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Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation

Nature Biotechnology volume 35, pages 653–658 (2017)Cite this article

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Abstract

Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut but commercial translation of technologies to accomplish this has been hindered by the susceptibility of synthetic genetic circuits to mutation and unpredictable function during extended gut colonization. Here, we report stable, engineered bacterial strains that maintain their function for 6 months in the mouse gut. We engineered a commensal murine Escherichia coli strain to detect tetrathionate, which is produced during inflammation. Using our engineered diagnostic strain, which retains memory of exposure in the gut for analysis by fecal testing, we detected tetrathionate in both infection-induced and genetic mouse models of inflammation over 6 months. The synthetic genetic circuits in the engineered strain were genetically stable and functioned as intended over time. The durable performance of these strains confirms the potential of engineered bacteria as living diagnostics.

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Figure 1: Engineering a tetrathionate-responsive memory device in E. coli NGF-1.
Figure 2: Tetrathionate-sensing of inflammation in vivo.
Figure 3: PAS638 does not accrue mutations in synthetic elements over long periods.

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Acknowledgements

We thank A. Graveline and L. Bry for discussions and assistance with mouse experiments and A. Verdegaal for experimental assistance. S. typhimurium TT22470 was a gift from J. Roth. We thank Dana-Farber/Harvard Cancer Center in Boston for the use of the Rodent Histopathology Core, which provided histology preparation service. Dana-Farber/Harvard Cancer Center is supported in part by a NCI Cancer Center Support Grant # NIH 5 P30 CA06516. D.T.R. was supported by a Human Frontier Science Program Long-Term Fellowship and an NHMRC/RG Menzies Early Career Fellowship from the Menzies Foundation through the Australian National Health and Medical Research Council. T.W.G. was supported by a Leopoldina Research Fellowship (LPDS 2014-05) from the German National Academy of Sciences Leopoldina. The research was funded by Defense Advanced Research Projects Agency Grant HR0011-15-C-0094 (P.A.S.) and the Wyss Institute for Biologically Inspired Engineering.

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Author notes

  1. S Jordan Kerns & Jonathan W Kotula

    Present address: Present addresses: Emulate Inc., Boston, Massachusetts, USA (S.J.K.) and SynLogic, Cambridge, Massachusetts, USA (J.W.K.).,

Authors and Affiliations

  1. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    David T Riglar, Tobias W Giessen, Michael Baym, S Jordan Kerns, Matthew J Niederhuber, Jonathan W Kotula & Pamela A Silver

  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA

    David T Riglar, Tobias W Giessen, S Jordan Kerns, Matthew J Niederhuber, Jonathan W Kotula, Jeffrey C Way & Pamela A Silver

  3. Department of Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, USA

    Michael Baym

  4. Department of Microbiology and Immunology, Harvard Medical School, Boston, Massachusetts, USA

    Roderick T Bronson

  5. Department of Pathology, Massachusetts Host-Microbiome Center, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Georg K Gerber

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Contributions

D.T.R., T.W.G., M.B., S.J.K., G.K.G., J.C.W. and P.A.S. designed experiments. D.T.R. and M.J.N. performed and analyzed in vitro characterization. D.T.R. performed and analyzed all mouse experiments. T.W.G. performed and analyzed tetrathionate mass spectrometry. M.B. performed and analyzed whole genome sequencing. S.J.K. and J.W.K. generated strains and collected preliminary data for the study. R.T.B. performed all histology scoring. D.T.R. and P.A.S. wrote the manuscript.

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Correspondence to Pamela A Silver.

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Integrated supplementary information

Supplementary Figure 1 The inflammatory response in the mammalian gut leads to tetrathionate generation.

Cytokine signaling following an inflammatory insult leads to, among other responses, release of reactive oxygen species (ROS) into the gut lumen that inhibit microbial growth, and can oxidize thiosulfate, generated from hydrogen sulfide, to tetrathionate.

Supplementary Figure 2 PAS638 senses tetrathionate in a murine streptomycin-treated S. typhimurium colitis model

