Tools for Noninvasive Imaging and Control of Engineered Bacteria In Vivo

Citation

Buss, Marjorie Theresa (2024) Tools for Noninvasive Imaging and Control of Engineered Bacteria In Vivo. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/mvgg-ch02. https://resolver.caltech.edu/CaltechTHESIS:05292024-221307093

Abstract

Genetically engineered bacteria are promising new cell-based diagnostic and therapeutic agents due to their ability to sense and respond to unique signals, access and interface with hard-to-reach areas of the body, and deliver therapeutics directly to these areas. However, currently tools to noninvasively monitor and control their activity in vivo are limited. Optical imaging methods, which are based on fluorescent and luminescent reporter genes, and optogenetics, which are based on light-activated proteins, are widely used in cell culture and rodent studies. However, these optical methods suffer from the poor penetration depth of light in tissue which limits their use in larger animals or humans. On the other hand, nuclear imaging methods such as PET and SPECT have good imaging depth but rely on radioactive tracers whose synthesis can be complex and exposes patients to radiation. Here I present tools for imaging and control of bacteria that based on non-ionizing forms of energy that easily penetrate tissue: sound waves and magnetic fields.

The first two parts of my thesis focuses on imaging bacteria in vivo with ultrasound, which is a widely available imaging modality that does not use ionizing radiation and has tissue penetration depth of several centimeters. Bacteria can be imaged with ultrasound by expressing acoustic reporter genes (ARGs) which result in the production of gas vesicles (GVs), air-filled protein nanostructures that aquatic microbes use to regulate their buoyancy. However, the first-generation acoustic reporter genes expressed too poorly under in vivo conditions to enable ultrasound imaging of bacteria in therapeutically relevant contexts. Here, we present a new and improved ARG construct that produces high levels of robust gas vesicle expression in the probiotic bacterium E. coli Nissle (EcN), enabling ultrasound imaging of these cells with high sensitivity. This second-generation ARG construct, bARGSer, uses genes derived from Serratia sp. ATCC 39006 and was optimized for plasmid-based expression in EcN. We demonstrate that with bARGSer, we can visualize the spatial distribution of engineered EcN after they home to and colonize tumors upon systemic administration. We also demonstrate that the engineered EcN can be imaged with ultrasound when colonizing the gastrointestinal tract of mice after sensing dietary sugars as well as biomarkers of inflammation. By enabling monitoring of the precise spatial location of engineered probiotic bacteria inside the body, this technology could greatly improve the development and eventual clinical use of this emerging class of microbial cell-based theranostics.

The last part of my thesis focuses on control of bacteria in vivo with magnetic fields. Many bacteria have limited ability to selectively colonize specific targeted regions of the GI tract due to a lack of external control over their location and persistence. Magnetic fields are well suited to provide such control due to their ability to freely penetrate biological tissues, but they are difficult to apply with enough strength to directly manipulate magnetically labeled cells within deep tissue or viscous environments such as in the GI tract. Here, we show that ingestible micron-sized magnetic particles, combined with an externally applied magnetic field, act as in vivo magnetic field gradient amplifiers, enabling the trapping and retention of orally administered probiotic E. coli within the mouse GI tract. This technology improves the ability of these probiotic agents to accumulate at specific locations and stably colonize without antibiotic treatment. By enhancing the ability of GI-targeted cellular agents to be at the right place at the right time, cellular localization assisted by magnetic particles (CLAMP) adds external physical control to an important emerging class of biotherapeutics.

Thesis Files