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

CDI is a leading cause of healthcare-associated diarrhoea with high incidence, mortality, and costs. Recurrence is common and current treatment paradoxically relies on antibiotics, which are a major risk factor because they induce gut dysbiosis. CDI pathogenesis depends on spore germination into toxin-producing vegetative cells; antibiotics-induced dysbiosis disrupts microbiome-mediated bile salt metabolism, leading to accumulation of conjugated bile salts (e.g., taurocholate) that trigger C. difficile spore germination, and depletion of unconjugated/secondary bile acids that inhibit colonization. The authors hypothesize that dynamically restoring bile salt metabolism in response to dysbiosis can inhibit both germination and vegetative growth of C. difficile and thereby reduce CDI severity and recurrence. The study aims to engineer a probiotic with a dysbiosis-responsive genetic circuit (sensor–amplifier–actuator) to modulate bile salt profiles in the intestine.

Prior work establishes: (1) the gut microbiota transforms primary conjugated bile salts into unconjugated and secondary bile acids via deconjugation and 7α-dehydroxylation; (2) antibiotic-induced dysbiosis perturbs these transformations, increasing germinants like taurocholate and decreasing inhibitory bile acids, thereby promoting C. difficile germination and outgrowth; (3) taurocholate is a potent germinant for C. difficile spores, whereas unconjugated/secondary bile acids inhibit colonization; (4) sialic acid levels rise post-antibiotics due to altered community metabolism, serving as a proxy of dysbiosis and a nutrient for pathogens; (5) strategies targeting germination/sporulation have been proposed, and microbiome restoration approaches (e.g., FMT) are clinically recommended for rCDI. These findings motivated using bile salt hydrolase-mediated deconjugation and dysbiosis-responsive control to limit CDI.

- Enzyme selection and in vitro validation: Selected bile salt hydrolase Cbh from Clostridium perfringens for preference toward taurocholate and activity near physiological pH. Expressed and purified His-tagged Cbh in E. coli; quantified deconjugation of taurocholate/glycocholate to cholate via HPLC/LC-MS; assessed effects on C. difficile spore germination and vegetative growth in BHIS under anaerobic conditions. - Probiotic host: Engineered auxotrophic E. coli Nissle 1917 (EcN) requiring D-alanine for survival to aid selection, plasmid stability, and biocontainment. - Dysbiosis sensor construction: Built sialic acid-responsive sensor using pNanA promoter and NanR regulator; optimized NanR expression using constitutive promoters (e.g., J23113) and RBS variants to maximize dynamic range; characterized glucose-dependent repression (CAP site) to align with low-glucose colon regions; dynamic GFP reporter assays and flow cytometry. - Amplifier–actuator design: Added amplifier module with cadC under pNanA to activate pCadBA and amplify actuator expression; actuator is Cbh. Compared sensor-only vs sensor-amplifier using GFP and immunoblot of Cbh; verified pH-sensitive CadC behavior. Final circuit: J23113r4-nanR–pNanA–cadC–pCadBA–cbh (EcN-Cbh). - In vitro functional assays: Incubated EcN-Cbh with taurocholate ± sialic acid; measured conversion to cholate; evaluated endospore germination reduction and vegetative growth inhibition; assessed extracellular vs intracellular deconjugation; quantified C. difficile toxin A (TcdA) in culture supernatants; tested Caco-2 viability with treated supernatants (MTT assay). - In vivo murine CDI model: Male C57BL/6 mice pretreated with antibiotic cocktail (day −6 to −3), clindamycin (day −1), infected orally with C. difficile VPI10463 (10^7 CFU, day 0). Probiotics (10^9 CFU) administered day −3. Groups: treatment (EcN-Cbh), no-sensor (constitutive cbh), no-amplifier (sensor-only cbh), no-actuator (sensor-amplifier with inactive cbh), EcN wild-type, and infection control. Monitored survival (Kaplan–Meier), weight, Clinical Sickness Score (CSS). Histopathology of colon (H&E, blinded HIS scoring). 16S rRNA sequencing of fecal microbiome (V3–V4, QIIME2) to assess C. difficile abundance and Shannon diversity. - Host metabolite measurements: Quantified fecal sialic acid (DMB derivatization, HPLC) pre/post antibiotic treatment. Quantified fecal bile acids (taurocholate, cholate, glycocholate, chenodeoxycholate, lithocholate, deoxycholate) by UPLC-ESI-MS/MS across timeline and groups. Statistical analyses included Student’s t-tests, Dunnett’s tests, mixed-model ANOVA, and log-rank tests.