a) Timecourse of PAS638 switching during infection of C57Bl/6 mice with wt and ΔttrR S. typhimurium (S.tm) (n= 6 for control and 7 for S. typhimurium infected groups). Switching was only apparent in colonies from mice co-infected with S.typhimurium ΔttrR and on days 4 and/or 5 post administration. b) Enumeration of PAS638 E. coli and c) S. typhimurium variant levels by selective plating showed relatively consistent levels across experimental groups. d) LCN-2 quantification demonstrated inflammation in both S. typhimurium ΔttrR and wt administered mice. Graphs show individual mouse values and mean. * p=0.02, ** p=0.001 *** p<0.0001, F(2,17) = 25.96, using one way ANOVA with Tukey’s multiple comparisons test. e) Histology scoring of cecum, proximal and distal colon, showed inflammation in the presence of both S. typhimurium variants decreasingly apparent from the caecum to the distal colon. f) Scoring used a 0-4 point scale with example images of each score by histology provided. For cecal samples, only severity 3 and 4 were observed during S. typhimurium infection, characterized by accumulation of neutrophils (N) in epithelial tissues, edema (E), mucosal thickening and at times low-level presence of neutrophils in exudate at severity 3 and edema (E), extensive neutrophils present in exudate and/or epithelia (N), mucosal thickening and signs of crypt damage or regeneration (C) at severity 4. Proximal and distal colon showed signs of low-level inflammation (I) at severity 1, more extensive inflammation (I) and neutrophils in exudate (N) at severity 2. Severity 3 was noted in the proximal colon only and showed signs of ulceration (U), inflammatory cell migration (I), and neutrophils commonly present in exudate. Scale bars = 10μm.

Supplementary Figure 3 PAS638 senses tetrathionate in cybb−/−, IL10−/−, and 129X1/SvJ mouse models.

a) Weight of cybb-/- and C57Bl/6 control mice was measured following administration of PAS638 ± S. typhimurium (S.tm) ΔttrR. Graph shows percentage of pre-administration weight of individual mice (dotted) along with group averages (solid line). b) Elevated LCN-2 levels were apparent in S. typhimurium ΔttrR infected mice and cybb-/- uninfected controls. Graph shows maximum measurements from days 3-5 following bacterial administration. Means are marked. *p=0.01, **p=0.009, F(3,16) = 6.575, using one-way ANOVA analysis with Tukey’s multiple comparison correction. For all other comparisons p>0.99. c) LCN-2 levels were elevated in IL10-/- compared to control mice in the week following PAS638 administration therefore PAS638 measurements were taken >1-week following administration. Values are averages of 2-3 measurements from individual mice, with mean shown. *p=0.02, **p=0.01, F(2,20) = 6.48 using one-way ANOVA test with Tukey’s multiple comparison correction. >week 1 IL10-/- samples were not significantly increased over C57Bl/6 controls (p=0.9) d) A subset of IL10-/- mice administered PAS638 without streptomycin pre-treatment (n=10) showed elevated PAS638 switching. Graph shows maximum switching percentage from 4 measurements in the 12 days following administration. Mean is marked. e) When administered without streptomycin pre-treatment no indication of elevated LCN-2 levels was seen following bacterial administration. Graph shows pre-administration levels (1 measurement) and average levels from 4 measurements in the 12 days following administration. Means are marked. n.s. = non-significant (p = 0.6) using a two-tailed Mann-Whitney test. f). PAS638 administered to 129X1/SvJ mice (n=5) without streptomycin pre-treatment colonized the gut successfully. Graph shows CFU values from individual mice (dotted lines) and average across the group (solid line). g) The absence of streptomycin pre-treatment did not affect PAS638 switching in these mice. Graph shows average switching from 4 measurements in the 12 days following bacterial administration. h) Histology scoring of small intestine, cecum, proximal and distal colon showed no signs of overt inflammation in 18 129X1/SvJ mice, including all that showed considerable switching by PAS638.

Supplementary Figure 4 Long-term stability of PAS638 during colonization of the mouse gut.

a) Essentially all PAS638 colonies tested following 200 days colonized in the 129X1/SvJ mouse (corresponding to Fig. 3e-f) retained ex vivo function when grown in the presence of sodium tetrathionate under anaerobic conditions. PAS638 colonies isolated after 200 days colonization were administered to C57Bl/6 mice ± S. typhimurium (S.tm) ΔttrR. b) Histology scoring of cecum and colon using a 0-4 point scale documented in Supplementary Fig. 2f detected elevated signs of inflammation in S. typhimurium ΔttrR co-infected mice. c) S. typhimurium ΔttrR infected mice also lost weight following infection (graph shows single mouse values (dotted lines) as percentage of pre-administration weight along with group averages (solid lines)) and d) showed elevated LCN-2 values. Graph shows maximum values measured from d1-5 post bacterial administration. ****p<0.0001 using separate two-tailed T-tests for the two individual experiments (t(8) = 16.55 and t(8) = 19.38 respectively). Means are shown.

Supplementary Figure 5 MS/MS fragmentation spectrum of tetrathionate

Exemplary fragmentation spectrum (MS/MS) of tetrathionate (M = S4O6H2) detected in a cecum sample (see Fig. 2c). Negative ion mode and collision induced dissociation (CID) with nitrogen gas at 10 eV was used. The parent ion was isolated as [M-H] at m/z = 224.8661. The corresponding chemical structures for the resulting fragments ([S3O3H]- and [SO3H]-) are shown in the spectrum.

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Riglar, D., Giessen, T., Baym, M. et al. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation. Nat Biotechnol 35, 653–658 (2017). https://doi.org/10.1038/nbt.3879

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