Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts

Abstract

The mammalian microbiome has many important roles in health and disease1,2, and genetic engineering is enabling the development of microbial therapeutics and diagnostics3,4,5,6,7. A key determinant of the activity of both natural and engineered microorganisms in vivo is their location within the host organism8,9. However, existing methods for imaging cellular location and function, primarily based on optical reporter genes, have limited deep tissue performance owing to light scattering or require radioactive tracers10,11,12. Here we introduce acoustic reporter genes, which are genetic constructs that allow bacterial gene expression to be visualized in vivo using ultrasound, a widely available inexpensive technique with deep tissue penetration and high spatial resolution13,14,15. These constructs are based on gas vesicles, a unique class of gas-filled protein nanostructures that are expressed primarily in water-dwelling photosynthetic organisms as a means to regulate buoyancy16,17. Heterologous expression of engineered gene clusters encoding gas vesicles allows Escherichia coli and Salmonella typhimurium to be imaged noninvasively at volumetric densities below 0.01% with a resolution of less than 100 μm. We demonstrate the imaging of engineered cells in vivo in proof-of-concept models of gastrointestinal and tumour localization, and develop acoustically distinct reporters that enable multiplexed imaging of cellular populations. This technology equips microbial cells with a means to be visualized deep inside mammalian hosts, facilitating the study of the mammalian microbiome and the development of diagnostic and therapeutic cellular agents.

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Figure 1: Genetic engineering of acoustic reporter genes.
Figure 2: Imaging dilute bacterial populations and dynamically regulated gene expression.
Figure 3: Multiplexed imaging of genetically engineered reporter variants.
Figure 4: Ultrasound imaging of bacteria in the gastrointestinal tract.
Figure 5: High-throughput screening of acoustic phenotypes.

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Acknowledgements

We thank F. S. Foster, D. Maresca, A. Mukherjee, M. Din, T. Danino, J. Willmann and S. K. Mazmanian for discussions, and A. McDowall for assistance with electron microscopy. This research was supported by the National Institutes of Health grant R01-EB018975, the Canadian Institute of Health Research grant MOP 136842 and the Pew Scholarship in the Biomedical Sciences. A.L. is supported by the NSF graduate research fellowship (award 1144469) and the Biotechnology Leaders Program. A.F. is supported by the NSERC graduate fellowship. S.P.N. was supported by the Caltech Summer Undergraduate Research Fellowship. Research in the Shapiro laboratory is also supported by the Heritage Medical Research Institute, the Burroughs Wellcome Career Award at the Scientific Interface and the David and Lucile Packard Fellowship for Science and Engineering.

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Contributions

R.W.B. and M.G.S. conceived and designed the study. R.W.B., A.L., A.L.-G., A.F. and S.P.N. prepared genetic constructs in E. coli. R.W.B., A.L., A.L.-G., S.P.N. and A.F. conducted in vitro ultrasound experiments. A.L.-G. and R.W.B. performed in vivo ultrasound experiments. A.L., A.F. and A.L.-G. conducted metabolic burden experiments in Nissle 1917 cells. R.W.B. and S.R.K. prepared genetic constructs in S. typhimurium. R.W.B. and A.L. obtained TEM images. R.W.B., A.L.-G. and M.G.S. analysed ultrasound data. R.W.B. and M.G.S. wrote the manuscript with input from all authors. M.G.S. supervised the research.

Corresponding author

Correspondence to Mikhail G. Shapiro.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks C. Caskey, O. Couture, P. Silver and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Sequence homology of GvpA/B.

Amino acid sequence alignment of the primary gas vesicle structural protein GvpB from B. megaterium (the GvpA analogue in this species) and GvpA from A. flos-aquae.

