Saccharomyces boulardii promoters for control of gene expression in vivo.

BACKGROUND: Interest in the use of engineered microbes to deliver therapeutic activities has increased in recent years. The probiotic yeast Saccharomyces boulardii has been investigated for production of therapeutics in the gastrointestinal tract. Well-characterised promoters are a prerequisite for robust therapeutic expression in the gut; however, S. boulardii promoters have not yet been thoroughly characterised in vitro and in vivo. RESULTS: We present a thorough characterisation of the expression activities of 12 S. boulardii promoters in vitro in glucose, fructose, sucrose, inulin and acetate, under both aerobic and anaerobic conditions, as well as in the murine gastrointestinal tract. Green fluorescent protein was used to report on promoter activity. Promoter expression was found to be carbon-source dependent, with inulin emerging as a favourable carbon source. Furthermore, relative promoter expression in vivo was highly correlated with expression in sucrose (R = 0.99). CONCLUSIONS: These findings provide insights into S. boulardii promoter activity and aid in promoter selection in future studies utilising S. boulardii to produce therapeutics in the gut.

Cold Exposure and Oral Delivery of GLP-1R Agonists by an Engineered Probiotic Yeast Strain Have Antiobesity Effects in Mice.

Advanced microbiome therapeutics (AMTs) holds promise in utilizing engineered microbes such as bacteria or yeasts for innovative therapeutic applications, including the in situ delivery of therapeutic peptides. Glucagon-like peptide-1 receptor agonists, such as Exendin-4, have emerged as potential treatments for type 2 diabetes and obesity. However, current administration methods face challenges with patient adherence and low oral bioavailability. To address these limitations, researchers are exploring improved oral delivery methods for Exendin-4, including utilizing AMTs. This study engineered the probiotic yeast Saccharomyces boulardii to produce Exendin-4 (Sb-Exe4) in the gastrointestinal tract of male C57BL/6 mice to combat diet-induced obesity. The biological efficiency of Exendin-4 secreted by S. boulardii was analyzed ex vivo on isolated pancreatic islets, demonstrating induced insulin secretion. The in vivo characterization of Sb-Exe4 revealed that when combined with cold exposure (8 °C), the Sb-Exe4 yeast strain successfully suppressed appetite by 25% and promoted a 4-fold higher weight loss. This proof of concept highlights the potential of AMTs to genetically modify S. boulardii for delivering active therapeutic peptides in a precise and targeted manner. Although challenges in efficacy and regulatory approval persist, AMTs may provide a transformative platform for personalized medicine. Further research in AMTs, particularly focusing on probiotic yeasts such as S. boulardii, holds great potential for novel therapeutic possibilities and enhancing treatment outcomes in diverse metabolic disorders.

Changes in liver metabolic pathways demonstrate efficacy of the combined dietary and microbial therapeutic intervention in MASLD mouse model.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease globally, yet no therapies are approved. The effects of Escherichia coli Nissle 1917 expressing aldafermin, an engineered analog of the intestinal hormone FGF19, in combination with dietary change were investigated as a potential treatment for MASLD. METHODS: MASLD was induced in C57BL/6J male mice by American lifestyle-induced obesity syndrome diet and then switched to a standard chow diet for seven weeks. In addition to the dietary change, the intervention group received genetically engineered E. coli Nissle expressing aldafermin, while control groups received either E. coli Nissle vehicle or no treatment. MASLD-related plasma biomarkers were measured using an automated clinical chemistry analyzer. The liver steatosis was assessed by histology and bioimaging analysis using Fiji (ImageJ) software. The effects of the intervention in the liver were also evaluated by RNA sequencing and liquid-chromatography-based non-targeted metabolomics analysis. Pathway enrichment studies were conducted by integrating the differentially expressed genes from the transcriptomics findings with the metabolites from the metabolomics results using Ingenuity pathway analysis. RESULTS: After the intervention, E. coli Nissle expressing aldafermin along with dietary changes reduced body weight, liver steatosis, plasma aspartate aminotransferase, and plasma cholesterol levels compared to the two control groups. The integration of transcriptomics with non-targeted metabolomics analysis revealed the downregulation of amino acid metabolism and related receptor signaling pathways potentially implicated in the reduction of hepatic steatosis and insulin resistance. Moreover, the downregulation of pathways linked to lipid metabolism and changes in amino acid-related pathways suggested an overall reduction of oxidative stress in the liver. CONCLUSIONS: These data support the potential for using engineered microbial therapeutics in combination with dietary changes for managing MASLD.

