By Raymond W. Bourdeau and Mikhail G. Shapiro, ShapiroLab @ Caltech
Behind the paper: Bourdeau RW, Lee-Gosselin A, Lakshmanan A, Farhadi A, Ravindra Kumar S, Nety SP, Shapiro MG. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature 553, 86–90 (2018). article | news and views
In the Shapiro lab at Caltech, in one corner of the brainstorming room, below the whiteboard, sits a framed Nobel poster from 2008 commemorating the discovery and development of the green fluorescent protein. This poster tells the story of how the gene for GFP was cloned from jellyfish and introduced into various cell types to visualize gene expression using light. For most biologists, GFP, or some analog thereof, has been an irreplaceable tool for life science research.
When a small group of us started the lab back in 2014 we hoped to create the next generation of tools that scientists could use to understand, diagnose, and treat human disease. These tools had to address one big limitation of fluorescent proteins: humans are opaque. Because light gets scattered as it moves through living biological tissue, imaging organisms thicker than a millimeter is very challenging. We therefore decided to focus our efforts on forms of energy that can more easily penetrate the human body, including ultrasound.
Ultrasound is one of the most widely used technologies in medical imaging. It is low cost, non-invasive, non-ionizing and portable. Could we create a “GFP” for ultrasound? In 2014 we published our discovery that a unique class of hollow protein nanostructures from buoyant cyanobacteria and haloarchaea, called gas vesicles, could scatter sound waves and therefore serve as ultrasound contrast agents1. Now, we wanted to show that the complex genetic machinery allowing gas vesicles to form in these organisms could be transferred to other species and make them visible to ultrasound. As our initial targets, we picked commensal and therapeutic microbes, including Escherischia coli Nissle 1917 and Salmonella typhimurium, which synthetic biologists are developing as engineered probiotics for diseases ranging from inflammatory bowel disease to cancer.
This project was initially a failure. Expressing the relevant genes from the cyanobacteria used in our 2014 paper yielded no gas vesicles, while a gene cluster from Bacillus megaterium, a closer genetic relative of E. coli, filled the cells with small nanostructures and no ultrasound contrast. The critical breakthrough came from our synthetic biology efforts to mix and match gas vesicle genes from different organisms. In the end, it turned out that co-expressing the two main structural genes from Anabaena flos-aquae with eight chaperone and assembly factor genes from B. megaterium produced gas vesicles that made E. coli “visible” under ultrasound. We named this construct arg1 as the first acoustic reporter gene.
Developing a reporter gene for this new modality required work across many disciplines including Synthetic Biology, Biochemistry, Engineering and Physics. We were fortunate to have assembled a team in our lab with expertise in all these areas (something uniquely fostered at Caltech). Our co-authors, Anupama Lakshmanan, Arash Farhadi, and Suchita Nety performed extensive genetic and biochemical work to understand the structure-function relationships in gas vesicles2. Audrey Lee-Gosselin developed in the in vivo proof-of-concept models that ultimately showed that we can image arg1-expressing microorganisms deep within biological tissue. And Priya Kumar helped with some initial expression experiments in S. typhimurium.
We also benefited from the expertise of fellow Shapiro Lab members Dan Piraner and Mohamad Abedi in the latest synthetic and molecular biology techniques, which they used to develop bacteria that can be controlled with ultrasound3, complementing our work on bacterial imaging. David Maresca and Nikita Reznik assisted with the development of ultrasound imaging protocols optimized for gas vesicles based on their knowledge of ultrasound physics4. And we benefited greatly from input provide by our collaborators in Stuart Foster’s lab at the University of Toronto.
Meanwhile, the GFP poster in our brainstorming room continues to inspire our work, pushing us to develop acoustic reporter genes as a truly valuable tool for researchers aiming to study and improve human health. Just as GFP spawned a fruitful rainbow of technologies (mCherry, mOrange, etc), we hope that our lab and others will innovate on our initial acoustic reporter genes to produce an equally colorful, and useful, set of genetic tools.
1. Shapiro, M.G. et al. Biogenic gas nanostructures as ultrasonic molecular reporters. Nat. Nanotechnol. 9, 311-316 (2014).
2. Lakshmanan, A. et al. Molecular Engineering of Acoustic Protein Nanostructures. ACS Nano 10, 7314–7322 (2016).
3. Piraner, D.I., Abedi, M.H., Moser, B.A., Lee-Gosselin, A. & Shapiro, M.G. Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nature Chem Biol 13, 75-80 (2017).
4. Maresca, D. et al. Nonlinear Ultrasound Imaging of Nanoscale Acoustic Biomolecules. Appl. Phys. Lett. 110 (2017).