubes (in vitro) to calculate average radiance (p/sec/cm2/sr) using Living Image software (PerkinElmer, Version 4.5.2). Flow cytometry determination of B. subtilis viability The effects of coating and heating on B. subtilis viability was assessed using flow cytometry. B. subtilis was coated as described above with all three nanoparticle variations (+/- AMF). Following treatment cells were resuspended in 100 µL of 150 mM NaCl and stained using a viability/cytotoxicity assay kit for live and dead bacteria (Biotium, #30027) according to 58 the manufacturers protocol. Following staining cells were collected by centrifugation and washed once with flow buffer (1X PBS, 0.5% bovine serum albumin) followed by fixation with 4% paraformaldehyde for 10 minutes. Cells were then resuspended in 100 µL flow buffer for analysis using the Cytek Aurora flow cytometer. Unstained dead (heat treated; 98°C), live (uncoated; untreated) and live (coated; plain, carboxyl, amine) plus single stained DMAO (live/dead, FITC) and Ethidium Homodimer III (EthD-III; dead, Cy3) were used as controls. EthD-III dead cells were gated on the DMAO+ cell population. Data were analyzed using FCS express software (De Novo Software, CA, USA; version 7.12.0005). A one-way ANOVA was used to determine any significance between treatments’ potential impact on viability. The data presented herein were obtained using instrumentation in the MSU Flow Cytometry Core Facility. The facility is funded in part through the financial support of Michigan State University’s Office of Research & Innovation, College of Osteopathic Medicine, and College of Human Medicine. RNA extraction and RT-qPCR Technical replicates from thermal inductions were pooled for RNA extractions. B. subtilis was lysed using LETS buffer (100mM LiCl, 10mM EDTA, 10mM Tris pH 7.8, 1% SDS) and bead beating (0.1mm zirconium beads, 3 cycles of 60 sec at max speed). Total RNA was extracted using RNeasy miniprep kit (QIAGEN). Samples were cleaned and made into cDNA with QuantiTect Reverse Transcription kit (QIAGEN). The resulting cDNA was diluted 1:20 in RNAse free water for qPCR. QuantiTect SYBR Green PCR kit (QIAGEN) was used to prepare 20 µL reactions according to instructions. Primers for luxB, 16s, and tlpa39R were created using NCBI Primer BLAST and used for all samples. No-template controls of RNase-free water were run in triplicate for each primer set. Reactions were run in triplicate for each sample. Data was screened for validity using melting curves and then analyzed for relative quantification using the 59 2-ΔΔCt method.98 The expression levels for luxB and tlpa39R was calculated relative to the 16s rRNA housekeeping gene and the experimental groups (37, 39, 42°C). Confidence intervals of 95% were calculated using the mean and standard deviation for all cycle threshold values of a given sample (n=6) and then converting to fold change using the above 2-ΔΔCt method. In vitro thermocycler inductions This protocol was adapted from Piraner et al., 2017.42 Cultures of B. subtilis were grown in LB for 36 h at 25°C and 250 RPM under spectinomycin (100 µg/mL) selection. These cultures were then diluted to optical density of 0.1 at 600 nm (OD600) in LB with appropriate antibiotic and grown until they reached OD600 of 0.25. Twenty-five microliters of the samples were aliquoted into 96-well PCR plates and sealed. Thermal inductions were carried out in Biorad C- 100 thermocyclers at 25, 37, 39, and 42°C for either 12 h or 1 h. After thermal induction, the samples were diluted 1:4 in LB and 90 µL was transferred to a 96-well, black Costar plate. The OD600 and bioluminescence output was measured. Controls for the measurements were: growth media only, empty vector strain, PTlpA39 constitutive strain without the repressor, and experimental PTlpA39 repressed strain. After bioluminescent imaging and optical density measurements (method above), a one-way ANOVA with Tukey’s post-hoc was used to determine any significance between temperatures within a strain. HYPER theranostic hyperthermia system Magnetic hyperthermia was performed using the HYPER Theranostic Hyperthermia System (Magnetic Insight). Magnetothermal heating is localized using a Field Free Point (FFP) to direct radiofrequency (RF) energy. HYPER was programmed to apply AMF to the coated B. subtilis strains by using a 0.66 T/m magnetic field gradient strength, a RF amplitude of 14.5 or 16.0 mT, 350 kHz excitation and a RF amplitude application time of 60 seconds with a 1 second 60 cool down time. This programmed AMF cycle would be repeated such that the AMF was applied for the desired total run time of either 1 or 12 h. Optimal parameters for each particle were determined when bacteria were normalized to OD600 = 1 or 2. For each run the following strains and replicates were included: B. subtilis PTlpA39 luxA-E +TlpA39R coated with one of the three variations of Synomag-D were divided into PCR tubes in two 50 µL aliquots where one aliquot would be placed in the AMF (+AMF) and outside the AMF (-AMF) in biological replicates (n=7). The same process was repeated for the B. subtilis PTlpA39 luxA-E -TlpA39R strain (n=3) to be run in same conditions with +TlpA39R strain. Optimal HYPER parameters for each variation of particle mentioned above with bacteria normalized to OD 600 = 2 were utilized to determine temperature increase in LB during the heating of B. subtilis with the three variations of coating. Fiber-optic temperature probes (Weidmann-Optocon, standard TS2 probes) were placed into the LB throughout the 12 h of heating to track temperature through the HYPER software. Temperature readings were recorded every AMF application cycle (60 readings per cycle were treated as technical replicates) with reads at each 30 min time point plotted for visualization. Unpaired Student or Welch’s t-test was used to determine statistical significance between samples with and without AMF treatment. In vivo magnetothermal heating and imaging Female BALB/c mice (6-8 weeks; Jackson Laboratories USA) were obtained and cared for in accordance with the standards of Michigan State University Institutional Animal Care and Use Committee. B. subtilis were coated with plain Synomag-D as described above. Mice (n=6) were anesthetized with isoflurane administered at 2% in oxygen followed by hair removal on each thigh using a depilatory. An intramuscular (IM) injection of 1x108 iron-coated bacteria in 25 µL PBS was performed into the left thigh followed by BLI (IVIS Spectrum; post-injection 61 timepoint) using auto-exposure settings (time = 30-120 sec, binning = medium, f/stop = 1, emission filter = open). One mouse was imaged using MPI using the default setting, as described above. No signal was detected and no further mice were imaged by MPI. Following imaging, mice were either placed into the HYPER system for magnetothermal heating (+AMF; 16 mT for 1 h, n=3) or maintained at room temperature in cage (-AMF, n=3). BLI was performed as above, after AMF application, or 1 h for mice which were not subjected to AMF (post-treatment timepoint). After the final imaging time point mice were sacrificed and thigh muscle from the IM injected side and the contralateral not injected side were excised followed by sectioning for histological staining and microscopy (see below for detailed methods). Two-way repeated measures ANOVA and Tukey’s post-hoc was used to determine statistical significance between AMF treatments and timepoints for bioluminescence. Histological analysis Thigh muscle samples were fixed in 4% paraformaldehyde for 24 h followed by cryopreservation through serial submersion in graded sucrose solutions (10%, 20% and 30%). Samples were then frozen in optimal cutting temperature compound (Fisher HealthCare, USA). Tissues were sectioned using a cryostat (6 µm sections). Sections were stained with a modified Gram stain as described by Becerra et al., 2016,90 followed by Perls’ Prussian Blue (PPB)89 on the same sections for detection of bacteria and detection of ferric iron. CitriSolv (Decon Labs, Inc., King of Prussia, PA, USA; Cat.