Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.
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The ARQiv software package is available on the GitHub open-source website (https://github.com/mummlab/ARQiv). All other freely available R code functions used are as listed in the Statistics section.
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This work was supported by the following grants from the National Institutes of Health, R01OD020376 (J.S.M. and D.F.A.), P30EY001765 (J.S.M., Animal Module and Imaging Cores), R01NS095355 (A.D.L. and A.L.C.), and R01HG009518 (H.J.). Additional support was provided by a Royal Society of New Zealand Marsden grant (contract VUW1902; D.F.A.) and additional consumables support was provided by an HRC Explorer grant (contract 19/750 from the Health Research Council of New Zealand; D.F.A. and J.S.M.). This study was also supported by an unrestricted departmental grant to the Wilmer Eye Institute from Research to Prevent Blindness. The authors wish to thank M. Williams, B. Bich, N. Lu, and G. Lee for technical assistance and J. Fadool and M. Halpern for providing antibodies and a QUAS reporter/effector plasmid, respectively.
J.S.M. has been awarded patents for the creation (US patent no. 7,514,595) and use of zebrafish expressing nitroreductase enzymes for gene (US patent no. 8,071,838) and drug discovery (US patent no. 8,431,768) applications. M.T.S. is the President and Scientific Director at Luminomics, a biotechnology start-up that offers ARQiv-based screening services. M.T.S. owns stock in Luminomics and J.S.M. serves as a consultant at Luminomics. The remaining authors declare no competing interests.
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Merged GFP (green), mCherry (magenta), and brightfield image of the MTZ-treated 60:40 co-culture shown in Fig. 2, n = 3 biologically independent experiments. Zoomed brightfield and mCherry images of the boxed region show the remaining mCherry fluorescence corresponds to small, round, phase-bright material suggestive of dead or dying cells adhering to healthy GFP-expressing cells. Scale bar = 100 microns.
a-d, Transgenic zebrafish larvae co-expressing YFP and NTR 2.0 in rod photoreceptors were treated ±400 μM MTZ for 24 h (5-6 dpf). Retinas were then fixed at 7 dpf, sectioned and labeled with the nuclear stain DAPI (blue cells) and an α-rhodopsin antibody (α-rho, also known as 1D1) specific to rods (red cells, a), or an α-arrestin 3a antibody (α-arr3a, also known as zpr-1) specific to cones (red cells, c). Representative confocal images of YFP and antibody labeling show effective ablation of NTR 2.0-expressing rod photoreceptors (a, n = 10 retinas imaged per condition) and maintenance of neighboring cone photoreceptors (c, n = 15 and 14 retinas imaged for the 0 and 400 μM MTZ conditions, respectively). Manual quantification of cell numbers confirmed MTZ-induced loss of rod cells (b) and maintenance of neighboring cones (d). Violin plots show first quartiles (25th percentile), medians, third quartiles (75th percentile), and the full distribution of the data, with individual data points (number of measurements per condition) overlaid as a dot plot. A two-tailed nested t test (GraphPad, Prism 9) was used to calculate p-values comparing MTZ-treated and control larvae. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50 microns.
a-f, Transgenic adult zebrafish larvae co-expressing YFP and NTR 2.0 in rod photoreceptors were treated ±1 mM MTZ for 3 (a,b) or 7 days (c-f). Retinas were then fixed, sectioned and labeled with the nuclear stain DAPI (blue cells) and antibodies specific to rods, 4C12 (a and e) or α-rho (c). Representative confocal images of YFP (yellow cells) and antibody labeling (red cells) show effective MTZ-induced ablation of NTR 2.0/YFP-expressing rod photoreceptors and concomitant loss of rod-specific antibody labeling (a, c, and e, n = 2 retinas imaged per condition). Manual quantification of cell numbers confirmed MTZ-induced loss of rod cells (b, d, and f. Violin plots show first quartiles (25th percentile), medians, third quartiles (75th percentile), and the full distribution of the data, with individual data points (number of measurements per condition) overlaid as a dot plot). A two-tailed nested t test (GraphPad, Prism 9) was used to calculate p-values comparing MTZ-treated and control larvae. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50 microns.
a, Transgenic zebrafish larvae co-expressing YFP and NTR 2.0 in neurons (NRSE:KalTA4;UAS:YFP-NTR2.0) were exposed to the indicated concentrations of NFP for 24 h (5- 6 dpf) and YFP levels were quantified by plate reader at 7 dpf (n = 3 biologically independent experiments, dot plots show the number of larvae examined). Box plots show first quartiles (25th percentile), medians, third quartiles (75th percentile), and whiskers = SD, with individual data points (larvae) overlaid as a dot plot. Fully detailed statistical comparisons (absolute effect sizes, 95% confidence intervals, Bonferroni-corrected p′-values derived from two-tailed t tests, and sample sizes) between NFP-treated and control conditions in graphs b and d are provided in Supplementary Table 5.Symbols: #p′ > 0.05, *p′ ≤ 0.05, ****p′ ≤ 0.0001; ○ = outlier data points.
