(H-304) Non-invasive imaging of gut microbes with reversibly switchable photoacoustic protein

Introduction::

Imaging serves as an effective method for exploring the colonization and spatial distribution of gut microorganisms. While genetic markers such as fluorescent proteins and luciferases are commonly used, their limited depth penetration constrains their use in in vivo gut imaging. Photoacoustic imaging, possessing both the deep penetration capabilities of ultrasound imaging and the high resolution of optical imaging, presents itself as a potential tool for investigating gut microbiota in animals. Widely utilized photoacoustic contrast agents include metal nanoparticles that absorb near-infrared light and organic dyes. However, due to the challenging cultivation of various gut microorganisms and the poor biocompatibility of photoacoustic imaging agents, marking gut microorganisms with exogenous contrast agents poses a significant challenge.

Bacterial Phytochrome (BphP), a class of biliverdin-binding proteins that absorb near-infrared light, hold potential as genetic labels for photoacoustic imaging. A category of BphP exhibits a photoswitch effect, which can be employed to suppress the photoacoustic background signals within the tissue. Here, we have engineered gut bacteria to express the light-switch ReBphP protein and conducted photoacoustic imaging. Our in vivo imaging results demonstrate that the photoswitchable BphP can serve as a tool for marking gut bacteria for photoacoustic imaging.

Materials and Methods::

We expressed the ReBphP and heme oxygenase (HO-1) genes in Escherichia coliBL21(DE3) and Bacteroides thetaiotaomicron. HO-1 is utilized to convert heme to biliverdin within the bacteria. The bacteria were cultured either in the presence of air or under anaerobic conditions.

We performed photoacoustic imaging with these engineered bacteria placed either within agar phantoms or within mice. We used the PHOCUS MOBILE system and a near-infrared laser (from OPOTEK), with the Verasonics ultrasound imaging acquisition system for this process.

Results, Conclusions, and Discussions::

ReBphP can be normally expressed in E. coli cultured in air. The bacteria expressing ReBphP show near-infrared absorption peaks and green features. E. coli expressing ReBphP can produce photoacoustic signals, and these bacteria can be located within agar phantoms as well as within mice. The ReBphP protein possesses a light-switch effect. Under laser irradiation at 780 nm, its photoacoustic signal decays within a second. However, after exposure to 700 nm laser light, the 780 nm photoacoustic signal can be restored. This light-switching can be repeated multiple times. As the photoacoustic signals from animal skin, tissue, blood, and other background sources lack decay, simple signal processing can be used to exclude these background signals.

The E. coli and B. theta cultured in an anaerobic environment did not exhibit photoacoustic signals or near-infrared absorption peaks. This may be due to the fact that the maturation of ReBphP relies on BV, which requires oxygen to be produced. This might limit the long-term imaging of ReBphP in the oxygen-deprived gut. Going forward, we will focus on studying whether ReBphP can mature by relying on the oxygen in the intestinal wall mucus, or use ReBphP-engineered bacteria to detect abnormal increases in oxygen content in the gut.

Acknowledgements (Optional): :

References (Optional): : Gao, R., Liu, F., Liu, W., Zeng, S., Chen, J., Gao, R., ... & Liu, C. (2022). Background-suppressed tumor-targeted photoacoustic imaging using bacterial carriers. Proceedings of the National Academy of Sciences, 119(8), e2121982119.

Mishra, K., Stankevych, M., Fuenzalida-Werner, J. P., Grassmann, S., Gujrati, V., Huang, Y., ... & Stiel, A. C. (2020). Multiplexed whole-animal imaging with reversibly switchable optoacoustic proteins. Science Advances, 6(24), eaaz6293.