Inflammatory bowel disease (IBD) has been sporadically observed since ancient times, and has emerged as a growing problem in industrialized nations. Since the middle of the twentieth century, the incidence of ulcerative colitis and Crohn’s disease has increased in North America, Europe, Australia and New Zealand. At the turn of the twenty-first century, the overall incidence of IBD plateaued in certain regions of the Western world, whereas IBD in specific populations (for example, paediatric-onset IBD) continued to show a rise in incidence [1]. IBD has affected approximately 2 million people in North America and Europe, and the incidence continues to increase [2]. Similarly, the incidence rate of IBD has also increased in China and other Asian countries in recent decades [3].

Inflammatory bowel disease (IBD) has a profound impact on patients and society as a whole. The chronic and unpredictable nature of the condition can lead to significant physical and emotional distress, affecting every aspect of a patient's life. The symptoms of IBD, such as diarrhea, abdominal pain, and fatigue, can be debilitating, making it difficult for individuals to maintain their daily routines, work, or participate in social activities [1,4,5]. Moreover, the emotional toll of IBD can be overwhelming, leading to anxiety, depression, and feelings of isolation [4,5].

Beyond the individual level, IBD also has substantial economic and societal implications. The disease is associated with significant healthcare costs, lost productivity, and absenteeism, resulting in a substantial economic burden on patients, families, and society. In the United States alone, the annual direct and indirect costs of IBD are estimated to be over $12 billion [1]. Furthermore, IBD can limit educational and career opportunities, leading to a long-term impact on personal and professional development.

Additionally, IBD can also have a profound impact on families and caregivers, who often take on a significant emotional and financial burden in supporting their loved ones.

New therapeutic strategies in inflammatory bowel disease (IBD) have shifted from symptom control towards treat-to-target algorithms in order to optimize treatment results. The treatment of IBD has evolved with the development of tumor necrosis factor-α inhibitors (TNF-α), such as infliximab, adalimumab, golimumab, and certolizumab pegol, which were introduced in the late 1990s to induce and maintain remission [6,7,8], beyond the conventional therapies. However, in spite of their long-term effectiveness, almost two thirds of patients do not respond to or cannot sustain treatment with these drugs, which have various side effects.

The key challenges in the current treatment of IBD include limited treatment efficacy, risk of relapse，and heterogeneity in treatment response and individualized needs. These issues highlight the need for further research and the development of more effective and safer therapeutic strategies for IBD.

New mechanisms of action and innovative therapies, such as modulating the gut microbiome and targeting specific immune pathways, should be explored, while evaluating the synergistic effects of various interventions to seek the optimal combination.

Engineered biological systems are currently being developed that use synthetic genetic circuits to generate a wide variety of biological behaviors. Intelligent biosensors developed via synthetic biology show a promising noninvasive strategy and has been used as a diagnostic tool for sensing disease biomarkers in the gut[9,10,11,12]. Bacterial sensors based on two-component systems, ThsS/R, have been constructed in Escherichia coli Nissle 1917 (EcN) to sense and respond to thiosulfate, which are promising biomarkers of gut inflammation.[13] What's more, we describe the construction and testing of another synthetic genetic circuit capable of detecting and responding to nitric oxide, an important marker of inflammation in inflammatory bowel disease (IBD). Thus, this biosensor can be used to diagnose colonic inflammation.

Herein, we developed a high-sensitive biomarker-responsive probiotic system to diagnose and treat IBD.

Mode:

The ultimate goal of our project is to design an effective whole-cell probiotic system to diagnose and treat inflammatory bowel disease

Overview

Figure 1. About our our system

We developed a whole-cell engineered bacteria, based on the integration of three modules (inflammatory reporting, drug secretion and safety module) to simultaneously diagnose and ameliorate inflammatory bowel disease(Figure 1).

The diagnosis and therapy part

Figure2. A brief overview of the diagnosis and therapy part

Figure3.How does our diagnosis part work

Studies have shown that sulfate‐reducing bacteria (SRB) present in the colon produce hydrogen sulfide (H2S) from oxidized sulfur species derived from the host and diet, which is a process that has been suggested to be involved in colitis [14]. Host enzymes detoxify H2S to thiosulfate. And by using a Salmonella typhimurium mouse model Winter and colleagues have shown that reactive oxygen species (ROS) produced by the host during inflammation convert thiosulfate to tetrathionate, which this pathogen consumes to establish a foothold for infection [15]. A two-component system, ThsS/R, have been constructed in Escherichia coli Nissle 1917 (EcN) as an ideal system to sense and respond to thiosulfate, which is a promising biomarker of gut inflammation.[16]

Therefore, we have designed a biotherapeutic strain of engineered probiotics specifically for the treatment of inflammatory bowel disease (IBD) (Figure 2), which possesses two key molecules, ThsS and ThsR, forming a high-efficiency signaling network to precisely recognize and respond to excessive amounts of thiosulfate, a key biomarker in the gut of IBD patients through phosphorylation and dephosphorylation [18,19].

Considering levels of many disease-related biomarkers are dynamic and difficult to capture and monitor in native environments, we added a CRISPR-based genome editing system that based on the second-generation base editor (CBE) to amplify the inflammatory signal reaction. In this added system, the promoter of drug protein was replaced with a CBE-editable ACG-tag sequence, which silenced the translation of our drug protein. CBE was expressed from PphsA to respond to thiosulfate levels in native environment. In the presence of thiosulfate, BE2 edited the ACG-tag sequence to ATG (molecular recording), which activated the translation of drug protein (Figure 3).

Once sulfide is detected, ThsS undergoes rapid phosphorylation, which in turn activates ThsR. This cascade of biochemical reactions triggers the transcription of downstream genes, including the AvCystatin protein from the parasite. AvCystatin plays a crucial role in regulating macrophage immune responses, activating cellular MAP kinase and inducing the expression of IL-10 and IL-12/23p40, thereby effectively alleviating gut inflammation [17,19].

The suicide part

Figure 4. How does our suicide part work

We then added a ‘suicide switch’ into our system to control drug expression (Figure 4). As the patient's symptoms gradually improve, the level of nitric oxide, which has been proved to be another promising biomarker of IBD [20], in the gut decreases. Then, the "suicide switch" within the probiotic is activated [18,19]. The PnorV promoter is inhibited, controlling the expression of CI protein, while the PL promoter is derepressed, inducing the expression of the MazF toxin protein [21]. As a toxin protein, MazF cleaves RNA to block protein synthesis, ultimately leading to the self-apoptosis of the probiotic cells. Moreover, MazF imparts resistance to human immunodeficiency virus (HIV) in primate and human T cells without affecting normal cell growth, demonstrating its potential for in vivo applications [22,23].

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