#1601) was used as a safe alternative for xylene in the final step of the modified Gram stain. Eosin was used as a counterstain in the Perls’ Prussian Blue protocol. Sequential sections of the tissue were stained with the modified Gram stain only and hematoxylin and eosin staining only to verify Gram status of the B. subtilis and confirm intramuscular injections, respectively. Microscopy was performed on the sections using a Nikon 62 Eclipse Ci microscope equipped with a Nikon DS-Fi3 camera (Nikon, Tokyo, Japan)) for color image acquisition and NIS elements BR 5.21.02 software (Nikon). Microscopy images were prepared using the auto-white feature on NIS elements and Fiji (ImageJ, version 2.0.0-rc- 69/1.52i). Image analysis Living Image software (PerkinElmer, Version 4.5.2) was used to quantify bioluminescent signals. An 8x12 grid region of interest (ROI) was used for 96-well plates (in vitro thermocycler induction) or ellipse ROIs with standardized area for all tubes (in vitro) or on the injection site of the mouse thigh (in vivo) to calculate average radiance (p/sec/cm2/sr). MPI data sets were visualized and analyzed utilizing Horos imaging software (Horos is a free and open-source code software program that is distributed free of charge under the LGPL license at Horosproject.org and sponsored by Nimble Co LLC d/b/a Purview in Annapolis, MD USA). Fixed ROIs were used to identify all samples and total MPI signal was determined (area x mean signal). Calibration standard curves were created by imaging different amounts of iron and plotting signal (y) versus iron content (x) with the y-intercept (b) set to zero. The slope (m) of the data was found using a simple linear regression and quantification of iron content was calculated using the trendline equation (y=mx+b). Standard curves were created using matched imaging parameters (default or high sensitivity) dependent on the data set being analyzed. Inductively coupled plasma-mass spectrometry (ICP–MS) After B. subtilis + TlpA39R was coated with the three SPION variations at OD600 = 2 then the cells were pelleted (centrifugation at 10,000xg) and resuspended in phosphate buffered saline (PBS; pH 7.4). Three technical replicates of the coating procedure were pooled in a final volume of 750 µL PBS. One sample of untreated B. subtilis + TlpA39R (OD600 = 2) from the 63 coating process (no SPION control) was also prepared and resuspended in 250 µL PBS. The cells were digested in concentrated nitric acid (J.T. Baker, USA; 69-70%) overnight, and diluted 25-fold with a solution containing 0.5% EDTA and Triton X-100, 1% ammonium hydroxide, 2% butanol, 5 ppb of scandium, and 7.5 ppb of rhodium, indium, and bismuth as internal standards (Inorganic Ventures, VA, USA). The samples were analyzed on an Agilent 7900 ICP mass spectrometer (Agilent, CA, USA). Elemental concentrations were calibrated using a 5-point linear curve of the analyte-internal standard response ratio. Bovine liver (National Institute of Standards and Technology, MD, USA) was used as a control. Statistical analysis and visualization Statistical analyses were performed using Prism software (9.2.0, GraphPad Inc., La Jolla, CA). Statistical tests are identified for each method. Significance was considered as p<.05 Plotting was performed using R version 4.0.4 with the following packages: ggplot2, dplyr, reshape2, ggsignif, ggpubr and plotrix. Availability of data and materials All raw data, Bacillus subtilis constructs and R scripts will be made available upon request by the corresponding author. Plasmids used to produce B. subtilis constructs will be submitted to Addgene after manuscript publication. All R scripts were written with established packages. Competing interests The authors declare that they have no competing interests. Acknowledgments The authors would like to acknowledge A. Withrow, C. Flegler, and S. Flegler at the MSU Center for Advanced Microscopy, the MSU Flow Cytometry Core, S. Rebolloso at the 64 MSU Veterinary Diagnostic Laboratory, L. Kroos, M. Witte, A. Tundo, and K. Conner at MSU, M. Huebner and J. Tait at MSU Center for Statistical Training and Consulting. Figure 2.1 was created using Biorender.com. Funding The authors would like to acknowledge the James and Kathleen Cornelius Endowment Fund, the College of Engineering Dissertation Completion Fellowship supported C. S. Madsen and the College of Natural Sciences Dissertation Continuation and Completion Fellowships supported E. M. Greeson. Affiliations Department of Biomedical Engineering, Michigan State University, East Lansing, MI, USA. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA. Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, MI, USA. Contributions E.M. Greeson and C.S. Madsen contributed equally to experimental design, performing experiments, data analysis and manuscript writing. A.V. Makela contributed to experimental design, performed in vivo studies and contributed to manuscript writing. C.H. Contag contributed to experimental design and data analysis, and manuscript writing. All authors have given approval to the final version of the manuscript. ‡E.M. Greeson and C.S. Madsen contributed equally. 65 REFERENCES 66 REFERENCES (1) Mahmoudi, M., Hofmann, H., Rothen-Rutishauser, B. & Petri-Fink, A. Assessing the in vitro and in vivo toxicity of superparamagnetic iron oxide nanoparticles. Chem. Rev. 112, 2323– 2338 (2012). (2) Prijic, S. & Sersa, G. Magnetic nanoparticles as targeted delivery systems in oncology. Radiol. Oncol. 45, 1–16 (2011). (3) Cardoso, V. F. et al. 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Methods 25, 402–408 (2001). 74 CHAPTER 3 ENGINEERED BACILLUS SUBTILIS CAPABLE OF EPITHELIAL CELL INVASION 75 ABSTRACT Epithelial tissue malignancies represent 80-90% of all cancer cases making epithelial cells an important target for cancer therapies. Bacterial-based therapies have been explored for cancers, such as Bacille Calmette-Guerin being used as the standard of care for early-stage bladder cancer. Current clinic trials of new cancer therapies target cell cycle regulators and tumor suppressors which have demonstrated efficacy; however, delivery of these proteins is a limitation. To address this limitation, B. subtilis llo was engineered to express internalin A (InlA), a protein native to Listeria monocytogenes. Internalin A is an adhesin that binds to the E- cadherin host cell receptor found in epithelial cells. In this chapter, Western blot analysis shows that B. subtilis llo inlA expresses InlA and that E-cadherin is present in 4T1s a mouse mammary carcinoma cell line and murine trophoblast stem cells (TSCs). TSCs were used as a model for InlA-mediated invasion via the “zipper” mechanism to be used for downstream cellular reprogramming applications. To demonstrate cytosolic persistence the engineered bacteria were co-incubated at a 100:1 MOI with TSCs, extracellular bacteria were washed away, and cells were fixed and imaged via confocal microscopy. Maximum intensity projections, orthogonal cross- sections, and three-dimensional cross-sections with cutaways all confirmed the +InlA bacteria were cytosolic and the -InlA bacteria were extracellular and beneath the host cells. Flow cytometry was utilized to measure the mean fluorescent intensity of the Cell Tracker Orange bacterial stain in each TSC population. The dot plots for the negative controls indicated an error that led to a false negative with zero TSCs with internal bacteria. This chapter is an ongoing study and so future directions include repeating several experiments including flow cytometry, as well as expanding the model to include the delivery of a protein after successful invasion. 76 INTRODUCTION Epithelial cancers, categorized as carcinomas by the International Classification of Diseases for Oncology, Third Edition, is malignant growth in the internal and external lining of the body.1 According to the National Cancer Institute these epithelial tissue malignancies make up 80-90% of all cancer cases.2 Epithelial tissue is present in skin and the linings and coverings of internal passageways and organs.1,2 This makes epithelial tissues and more specifically, carcinomas, an important target for bacterial cancer therapies. Current approaches to carcinoma treatments include surgery, chemotherapy, radiation therapy, bone marrow transplant, immunotherapy, hormone therapy, targeted drug therapy, cryoablation, radiofrequency ablation, and clinical trials of new treatments such as cytokine-based3 and bacterial-based therapy.4–9 Cell cycle regulators and tumor suppressors are often targets of these clinical trials; however, delivery methods remain a limitation.10–16 Bacterial cancer therapy with Bacille Calmette-Guerin (BCG, the Mycobacterium bovis strain used as a tuberculosis vaccine) is the standard of care for early- stage bladder cancer17 and there are many other approaches under investigation and FDA review for various human cancers.5,7–9,18 In general, bacteriotherapies are being studied for a variety of targets currently including treatment of hyperammonemia,19 phenylketonuria,20 in situ microbiome engineering,21 recurrent streptococcal pharyngotonsillitis,22 and adenoidectomy prevention.23 The study of pathogens and virulence factors can inform the development of bacteriotherapies for the treatment of cancers and other disease states. This study will address the delivery method limitations with a focus on targeting epithelial cells and in some cases carcinomas. Bielecki et al. transferred the hlyA gene (LLO protein) from Listeria monocytogenes into B. subtilis and studied bacterial invasion of phagocytic macrophage/monocyte cells 77 (J774A.1).24–26 Previous work has shown that this chassis organism can be coupled with different proteins to function as a modular bacteriotherapy within these phagocytic cells. 