Engineered E. coli Nissle 1917 for delivery of bioactive IL-2 for cancer immunotherapy.

In this study we performed a step-wise optimization of biologically active IL-2 for delivery using E. coli Nissle 1917. Engineering of the strain was coupled with an in vitro cell assay to measure the biological activity of microbially produced IL-2 (mi-IL2). Next, we assessed the immune modulatory potential of mi-IL2 using a 3D tumor spheroid model demonstrating a strong effect on immune cell activation. Finally, we evaluated the anticancer properties of the engineered strain in a murine CT26 tumor model. The engineered strain was injected intravenously and selectively colonized tumors. The treatment was well-tolerated, and tumors of treated mice showed a modest reduction in tumor growth rate, as well as significantly elevated levels of IL-2 in the tumor. This work demonstrates a workflow for researchers interested in engineering E. coli Nissle for a new class of microbial therapy against cancer.

Candida expansion in the gut of lung cancer patients associates with an ecological signature that supports growth under dysbiotic conditions.

Candida species overgrowth in the human gut is considered a prerequisite for invasive candidiasis, but our understanding of gut bacteria promoting or restricting this overgrowth is still limited. By integrating cross-sectional mycobiome and shotgun metagenomics data from the stool of 75 male and female cancer patients at risk but without systemic candidiasis, bacterial communities in high Candida samples display higher metabolic flexibility yet lower contributional diversity than those in low Candida samples. We develop machine learning models that use only bacterial taxa or functional relative abundances to predict the levels of Candida genus and species in an external validation cohort with an AUC of 78.6-81.1%. We propose a mechanism for intestinal Candida overgrowth based on an increase in lactate-producing bacteria, which coincides with a decrease in bacteria that regulate short chain fatty acid and oxygen levels. Under these conditions, the ability of Candida to harness lactate as a nutrient source may enable Candida to outcompete other fungi in the gut.

Biocontainment strategies for in vivo applications of Saccharomyces boulardii.

The human gastrointestinal tract is a complex and dynamic environment, playing a crucial role in human health. Microorganisms engineered to express a therapeutic activity have emerged as a novel modality to manage numerous diseases. Such advanced microbiome therapeutics (AMTs) must be contained within the treated individual. Hence safe and robust biocontainment strategies are required to prevent the proliferation of microbes outside the treated individual. Here we present the first biocontainment strategy for a probiotic yeast, demonstrating a multi-layered strategy combining an auxotrophic and environmental-sensitive strategy. We knocked out the genes THI6 and BTS1, causing thiamine auxotrophy and increased sensitivity to cold, respectively. The biocontained Saccharomyces boulardii showed restricted growth in the absence of thiamine above 1 ng/ml and exhibited a severe growth defect at temperatures below 20°C. The biocontained strain was well tolerated and viable in mice and demonstrated equal efficiency in peptide production as the ancestral non-biocontained strain. In combination, the data support that thi6∆ and bts1∆ enable biocontainment of S. boulardii, which could be a relevant chassis for future yeast-based AMTs.

Characterization of Eight Bacterial Biosensors for Microbial Diagnostic and Therapeutic Applications.

The engineering of microbial cells to produce and secrete therapeutics directly in the human body, known as advanced microbial therapeutics, is an exciting alternative to current drug delivery routes. These living therapeutics can be engineered to sense disease biomarkers and, in response, deliver a therapeutic activity. This strategy allows for precise and self-regulating delivery of a therapeutic that adapts to the disease state of the individual patient. Numerous sensing systems have been characterized for use in prokaryotes, but a very limited number of advanced microbial therapeutics have incorporated such sensors. We characterized eight different sensors that respond to physiologically relevant conditions and molecules found in the human body in the probiotic strain Escherichia coli Nissle 1917. The resulting sensors were characterized under aerobic and anaerobic conditions and were demonstrated to be functional under gut-like conditions using the nematode Caenorhabditis elegans as an in vivo model. We show for the first time how a biosensor is able to detect in vivo the bile acid-like molecule Δ4-dafachronic acid, a small molecule in C. elegans that regulates lifespan. Furthermore, we exemplify how bacterial sensors can be used to dynamically report on changes in the intestinal environment of C. elegans, by demonstrating the use of a biosensor able to detect changes in lactate concentrations in the gut lumen of individual C. elegans. The biosensors presented in this study allow for dynamic control of expression in vivo and represent a valuable tool in further developing advanced microbiome therapeutics.

