Genomically mined acoustic reporter genes enable real-timein vivomonitoring of tumors and tumor-homing probiotics

Abstract

A major outstanding challenge in the fields of biological research, synthetic biology and cell-based medicine is the difficulty of visualizing the function of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and a spatial resolution below 100 um. Recently, the first genetically encoded reporter molecules were developed based on bacterial gas vesicles to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging in the in vivo context. Here, we describe second-generation ARGs with greatly improved acoustic properties and expression characteristics. We identified these ARGs through a systematic phylogenetic screen of candidate gas vesicle gene clusters from diverse bacteria and archaea. The resulting constructs offer major qualitative and quantitative improvements, including the ability to produce nonlinear ultrasound contrast to distinguish their signals from those of background tissues, and a reduced burden of expression in probiotic hosts. We demonstrate the utility of these next-generation ARGs by imaging the in situ gene expression of tumor-homing probiotic bacteria, revealing the unique spatial distribution of tumor colonization by these cells noninvasively in living subjects. *equal contribution

New Insights for Biosensing: Lessons from Microbial Defense Systems

“…GVs act as acoustic sensors and MRI sensors, respectively. (A–D) GVs act as a biomolecular acoustic sensor for (A) protein detection, (B) bacteria detection, (C) mammalian cells, and (D) mouse imaging . (E–H) GVs act as a biomolecular magnetic resonance imaging (MRI) sensor for the imaging of (E) mouse, (F) brain tissue, (G) mammalian cells, and (H) GVs …”

The Future Potential of Biosensors to Investigate the Gut-Brain Axis

“…Genetic engineering has tuned the sensitivity of GVs to varied acoustic pressure (Figure 2Biv) (Shapiro et al, 2014). Therefore, distinct GVs can be distinguished by their sensitivity to acoustic pressure (Lakshmanan et al, 2016;Hurt et al, 2021) to monitor two signals simultaneously. The ARGs have been engineered with improved expression (Lakshmanan et al, 2017;Hurt et al, 2021) and various acoustic properties (Bourdeau et al, 2018;Hurt et al, 2021) in different cell types, such as E. coli and Salmonella typhimurium (Bourdeau et al, 2018).…”

“…Therefore, distinct GVs can be distinguished by their sensitivity to acoustic pressure (Lakshmanan et al, 2016;Hurt et al, 2021) to monitor two signals simultaneously. The ARGs have been engineered with improved expression (Lakshmanan et al, 2017;Hurt et al, 2021) and various acoustic properties (Bourdeau et al, 2018;Hurt et al, 2021) in different cell types, such as E. coli and Salmonella typhimurium (Bourdeau et al, 2018). The engineered bacteria with ARGs were also tested deep inside a mammalian host within the tumors (Hurt et al, 2021) and gastrointestinal tract (Bourdeau et al, 2018).…”

“…The ARGs have been engineered with improved expression (Lakshmanan et al, 2017;Hurt et al, 2021) and various acoustic properties (Bourdeau et al, 2018;Hurt et al, 2021) in different cell types, such as E. coli and Salmonella typhimurium (Bourdeau et al, 2018). The engineered bacteria with ARGs were also tested deep inside a mammalian host within the tumors (Hurt et al, 2021) and gastrointestinal tract (Bourdeau et al, 2018). ARGs thus present maturing diagnostic technologies for real-time reporting of gut-brain axis dysregulation.…”

Ultrastructure of the Gas Vesicle Protein Shell

“…Production of GVs reduces cytoplasm density, allowing cell flotation, and consequently maintaining optimal access to light and nutrients. The unique physical properties of GVs allowed to utilize them as genetically encodable ultrasound and MRI contrast agents, enabling deep tissue imaging of cells such as microbes and tumors with high spatial and temporal resolution [2][3][4]. Although bioengineering of GVs is rapidly progressing, it is hindered by our limited knowledge about their basic structure and assembly.…”