27 One mechanism of invasion into non-phagocytic host cells that L. monocytogenes utilizes is the “zipper” mechanism between the bacterial ligand internalin A (InlA) and the host cell receptor E- cadherin.28 The “zipper” mechanism is broadly described with four steps in L. monocytogenes, infection, receptor binding, membrane engulfment, and vacuole disruption.60 Receptor binding for L. monocytogenes in epithelial cells is between the adhesin molecule internalin A (InlA; inlA) and the host cell receptor E-cadherin.28–30 Once fully engulfed in the vacuole, L. monocytogenes uses listeriolysin O (LLO, hlyA) to disrupt the vacuole membrane to escape into the cytoplasm of the host epithelial cell.28 Pathogenicity, host cell toxicity, and an excess of virulence factors are limitations to using known bacterial pathogens as chassis organisms for therapies. 31,32 Synthetic biology techniques create an opportunity to selectively choose one or more virulence factors and move them into bacteria more amenable to treatments.33,34 B. subtilis physiology is ideal for a bacteriotherapy chassis as it is a generally recognized as safe (GRAS), non-pathogenic, Gram- positive, soil bacterium that respires as a facultative anaerobe35–37 and does not have a lipopolysaccharide- (LPS) mediated immune response.38 Once the method of bacterial invasion into epithelial cells is addressed with synthetic biology the cytosolic bacteria can deliver transcription factor or other relevant proteins as treatments. To understand the possibilities for this modular therapy the field of regenerative medicine can provide insight to the current approaches and areas for improvement. Regenerative medicine is a branch of translational research in tissue engineering and molecular biology which deals with the process of replacing, engineering or regenerating human or animal cells, tissues or organs to restore or establish normal function.39 Cell therapies include delivery of cells, often 78 from a donor, to the patient, as a therapeutic approach.40 Specifically, cellular reprogramming is important to the field because iPSCs and their derivatives allow for autologous cell replacement of immune-compatible cells.41 However, large-scale productions are difficult with the current systems.41 An engineered bacterium can help make these types of therapies scalable by providing a means of studying and controlling differentiation conditions to inform defined, reproducible conditions. Cellular reprogramming is the reconfiguration of a cellular gene expression program to drive the cell to a different state in a more or less deterministic fashion, including modification of the epigenetic marks of mammalian germ cell development.42 The field of cell biology was dramatically changed when Gurdon described nuclear reprogramming43 and led to Takahashi and Yamanaka publishing four transcription factors that are necessary and sufficient to drive a terminally differentiated cell, such as a fibroblast, to an iPSC.44 These four factors known now as OSKM factors (i.e. Yamanaka factors) are Oct3/4, Sox2, Klf4, and c-Myc.5 Currently, systems of reprogramming include viral transduction (e.g. lentivirus, adeno-associated virus, etc.), DNA transfection (e.g. plasmid, minicircle, transposon), OSKM protein transduction, synthetically modified RNA transfection (e.g. microRNA, in vitro transcribed-RNA, etc.), and chemical/small molecule-mediated reprogramming.41 Most of these systems have a low efficiency, on average 1%, and require 4-6 weeks to complete reprogramming.45 Previous work demonstrates that trophoblast stem cells (TSCS) which are multipotent46 can be driven to pluripotency using two transcription factors (Oct3/4, Sox2) in 16 days.47,48 This indicates that TSCs are a good candidate for in vitro cellular reprogramming with a new transcription factor delivery method. A recent study investigated using Pseudomonas aeruginosa to deliver transcription factors to iPSCs via a type III secretion system to drive differentiation to cardiomyocytes. 49 This 79 study demonstrates that a pathogenic, extracellular bacterium can deliver transcription factors to drive mammalian cell fate. Previous work demonstrated the ability of a non-pathogenic engineered endosymbiont (EES) technology to modulate immune cells.27 This study will expand on the field and delivering proteins in a controlled fashion to epithelial cells with a potential model to alter cell fate. For this study, an EES will be defined as a normally extracellular, non-pathogenic bacterium that can be genetically and physically manipulated to persist viably in the cytoplasm of a mammalian cell.27 With the EES system outlined, both applied and basic science questions can be examined with a model of B. subtilis llo inlA invasion into epithelial cells. This tool can be applied not only to cellular reprogramming and tissue regeneration, but also more broadly to the field of regenerative medicine (e.g. immunomodulation therapy, delivery of cancer therapeutics, etc.).27,50 To expand the model and demonstrate the modular nature of this approach mouse mammary carcinoma cells (4T1s) will be studied to show the translatable nature of the invasion mechanism. B. subtilis llo within J774A.1 cells, was first studied to understand the behavior of LLO and its role in evading phagosome destruction.24–26 The current mammalian cell type being explored as a host cell is trophoblast stem cells (TSCs) derived from a female C57B6 mouse. TSCs were chosen for their ability to be reprogrammed in half the time of terminally differentiated cells47,48 (i.e. macrophages) as well as their tendency to take up extracellular bacteria.51–53 Previous work has shown that β-gal can be effectively produced, secreted, and trafficked from intracellular B. subtilis llo to J774A.1 nuclei.27 Our basic EES system involves a co-incubation of bacteria and mammalian cells to promote an interaction allowing the bacteria entry into the host mammalian cell (Fig. 3.1, 1).27,50 80 The main mechanisms of invasion utilized in this setup are first, an E-cadherin-mediated entry into murine epithelial cells facilitated by the inlA gene encoding internalin A (InlA; Fig. 3.1, 2) followed by vacuole disruption with the hlyA gene encoding listeriolysin O (LLO; Fig. 3.1, 3) and persistence in the host cell cytoplasm (Fig. 3.1, 4).29 A secondary mechanism of invasion which may contribute to host cell entry is phagocytosis and vacuole disruption with LLO (Fig. 3.1A-C).24 This requires the host cell type to exhibit enough phagocytic activity to engulf B. subtilis which is a limitation mitigated by the inclusion of InlA in the model. Once inside the host cell, an engineered bacterium can be induced to secrete folded proteins to the nucleus which allows for control of host cellular functions.27 The bacterium is described as a chassis organism, emphasizing the modular nature of the prokaryotic components. The chassis organism can be genetically modified using synthetic biology techniques to include operons encoding mammalian transcription factors and bacterial secretion peptide tag.27,54 Additionally, transcriptional control will be incorporated into the approach to ensure precise, inducible timing for protein production. 81 Figure 3.1. Model of engineered B. subtilis invasion into epithelial cells (1-4) Primary method of uptake via internalin A “zipper” mechanism of internalization illustrating receptor binding with E-cadherin, membrane engulfment facilitated by G- and F- actin, endosomal escape with listeriolysin O (LLO), and persistence in the cytoplasm. (A-C) Secondary method of uptake via LLO alone with phagocytosis, endosomal escape, and persistence in the cytoplasm. 