Screening for effective cell-penetrating peptides with minimal impact on epithelial cells and gut commensals in vitro.

One of the biggest challenges for oral drug absorption is the epithelial barrier of the gastrointestinal tract. The use of cell-penetrating peptides (CPPs) to modulate the epithelial barrier function is known to be an effective strategy to improve drug absorption and bioavailability. In this study we compare side-by-side, 9 most promising CPPs to study their cytotoxicity (Cytotox Red dye staining) and cell viability (AlamarBlue staining) on epithelial cells and their effects on paracellular permeability of the intestinal barrier in vitro in a differentiated Caco-2 epithelial monolayer model. The data revealed that 4 out of 9 well-studied CPPs significantly improved Caco-2 paracellular permeability without compromising on cellular health. To assess the impact of CPPs on the human microbiota we studied the antimicrobial effects of the 4 effective CPPs from our permeation studies against 10 representative strains of the gut microbiota in vitro using microbroth dilution. Our data revealed that these 4 CPPs affected the growth of almost all tested commensal strains. Interestingly, we found that two synthetic CPPs (Shuffle and Penetramax) outperformed all the other CPPs in their ability to increase intestinal paracellular permeability at 50 µM and had only a small to moderate effect on the tested gut commensal strains. Based on these data Shuffle and Penetramax represent relevant CPPs to be further characterized in vivo for safe delivery of poorly absorbed therapeutics while minimizing negative impacts on the gut microbiota.

Effects of broad-spectrum antibiotics on the colonisation of probiotic yeast Saccharomyces boulardii in the murine gastrointestinal tract.

Mouse models are commonly used to study the colonisation profiles of microorganisms introduced to the gastrointestinal tract. Three commonly used mouse models include conventional, germ-free, and antibiotic-treated mice. However, colonisation resistance in conventional mice and specialised equipment for germ-free mice are usually limiting factors in their applications. In this study, we sought to establish a robust colonisation model for Saccharomyces boulardii, a probiotic yeast that has caught attention in the field of probiotics and advanced microbiome therapeutics. We characterised the colonisation of S. boulardii in conventional mice and mice treated with a cocktail of broad-spectrum antibiotics, including ampicillin, kanamycin, metronidazole and vancomycin. We found colonisation levels increased up to 10,000-fold in the antibiotic-treated mice compared to nonantibiotic-treated mice. Furthermore, S. boulardii was detected continuously in more than 75% of mice for 10 days after the last administration in antibiotic-treated mice, in contrast to in nonantibiotic-treated mice where S. boulardii was undetectable in less than 2 days. Finally, we demonstrated that this antibiotic cocktail can be used in two commonly used mouse strains, C57BL/6 and ob/ob mice, both achieving ~ 108 CFU/g of S. boulardii in faeces. These findings highlight that the antibiotic cocktail used in this study is an advantageous tool to study S. boulardii based probiotic and advanced microbiome therapeutics.

Identification and Optimization of Novel Small-Molecule Cas9 Inhibitors by Cell-Based High-Throughput Screening.

CRISPR/Cas9 has revolutionized several areas of life science; however, methods to control the Cas9 activity are needed for both scientific and therapeutic applications. Anti-CRISPR proteins are known to inhibit the CRISPR/Cas adaptive immunity; however, in vivo delivery of such proteins is problematic. Instead, small-molecule Cas9 inhibitors could serve as useful tools due to their permeable, proteolytically stable, and non-immunogenic nature. Here, we identified a small-molecule ligand with anti-CRISPR/Cas9 activity through a high-throughput screening utilizing an Escherichia coli selection system. Extensive structure-activity relationship studies, which involved a deconstruction-reconstruction strategy, resulted in a range of analogues with significant improvements in the inhibitory activity. Based on NMR and electrophoretic mobility shift assays, we propose that the inhibitory action of these compounds likely results from direct binding to apo-Cas9, preventing Cas9:gRNA complex formation. These molecules may find use as Cas9 modulators in various applications.

