Acoustic Reporter Genes for High-Resolution Ultrasound Imaging of Engineered Bacteria In Vivo

More than 90% of the cells in a typical human body are microbes, and there is a rapidly growing appreciation of the crucial roles these organisms play in human health and disease. Additionally, advances in synthetic biology have enabled the development of genetically engineered bacterial therapies and diagnostics that are starting to advance into the clinic. These diverse activities of natural and engineered microbes depend critically on their anatomical location within host organisms, such as the various regions of the mammalian gastrointestinal tract. However, no effective methods are currently available to image microbes inside mammalian hosts with high spatial and temporal resolution. The development of such methods would transform the study of microbial-host interactions and facilitate the development of an entirely new class of bacterial diagnostics.

We address this challenge by developing acoustic reporter genes (ARGs) to enable the imaging of microbes with ultrasound. Unlike optical techniques, which have poor spatial resolution in vivo due to the scattering of light by tissue and are not commonly used in the clinic, ultrasound is capable of very high spatial resolution in vivo (tens of microns in mice) and is used widely in medicine.  We recently discovered a unique class of protein nanostructures encoded in the genomes of certain photosynthetic microbes that can serve as imaging agents for ultrasound¹. These nanostructures, called gas vesicles (GVs), comprise all-protein shells that are ~ 250 nm in size and are hollow on the inside. We showed that the hollow interior and unique mechanical properties of GVs allow them to scatter sound waves and thereby serve as sensitive in vivo imaging agents for ultrasound.

Now, we have succeeded in adapting the gene clusters encoding GV formation for heterologous expression as ARGs in Escherichia coli and Salmonella typhimurium, two important model organisms and chasses for synthetic biology. ARG expression allows us to image these bacteria with ultrasound in vitro and in vivo.  Cells can be detected at concentrations below 10^8 cells/ml. In addition, engineering of the GV gene cluster has yielded variants that produce non-linear ultrasound contrast and can be “erased” with acoustic pressures above specific thresholds, thus enabling more sensitive detection within biological tissues. Furthermore, the engineering of ARGs with different thresholds for acoustic collapse enables multiplexed imaging of more than one cellular population.

ARGs will allow microbes to be imaged dynamically in vivo with unprecedented precision, unlocking a wide range of new biological research and the development of spatially informative bacterial diagnostics.

1.         Shapiro, M.G. et al. Biogenic gas nanostructures as ultrasonic molecular reporters. Nature nanotechnology 9, 311-316 (2014).