82 B. subtilis has several options for inducible transcription systems because it is an environmental organism that maintains fitness by responding to dynamic stimuli in the soil environment.55 Some well-defined inducible systems for B. subtilis expression include D- xylose,56 L-rhamnose,57 D-mannose,58 and IPTG59 for chemical control. Chemical control relies on transport into the mammalian cell to reach the chassis organism and literature shows transport into mammalian cells of mannose60,61 and IPTG.62,63 Xylose is a plant-derived sugar64 and while it does not have a dedicated transporter in mammalian cells, studies indicate D-xylose can be transported by facilitated diffusion and as a D-glucose analog.65,66 The field of regenerative medicine has advanced greatly since the discovery of OSKM factors. Unfortunately, the need for tissue and organ donations in the United States is ever present and despite the increase in transplants occurring annually, there is still a deficit. Organogenesis, tissue regeneration, and cellular reprogramming could all be performed via an EES. Our EES model involves B. subtilis llo inlA with a Tat secretion system to deliver proteins of interest to the host cell (e.g. TSCs, 4T1s) nucleus to alter cell fate. While developing such an EES system, it is possible to deliver a variety of proteins intracellularly such as pro-drug converting enzymes, transcription factors, tumor suppressors, and cell cycle regulators highlighting the modular nature of this bacteriotherapy. The scope of this study is to engineer B. subtilis physiology by adding virulence factors under transcriptional control to develop an in vitro tool for cellular reprogramming and, more broadly, as a tool for a variety of medical therapeutics. 83 RESULTS Protein expression Engineered B. subtilis llo inlA was analyzed via Western blot to confirm protein expression of internalin A (InlA) in the bacterial cell pellet (Fig. 3.2). Xen32, a bioluminescent strain of L. monocytogenes which is the native source of InlA, was used as a positive control and showed a faint band at the correct molecular weight, 87 kD. The B. subtilis llo inlA strain showed a strong band at 87 kD. Negative controls of B. subtilis llo and Xen32 ΔinlAΔinlB were included and showed no bands in the relevant molecular weight range. Growth curves of the background strain B. subtilis 168 (168), the -InlA strain B. subtilis llo (LLO), and the new construct B. subtilis llo inlA (LLO InlA) were conducted (Fig. 3.3). All three strains appear to be in a logarithmic growth phase between 2 and 4 hours of growth, with variable growth rates. The LLO InlA strain reached the highest optical density (580 nm) of the three strains. The LLO strain grows similarly to the LLO InlA strain but diverges when it reaches stationary phase around OD580 = 0.5 and the LLO InlA strain continues increasing to OD580 = 0.6. The 168 strain has the lowest growth rate of the three strains but reaches the same final OD580 = 0.5 at 7.5h as the LLO strain. Two mouse cell lines were analyzed via Western blot to confirm E-cadherin expression, the binding partner of murinized InlA (Fig. 3.4). Mouse mammary carcinoma cells (4T1s) were evaluated after EDTA (Fig. 3.4, lane 1-3) and trypsin (Fig. 3.4, lane 4-6) treatment and showed E-cadherin in the EDTA treatment, but an absence of bands in the trypsin lanes. TSCs were evaluated after treatment with Accutase detachment solution (Fig. 3.4, lane 7-9) and showed the presence of E-cadherin. GAPDH was a loading control and showed similar levels of protein across all lanes (Fig. 3.4, lane 1-9). 84 Figure 3.2. Western blot showing expression of InlA in B. subtilis Internalin A (87 kD) is present in B. subtilis llo inlA pellet lysates. A faint band is present in Xen32 a strain of Listeria monocytogenes a native source of internalin A. B. subtilis llo and Xen32 ΔinlAΔinlB lanes are negative controls and do not have internalin A. Figure 3.3. Growth curves of B. subtilis strains Growth rates measured as optical density at 580 nm of the B. subtilis background strain (168), llo strain (LLO), and llo inlA (LLO InlA) were evaluated for any differences. Data plotted is mean ± standard deviation; n = 3 biological replicates. 85 Figure 3.4. Western blot showing expression of E-cadherin in epithelial cells E-cadherin (120 kD) is present in 4T1 murine mammary carcinoma cells treated with EDTA (1- 3) and absent in 4T1 cells treated with trypsin (4-6). Trophoblast stem cells show the presence of E-cadherin when treated with accutase (7-9). GAPDH (36 kD) was used as a loading control in all samples (1-9). 86 B. subtilis internalization A functional assay was developed and carried out to demonstrate uptake of B. subtilis llo inlA (+InlA) by mouse TSCs, a non-phagocytic, E-cadherin positive cell type as an in vitro model for other murine epithelial cells. TSCs were co-incubated with B. subtilis llo with and without InlA (±InlA) at a 100:1 MOI for 2.5 h before gentamicin was added to clear the extracellular bacteria. The cells were fixed 1 h post-addition of gentamicin and subsequently imaged via confocal which showed the spatial relationships between F-actin, nuclei, and ±InlA engineered B. subtilis (Fig. 3.5). The maximum intensity projections of the +InlA strain showed more bacteria than the -InlA strain (Fig. 3.5A-B). The orthogonal cross-sections show the +InlA strain colocalized with the nuclei and F-actin as indicated by the white arrows pointing to the same bacterial signal in x-y, z-x, and z-y planes (Fig 3.5C). The -InlA strain indicates bacteria in a different z-depth than the F-actin and nuclei when observing the white arrows pointing to the same bacterial signal in x-y, z-x, and z-y planes (Fig. 3.5D). Three-dimensional cross-sections and cutaway images were rendered to examine the interaction between the TSCs and the ±InlA conditions more closely. The +InlA condition shows colocalization of the bacterial signal with the F-actin and nuclei signals as indicated by the white arrows (Fig. 3.6A,C). The -InlA condition in the three dimensional rendering shows the bacterial signal away from the F-actin and nuclei signals as indicated by the white arrows (Fig. 3.6B,D). Flow cytometry was utilized to identify and quantify the TSCs that had internal B. subtilis and to elucidate the variation between the number of bacteria per mammalian cell. This experiment measured Cell Tracker Orange (CTO) signal for each condition of uptake but is in the process of being repeated so the results presented here will not be complete. CTO signal was plotted versus count (Fig. 3.7A) and showed distinct difference between the ±InlA conditions 87 and the negative control with the unstained bacteria. The population of TSCs that was stained with the CTO wash supernatant was distinct from the unstained population and showed a mean fluorescence intensity (MFI) above that of the experimental conditions (±InlA). The dashed blue lines indicate the CTO positive TSC population which contains a count of zero cells. The flow cytometry dot plots with forward scatter (FSC-A) versus CTO signal (Fig 3.7B,C) the two negative controls can be compared. The TSCs + unstained B. subtilis (Fig 3.7B) and the TSCs + CTO wash supernatant (Fig. 3.7C) have similar forward scatter signatures while having different CTO profiles, approximately 1x103 and 5x105, respectively. 88 Figure 3.5. Uptake of B. subtilis into trophoblast stem cells Confocal microscopy showing maximum intensity projections of (A) B. subtilis llo inlA (+InlA; orange) overlayed with F-actin (green) and cell nuclei (blue) and (B) B. subtilis llo (-InlA; orange) as a negative control for internalin A based internalization. An orthogonal cross-section of the +InlA (C) and –InlA (D) conditions with white arrows indicating bacterial regions in the cross-sections of z-x and z-y planes. Scale bar = 20 µm. 89 Figure 3.6. Three-dimensional cross-sections of B. subtilis uptake Confocal microscopy showing three dimensional cross-sections of (A,C) B. subtilis llo inlA (+InlA; orange) overlayed with F-actin (green) and cell nuclei (blue) and (B,D) B. subtilis llo (- InlA; orange) as a negative control for internalin A based internalization. White arrows indicate bacteria in the cross-sections. Magnification = 60X with 1.5 zoom. 90 Figure 3.7. Characterization of B. subtilis interaction with trophoblast stem cells Flow cytometry was used to identify and characterize CTO (Cell Tracker Orange) positive trophoblast stem cells (TSC), relating to the presence of fluorescently labeled bacterial strains (±InlA) within the cells. (A) Median fluorescence intensity (MFI) of CTO demonstrates the shift in CTO intensity between the unstained and stained supernatant negative controls and the ±InlA sample populations. Flow cytometry dot plots identify CTO intensity in TSCs + unstained bacteria (B, low) and TSCs + stained supernatant from the bacterial wash step (C, high). n = 2 biological replicates. 