Guide, Supplementary Methods, Primer Table, Transgenic Line Table.
Supplementary Table 1 Statistical analysis of Fig. 3. Experimental conditions were compared to a signal window established by normalizing all individual sample values to the averages of the non-ablated 0 MTZ control (set at 100%) and Non-Tg control (set at 0%). Standard R code functions were used to calculate absolute effect sizes, 95% confidence intervals, and Bonferroni-corrected P values (P′ values) derived from two-tailed t tests (see Methods for details). The last row (N) shows the number of larvae analyzed per condition. The number of biologically independent experiments were as follows. 24 h MTZ treatments: 0 µM (n = 6), 12.5 μM (n = 2), 25 μM (n = 4), 50 μM (n = 4), 100 μM (n = 6), 200 μM (n = 2), Non-Tg (n = 6). 48 h MTZ treatments: 0 µM (n = 3), 12.5 μM (n = 2), 25 μM (n = 3), 50 μM (n = 3), 100 μM (n = 3), 200 μM (n = 2), Non-Tg (n = 3). 2 h MTZ treatments: 0 mM (n = 8), 0.5 mM (n = 5), 1 mM (n = 7), 10 mM (n = 7), Non-Tg (n = 8). 24 h MTZ treatments: 0 mM (n = 8), 0.5 mM (n = 4), 1 mM (n = 4), 10 mM (n = 4), Non-Tg (n = 8).
Supplementary Table 2 Statistical analysis of Fig. 4. Experimental conditions were compared to a signal window established by normalizing all individual sample values to the averages of the non-ablated 0 MTZ control (set at 100%) and Non-Tg control (set at 0%). Standard R code functions were used to calculate absolute effect sizes, 95% confidence intervals, and Bonferroni-corrected P values (P′ values) derived from two-tailed t tests (see Methods for details). The last row (N) shows the number of larvae analyzed per condition, n = 4 biologically independent experiments per condition.
Supplementary Table 3 Statistical analysis of Fig. 5. Experimental conditions were compared to 0 mM MTZ control conditions. Data was subjected to ANOVA followed by Dunnett’s multiple comparisons test, depending on the outcome of Levene’s test. Standard R code functions were used to calculate absolute effect sizes, 95% confidence intervals, and Bonferroni-corrected P values (P′ values) derived from two-tailed t tests (see Methods for details). The last row (N) shows the number of successful matings analyzed per condition over n = 3 independent mating sessions.
Supplementary Table 4 Statistical analysis of Fig. 6. Experimental conditions were compared to 0 mM MTZ control conditions. Standard R code functions were used to calculate absolute effect sizes, 95% confidence intervals, and Bonferroni-corrected P values (P′ values) derived from two-tailed t tests (see Methods for details). The last row (N) shows the number of larval images quantified per condition, n = 3 biologically independent experiments.
Supplementary Table 5 Statistical analysis of Ext. Data Fig. 4. Experimental conditions were compared to 0 mM nifurpirinol and non-transgenic control conditions. Standard R code functions were used to calculate absolute effect sizes, 95% confidence intervals, and Bonferroni-corrected P values (P′ values) derived from two-tailed t tests (see Methods for details). The last row (N) shows the number of larval images quantified per condition, n = 3 biologically independent experiments.
DNA sequences of NTR variants and plasmids used or created for this study.
Data used to generate graphs in Fig. 1.
Data used to generate graphs in Fig. 2.
Data used to generate graphs in Fig. 3.
Data used to generate graphs in Fig. 4.
Data used to generate graphs in Fig. 5.
Data used to generate graphs in Fig. 6.
Data used to generate graphs in Extended Data Fig. 2.
Data used to generate graphs in Extended Data Fig. 3.
Data used to generate graphs in Extended Data Fig. 4.
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Sharrock, A.V., Mulligan, T.S., Hall, K.R. et al. NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation. Nat Methods 19, 205–215 (2022). https://doi.org/10.1038/s41592-021-01364-4