ACOUSTIC BIOSENSORS FOR ULTRASOUND IMAGING OF ENZYME ACTIVITY
3.3 Results
3.3.5 Ultrasound imaging of intracellular ClpXP activity in vivo
= 5 biological replicates for (f). Individual dots represent each N and solid line represents the mean of all the replicates for a, c, d, e and f.
A major application of dynamic sensors in cells is to monitor the activity of natural or synthetic gene circuits38-40. To test if our acoustic sensors could be used to track the output of a synthetic gene circuit in cells, we co-transformed WT Nissle cells with ASGClpXP, and a separate wild-type gvpC gene controlled by anhydrotetracycline (aTc) (Fig. 3.4e). Our hypothesis was that induction of this gene circuit only with IPTG would result in the production of GVs with ClpXP-degradable GvpC, resulting in nonlinear contrast, whereas the additional input of aTc would result in the co-production of non-degradable wild-type GvpC, which would take the place of any degraded engineered GvpC on the biosensor shell and lead to reduced nonlinear scattering (Fig. 3.4e). Indeed, when we induced cells with just IPTG we observed strong nonlinear contrast. However, when aTc was added to the cultures after IPTG induction, this contrast was reduced by approximately 10 dB (Fig. 3.4f-g and Extended Data Fig. 3.5). These results, together with our findings in DclpXP cells with inducible ClpXP, show that acoustic biosensors can be used to visualize the output of synthetic gene circuits.
To demonstrate the ability of acoustic biosensors to produce nonlinear ultrasound contrast within the in vivo context of the mouse GI tract, we first co-injected WT Nissle cells expressing ASGClpXP and ARGWT into the mouse colon (schematic shown in Extended Data Fig. 3.6), distributing one cell population along the lumen wall and the other in the lumen center. In these proof-of-concept experiments, the cells are introduced into the colon in a rectally-injected agarose hydrogel to enable precise positioning and control over composition. Using nonlinear ultrasound imaging, we could clearly visualize the unique contrast generated by the protease-sensitive ASGs as a bright ring of contrast lining the colon periphery (Fig. 3.5a).
When the spatial arrangement was reversed, the bright nonlinear contrast was concentrated in the middle of the lumen (Extended Data Fig. 3.7). A comparison of ultrasound images acquired before and after acoustic collapse of the GVs, using a high-pressure pulse from the transducer, confirmed that the bright ring of nonlinear contrast was emanating from ASGClpXP -expressingcells (Fig. 3.5a), and this result was consistent across independent experiments in 9 mice (Fig. 3.5b).
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Figure 3.5: Ultrasound imaging of bacteria expressing acoustic sensor genes in the gastrointestinal tract of mice. (a) Transverse ultrasound image of a mouse whose colon contains WT Nissle cells expressing ARGWT at the center of the lumen and the same strain expressing ASGClpXP at the periphery of the lumen. These imaging experiments were independently repeated 9 times with similar results. (b) B-mode and xAM contrast-to-noise ratio (CNR) in vivo, for WT Nissle cells expressing ARGWT or ASGClpXP. N = 9 mice. P = 7.8E-5 for x-AM signal from cells expressing ASGClpXP versus the ARGWT control and P = 0.2890 for B-mode signal. (c) Transverse ultrasound image of a mouse whose colon contains DclpXP Nissle cells expressing ASGClpXP with L-ara induction of ClpXP protease expression at the center and without L-ara induction at the periphery of the lumen. These imaging experiments were independently repeated 7 times with similar results. Cells were injected in agarose gel at a final concentration of 1.5E9 cells ml-1 for a and c. Nonlinear (x-AM) images of the colon, acquired at 1.27 MPa for (a) and 1.56 MPa for (c) before and after acoustic collapse (hot color map), are superimposed on linear (B-mode) anatomical images (bone colormap). Color bars represent relative ultrasound signal intensity on the dB scale. Scale bars represent 2 mm for a and c. (d) B-mode and xAM CNR in vivo, for DclpXP Nissle cells expressing ASGClpXP with or without L-ara induction of ClpXP expression. N = 7 mice. P = 1.8E-5 for x-AM signal from cells expressing ASGClpXP with ClpXP protease expression induced versus non- induced and P = 0.8293 for B-mode signal. Individual dots represent each N, and the thick horizontal line indicates the mean. Error bars indicate SEM. P-values were calculated using a two-tailed paired t-test.