91 DISCUSSION Due to the in silico engineering of B. subtilis llo inlA to include a murinized version of InlA capable of membrane anchoring and binding with mouse E-cadherin rigorous confirmation was conducted. Sanger sequencing confirmed the construct was built correctly (data not shown) and a Western blot was used to check for protein expression in the bacterial cell pellet (Fig. 3.2). The engineered B. subtilis llo inlA showed a higher expression level than the native source of InlA, Xen32, a bioluminescent strain of L. monocytogenes. This higher level of expression of the experimental strain compared to the positive control can be explained by a difference in promoter strength56,67,68 and codon optimization.69,70 The negative control strains further confirmed the antibody specificity by ruling out the background of the B. subtilis llo strain24 and the Xen32 knockout strain lacking InlA and InlB.71 Following confirmation of the protein expression the viability of the B. subtilis constructs were compared by measuring and plotting growth curves. Growth curves of the three strains 168, LLO, and LLO InlA showed some differences in growth rate, although the samples demonstrated an exponential growth phase during the same time frame (Fig. 3.3). Due to the transcriptional control elements engineered into the LLO and LLO InlA constructs chemical inducers were added to these cultures which most likely influenced the growth of these samples compared to 168.72 The LLO InlA strain reached the highest OD580 of the three strains which agrees with the addition of 1% D-xylose to the system.56 The 168 strain only had access to the nutrients in standard LB Miller broth with no additives. In the future, adding 1% D-xylose and 500 µM IPTG to all the cultures regardless of the necessity for transcriptional regulation should negate this variable. 92 Once the bacterial strains were confirmed as expressing InlA and growing well it was necessary to investigate potential cell lines with the correct receptor, E-cadherin. Two mouse cell lines were chosen to explore as candidates for bacterial internalization via E-cadherin and InlA interaction. Specifically, 4T1s and TSCs were shown to have E-cadherin present in cell lysates with Western blotting when treated with EDTA and Accutase, respectively (Fig. 3.4). The 4T1 cell lysate treated with trypsin to detach the cells did not have a band in the E-cadherin molecular weight range. Trypsin is a proteolytic enzyme that dissociates adherent cells from the cell culture flask by breaking down proteins that allow the cells to adhere.73 Trypsin has been reported to disrupt the proteome when used for mammalian cell subculture.74 The cadherin family, including E-cadherin, are a group of adhesive, cell membrane proteins that act as anchors connecting the extracellular matrix and the cytoskeleton to help regulate cell adhesion and migration which makes them a target of trypsin proteolysis.74,75 This result suggests that Accutase or EDTA should be used for subculturing mammalian cells when downstream experiments require the measurement or functionality of E-cadherin such as in this study. The +InlA condition showing more bacterial signal than the -InlA condition after the addition of gentamicin suggests internalization of the +InlA bacteria since the extracellular bacteria should be washed away (Fig. 3.5A-B). 24,76 The colocalization of the +InlA strain signal (orange) with the nuclei and F-actin (blue and green, respectively) supports internalization of the B. subtilis llo inlA (Fig. 3.5C). The -InlA strain supports bacteria being extracellular since they are in a different z-depth than the F-actin and nuclei (Fig. 3.5D). The ±InlA condition have some extracellular bacteria that remained after the gentamicin wash step. One explanation is the bacteria appear to be below the TSCs in the image which matches the observation during the 93 experiment of the cells starting to lift slightly along the edges at the 100:1 MOI before fixation (data not shown). The three-dimensional cross-sections and cutaway renderings further confirm the internalization of the +InlA bacteria by the TSCs when compared to the -InlA condition. The +InlA condition shows colocalization of the three signals (white arrows) which demonstrates the bacteria are cytosolic (Fig. 3.6A,C). The -InlA condition further illustrates the point that the bacteria are below the TSCs and remain extracellular partially protected from the gentamicin and wash steps as the bacterial signal is located by itself (white arrows) and not colocalized with the other structures (Fig. 3.6B,D). The CTO flow cytometry experiment indicated that there were zero CTO positive cells meaning no bacteria were internalized by the TSCs in any experimental condition (±InlA; Fig 3.7A). When further examined the negative control of the TSCs + the CTO wash supernatant showed higher MFI than the experimental conditions (±InlA). The CTO positive range is gated based on the negative controls so the high MFI from the supernatant control possibly led to a false negative. This is further confirmed by the flow cytometry dot plots of the two negative control populations (Fig. 3.7B,C). The forward scatter is a strong indicator of cell size77 and in this study shows that the cells are similarly sized which concurs with the TSCs all being subcultures prior to the experiment. The TSCs + CTO wash supernatant (Fig 3.7C) have an approximately 500-times higher CTO signal when compared to the TSCs + unstained B. subtilis (Fig. 3.7B) which is suspicious. When critically assessing the methods used for this experiment the CTO wash supernatant was reserved after the first wash in the bacterial staining process. This is most likely inflating the amount of CTO signal considered background when setting the CTO positive bounds. By taking the final wash step supernatant and repeating the experiment the CTO 94 positive cells should reflect the results seen in the confocal microscopy (Fig. 3.5, 3.6). This amended method also agrees with the protocol used in previous studies with B. subtilis llo and phagocytic cells.27 TSCs can be reprogrammed to pluripotency with the over expression of Oct3/4 (Pouf51) and Sox2 (Sox2) in as little as 16 days in vitro as reported in previous work.47,48 This will be the next step in the continuation of this study. A B. subtilis llo inlA Pou5f1 Sox2 construct is being built and will be used to allow persistence in the TSC cytoplasm and then deliver Oct3/4 and Sox2 to the nucleus to drive TSCs towards pluripotency. The characterization of this transition to pluripotency will be tracked with Cdx2, Klf4, and Nanog markers.47,48 Cdx2 is a TSC specific marker that is lost as the cells become pluripotent, Klf4 increases expression with pluripotency, and Nanog only a marker of pluripotency that is not expressed in TSCs.78–81 95 MATERIALS AND METHODS Data and code availability All raw data, Bacillus subtilis constructs and R scripts will be made available upon request by the corresponding author. Plasmids used to produce B. subtilis constructs will be submitted to Addgene after manuscript publication. All R scripts were written with established packages. Mammalian cell culture Trophoblast stem cells (TSCs) originally isolated from female C57BL/6 mice according to the protocol established by Tanaka et al.82 were maintained at 37⁰C and 5% CO2 in RPMI 1640 (GIBCO, catalog# 61870036) media supplemented with 20% fetal bovine serum, 1 µM sodium pyruvate, 35 μg/mL fibroblast growth factor 4 (FGF-4; GenScript, catalog# Z02984), 1 μg/mL heparin, and 10 ng/mL activin-A (R&D Systems, catalog# 338-AC).82–84 Murine mammary carcinoma cells (4T1; ATCC-CRL-2539, ATCC, Manassas, VA) were maintained at 37⁰C and 5% CO2 in RPMI 1640 media supplemented with 10% fetal bovine serum. All cells tested negative for mycoplasma using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza, catalog# LT07-703). Cells were passaged with Accutase cell detachment solution (5 min at 37⁰C; Corning, catalog# MT25058CI) and cell scraping unless otherwise specified. B. subtilis constructs The internalin A protein sequence originated from Listeria monocytogenes on the UniProt database (Accession # P0DJM0) and then was murinized in silico according to previous studies by substituting serine 192 to asparagine and tyrosine 369 to serine.85,86 This was then back translated with B. subtilis codon usage parameters to obtain the coding DNA sequence.69,70 A ribosome binding site was added to the 5’ end, a stop codon was added to the 3’ end, and NheI 96 overhangs were added to both sides before gBlock gene fragment synthesis (Integrated DNA Technologies, Inc.; IDT). The xylose-inducible system amplified from B. subtilis 168 strain genome56 was cloned into the pDR111 plasmid to replace the Phyper-spank promoter and LacI regulator using Gibson assembly.33 The murininized InlA gene fragment (inlAm) was inserted into in the NheI restriction site of the pDR111 backbone by Gibson assembly to create the new pDR111 xylR Pxyl inlAm E. coli construct. UniProt sequences for mouse Oct3/4 (Pou5f1; accession P20263) and Sox2 (accession P48432) were back translated70 and a ribosome binding site was added to the 5’ end and a stop codon was added to the 3’ end before gBlock gene fragment synthesis (IDT). Restriction enzyme cut sites and PhoD signal peptide54 were built into the gBlocks for cloning and protein secretion through the twin-arginine translocation (Tat) pathway,54 respectively. Pou5f1 had SbfI on the 5’ end and EagI on the 3’ end, while Sox2 had EagI on the 5’ end and BmtI on the 3’ end. The mannose-inducible system amplified from B. subtilis 168 strain genome58 was cloned into the pDR111 plasmid to replace the Phyper-spank promoter and LacI regulator using Gibson assembly. A SbfI cut site was introduced into the multiple cloning site by inverse PCR then digesting both ends with SbfI and re-ligating the pDR111 mannose plasmid. The Oct3/4 and Sox2 gene fragments was inserted sequentially into the pDR111 backbone by restriction enzyme cloning with SbfI/EagI and EagI/BmtI, respectively, followed by T4 ligation (New England Biolabs, catalog# M0202S) to create the new pDR111 Pman Pou5f1Sox2 E. coli construct. Constructs were inserted into the genome of B. subtilis at the amyE locus using a homologous recombination plasmid (pDR111,87 a gift from Dr. Lee Kroos). The pDR111 plasmid was transformed into B. subtilis using a natural competence protocol and constructs were selected for by spectinomycin then confirmed by PCR amplification out of the genome.88 Two B. subtilis strains were created with the pDR111 vector 97 in the B. subtilis ZB307 llo background strain24,58 with Pxyl inlAm and Pman Pou5f1 Sox2. A third construct, Pxyl inlAm Pman Pou5f1Sox2 with the llo background is being created in the pDR111 vector for further studies. All constructs were confirmed by PCR, restriction enzyme digestion, functional assays (when applicable), protein expression (when applicable), and Sanger sequencing (Azenta Life Sciences). Bacterial growth conditions B. subtilis constructs with amyE locus recombination were grown in Luria-Bertani Miller broth (LB; Sigma Aldrich, catalog# L3522) with spectinomycin (100 µg/mL). B. subtilis llo24 was grown in LB with chloramphenicol (10 µg/mL) and B. subtilis 168 (ATCC-23857, ATCC, Manassas, VA, USA) was grown in LB with no antibiotic selection. Xen3271 strains were grown in Brain Heart Infusion broth (Millipore, catalog# 53286). The overnight cultures were grown for 16 h at 37°C and 250 RPM unless otherwise specified. Growth curves of B. subtilis B. subtilis 168 background strain, LLO, and LLO InlA overnight cultures were grown for 16 h at 37°C and 250 RPM in triplicate (n = 3). All sugar inducers (500 µM IPTG, 1% D-xylose) required for protein expression in the engineered strains were included throughout the growth curves. All cultures were diluted 1:20 then allowed to grow into logarithmic phase for 3 h. Subsequently the cultures were normalized to OD580 = 0.1 then 100 µL was transferred into columns of a 96 well plate (Falcon, #351172) for a total of 24 replicates (n = 3 biological, n = 8 technical). Cultures were grown in a PerkinElmer VICTOR Nivo plate reader at 37°C and 300 RPM with OD580 measurements (580 nm was used due to plate reader limitations) taken every 30 min. Measurements were performed until growth rates began to slow in late logarithmic phase in 98 the 100 µL (8 h for all samples). All replicates were plotted to visualize differences in growth rates between strains as mean ± SD. Bacterial protein extraction This bacterial protein extraction method is adapted from Johnson and Hecht, 1994.89 Five milliliters of overnight cultures (grown as described above) for the following samples: B. subtilis llo, llo inlA, Xen32, and Xen32 ΔinlA ΔinlB were pelleted at 10,000xg for 10 min at 4°C and the supernatant was discarded. The pellets were frozen by submerging the tubes in a dry ice/ethanol bath for 2 min followed by thawing in an ice/water bath for 8 min. This was repeated for a total of three free-thaw cycles. Fifty microliters of 20 mM, pH 8 Tris-HCl with protease inhibitors (cocktail set I, II, III from Sigma Aldrich; catalog#539131, 539132, 539134) was added and the samples were resuspended gently by tapping the bottom of the tube for 30 seconds. Resuspended samples were transferred to an ice/water bath for an additional 30 min then centrifuged at 4°C for 10 min at 10,000xg. The supernatant was removed and saved without disturbing the pellet, samples were stored at 4°C until downstream analysis (described below). Mammalian cell protein extraction Approximately 1x106 cells (4T1s and TSCs) were incubated with either 2.5 mM EDTA in PBS, 0.25% trypsin-EDTA, or Accutase cell detachment solution for 5-10 min. Any remaining cells were scraped with a cell scraper and centrifuged at 2,000xg for 5 min. The cell pellets were washed with PBS and centrifuged at 2,000xg for 5 min and then resuspended in 1 mL ice cold lysis buffer containing 1 tablet of protease inhibitor (Thermo, cat #A32955) & 1 tablet of phosphatase inhibitor (Thermo, cat #A32957) in 10 mL modified mRIPA buffer (0- 0.1% SDS) and transferred to a 1.5 mL microcentrifuge tube. Samples were agitated by shaking 99 for 30 min at 4°C and then centrifuged at 16,000xg for 20 min in 4°C. Supernatant was collected without agitating the pellet and stored at 4°C. Western blot Protein levels were measured with the Qubit protein quantification assay (Qubit 2.0; Thermo Fisher Scientific catalog# Q33211) and adjusted to the same total protein concentration for loading. For E-cadherin samples ~20 µg was loaded and for internalin A samples ~2 µg was loaded into each lane. Samples were prepared for gel electrophoresis by mixing protein with 4X sample buffer (Expedeon, cat #NXB31010), 10X reducing buffer (Thermo, cat #NP004) and deionized water and heating to 90°C for 5 min. SDS-PAGE was performed with the BioRad Mini-PROTEAN tetra system using Mini-PROTEAN TGX Stain-free Precast gels (BioRad, cat #4568093) and 1X SDS Running Buffer solution (BioRad, cat #1610732). Ten microliters of Precision Plus Protein All Blue Standards (BioRad, cat #1610373) was added for protein sizing and 10 µL of adjusted protein sample loaded into each lane. Gels were run at 85V for approximately 90 min. The E-cadherin blot transfer used a wet, tank system (100V for 1h) and the Internalin A blot transfer used a semi-dry membrane transfer (25V, 30 min; BioRad Trans- Blot Turbo Transfer System). Blots were blocked with 5 mg/ml of ECL Blocking Agent (Sigma Aldrich, catalog#GERPN2125) in TBST for 1h at room temperature. Blots were incubated with primary antibodies at 4°C overnight with slow rocking and secondary antibodies for 1h, shaking at room temperature. Rat IgG1 anti-mouse CD324 (E-cadherin) monoclonal antibody (Biolegend, catalog#147301; 1:1,000 dilution), mouse IgG2b anti-GAPDH monoclonal antibody (Proteintech, catalog# 60004-1; 1:5,000 dilution), Rabbit anti-Listeria monocytogenes inlA polyclonal antibody (CUSABio, catalog# CSB-PA758331HA01AAD; 1:2,000 dilution) were used as primary antibodies. Goat anti-rabbit IgG HRP-linked monoclonal antibody (Cell 100 signaling, catalog#7074; 1:2,000 dilution), horse anti-mouse IgG HRP-linked monoclonal antibody (Cell signaling, catalog#7076; 1:2,000), goat anti-rat IgG HRP-linked monoclonal antibody (Cell signaling, catalog#7077; 1:2,000) were used as secondary antibodies. ECL prime Western blotting detection reagent (VWR, catalog#RPN2232) was used according to directions and blots were imaged with the Gel Doc XR+ system (Bio-Rad Laboratories, Inc.) using autoexposure and chemiluminescence for the samples and 635 nm with autoexposure settings for the ladder. B. subtilis uptake assay This protocol was modified from Madsen, et al.27 for TSCs. The following conditions were utilized to induce B. subtilis invasion, unless otherwise described. TSCs were seeded onto a 96-well black glass-bottom plate (40,000 cells/well; Greiner Bio-One, Austria, cat# 655892) and allowed to adhere overnight when an estimation of total number of cells was made based on confluency. Bacteria were pre-stained using a TRITC fluorescent dye, Cell Tracker Orange CMRA Dye (Invitrogen, C34564). The B. subtilis samples was spun down (10,000 x g) for 2 min then resuspended in Cell Tracker Orange CMRA Dye (4 µM) in PBS then incubated at 37⁰C and 250 RPM for 25 min. Afterwards, the bacteria were spun down (10,000 x g) and washed three times before adding to TSCs. B. subtilis strains were added at an optimized 100:1 multiplicity of infection (MOI) for all experiments, along with IPTG (500 µM) to induce expression of LLO with or without protein of interest. For xylose control of InlA expression, 1% (w/v) D-xylose was added. For D-mannose control of the Pou5f Sox2 operon (OS operon), 1% (w/v) D-mannose is added. B. subtilis and TSCs were then co-incubated at 37⁰C and 5% CO2 for 2.5h. TSCs were then washed once with PBS and new medium was added containing gentamicin (8 µM) to 101 