- Unconjugated bile acid cholate suppressed C. difficile: reduced spore germination by up to 81% and vegetative growth by up to 82% across strains at 2 mM. - Purified Cbh efficiently deconjugated bile salts: converted 99.2% of taurocholate to cholate; deconjugated glycocholate similarly, yielding >99% reduction of conjugated bile salts; decreased endospore germination by 96% and vegetative cells by 89% vs taurocholate control. - Sialic acid biosensor optimization: pNanA–NanR with J23113 promoter and rbs4 provided highest dynamic range; glucose repressed induction (CAP-dependent), aligning with low-glucose distal gut sites. - Amplifier improved actuator expression: CadC/pCadBA amplifier under pNanA increased Cbh expression to levels comparable to purified enzyme, overcoming low efficacy of sensor-only Cbh expression. - Engineered probiotic function in vitro: EcN-Cbh fully converted taurocholate to cholate under sialic acid; reduced spore germination by 98%. Without sialic acid, basal activity still achieved 45% conversion and 90% germination reduction; growth inhibition of vegetative cells attributable to cholate; markedly reduced TcdA levels and improved Caco-2 cell viability. - In vivo efficacy in murine CDI: EcN-Cbh conferred 100% survival versus 60–14.3% across controls; minimized weight loss, especially days 2–4; achieved lowest mean CSS (4.42, mild) vs controls (7.73–10.00, moderate–severe). Only EcN-Cbh decreased fecal C. difficile abundance; sensor-bearing Cbh groups increased Shannon diversity. - Histopathology: EcN-Cbh markedly reduced histologic injury scores of colon compared to controls, indicating ameliorated epithelial damage, edema, and neutrophil infiltration. - Host metabolite dynamics: Fecal sialic acid increased ~6-fold after antibiotics (day −6 to −3), supporting sensor choice. EcN-Cbh decreased taurocholate and increased cholate pre-challenge and early post-infection; Cbh-expressing groups elevated deoxycholate and chenodeoxycholate/lithocholate levels. - Statistical significance: Survival improvements (log-rank) and CSS reductions (Dunnett’s) were significant versus controls; multiple metabolite and microbiome measures showed significant group/time effects.

The study demonstrates that dynamically restoring bile salt metabolism via an engineered probiotic can target CDI pathogenesis by simultaneously preventing spore germination (via deconjugation of conjugated bile salts) and inhibiting vegetative growth (via cholate). The dysbiosis-responsive sensor allowed spatially and temporally appropriate actuator expression in the distal gut, further tuned by glucose and pH (CadC), and the amplifier ensured sufficient enzyme levels. In vivo, only the complete sensor–amplifier–actuator circuit yielded comprehensive protection: full survival, improved clinical scores, reduced pathogen abundance, and ameliorated histopathology. These findings support a host–pathogen microenvironment modulation strategy, complementing antibiotics and aligning with therapeutic paradigms that restore microbiome functions (e.g., FMT). The results also highlight that probiotic deployment must be precisely regulated; constitutive or insufficient expression did not improve outcomes and, consistent with prior reports, certain probiotic interventions may impair microbiome recovery if not properly controlled.

Engineered E. coli Nissle 1917 carrying a sialic acid-responsive sensor, CadC-based amplifier, and bile salt hydrolase actuator (Cbh) reprograms intestinal bile salt metabolism to inhibit C. difficile spore germination and vegetative growth. This dynamic, microenvironment-targeted approach achieved robust in vitro activity and, in a murine CDI model, delivered 100% survival, improved clinical and histological outcomes, decreased C. difficile abundance, and partially restored microbiome diversity. The work establishes modulation of bile acid metabolism as a mechanism of action for CDI therapy and suggests practical regimens: co-administration with standard antibiotics and prophylaxis in high-risk patients undergoing antibiotic therapy. Future directions include mechanistic dissection of circuit module interplay, broader strain coverage (including spore-focused testing), comprehensive metabolomic and microbiome profiling to identify additional protective factors, and optimization of dosing and treatment duration.

- Hydrolase expression and taurocholate-to-cholate conversion correlated with enzyme presence but did not linearly correlate with protection across all groups; only the complete circuit achieved strong infection inhibition despite similar conversion in some controls. - The necessity of the full sensor–amplifier–actuator circuit indicates incomplete understanding of the mechanistic requirements and interactions among modules; further mechanistic elucidation is needed for clinical translation. - Efficacy should be validated across additional C. difficile strains and focused spore challenges. - Engineered deconjugation likely shifts broader intestinal metabolite profiles beyond bile acids, potentially contributing to protection; these were not comprehensively profiled. - Optimal dosing, timing, and duration of probiotic administration remain to be determined.