Extended Data Figure 2 Ultrasound contrast from buoyancy-enriched cells.

a, Diagram of centrifugation-assisted enrichment of buoyant cells. b, Image of arg1 E. coli culture 22 h after induction and 4 h of centrifugation at 350g, showing the presence of buoyant cells. Arrowhead points to the meniscus layer containing buoyant cells. Experiment repeated three times with similar results. c, Ultrasound images of E. coli expressing arg1 at various cellular concentrations, with and without buoyancy enrichment. Experiment was repeated three times with similar results. d, Ultrasound contrast from E. coli expressing arg1, with and without buoyancy enrichment, and GFP at various cell densities. Data are from three biological replicates; lines represent the mean.

Extended Data Figure 3 Time course of acoustic reporter gene contrast after induction.

a, Ultrasound images of arg1-expressing E. coli at various times after induction with IPTG. Experiment repeated four times with similar results. b, Ultrasound contrast at each time point. Data are from four biological replicates; line represents the mean. Cell concentration, 5 × 108 cells ml−1. Scale bar, 2 mm.

Extended Data Figure 4 Acoustic reporter gene expression and ultrasound imaging does not affect cell viability.

a, Growth curves of E. coli containing the arg1 or GFP expression plasmid, with or without induction using 0.4 mM IPTG. Data are from three biological replicates per sample; lines represent the mean. b, Representative TEM images of whole E. coli cells expressing arg1 with and without exposure to acoustic collapse pulses, and E. coli cells expressing GFP. Images were acquired from three biologically independent samples for arg1, two for arg1 with ultrasound collapse and one for GFP (more than 50 cells imaged per sample) with similar results. c, Dark-field optical image of agar plate containing colonies of E. coli expressing arg1 14 h after seeding. d, Image of the same plate after the right half of the plate was insonated with high-pressure ultrasound. e, Image of the same plate 20 h after insonation. f, Image after the right half of the plate in e was insonated with high-pressure ultrasound. Zoomed in images of representative colonies shown below each plate image. Scale bars, 500 nm. Experiment was repeated three times with similar results.

Extended Data Figure 5 Multiplexed imaging of genetically engineered reporter variants.

a, Image of arg2 E. coli culture 22 h after induction showing the presence of buoyant cells (top). Experiment repeated three times with similar results. Mass fraction of gas vesicles produced 22 h after induction (bottom). Line represents the mean. b, Ultrasound contrast from the whole population of cells expressing arg1, arg2 or GFP. Lines represent the mean. c, Ultrasound contrast from the buoyancy-enriched population of cells expressing arg1, arg2 or GFP. Lines represent the mean. d, Normalized optical density (representing the intact fraction) of gas vesicles isolated from E. coli expressing arg1 or arg2 as a function of applied hydrostatic pressure. e, Normalized ultrasound intensity as a function of peak positive pressure from 0.6 to 4.7 MPa for E. coli expressing arg1 or arg2. f, Acoustic collapse spectra derived by differentiating the data and curves in e with respect to applied pressure. af, Data are from three biological replicates per sample. df, Curves represent fits of the data using the Boltzmann sigmoid function to assist visualization.

Extended Data Figure 6 Anatomical ultrasound images of acoustic bacteria in the gastrointestinal tract.

Raw images underlying the difference maps shown in Fig. 4e, g. The cyan outline identifies the colon region of interest for difference processing. This experiment was repeated three times with similar results.

Extended Data Figure 7 Ultrasound imaging of ARG-expressing cells in the mouse colon.

a, Transverse ultrasound images of mice whose colon contains BL21 E. coli expressing either arg2 or GFP at a final concentration of 109 cells ml−1. A difference heat map of ultrasound contrast within the colon region of interest before and after acoustic collapse is overlaid on a grayscale anatomical image. b, Signal intensity in mice with E. coli expressing either arg2 or GFP. Data are from 5 biological replicates per sample. P value = 0.02 using two-sided heteroscedastic t-test. Scale bar, 2 mm. Source data

Extended Data Figure 8 Effect of arg1 and lux expression on ECN cell growth, viability and microcin release.

