Engineering probiotics to inhibit *Clostridioides difficile* infection by dynamic regulation of intestinal metabolism

*Clostridioides difficile* infection (CDI) is a significant healthcare problem globally, causing substantial morbidity and mortality, particularly in hospitalized patients. The high recurrence rate of CDI, often following antibiotic treatment, poses a major challenge. Antibiotic therapy, while necessary to treat CDI, also disrupts the intestinal microbiome, leading to dysbiosis—a state characterized by reduced bacterial diversity and altered production of microbiome-derived metabolites. This dysbiosis creates an environment favorable for *C. difficile* colonization and recurrence. *C. difficile*'s ability to switch between active vegetative cells and dormant, antibiotic-resistant endospores is central to its pathogenesis. Endospores persist even after antibiotic treatment, and dysbiosis triggers their germination, leading to infection. Vegetative *C. difficile* cells then release toxins (TcdA and TcdB), causing colonic inflammation and tissue damage. The persistence of dysbiosis following treatment is a key factor in recurrent CDI (rCDI). Recent research has highlighted a strong link between CDI and dysregulation of bile salt metabolism mediated by the gut microbiome. Bile salts, synthesized and conjugated in the liver, undergo modifications by gut bacteria, resulting in a complex mixture of primary and secondary bile acids. Antibiotic-induced dysbiosis disrupts this process, leading to an accumulation of conjugated bile salts (e.g., taurocholate), which are known germinants of *C. difficile* spores. Conversely, levels of unconjugated and secondary bile salts, which inhibit *C. difficile* colonization, are reduced. This study hypothesizes that manipulating microbiome-mediated bile salt metabolism can be a powerful strategy to combat CDI pathogenesis.

The literature extensively documents the burden of *C. difficile* infection and the role of antibiotics in its pathogenesis. Studies show the global prevalence of CDI and its substantial impact on healthcare systems. The high recurrence rate of CDI, often within 30 days of initial treatment, highlights the need for innovative therapeutic strategies. The disruptive effects of antibiotics on the gut microbiome, leading to dysbiosis and impaired colonization resistance, have been well-established. Furthermore, research indicates the importance of the bile salt metabolism pathway in CDI development. Studies have demonstrated the role of conjugated bile salts as germinants for *C. difficile* spores and the protective effects of specific unconjugated and secondary bile salts. Previous work has demonstrated successful manipulation of the microbiome to restore bile acid-mediated resistance to *C. difficile* using precise microbiome reconstitution techniques, such as fecal microbiota transplantation (FMT). These studies support the idea that microbiome modulation can effectively counter CDI pathogenesis. However, FMT is not without drawbacks, with accessibility and standardization issues being two of the major ones. This study aims to address these drawbacks by engineering probiotics to dynamically regulate bile acid metabolism, providing a more accessible and controllable approach for CDI treatment.

The researchers engineered *E. coli* Nissle 1917, a well-studied probiotic, to dynamically modulate bile salt metabolism in response to dysbiosis. They employed a genetic circuit consisting of three core components: a sensor, an amplifier, and an actuator. The sensor, based on the pNanA promoter responsive to sialic acid (a proxy for dysbiosis), detected elevated sialic acid levels associated with antibiotic-induced dysbiosis. The amplifier module, incorporating the *cadC* gene, amplified the signal from the sensor to further enhance the expression of the actuator. The actuator module produced bile salt hydrolase (Cbh) from *Clostridium perfringens*, which deconjugates conjugated bile salts (like taurocholate) into their unconjugated forms (like cholate). The unconjugated forms of bile salts have been shown to inhibit *C. difficile* spore germination and vegetative growth. Initially, the researchers validated the effect of taurocholate and its deconjugated form, cholate, on *C. difficile* germination and growth *in vitro*. They demonstrated that cholate significantly reduced both processes. The effectiveness of the recombinant Cbh in converting taurocholate to cholate was also confirmed. The engineered probiotics (EcN-Cbh) were then tested *in vitro* to assess their ability to deconjugate taurocholate and inhibit *C. difficile* germination and growth. The impact on *C. difficile* toxin (TcdA) production and the viability of human intestinal epithelial cells (Caco-2 cells) were also evaluated. Finally, the efficacy of the engineered probiotics was assessed *in vivo* using a mouse model of CDI. Mice were pre-treated with antibiotics to induce dysbiosis, followed by administration of the engineered probiotics before infection with *C. difficile*. The survival rate, weight change, clinical symptoms, and *C. difficile* abundance in the gut were monitored. Histopathological analysis of colon tissues was performed to assess the extent of tissue damage. Bile salt profiles and sialic acid levels in the faeces were measured to confirm the in vivo activity of the engineered probiotics. Microbiome sequencing was conducted to analyze the impact on gut microbiota diversity.

