This dissertation describes the construction of engineered endosymbionts (EES) as a platform technology for modulating macrophage function for therapeutic applications. Dr. Ashley Makela and I worked closely to advance the EES technology. Dr. Makela focused on the characterization of the EES ability to change macrophage function and I focused on developing the EES technology and working with Dr. Makela on characterization and using the EES in applications (Chapter 2 and 3). In Chapter 2, Bacillus subtilis was developed as a chassis organism for EES that escape phagosome destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to impact host cell response and function. Two synthetic operons encoding either the mammalian transcription factors (TFs) Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the genome of B. subtilis expressing listeriolysin O (LLO) from Listeria monocytogenes and expressed from regulated promoters. Controlled expression of the mammalian proteins from B. subtilis LLO in the cytoplasm of J774A.1 macrophage/monocyte cells altered surface marker, cytokine and chemokine expression. Once the EES platform was developed and initially tested in vitro with a macrophage cell line, translating the EES to applications became the next step to understand the capacity of the new technology (Chapter 3). For increased translatability, the effect of the engineered B. subtilis LLO TF strains on murine bone marrow-derived macrophages (BMDMs) function was characterized. The TF strains shifted BMDM production of cytokines, chemokines and metabolic patterns. RNA-seq is still being analyzed to elucidate effects on gene expression. Furthermore, the ability of the B. subtilis LLO TF strains to alter the tumor microenvironment was characterized in a murine 4T1 orthotopic breast cancer model. The B. subtilis LLO strains altered the tumor microenvironment by promoting immune cell invasion, altering the functional metabolism of cells within the tumor, and causing tumor growth stabilization. Additionally, safety of this EES platform was observed as multiple doses at bacterial concentrations 100-fold more than other bacterial therapies were injected without affecting the health of mice. Yet, during the development and characterization of the EES, the sugar (D-mannose) that was used to induce transcription in the EES once inside the host cell was observed to significantly impact macrophage physiology which created additional complexity and was not ideal for in vivo applications. Accordingly, Emily Greeson and I worked on developing a mechanism for non-invasive localized control of gene expression in vivo. Emily Greeson engineered B. subtilis with temperature sensitive repressors (TSRs) and characterized this new genetic switch. I then coated B. subtilis with superparamagnetic iron oxide nanoparticles (SPIONs) which could be stimulated by an alternating magnetic field (AMF) to generate thermal energy. Chapter 4 discusses this new approach, and we investigated the ability of magnetic hyperthermia to regulate 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 B. 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 9-fold (12 h) and 11.1-fold (1 h) increase in bioluminescence. To develop magnetothermal control, B. subtilis cells were coated with three SPION variations which was confirmed by electron microscopy coupled with energy dispersive X-ray spectroscopy. Furthermore, using long duration AMF, we demonstrated magnetothermal induction of the TSRs in SPION-coated B. subtilis with a maximum of 4.6-fold increases in bioluminescence. Pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to improved EES transcriptional control. The research described in this dissertation demonstrates a multi-disciplinary approach towards developing a new modular technology to alter mammalian cell function with the specific focus on macrophages.
Read
