Engineered yeast-based eATP precisely controlled system for the treatment of inflammatory bowel disease

Article summary

Increased levels of extracellular ATP (eATP) are recognized as a crucial mediator in inflammatory bowel disease (IBD) and other inflammatory diseases [1]. How to fine-tune the balance between eATP and its metabolite adenosine (ADO) to control inflammation and avoid the adverse effects is a big challenge. In the study performed by Scott et al. [2], an engineered yeast-based closed-loop control system [3] was used to regulate the levels of eATP acting at purinergic P2Y₂ receptors in order to protect mice from intestinal inflammation and developing intestinal fibrosis. This system provides a novel and promising strategy for the treatment of IBD by means of a probiotic delivery system together with the ligand-receptor interaction model of purinergic signalling.

Commentary

Inflammatory bowel disease (IBD) is a very common type of chronic gastrointestinal disorder, which includes two conditions (Crohn’s disease and ulcerative colitis), characterized by chronic and relapsing mucosal inflammation of the intestinal tract and affects thousands of people worldwide. IBD patients commonly present with abdominal cramping, pain, diarrhea, anemia, and weight loss. More seriously, IBD patients are at higher risk for the development of colon cancer. Due to its complicated pathogenesis, IBD is difficult to completely cure with currently available clinical drugs. Several types of therapies such as NSAIDs, corticosteroids, and immunomodulators are used to treat inflammation and other symptoms. However, in the long-term, these therapies display side-effects, such as tolerance, gastrointestinal and cardiovascular toxicity, and suppression of the immune system. Hence, there is an urgent need to develop novel small molecule or precision therapy for IBD [4, 5].

Intestinal microbiota or the microbiome is associated with changes in the immune system in IBD patients, and the probiotics may offer an alternative approach to treat IBD [6]. However, unmanipulated probiotics may enhance the host-commensal interaction in the healthy gut but not in the context of pathologic inflammation [7]. In recent years, research has focussed on the development of synthetic biological engineering of probiotics to deliver therapeutic proteins in response to disease-associated signals. One of the important signals relevant to IBD is eATP, which is released by activated immune cells and commensal bacteria. eATP signals via a set of purinergic receptors and P2Y₂ receptors especially boost pro-inflammatory cytokine production and activate effector T cell, suppress regulatory T cell (Treg) responses, and promote enteric neuron apoptosis, among other biological responses thought to contribute to IBD pathology. Pathological deregulation of purinergic signalling contributes to various diseases including IBD and cancer. Thus, purinergic signalling is an important potential therapeutic target for IBD [8, 9].

Saccharomyces cerevisiae yeast species are considered safe probiotics, which have well-defined signal transduction pathways that can be functionally linked to human G protein–coupled receptors (GPCRs) and could possibly control the expression of proteins produced in response to stimuli relevant to human physiology. Based on the role of eATP in intestinal inflammation, Scott and his coworkers [1] used synthetic biological approaches to develop S. cerevisiae probiotics, which, in response to eATP detected via an engineered human P2Y₂ GPCR, secrete the CD39-like eATP-degrading enzyme apyrase. These self-tunable engineered probiotic yeasts suppressed experimental intestinal inflammation in mice and could provide a new therapeutic platform for IBD and other inflammatory disorders.

In the current study, the researchers used different techniques such as yeast microscopy, flow cytometry, quantitative PCR, homology modeling of P2Y₂ receptors, integration of P2Y₂ receptor mutants with CRISPR/Cas9, apyrase genome mining, western blotting, and gene-expression analysis by NanoString. In addition, several different experimental mouse models of colitis (trinitrobenzenesulfonic (TNBS) acid–induced colitis, dextran sodium sulfate–induced colitis, anti-CD3 antibody–induced enteritis) were used. Firstly, they engineered the human P2Y₂ receptor to increase its sensitivity to eATP when expressed in yeast. For this purpose, they combined the human P2Y₂ receptor to the yeast mating pathway via a chimeric yeast Gpa1–human Gαi3 protein and monitored pathway activation using a fluorescent mCherry reporter controlled by the mating-responsive FUS1 promoter. Yeast expressing wild-type (WT) P2Y₂ receptors showed a weak response to 100 μM eATP, as determined by flow cytometry analysis of mCherry expression. Thus, human P2Y₂ purine receptor mutant proteins were expressed in yeast, which enhanced eATP sensitivity by up to 1,000-fold compared to the WT human P2Y₂ receptor. To achieve this goal, they established a plasmid library of human P2Y₂ receptor mutants using error-prone PCR. They incorporated the sensing P2Y₂ receptors and responding RROP1 (Solanum tuberosum ATP-diphosphohydrolase, GenBank accession U58597.1) components into the genome of the same yeast strains using a CRISPR–Cas9-based method. Moreover, eATP-induced ATPase enzymatic activity was higher in culture supernatants from yeast strains containing the P2Y₂–RROP1 gene circuit than that containing the WT receptor. Further, they confirmed the molecular mechanism for the enhancement of sensitivity in the selected P2Y₂ receptor mutants and also confirmed the responsible mechanism responsible for the differential activity of P2Y₂ purine receptor mutants alone or in combination.

The local eATP levels are increased in intestinal inflammation. To determine the activation of P2Y₂ receptor signaling in engineered yeasts during experimental colitis, they employed a strain in which the activation of mutant TM-3 P2Y₂ receptors induced mCherry expression in the caecum and colon, associated with increased local eATP levels in TNBS acid–treated mice. For the evaluation of therapeutic efficacy, engineered yeast administered orally ameliorated the TNBS-induced colitis. The engineered yeast produced similar effects in the dextran sodium sulfate–induced colitis, and anti-CD3 antibody–induced enteritis models. Thus, these data establish that eATP-responsive yeasts harboring a synthetic P2Y₂-PROP1 gene circuit ameliorate gastrointestinal tract inflammation. eATP-inducible engineered yeast strain significantly reduced the fibrosis in treated mice. This establishes purinergic signalling as a key player in managing intestinal inflammation and avoiding untoward side-effects associated with fibrotic lesions and microbiome dysregulation.

Probiotics are therapeutic agents that can change the luminal microflora, enhance epithelial barrier function, and modify the response of the mucosal and systemic immune systems. To investigate the apyrase production by engineered yeast strains and their effects on the gut microbiome, they performed sequencing of 16S rRNA of fecal samples collected from control and disease model mice. Both healthy and TNBS-induced colitis mice treated with engineered yeasts harbored a similar microbiome. These results suggested that the engineered yeast strain in which apyrase expression is induced by eATP re-generates the intestinal microbiome.

The engineered yeasts incorporating with human P2Y₂ purine receptors described in this study represent a major advance in the application of directed evolution and synthetic biology to develop self-tunable anti-inflammatory probiotics. eATP enhances intestinal inflammation in other diseases besides IBD, such as irradiation-induced small intestinal fibrosis and graft-versus-host disease. Moreover, the microbiome may control inflammation of other tissues beyond the intestinal system such as the central nervous system. Thus, the engineered yeast-based eATP precisely controlled system provides a novel and promising therapeutic approach for IBD and other inflammatory disorders throughout the body [10–12].

Funding

This work was financially supported by the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (ZYYCXTD-D-202003) and Sichuan Science and Technology Program (2019YFH0108, 22GJHZ0007).

Data availability

Not applicable.

Declarations

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Not applicable.

Conflicts of interest

The authors declare no competing interests.