*In vitro* studies confirmed that cholate, the deconjugated form of taurocholate, significantly inhibited *C. difficile* spore germination and vegetative cell growth. Recombinant Cbh effectively converted taurocholate to cholate. The engineered probiotics (EcN-Cbh) showed a strong ability to deconjugate taurocholate and inhibit *C. difficile* in vitro, reducing toxin production and protecting Caco-2 cells. In the mouse model of CDI, the EcN-Cbh treatment group showed a 100% survival rate compared to significantly lower survival rates in control groups. The EcN-Cbh-treated mice exhibited significantly less weight loss and lower clinical sickness scores, indicating milder disease severity. Metagenomic sequencing revealed a significant reduction in *C. difficile* abundance in the EcN-Cbh group. Histopathological analysis showed reduced colonic tissue damage in EcN-Cbh-treated mice. Analysis of fecal samples confirmed that EcN-Cbh successfully modulated bile salt profiles, decreasing taurocholate and increasing cholate levels in vivo. The complete sensor-amplifier-actuator circuit was critical for achieving this effectiveness, as control groups lacking one or more components showed reduced efficacy.

The findings strongly support the hypothesis that restoring intestinal bile salt metabolism can effectively inhibit CDI. The engineered probiotics, through dynamic regulation of bile salt hydrolase activity, successfully targeted the host-pathogen microenvironment to limit *C. difficile* infection. The success of this targeted microbiome modulation strategy offers a promising alternative to traditional antimicrobial approaches, which often cause dysbiosis and exacerbate infection. The results highlight the potential of using genetically modified probiotics as adjuvants to standard antibiotic therapies or as preventative measures for high-risk individuals. The study’s success in enhancing gut microbiome diversity in the context of CDI adds another dimension to its significance. The use of a sensor-amplifier-actuator system is particularly noteworthy, indicating that precise regulation of the hydrolase activity is crucial for therapeutic efficacy. This contrasts with constitutively expressing the hydrolase, where significant improvement was not observed.

This study demonstrates the successful engineering of probiotics to inhibit *C. difficile* infection by dynamically regulating intestinal bile salt metabolism. The engineered probiotics, with their sensor-amplifier-actuator circuit, showed remarkable efficacy in a mouse model, achieving 100% survival and significant improvement in clinical outcomes. This work provides a strong foundation for developing novel microbiome-based therapies for CDI, offering potential adjunctive or preventative strategies for combating this important healthcare challenge. Future research should focus on optimizing the circuit design, exploring additional microbial and metabolic interactions, and performing clinical trials to evaluate efficacy in humans.

While the study demonstrated significant efficacy, some limitations should be noted. The correlation between hydrolase expression, bile salt conversion, and infection inhibition was not perfectly linear across all groups, suggesting potential additional factors influencing the overall effect. The complete sensor-amplifier-actuator system was crucial for maximal efficacy, emphasizing the complexity of the interaction and necessitating further investigation to fully understand the underlying mechanisms. The study used a single *C. difficile* strain in the mouse model, warranting further research on broader strain applicability. Further studies are needed to explore potential effects on other intestinal metabolites and to optimize the dosage and administration regimen for clinical translation.