ENGINEERING B. SUBTILIS TRANSCRIPTIONAL CONTROL AND PHYSIOLOGY FOR THE ADVANCEMENT OF BACTERIOTHERAPIES

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.