To demonstrate in vivo imaging of enzyme activity, we introduced DclpXP Nissle cells expressing ASGClpXP into the mouse colon, with and without transcriptionally activating intracellular ClpXP (schematic shown in Extended Data Fig. 3.6) . As above, the cells were contained in an agarose hydrogel. Cells induced to express this enzyme showed enhanced nonlinear contrast compared to cells not expressing ClpXP (Fig. 3.5c). Acoustic collapse confirmed the acoustic biosensors as the primary source of nonlinear signal (Fig. 3.5c). This performance was consistent across 7 mice and 2 spatial arrangements of the cells (Fig. 3.5d). These results demonstrate the ability of acoustic biosensors to visualize enzyme activity within the context of in vivo imaging.
Extended Data Figure 3.7 | Ultrasound imaging of bacteria expressing acoustic sensor genes in the
gastrointestinal tract of mice. (a) Schematic illustrating two orientations of the wild type (WT) E. coli Nissle cells expressing ARGWT or ASGClpXP introduced into the mouse colon as a hydrogel. (b, c) Representative transverse ultrasound images of the colon for two mice used in the in vivo imaging experiments, with orientation #1 (b) and with orientation #2. (c). Cells are injected at a final concentration of 1.5E9 cells ml-1. B-mode signal is displayed using the bone colormap and x-AM signal is shown using the hot colormap. Color bars represent B-mode and x-AM ultrasound signal intensity in the dB scale. Scale bars represent 2 mm. (d, e) B-mode and xAM contrast-to-noise ratio (CNR) in vivo, for WT Nissle cells expressing ARGWT or ASGClpXP in orientation #1 (d) and orientation #2. (e).
N = 5 mice for orientation #1 (b, d) and N = 4 mice for orientation #2 (c, e). Error bars indicate SEM. P = 0.0014 for x-AM signal from cells expressing ASGClpXP versus the ARGWT control in orientation #1, and P = 0.0016 for that in orientation #2. P = 0.0570 for B-mode signal in orientation #1 and P = 0.3445 in orientation #2. P-values were calculated using a two-tailed paired t-test. Individual dots represent each N and horizontal line indicates the mean.
Besides molecular sensing, one additional benefit of the nonlinear contrast generated by ASGClpXP
-expressingcells is to make the cells easier to detect relative to background tissue compared to linear B- mode imaging. Indeed, the nonlinear contrast of WT Nissle cells expressing ASGClpXP had a significantly higher contrast-to-tissue ratio than either the nonlinear contrast of ARGWT-expressing cells, or the B-mode contrast of either of these two species (Extended Data Fig. 3.8).
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Extended Data Figure 3.8 | ASGClpXP -expressingcells showed higher contrast to tissue with nonlinear imaging. B-mode and xAM contrast-to-tissue ratio (CTR) in vivo, for WT Nissle cells expressing ARGWT or ASGClpXP in both orientations. P = 7.8E-5 for the CTR from xAM imaging of cells expressing ASGClpXP versus CTR from xAM imaging of cells expressing ARGWT. P = 1.4E-6 for the CTR from xAM imaging of cells expressing ASGClpXP versus CTR from B-mode imaging of cells expressing ASGClpXP and P = 4.9E-7 for the CTR from xAM imaging of cells expressing ASGClpXP versus CTR from B-mode imaging of cells expressing ARGWT. Individual dots represent each N, and the thick horizontal line indicates the mean. Error bars indicate SEM. N = 9 mice. P-values were calculated using a two-tailed paired t-test for each comparison independently. Individual dots represent each N and horizontal line indicates the mean.