Description

Our project focuses on developing intelligent engineered probiotics for the precise detection and treatment of inflammatory bowel disease (IBD). Through three core systems—sensing, therapeutic, and suicide systems—we achieve an integrated approach from diagnosis to treatment. The sensing system detects inflammatory markers —— thiosulfate in the gut and indicates inflammation through color changes. The therapeutic system promotes the repair of intestinal mucosa by secreting epidermal growth factor (EGF). The suicide system ensures that the engineered bacteria automatically die after completing their tasks, preventing long-term survival in the body or the environment, thereby ensuring biosafety.

This project not only offers a convenient home-based method for monitoring enteritis but also features the advantages of low cost, in situ treatment, and high safety. With this innovative treatment system, patients can reduce their dependence on invasive examinations, improve their quality of life, and lower treatment costs, making it especially promising in regions with limited medical resources！

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the intestines with an unknown cause, and it includes ulcerative colitis (UC) and Crohn's disease (CD). The exact mechanism of IBD remains unclear, with contributing factors including genetics, environment, gut microbiota, and immune response [1]. The main symptoms of IBD are chronic abdominal pain, diarrhea, bloody stools, and weight loss. Once diagnosed with IBD, patients often require long-term medication to control the disease, and it is prone to recurrence. Crohn's disease, in particular, is difficult to treat, expensive, and prone to complications. UC typically affects the rectum and sigmoid colon (located in the lower left abdomen), and a colonoscopy can reveal extensive bleeding, edema, erosion, and superficial ulcers in the intestinal mucosa. In severe cases, the entire colon may be affected. CD primarily affects the terminal ileum and cecum (the lower right abdomen, around the appendix), characterized by persistent right lower abdominal pain, often accompanied by weight loss and diarrhea [2].

It is important to note that enteritis can be classified into two types: acute enteritis and chronic enteritis. Acute enteritis is usually short-term, caused by infection or food poisoning, and can be cured in a relatively short period. However, chronic enteritis, such as IBD, has a more complex pathogenesis and poses long-term health risks, requiring ongoing management and treatment[3]. Our research and solutions focus on chronic enteritis, particularly IBD, which poses significant health risks.

With globalization, IBD has spread from early industrialized countries to newly industrialized nations, becoming a global health issue affecting nearly 5 million people worldwide. As a populous country, China is also facing a rising number of IBD patients and a rapidly increasing incidence rate. Some studies predict that by 2025, the number of IBD patients in China will reach 1.5 million, posing a major public health challenge [4].

The inspiration for our project came from an unexpected incident. During a group discussion, one of our team members was rushed to the hospital due to acute abdominal pain and was eventually diagnosed with IBD. During a hospital visit, the doctor explained to us that the pathogenesis of IBD is complex, and many people miss the early signs, leading to delayed diagnosis, worsening of the condition, and even life-threatening complications. The doctor emphasized that early diagnosis and prevention can significantly improve outcomes. As a result, we decided to focus our research on the early diagnosis of IBD, aiming to explore ways to help more people detect and address gut health issues at an early stage.

Current diagnostic methods for IBD, such as colonoscopy and endoscopy, are often invasive and uncomfortable. Imaging tests, like Magnetic Resonance Imaging (MRI) and Computed Tomography (CT), tend to be expensive. These tests require bowel preparation, making the process not only complicated but also difficult for many patients to undergo [5]. Furthermore, these tests are typically used once the disease has progressed, making early diagnosis challenging and resulting in many patients only receiving treatment after their condition has worsened. While blood tests, such as C-Reactive Protein (CRP), or stool tests like Calprotectin, can monitor inflammation, their specificity is limited, making it difficult to distinguish from other gastrointestinal diseases [6].

Current treatments for IBD mainly focus on symptom control and inflammation reduction. Common treatment methods include anti-inflammatory drugs, such as corticosteroids and 5-aminosalicylic acid, which are used to manage acute flare-ups but have significant side effects with long-term use [7]. Immunosuppressants, such as azathioprine and methotrexate, can reduce immune responses but increase the risk of infection [8]. Biological agents, such as infliximab and adalimumab, target specific inflammatory pathways but are expensive and may lose effectiveness over time. Traditional Chinese medicines, such as Changyanning tablets, have shown some alleviating effects on IBD, but their mechanisms are complex, and there is significant individual variability, limiting their clinical application [9]. Surgery is often used for severe cases, especially Crohn's disease; however, even after surgery, there is still a high risk of recurrence [10].

