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Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease

Abstract

Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.

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Fig. 1: Directed evolution of the human P2Y2 receptor.
Fig. 2: Characterization of human P2Y2 receptor mutants.
Fig. 3: eATP-responsive secretion of ATPase by engineered yeast.
Fig. 4: eATP-responsive engineered yeasts ameliorate chemically induced colitis.
Fig. 5: eATP-responsive engineered yeasts ameliorate anti-CD3 antibody-induced enteritis.
Fig. 6: eATP-responsive engineered yeast probiotics limit fibrosis and dysbiosis.

Data availability

RNA-seq data were deposited in the GEO database under the accession number GSE152869. Sequencing data from microbiota 16S rRNA were submitted to the NCBI short-read archive under BioProject number PRJNA641709. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants NS102807, ES02530, ES029136 and AI126880 from the NIH; RG4111A1 from the National MS Society; PA-1604-08459 from the International Progressive MS Alliance; and NSERC 492911 from the Natural Sciences Engineering Research Council of Canada. B.M.S. was supported by an Ontario Graduate Scholarship and by the Professional Research Experience Program agreement between the National Institute of Standards and Technology (NIST) and the University of Maryland. C.G.-V. was supported by an Alfonso Martín Escudero Foundation postdoctoral fellowship and by postdoctoral fellowship ALTF 610-2017 from the European Molecular Biology Organization. J.P. was supported by grant 2019/04780-7 from the São Paulo Research Foundation (FAPESP). This paper is dedicated to the memory of Dan S. Tawfik. We thank Z. Kelman and J. Marino of the University of Maryland Institute for Bioscience and Biotechnology Research for use of their incubators and W. Shaw of Imperial College London for providing yeast codon-optimized sfGFP. We thank the Harvard Medical School Rodent Histopathology Core, which provided histopathology service. We thank the NeuroTechnology Studio at Brigham and Women’s Hospital for providing access to the Leica DMi8 fluorescent microscope and consultation on data acquisition and data analysis. B.M.S. is an International Associate of the NIST. The NIST notes that certain commercial equipment, instruments and materials are identified in this paper to describe an experimental procedure as completely as possible. In no case does this identification imply a recommendation or endorsement by the NIST, nor does it imply that the materials, instruments or equipment are necessarily the best available for the purpose. The opinions expressed in this article are the authors’ own and do not necessarily represent the views of the NIST.

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B.M.S., C.G.-V., L.M.S., J.A.d.S.P., A.P., P.H., M.O’.B. and S.K.C. performed in vitro and in vivo experiments; Z.L., L.M.C. and C.G.-V. performed bioinformatic analysis; P.M.M.-V. discussed and interpreted findings; B.M.S., C.G.-V. and F.J.Q. wrote the manuscript with input from co-authors; and B.S.W.C., S.G.P. and F.J.Q. contributed equally to the design and supervision of the study and editing the manuscript.

Corresponding author

Correspondence to Francisco J. Quintana.

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Competing interests

B.M.S., C.G.-V., B.S.W.C., S.G.P. and F.J.Q. filed a patent for the use of engineered yeast to treat inflammation. All other authors declare no competing interests.

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Peer review information Nature Medicine thanks Cathryn Nagler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Response to eATP over time of engineered mating pathway.

a,b, Yeasts from the BS016 strain transformed with plasmid pRS316 pTDH3 P2Y2 (WT human P2Y2 receptor) were incubated with 100 μM ATP in 300 μL (a) or 5 mL SC-URA media (b); and mCherry fluorescence was quantified. Data points represent the mean of 2 colonies. c, Engineered mating pathway response to UTP using the wild-type (WT) human P2Y2 receptor and various yeast strains with integrated Gpa1-Gα chimeras as follows: BS019 (G14), BS020 (Gq), BS016 (Gi3). Following incubation for 6h with the indicated UTP concentration, the activation of the mating pathway was monitored by quantifying mCherry fluorescence by flow cytometry. Data points represent the mean of 2 colonies.

Source data

Extended Data Fig. 2 Strategy for directed evolution of human P2Y2 receptor.

During each FACS sort the top ~1% of mCherry fluorescence was collected. ‘Recovered’ refers to the number of yeast colonies obtained after plating sorted cells on selective media.

Extended Data Fig. 3 Apyrase Genes and ATP concentration in yeast supernatants.

a, Sequence Alignment of Apyrase Genes. Human ENTPD1 (CD39), potato apyrase (RROP1) and wheat apyrase (TUAP1) were aligned using MUSCLE, in the MEGA6 alignment explorer. b, Measurement of a known ATP concentration in the presence of 5 μL yeast supernatant from strain CB008 (blue) or reaction buffer only (red). Yeast supernatant did not affect the measurement readout. n = 3 samples, error bars represent SD. c, To estimate the amount of active apyrase secreted by yeast, 50 μM ATP was incubated with the indicated concentration of commercial apyrase for 30 minutes at 30oC, with 5 μL supernatant from a culture of strain Ctrl, in a 50 μL reaction volume and residual ATP was quantified. No apyrase activity was observed when 31.3 pM commercial apyrase was added. n = 6 samples.

Source data

Extended Data Fig. 4 Engineered yeasts probiotics are viable in the mouse gut.

a, Colony forming units per mg of stool collected 6, 24 and 48h after oral gavage to the mice with either Ctrl KG, conAP KG or indAP KG yeast strains. n = 3 Ctrl, n = 5 conAP and n = 4 indAP samples per group. b, ATP relative levels in the specified portions of the gut of naïve and TNBS induced mice. n = 4 naïve, n = 6 TNBS mice per group. c, Gating strategy to measure mCherry positive yeasts in the fecal content. d, mCherry positive yeasts (% of total GFP yeast) measured by flow cytometry in the fecal content of the specified portion of the gut after 2h from oral gavage to naïve mice with ATP inducible strain indCherry KG. ATP levels were measured in the same portions of the gut. n = 3 samples for cytometry and n = 8 for eATP levels. e, mCherry positive yeasts (% of total GFP yeast) quantified by flow cytometry in the fecal content of the specified portion of the gut from TNBS treated mice 2 hours after oral gavage with indCherry KG or WTCherry KG yeast strains. f, Changes in body weight during the course of DSS-induced colitis in mice treated with engineered yeasts starting 3 days before DSS administration. n = 4 mice Healthy control and n = 3 otherwise. Two-way ANOVA followed by Tukey’s post-hoc test, ns = not significant.

Source data

Extended Data Fig. 5 Integration of P2Y2 mutants with CRISPR STAR Method. Plasmid pCAS AarI.

Custom multiple cloning site inserted at the XmaI and BglII sites in the pCAS plasmid, obtained from AddGene68. Image generated with CLC Sequence Viewer.

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Scott, B.M., Gutiérrez-Vázquez, C., Sanmarco, L.M. et al. Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease. Nat Med 27, 1212–1222 (2021). https://doi.org/10.1038/s41591-021-01390-x

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