The percent change in cell number was calculated by normalizing day 7 to day 5 image values per each fish. ?;2a2aCe; ?;3b3bCd, ?,g,g, and ?andi;i; ?;4a4aCb; ?;5a5aCc, ?,e,e, and ?andg;g; ?;6b6b and ?andd;d; Extended Data Figs. 2b and ?andd.d. All additional source data files generated during and/or analysed during the current study are available from the corresponding authors upon request. Abstract Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell ablation NH2-C2-NH-Boc 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 a 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 NH2-C2-NH-Boc 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. Editors Summary An engineered bacterial nitroreductase, NTR 2.0, improves chemically induced cell ablation, facilitating novel sustained ablation paradigms for testing the effects of chronic inflammation on regeneration, and modeling degenerative disease. INTRODUCTION: Bacterial nitroreductases (NTRs) are promiscuous enzymes capable of prodrug conversion via reduction of nitro substituents on aromatic rings1C4. This generates genotoxic products that rapidly kill the host cell, a mechanism exploited by anti-cancer and antibiotic prodrugs5. When expressed heterologously, NTRs sensitize vertebrate cells to such prodrugs6. The canonical NTR, NfsB (NfsB_Ec, NTR 1.0), has been widely tested in combination with the anti-cancer prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) as an enzyme-prodrug therapy for killing tumors1. Transgenic expression of NTR 1.0 in combination with CB1954 was previously advanced as a targeted cell ablation strategy for interrogating cell function in vertebrates7,8. However, CB1954 produces cell-permeable cytotoxins that kill nearby non-targeted cells, i.e., bystander cell death9, compromising its use for selective cell ablation. In contrast, the prodrug metronidazole (MTZ) ablates NTR-expressing cells without discernible bystander effects9. Importantly, fusion proteins between NTR and fluorescent reporters retain MTZ-inducible cell-specific ablation activity10. We therefore adapted the NTR/MTZ ablation system to zebrafish11 to expand studies of cellular regeneration12, reasoning that co-expression of NTR with reporters would enable visualization13,14 and quantification14C17 of MTZ-induced cell loss, and subsequent cell replacement, (NfsB_Vv F70A/F108Y, NTR 2.0) which improves MTZ-meditated cell ablation efficiency ~100-fold; i.e., robust ablation at 100 M MTZ versus typical 10 mM treatments. Additional data show that NTR 2.0 will expand the functionality of the NTR/MTZ system by allowing: 1) sustained interrogations of cell function, 2) effective ablation of resistant cell types, 3) prolonged cell loss, as novel tests of EFNB2 regenerative capacity, and 4) modeling of degenerative diseases caused by chronic cell loss. Accordingly, we have NH2-C2-NH-Boc created NH2-C2-NH-Boc a series of bipartite expression vectors and transgenic zebrafish lines co-expressing NTR 2. 0 and fluorescent reporters as versatile new toolsets for the research community. RESULTS Rational improvement of MTZ-activating NTR variants We previously compiled an gene library of 11 orthologs and used a DNA damage screen in host cells to monitor activation of SN33623, a PET imaging probe that shares a 5-nitroimidazole core structure with MTZ23. This same library was used here to evaluate ablation efficacy at higher SN33623 doses; relative growth of replicate cultures was assessed across a dilution series to establish EC50 values. Consistent with the previous DNA damage screen, the six most closely related orthologs of NTR 1.0 (NfsB_Ec-like, 50% amino acid identity with NfsB) were far less effective NH2-C2-NH-Boc at activating SN33623 than the other five enzymes in the panel (Fig. 1a,?,b).b). MTZ activation followed the same trend, with one notable exception: the ortholog (NfsB_Vv), despite being NfsB_Ec-like, was one of the most active MTZ-converting enzymes (Fig. 1c). Open in a separate window Fig. 1: Rationally engineered NfsB-family NTRs for improved activation of MTZ.a, Amino acid sequence identity cladogram of eleven NfsB orthologs, grouped according to percent shared amino acid identity with NfsB_Ec. The asterisk (*) marks where other NTR variants diverge from NfsB_Ec-like enzymes. b, host sensitivity conferred by NfsB variants to the compound SN33623, n=3 biologically independent experiments for all strains except those expressing NfsB_Pp, NfsB_Cs, FraseI_Vf, NfsB_Vh, YfkO_Bs and YdgI_Bs (n=4). c, host sensitivity conferred by NfsB variants to the compound MTZ, n=4 biologically.