The invasiveness of current diagnostic methods, the inadequacy of early detection, and the fact that treatment options are limited to symptom control result in many patients enduring the pain of recurrent flare-ups and facing high treatment costs, which severely impacts their quality of life. This underscores the urgency of improving diagnostic techniques and developing more effective treatment options to help patients receive more accurate detection and timely treatment in the early stages of the disease, thereby reducing the long-term burden of the illness and improving overall health outcomes.

Our product is an intelligent engineered probiotic capable of precise detection and in situ treatment of IBD. Through the design of three core systems (the sensing system, therapeutic system, and suicide system), it can detect inflammation signals in the intestines, release human epidermal growth factor (hEGF), and ensure that the engineered bacteria automatically die after completing their task. Specifically, the sensing system detects thiosulfate in the intestines, indicating intestinal inflammation through color changes; the therapeutic system secretes EGF to accelerate the repair of intestinal mucosal damage; and the suicide system ensures that the engineered bacteria do not survive long-term, enhancing safety. The overall design aims to achieve non-invasive, automated enteritis management, improving patients' quality of life.

Escherichia coli Nissle 1917 (EcN) is a well-studied probiotic that has been applied in clinical settings. Due to its safety and immunomodulatory properties, it was selected as the chassis microorganism for this project. The unique lipopolysaccharide (LPS) structure of EcN regulates the host's immune system without triggering toxic reactions. Moreover, the complete sequencing of EcN’s genome provides a solid foundation for precise genetic modifications, facilitating gene insertion, knockout, or editing [11]. This strain also has relatively simple cultivation requirements, making it suitable for large-scale production in both laboratory and industrial settings [12].

During inflammatory responses, the levels of sulfate-reducing bacteria in the patient's intestines increase, leading to a rise in the concentration of sulfur metabolites, particularly thiosulfate, associated with these bacteria. Based on this, we initially selected the TtrS/R two-component system, which senses tetrathionate, for detection. However, we later discovered that tetrathionate is rapidly metabolized by microorganisms in the gut, resulting in an unstable signal and an inability to consistently and effectively monitor inflammation. Therefore, we shifted to a thiosulfate sensing system, designing it using the ThsS/R two-component system [13].

In this system, ThsS undergoes autophosphorylation upon detecting thiosulfate and transmits the signal to the response regulator protein ThsR. Phosphorylated ThsR binds to the PphsA promoter, activating the expression of the lacZ gene. The lacZ gene encodes β-galactosidase, which breaks down X-gal, producing a blue color reaction that indicates the presence of intestinal inflammation. This design enables sensitive detection of enteritis with a visual color output, significantly enhancing the stability and reliability of the detection process [14].

During an IBD flare-up, the intestinal mucosal barrier is compromised, exposing the inner intestinal wall to harmful environments [15]. Rapid restoration of intestinal epithelial integrity becomes crucial for treatment. EGF is an important mucosal intervention agent, promoting cell migration and rapidly repairing mucosal wounds. However, when administered orally, EGF is easily degraded by digestive enzymes in the stomach, severely limiting its application in the treatment of enteritis. Therefore, we integrated EGF downstream of the bacterial sensor ThsS/R two-component system to achieve a combined detection and treatment approach.