eliminate any remnant extracellular bacteria.76 Co-incubation continued for 1h at 37⁰C and 5% CO2 prior to preparation for microscopy (described below). Cell fixation and counter staining Cells were permeabilized using 0.3% Triton X-100 (ThermoFisher) followed by a blocking step containing 0.3% Triton X-100 and 5% normal goat serum (ThermoFisher). Nuclei were counterstained by incubating cells with Hoechst 33342 (1 µg/mL) for 10 min at room temperature followed by an F-actin counterstain by incubating with fluorescein phallodin (11 nM; Invitrogen) for 30 min at room temperature. Wells were then washed with PBS three times before adding 150 µl of PBS and sealing the plate with parafilm and protecting from light before confocal microscopy (see below). Confocal microscopy Confocal microscopy was performed using a Nikon A1 CLSM (Nikon, NY, USA) microscope to determine B. subtilis persistence in the mammalian cytoplasm by imaging TSCs that had been treated with the B. subtilis strains with and without InlA (InlA). Imaging was performed using a 60x oil objective and 1.5x zoom and using filter sets for DAPI (Hoechst 33342 for DNA), FITC (fluorescein phalloidin for F-actin), and TRITC (Cell Tracker Orange CMRA dye for B. subtilis). Z-stacks were taken at 0.5 µM steps to confirm location of B. subtilis within host cells. Images were analyzed using NIS-Elements AR Software (Nikon) and background noise was reduced by using Nikon denoise.ai algorithm. The 3-dimensional volume images and cutaways were produced by the Alpha display mode. Flow cytometry with cell tracker orange Uptake of B. subtilis ± InlA by TSCs was assessed using flow cytometry after staining bacteria with CellTracker Orange CMRA Dye (CTO, Invitrogen, C34564, 4 µM incubated at 102 37°C and 250 RPM for 25 min). Following staining, the strains were washed two times before adding to TSCs at a 100:1 MOI for a 2.5 h incubation. After the first wash step the supernatant was reserved for use as a negative control to account for CTO staining of TSC; CTO fluorescence above this level would be due to the presence of CTO bacteria. Cells were collected, washed once with PBS, and incubated with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen; cat#L34961) for 30 min at 4°C. Cells were washed twice followed by fixation using 4% PFA and resuspended in 100 µL flow buffer for analysis using the Cytek Aurora Cytometer (Cytek Biosciences, CA, USA). All samples were assessed for cell viability. Cells which were incubated with CTO bacteria were assessed for percent CTO positive cells (indicating TSCs containing bacteria). TSC which were incubated with unstained B. subtilis or with the reserved supernatant from the CTO staining were used as negative controls to set the threshold for TSCs containing CTO bacteria. In the future, this experiment will be repeated with the final wash step reserved from the CTO staining to closely match the probable levels of residual stain. The data presented herein were obtained using instrumentation in the MSU Flow Cytometry Core Facility. The facility is funded in part through the financial support of Michigan State University’s Office of Research & Innovation, College of Osteopathic Medicine, and College of Human Medicine. Data visualization Images were analyzed using Fiji (ImageJ2, version 2.3.0/1.53q). Plotting was performed using R version 4.2.1 with the following packages: ggplot2, dplyr, reshape2, ggsignif, and plotrix. Competing interests The authors declare that they have no competing interests. 103 Acknowledgments The authors would like to acknowledge M. Frame at the MSU Center for Advanced Microscopy, the MSU Flow Cytometry Core, L. Kroos, A. Aguirre, J. Hardy, J. Kaletka, and K. Conner at MSU. Figure 3.1 was created using Biorender.com. Funding The authors would like to acknowledge the James and Kathleen Cornelius Endowment Fund, the College of Engineering Dissertation Completion Fellowship supported C. S. Madsen and the College of Natural Sciences Dissertation Continuation and Completion Fellowships supported E. M. Greeson. Affiliations Department of Biomedical Engineering, Michigan State University, East Lansing, MI, USA. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA. Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, MI, USA. Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI, USA. Contributions E.M. Greeson contributed to experimental design, performing experiments, data analysis and manuscript writing. C.S. Madsen contributed to experimental design, data analysis, and manuscript writing. E.E. Ural contributed to experimental design, Western blot experiments, data analysis, and contributed to manuscript writing. A.V. Makela performed flow cytometry experiments, data analysis, and contributed to manuscript writing. B.D. Unluturk contributed to experimental design and synthetic biology efforts, and manuscript review. V.A. Toomajian contributed to Western blot experiments, data analysis, and manuscript writing. C.H. Contag 104 contributed to experimental design and manuscript writing. All authors have given approval to the final version of the manuscript. 105 REFERENCES 106 REFERENCES (1) Organization, W. H. 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Biotechnol. 1994, 12, 1357–1360. 114 CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS 115 The field of regenerative medicine has advanced greatly since the discovery of OSKM factors.1–3 Unfortunately, the need for tissue and organ donations in the United States is ever present and despite the increase in transplants occurring annually, there is still a deficit. 4–6 Organogenesis, tissue regeneration, and cellular reprogramming could all be performed via an EES. Our EES model involves B. subtilis llo inlA with a Tat secretion system7 and SV40 nuclear localization8 signal to deliver proteins of interest to the host cell (e.g. TSCs, 4T1s) nuclei to alter cell fate. While developing such an EES system, it is necessary to consider how to tightly control the delivery of the proteins (e.g. pro-drug converting enzymes, transcription factors, tumor suppressors, cell cycle regulators). A temperature-sensitive repressor coupled with magnetic hyperthermia for B. subtilis transcriptional control was the topic of Chapter 2. With consideration to factors like growth temperatures9, ribosome binding sites, codon optimization,10,11 and non-monomeric plasmids12,13 for optimal B. subtilis gene expression, the TlpA39 TSR system14 was transferred from E. coli to B. subtilis. To show functionality as thermal switches the bacterial luminescent operon (luxA- E)15–17 was chosen as a proof of concept reporter to be paired with magnetothermal control via SPIONs. This was useful for cellular reprogramming because it demonstrated a non-invasive method of control over the transcription of an enzymatic operon. The engineered TSR system was tested both in vitro and in vivo. Using thermal energy that can be localized to specific regions and be limited in impact on host cells provides several advantages over commonly used methods of control such as sugar- inducible systems.18–21 The magnetothermal control demonstrated by using the TlpA39 regulatory system in B. subtilis to respond to thermal energy generated when an AMF was applied to SPION coated B. subtilis took the first step towards a method of controlling the EES 116 non-invasively and in a localized region in vivo. The TlpA39 promoter and regulator system was functional in B. subtilis and able to regulate an operon with a slight temperature shift from what was observed in Escherichia coli previously14 which was demonstrated by increased bioluminescence and transcript levels when heated in a thermocycler. Though, the TlpA39 promoter and regulator system could be further optimized in B. subtilis as was done previously in E. coli14 and B. subtilis.22 Further optimization by directed mutagenesis14,22 or other measures such as gene circuit optimization23–25 could improve the PTlpA39 genetic switch to have a more stringent on/off state which would improve responses during magnetothermal heating studies. When transitioning to magnetothermal heating, an approach was needed to facilitate thermal energy release when an AMF was applied. Accordingly, the SPION coatings provided the solution which was confirmed by SEM-EDS. The variations coated B. subtilis in different ways, but these observations were explainable by the DLVO (Derjaguin–Landau–Verwey– Overbeek) theory which has been shown to predict the potential interaction between a given nanoparticle and bacteria.26,27 Culminating the observed coating of the variations with the response of the bacteria to magnetothermal heating and heating of the culture medium, the plain- dextran SPION showed the most promise for reproducible localized heating that could be used in vivo. Magnetothermal energy over 12 h showed significant increase in reporter output but this change is still substantially less than that of the continuous heating in a thermocycler and this result is even more clear after only 1 h of heating. Therefore, the thermal energy diffusion from the SPIONs to B. subtilis needs to be improved because the 1 h of continuous heating demonstrated that the TSRs can produce a significant amount of transcription leading to a change in bioluminescence signal even within the constraints for small animal anesthesia. 