a, Optical density at 600 nm measured from 0 to 22 h after induction with 3 μM IPTG, or without induction, in ECN cells transformed with arg1 or lux. Data are from four biological replicates per time point, lines represent the mean. For comparisons between induced arg1 and induced lux values at 22 h P = 0.12. For comparisons between uninduced arg1 and uninduced lux at 22 h P = 0.04. For comparisons at all other time points P > 0.14. b, Colony-forming units (cfu) per millilitre culture per OD600nm after 22 h of induction with 3 μM IPTG, or uninduced growth, of ECN cells transformed with arg1 or lux. P ≥ 0.22. Data are from 7 biological replicates for arg1 samples and four biological replicates for lux samples. Lines represent the mean. c, Fraction of opaque, gas vesicle-producing colonies produced by plating arg1-transformed ECN cells 22 h after induction with 3 μM IPTG, or uninduced growth. Cells were plated on dual-layer IPTG induction plates, allowed to grow overnight at 30 °C, and imaged as in (Extended Data Fig. 4c–f, P = 0.12. data are from seven biological replicates, lines represent the mean. d, Microcin release assay using a uniform layer of the indicator strain E. coli K12 H5316 in soft agar, after 17-h incubation with filters containing microcin sources and controls, as indicated. ECN cells transformed with arg1 or lux were induced for 22 h with 3 μM IPTG, or grown without induction, before spotting. H5316* indicates H5316 cells transformed with mWasabi and cultured for 22 h as with ECN cells. All cells were washed before spotting to remove antibiotic. Experiment was performed four times with similar results. Amp, 100 mg ml−1 ampicillin; LB, LB medium. e, As in d, but with the indicator strain comprising H5316* cells and the agar containing 50 μg ml−1 kanamycin, 3 μM IPTG and 50 μM desferal, to show that microcin release also occurs during transgene expression. Note that the H5316* spot appears bright because the plate image is acquired with blue-light transillumination, resulting in mWasabi fluorescence. Experiment was performed four times with similar results. All P values were calculated using a two-sided heteroscedastic t-test.

Extended Data Figure 9 Ultrasound imaging of S. typhimurium in tumour xenografts.

a, Diagram of tumour imaging experiment. S. typhimurium expressing arg1 were introduced into the tumours of mice and imaged with ultrasound. b, Ultrasound images of a gel phantom containing S. typhimurium expressing arg1 or the lux operon. Cell concentration is 109 cells ml−1. Experiment repeated three times with similar results. c, TEM images of whole S. typhimurium cells expressing arg1 with and without exposure to acoustic collapse pulses. At least 20 cellular images were acquired for each sample type (from one biological preparation each) with similar results. d, Ultrasound images of mouse OVCAR8 tumours injected with 50 μl of 3.2 × 109 cells ml−1 arg1-expressing S. typhimurium, before and after acoustic collapse. Experiment repeated five times with similar results. e, Collapse-sensitive ultrasound contrast in tumours injected with arg1-expressing or lux-expressing cells. Data are from five animals, line represents the mean. P = 0.002 using a two-sided heteroscedastic t-test. Scale bars, 2 mm (b), 500 nm (c) and 2.5 mm (d). Source data

Extended Data Figure 10 High-throughput screening of acoustic phenotypes.

a, Ultrasound intensity histogram of 22 randomly picked colonies. Colonies with low contrast were predicted to contain the gene encoding GFP and those with high contrast to contain genes encoding arg1 or arg2 genes. b, Normalized change in ultrasound intensity (U) for each of the 15 arg1 or arg2 colonies after insonation at increasing pressures. At 4 MPa, colonies with signal above the indicated threshold were predicted to be arg1 and below to be arg2. This experiment was performed once; each colony was treated as a biological replicate.

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This file contains Supplementary Notes 1–5, Plasmid Sequences for ARG1 and ARG2 constructs, Supplementary References and Supplementary Table 1. (PDF 398 kb)

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Bourdeau, R., Lee-Gosselin, A., Lakshmanan, A. et al. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature 553, 86–90 (2018). https://doi.org/10.1038/nature25021

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