This system is regulated by the aforementioned sensing system, ensuring that EGF is only expressed and secreted upon detection of intestinal inflammation signals. This design not only prevents EGF degradation in the stomach but also enables in situ treatment within the intestines, ensuring that EGF acts directly at the inflammation site. To enhance the rapid secretion of EGF, we introduced the ⍺-Hemolysin A (HlyA) secretion system [16]. This system consists of the hlyB and hlyD proteins, which regulate the efficient secretion of EGF through the Hly signal peptide. As a transporter protein, HlyB provides energy for the system and recognizes the C-terminal of EGF, guiding it toward the transmembrane transport channel in the outer membrane [17]. HlyD, a key protein connecting the inner and outer membranes, provides a pathway for EGF to cross the membranes, ensuring smooth transport from the cytoplasm to the external intestinal environment. The outer membrane porin TolC binds to HlyD, forming a complete ⍺-hemolysin secretion system [18]. Through its β-barrel structure, this system facilitates the extracellular secretion of EGF, allowing it to exert its therapeutic effect.

To ensure the safe control of the engineered bacteria both inside and outside the body, we designed a suicide mechanism based on the MazE/F toxin-antitoxin system [19]. This system allows the engineered bacteria to survive temporarily in the intestines and complete their therapeutic task before automatically dying [20]. Additionally, the system can be controlled by external conditions to facilitate laboratory testing and cultivation. MazF is an mRNA endonuclease that inhibits protein synthesis by cleaving specific mRNAs, ultimately leading to bacterial death. Its counterpart, MazE, is an inhibitory protein that neutralizes MazF's toxicity, ensuring the normal survival of the bacteria [21]. We designed the constitutive expression of MazF under a continuous promoter, ensuring that the bacteria are in a potential suicide state without external intervention. The expression of MazE is regulated by a rhamnose-inducible promoter (pRHa), meaning MazE can only effectively neutralize MazF and prevent bacterial death when sufficient rhamnose is present.

In vivo control: When patients ingest the engineered bacteria, they simultaneously consume rhamnose, ensuring the bacteria's survival during treatment in the intestines. Once the treatment is completed and rhamnose is absorbed by the body, the expression of MazE decreases, and it can no longer suppress the toxic effects of MazF, causing the engineered bacteria to self-destruct, thereby preventing long-term survival in the body.

In vitro suicide: When the engineered bacteria are excreted through feces, the rhamnose content in the environment is extremely low. This lack of MazE leads to the bacteria's self-destruction, reducing the biosafety risks in the environment.

Laboratory control: During laboratory testing and cultivation, adding rhamnose to the culture medium induces the expression of MazE, thereby inhibiting the toxic effects of MazF and ensuring the bacteria's safe survival under laboratory conditions.

Based on the chassis strain EcN, we designed a sensing system, therapeutic system, and suicide system to work synergistically. The sensing system detects thiosulfate in the intestines through the ThsS/R two-component system. Upon detecting inflammation signals, ThsS autophosphorylates and transmits the signal to ThsR, activating the expression of the lacZ gene. The β-galactosidase encoded by lacZ breaks down X-gal, producing a blue color reaction, providing a visual inflammation detection signal. The therapeutic system is then activated, releasing EGF through the ⍺-hemolysin A secretion system, enabling in situ treatment of damaged intestinal areas. Finally, the suicide system, utilizing the MazE/F toxin-antitoxin system, ensures that the engineered bacteria self-destruct after completing their task, preventing long-term survival in the body or the environment, thereby ensuring biosafety.

The intelligent engineered probiotic in this project is primarily targeted at the following high-risk groups for IBD, providing non-invasive detection and treatment to help patients monitor and improve their gut health:

Elderly individuals: As people age, their intestinal function deteriorates, and their immune system weakens, making them more susceptible to enteritis and other digestive system diseases. The intelligent engineered probiotic offers a simple method for monitoring gut health in this population, reducing the invasiveness and discomfort of traditional examinations.

Individuals with a history of enteritis: For those who have previously suffered from enteritis or have a history of chronic enteritis, regular monitoring of gut health is crucial. The engineered probiotic helps these patients monitor their condition in real time through convenient stool color changes, allowing for timely detection of inflammation recurrence.

High-risk groups for digestive system diseases: People with conditions such as ulcerative colitis or Crohn's disease require frequent monitoring of their intestinal health. This project provides a continuous, non-invasive detection method that signals the presence of intestinal inflammation through changes in stool color.

