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Inflammatory Bowel Disease (IBD) is a familial disease characterized by chronic recurrent inflammation of the gastrointestinal tract[1], which can be categorized into two subtypes: Crohn's Disease (CD) and Ulcerative Colitis (UC), each presenting distinct manifestations and pathophysiology[2]. Historically, IBD has been sporadically observed and has become an increasingly severe issue in industrialized nations[3]. In 1875, Wilks and Moxon introduced the term "ulcerative colitis" into the medical lexicon. Since the mid-20th century, the incidence of both ulcerative colitis and Crohn's disease has risen, with the prevalence of IBD in the Western world reaching up to 0.5% of the general population. Due to its complex pathogenesis, recurrent nature, and the absence of a definitive cure, IBD has been referred to as a "green cancer."[4,5]
Current traditional treatment methods for IBD exhibit numerous shortcomings, primarily as patients frequently confront issues such as inflammatory relapse, exacerbated pain, and increased financial burden. Research has identified several notable characteristics of IBD:excessive production of active substances (NO,ROS), hyperreactive immune responses from CD8+T cells, dysbiosis of gut microbiota, and damage to intestinal epithelial cells.
Our team ingeniously proposed a probiotic-based therapeutic approach tailored to the specific features of IBD, engineering the probiotic Escherichia coli Nissle 1917 to detect and treat IBD, thereby alleviating patient suffering and reducing economic burden. Through this strategy, patients can significantly decrease the likelihood of inflammatory relapses and lessen their discomfort and financial strain by regularly taking probiotic capsule formulations.
Based on the characteristics of IBD, we designed six key modules for probiotic therapy targeting the condition. These modules include: 1. Sensing Module: SoxR/SoxS oxidative stress-responsive promoter 2. Antioxidant Module: Superoxide dismutase 3. Adhesion Module: Mussel foot protein 4. Immune Module: Programmed cell death ligand-1 (PD-L1) 5. Lysis Module: PhiX174E 6. Safety Module: MazF. These modules are intended to enhance the therapeutic efficacy of probiotics in addressing the complexities of IBD.These modules are intended to enhance the therapeutic efficacy of probiotics in addressing the complexities of IBD.
Among various chassis cells, we selected the more readily modifiable Escherichia coli Nissle 1917. It is well known that most E. coli strains contain endotoxins, which precludes their use in probiotic therapy; however, E. coli Nissle 1917 is an exception. This strain is a naturally occurring probiotic found in the human gut, notable for being one of the few E. coli strains devoid of endotoxins, while also possessing antimicrobial, anti-inflammatory, and gut microbiota-regulating properties.
In areas of intestinal inflammation, damage to intestinal epithelial cells and the gut wall leads to the excessive synthesis of nitric oxide (NO) by nitric oxide synthase located within the gut wall, resulting in elevated levels of NO in inflamed regions. We selected the SoxR/SoxS oxidative stress-responsive promoter; SoxR is a transcription factor containing a [2Fe-2S]2+ iron-sulfur cluster, while SoxS is a 53 bp promoter sequence. In high NO and ROS environments, SoxR binds to specific sequences of the SoxS promoter and undergoes a conformational change due to oxidative stress, causing the SoxS nucleic acid chain to bend (approximately 65°). This bending brings the -10 and -35 regions of the promoter closer together, facilitating the binding of RNA polymerase and other transcription factors, thereby regulating the expression of downstream genes. This mechanism enables our therapeutic module to activate and synthesize therapeutic proteins exclusively in regions of intestinal inflammation.
The pathogenesis of Inflammatory Bowel Disease (IBD) is complex; however, numerous studies indicate that reactive oxygen species (ROS) play a significant role in its progression. ROS include highly reactive free radicals (such as superoxide anions) and non-radicals (such as hydrogen peroxide). In the context of IBD, an excess of pro-inflammatory factors can activate immune cells and amplify inflammation, leading to the generation of substantial amounts of ROS. Excessive ROS further activate inflammatory responses through the NF-κB signaling pathway, resulting in the expression and secretion of pro-inflammatory factors. High levels of ROS exposure can cause mitochondrial oxidative damage, which in turn induces apoptosis in intestinal epithelial cells. Superoxide dismutase (SOD) can counteract and block the damage caused by oxygen free radicals to cells and facilitate the timely repair of damaged cells, making it a valuable therapeutic enzyme. SOD is classified into three types based on the metal cofactors it contains: Cu/Zn-SOD, Mn-SOD, and Fe-SOD.
We selected the commonly found Cu/Zn-superoxide dismutase (SOD) in cells as our target and conducted directed evolution on it. The mutagenesis of specific bases in proteins has been shown to be highly valuable in elucidating the contributions of individual amino acid side chains to protein properties. Therefore, we utilized computational methods to perform virtual amino acid mutations aimed at enhancing the protein-ligand complex affinity and thermal stability. This process identified 33 amino acid mutation targets within the active site, with the goal of increasing SOD activity. The predictions of these mutation targets can reduce the randomness associated with amino acid mutagenesis screening, guiding us to conduct rational amino acid mutations to enhance the enzyme's activity. We selected the better-performing mutants from the previous screening round as templates for subsequent iterative mutations, ultimately identifying the most effective SOD-1 variant.
Disruption of mucosal immune responses is associated with the pathogenesis of IBD, where persistent dysbiosis can lead to chronic and severe inflammation. Utilizing intestinal nitric oxide (NO) as a signaling molecule and leveraging the adhesion properties of Mfp, we aim to recruit engineered probiotics to inflamed sites. This approach induces the expression of the fusion protein Mfp5+Mfp3 to enhance intestinal adhesion, modulate gut microbiota, and protect damaged areas of the intestinal mucosa, ultimately facilitating repair and achieving therapeutic effects.
IBD is a secondary autoimmune disease characterized by excessive activation of CD8+ T cells at sites of intestinal inflammation, driven by the presence of numerous active substances and pro-inflammatory factors. This hyperactivation leads to the attack on normal self-cells, exacerbating inflammation. To mitigate immune dysregulation, we leverage the immune checkpoint PD-1/PD-L1 by employing programmed cell death ligand-1 (PD-L1). Additionally, we utilize the Lpp-OmpA surface display system to present PD-L1 on the surface of EcN, thereby downregulating the hyperactive immune response at the inflammatory sites.
To achieve optimal results in bacterial therapy, we designed a pLuxI-regulated PhiX174E lysis system. When the engineered probiotics detect NO, they synthesize Mfp, which facilitates the adhesion of EcN to the sites of intestinal inflammation, thereby establishing a dominant microbial population. However, since the therapeutic protein SOD cannot cross membranes, we exploit the principle of quorum sensing among bacteria. As the population density of EcN increases in inflamed regions, the quorum sensing system is activated, leading to the expression of the PhiX174E protein. This, in turn, induces bacterial lysis and releases the therapeutic protein, achieving optimal therapeutic effects.
Biosafety has always been a primary concern for biologists. To ensure biosafety and prevent potential issues such as gene contamination, we designed the pDawn-MazF safety module. pDawn is a blue light-inducible promoter that activates the expression of downstream genes under natural light or a specific blue light source. MazF is an RNAase that effectively degrades bacterial mRNA, thereby inhibiting bacterial growth, reproduction, and other vital activities.
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