28,29 An immediate change that could enhance the magnetic hyperthermia is increasing the RF amplitude 117 beyond the limitations of the HYPER system (>16.0 mT). However, an increase in RF amplitude will result in an increase in specific absorption rate (SAR),30 this would have to be further studied to prevent any biological effects. Another option is considering more variations of SPIONs that have been modified to improve magnetic hyperthermia properties. 31–34 Accordingly, these variations should be investigated for efficient coating of B. subtilis and improved magnetic hyperthermia after exposure to AMF. The Perls’ Prussian Blue staining35 and modified Gram stain36 demonstrated the presence of iron and B. subtilis at the same location, which provides the opportunity to utilize magnetic hyperthermia to control B. subtilis transcription in vivo. As mentioned above, further tuning of the genetic elements and characterizing the interaction of improved particles for magnetic hyperthermia with B. subtilis would enhance further in vivo studies. Other coatings of SPIONs such as with polymers, small molecules, lipids and composites can increase stability, water solubility and biocompatibility.32 For example, Fe3O4-oleic acid-Na-oleate nanoparticles34 increased stability in a transplanted carcinoma model and polycaprolactone-coated superparamagnetic iron oxide nanoparticles synthesized with a micellular conformation were used to increase cytocompatibility and thermosensitivity as a cancer therapy.32 Additionally, increasing RF amplitude in accordance with SAR requirements 30 and amount of iron associating with the bacteria could improve heating along with imaging properties in vivo. Yet, increases in bioluminescence were observed after AMF treatment with only ~1 ppm of Fe in the plain- dextran coated condition in vitro. This reduced the amount of Fe that is delivered compared to other typical magnetic hyperthermia applications, such as for tumor ablation,37 from 1 mg/cm3 to 13.6 ng/cm3. Accordingly, the bacteria act as a carrying mechanism for and a responsive mechanism to SPIONs where minimal SPIONs are needed to produce a desired therapeutic 118 outcome through controlling bacteriotherapies. Alternatively, other heating mechanisms could be used for magnetic hyperthermia such as ultrasound which was been shown previously 14,38,39 that could be paired with the SPION-coated B. subtilis strategy. Overall, the demonstration of magnetothermal control of B. subtilis transcription advances the translatability of the EES platform by increasing control of bacteria through a non-invasive measure during in vivo applications. Engineering B. subtilis to enter and affect epithelial cells was the topic of Chapter 3. TSCs were chosen as a host cell for their shortened time needed for reprogramming to pluripotency40,41 and their potential to host intracellular bacteria.42–44 Previous studies show that OSKM factors can reprogram differentiated cell types to iPSCs.1 Specifically, TSCs have been shown to be reprogrammed to iPSCs using only Oct3/4 (Pou5f1) over expression.41 The same study showing Oct3/4 alone was sufficient to reprogram TSCs also demonstrated a faster timeline with the addition of Sox2 as compared to only Oct4 and not significantly different than all four OSKM factors.41 Using only Oct3/4 and Sox2 instead of all four OSKM factors will reduce metabolic burden on B. subtilis and possibly reduce toxicity to the host cell. Based on previous Listeria monocytogenes studies with TSCs,45–48 B. subtilis llo inlA will be used as a chassis with an OS operon under D-mannose20 transcriptional control engineered into the construct to demonstrate cellular reprogramming to iPSCs.46,49 This study has set up a model of B. subtilis internalization via E-cadherin mediated by LLO and InlA in trophoblast stem cells. A murinized version of internalin A was designed in silico10,45,47,50 and engineered into B. subtilis llo to target mouse epithelial cells for uptake.46,51 Western blotting was used to confirm the presence of InlA in the B. subtilis llo inlA strain as well as to confirm E-cadherin, the corresponding host cell receptor in both TSCs and 4T1s. Growth 119 curves confirmed that the bacterial constructs are all metabolically active, although some differences between growth rates in the exponential phase warrant repeating the experiment. Confocal microscopy showed internalization of the +InlA condition with colocalization of bacterial, F-actin, and nuclei signals while the -InlA condition demonstrated the bacteria were outside of the TSCs. The CTO flow cytometry experiment contradicted the confocal microscopy images by indicating that there were no TSCs with cytosolic bacteria. However, this incongruity is most likely an issue with a flow cytometry negative control that can be solved by repeating the experiment.52 Additionally, B. subtilis constructs will be engineered to demonstrate delivery of transcription factors and eventually, cellular reprogramming. Future directions include expanding the uptake assay with confocal microscopy and CTO flow cytometry experiments to include 4T1 cells. Additionally, TSCs will be reprogrammed to pluripotency in vitro with transcription factors delivered from B. subtilis llo inlA and will be characterized with RT-qPCR and flow cytometry of differential markers. Specifically, Cdx2, Klf4, and Nanog markers will be used to track the degree of pluripotency in vitro.40,41 Cdx2 is a TSC specific marker that is lost as the cells become pluripotent, Klf4 increases expression with pluripotency, and Nanog only a marker of pluripotency that is not expressed in TSCs.53–56 With the continuation of this newly developed area of research there are future questions that should be considered. First, it should be estimated, if possible, how much protein is needed for a specific application to be viable. Transcription factors or proteins that trigger cascades to amplify the signal strength will need to be delivered at lower amounts than proteins without signal amplification. Second, to deliver the amount of protein needed how many bacteria per cell are required? To answer this question, it is necessary to determine how many bacteria the host 120 cell type can handle and the time frame this number can be maintained. This will require factoring in bacterial and host cell replication rates, an area of study that needs to be further investigated as another cellular target for control. Third, immune response should be considered when deciding what chassis organism and host cell configurations to utilize. For example, in chapter 3 the main host cell type being used is TSCs which have been shown to exhibit less of an innate immune response to bacteria compared to other more differentiated/developed cell types such as macrophages.57–60 Currently, it has not been investigated if an EES triggers innate immune responses in the different cells used in the work. Given what we know of these responses this is something to consider for tissue engineering platforms such as this. The expansion of the EES platform to non-phagocytic, epithelial cells and further advancements on transcriptional control creates a new approach for bacteriotherapies. By complementing the epithelial invasion model described in this study with cellular reprogramming and functional protein delivery it highlights the utility and modular nature of this platform. The temperature-sensitive repressors coupled with magnetic hyperthermia allow for future use of this tool in vivo as a way of non-invasive control of a theranostic. Ultimately, the engineering of B. subtilis transcriptional control and physiology creates a new modular approach to regenerative medicine, cellular reprogramming, and cancer therapy that can be used in human health applications. 121 REFERENCES 122 REFERENCES (1) Takahashi, K.; Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 2006, 126 (4), 663–676. https://doi.org/10.1016/J.CELL.2006.07.024/ATTACHMENT/A7BA2E0F-99EF-4A86-88AA- 418202149347/MMC1.PDF. (2) Yoshida, Y.; Yamanaka, S. Induced Pluripotent Stem Cells 10 Years Later. Circ. 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