Individuals with intestinal microbiota imbalance: Long-term use of antibiotics, irregular eating habits, or malnutrition can lead to an imbalance in gut microbiota, which may cause intestinal inflammation. By consuming the engineered probiotic, these individuals can restore the balance of gut flora and quickly receive diagnosis and treatment when inflammation occurs.

Our intelligent engineered probiotic capsule is composed of three parts: EcN lyophilized powder, X-gal, and rhamnose. Lyophilization technology enables long-term preservation of the engineered bacteria, which can be quickly activated in the intestines. X-gal is used for the color reaction, as the engineered bacteria express β-galactosidase upon entering the intestines, breaking down X-gal and causing a change in stool color. Rhamnose is used to control the suicide system. Once rhamnose is depleted in the intestines, the engineered bacteria will automatically die, preventing their long-term survival in the body or the environment, ensuring biosafety.

Target users can take the capsule regularly as advised by their doctor (for enteritis detection, patients can take the capsule once a month to regularly monitor gut health; for IBD treatment, it is recommended that patients take the capsule once a day). The engineered bacteria in the capsule will reach the intestines 4-5 hours after ingestion and begin detecting the inflammation marker thiosulfate. When the marker concentration increases, the stool will turn blue, allowing patients to monitor their intestinal health by observing the color change and take timely intervention measures. If the stool color gradually lightens, it indicates a reduction in inflammation, and patients may consider discontinuing the treatment under the advice of their doctor.

Currently, the laboratory is using E. coli DH5α, a strain that naturally lacks β-galactosidase, making it suitable for our research. However, for safety in practical applications, we need to switch to the endotoxin-free EcN, which is safe for oral administration. The challenge, however, is that EcN naturally contains the lacZ gene, which interferes with our detection system, as it continuously breaks down X-gal and produces a blue color, regardless of whether the patient has enteritis or not. To resolve this issue, we plan to use CRISPR-Cas9 gene-editing technology. We will design a gRNA that targets the lacZ gene, guiding the Cas9 enzyme to precisely excise the lacZ gene from EcN. Following this, we will culture the edited strains on plates containing X-gal and screen for colonies that no longer produce a blue color, indicating successful knockout of the lacZ gene in EcN.Through this modification, the engineered bacteria will avoid nonspecific reactions with X-gal, ensuring that our detection system only produces a blue product when enteritis occurs, thereby improving the accuracy and reliability of the detection [22].

Convenience

By using our product, patients can easily monitor their health at home by observing changes in stool color, eliminating the need for complex and invasive procedures like colonoscopies. This convenience significantly reduces patient discomfort and the frequency of hospital visits, making the daily management of enteritis much easier.

Cost-effectiveness

The system is designed as a low-cost solution, delivered in the form of lyophilized probiotic capsules, reducing the need for expensive hospital treatments and specialized equipment. This simplified production and administration method ensures that the system can be widely adopted at a lower cost, particularly benefiting patients in regions with limited healthcare resources.

Intelligence

The system uses a two-component sensing system (ThsS/R) to detect changes in the inflammatory marker thiosulfate in real-time. Once a signal is detected, the engineered EcN probiotics directly release epidermal growth factor (EGF) at the site of intestinal inflammation, enabling in situ treatment and precise repair of damaged intestinal tissue.

Facing the diagnosis and long-term treatment of enteritis, especially chronic enteritis（IBD）, patients and their families often endure significant stress. This is particularly true for elderly patients whose ability to care for themselves has diminished, leading their children to take on more caregiving responsibilities, which increases financial and time burdens and can even affect family harmony. To address these challenges, we have introduced the intelligent engineered probiotic treatment system, a non-invasive, low-risk therapy that can significantly reduce the risk of complications and enable gentle treatment with quick recovery. Patients can conduct both detection and treatment at home, eliminating the need for frequent hospital visits. Even while traveling, they can maintain their treatment regimen without compromising their quality of life. This technology not only enables precise detection and early intervention for enteritis but also incorporates an innovative suicide system that ensures the engineered bacteria self-destruct after completing their task, preventing long-term survival in the body and ensuring safety. The goal of this project is to alleviate the burden of IBD on patients and their families, improve quality of life, reduce the strain on the healthcare system, and promote overall societal well-being and health.