Escherichia coli Promoters with Consistent Expression throughout the Murine Gut.

Advanced microbial therapeutics have great potential as a novel modality to diagnose and treat a wide range of diseases. Yet, to realize this potential, robust parts for regulating gene expression and consequent therapeutic activity in situ are needed. In this study, we characterized the expression level of more than 8000 variants of the Escherichia coli sigma factor 70 (σ70) promoter in a range of different environmental conditions and growth states using fluorescence-activated cell sorting and deep sequencing. Sampled conditions include aerobic and anaerobic culture in the laboratory as well as growth in several locations of the murine gastrointestinal tract. We found that σ70 promoters in E. coli generally maintain consistent expression levels across the murine gut (R2: 0.55-0.85, p value < 1 × 10-5), suggesting a limited environmental influence but a higher variability between in vitro and in vivo expression levels, highlighting the challenges of translating in vitro promoter activity to in vivo applications. Based on these data, we design the Schantzetta library, composed of eight promoters spanning a wide expression range and displaying a high degree of robustness in both laboratory and in vivo conditions (R2 = 0.98, p = 0.000827). This study provides a systematic assessment of the σ70 promoter activity in E. coli as it transits the murine gut leading to the definition of robust expression cassettes that could be a valuable tool for reliable engineering and development of advanced microbial therapeutics.

Antibiotics create a shift from mutualism to competition in human gut communities with a longer-lasting impact on fungi than bacteria.

BACKGROUND: Antibiotic treatment has a well-established detrimental effect on the gut bacterial composition, but effects on the fungal community are less clear. Bacteria in the lumen of the gastrointestinal tract may limit fungal colonization and invasion. Antibiotic drugs targeting bacteria are therefore seen as an important risk factor for fungal infections and induced allergies. However, antibiotic effects on gut bacterial-fungal interactions, including disruption and resilience of fungal community compositions, were not investigated in humans. We analysed stool samples collected from 14 healthy human participants over 3 months following a 6-day antibiotic administration. We integrated data from shotgun metagenomics, metatranscriptomics, metabolomics, and fungal ITS2 sequencing. RESULTS: While the bacterial community recovered mostly over 3 months post treatment, the fungal community was shifted from mutualism at baseline to competition. Half of the bacterial-fungal interactions present before drug intervention had disappeared 3 months later. During treatment, fungal abundances were associated with the expression of bacterial genes with functions for cell growth and repair. By extending the metagenomic species approach, we revealed bacterial strains inhibiting the opportunistic fungal pathogen Candida albicans. We demonstrated in vitro how C. albicans pathogenicity and host cell damage might be controlled naturally in the human gut by bacterial metabolites such as propionate or 5-dodecenoate. CONCLUSIONS: We demonstrated that antibacterial drugs have long-term influence on the human gut mycobiome. While bacterial communities recovered mostly 30-days post antibacterial treatment, the fungal community was shifted from mutualism towards competition. Video abstract.

Predictable modulation of cancer treatment outcomes by the gut microbiota.

The gut microbiota has the potential to influence the efficacy of cancer therapy. Here, we investigated the contribution of the intestinal microbiome on treatment outcomes in a heterogeneous cohort that included multiple cancer types to identify microbes with a global impact on immune response. Human gut metagenomic analysis revealed that responder patients had significantly higher microbial diversity and different microbiota compositions compared to non-responders. A machine-learning model was developed and validated in an independent cohort to predict treatment outcomes based on gut microbiota composition and functional repertoires of responders and non-responders. Specific species, Bacteroides ovatus and Bacteroides xylanisolvens, were positively correlated with treatment outcomes. Oral gavage of these responder bacteria significantly increased the efficacy of erlotinib and induced the expression of CXCL9 and IFN-γ in a murine lung cancer model. These data suggest a predictable impact of specific constituents of the microbiota on tumor growth and cancer treatment outcomes with implications for both prognosis and therapy.