Dual engineered bacteria improve inflammatory bowel disease in mice.

Currently, there are many different therapies available for inflammatory bowel disease (IBD), including engineered live bacterial therapeutics. However, most of these studies focus on producing a single therapeutic drug using individual bacteria, which may cause inefficacy. The use of dual drugs can enhance therapeutic effects. However, expressing multiple therapeutic drugs in one bacterial chassis increases the burden on the bacterium and hinders good secretion and expression. Therefore, a dual-bacterial, dual-drug expression system allows for the introduction of two probiotic chassis and enhances both therapeutic and probiotic effects. In this study, we constructed a dual bacterial system to simultaneously neutralize pro-inflammatory factors and enhance the anti-inflammatory pathway. These bacteria for therapy consist of Escherichia coli Nissle 1917 that expressed and secreted anti-TNF-α nanobody and IL-10, respectively. The oral administration of genetically engineered bacteria led to a decrease in inflammatory cell infiltration in colon and a reduction in the levels of pro-inflammatory cytokines. Additionally, the administration of engineered bacteria did not markedly aggravate gut fibrosis and had a moderating effect on intestinal microbes. This system proposes a dual-engineered bacterial drug combination treatment therapy for inflammatory bowel disease, which provides a new approach to intervene and treat IBD. KEY POINTS: • The paper discusses the effects of using dual engineered bacteria on IBD • Prospects of engineered bacteria in the clinical treatment of IBD.

Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan.

Heparosan is a crucial-polysaccharide precursor for the chemoenzymatic synthesis of heparin, a widely used anticoagulant drug. Presently, heparosan is mainly extracted with the potential risk of contamination from Escherichia coli strain K5, a pathogenic bacterium causing urinary tract infection. Here, a nonpathogenic probiotic, E. coli strain Nissle 1917 (EcN), was metabolically engineered to carry multiple copies of the 19-kb kps locus and produce heparosan to 9.1 g/L in fed-batch fermentation. Chromosome evolution driven by antibiotics was employed to amplify the kps locus, which governed the synthesis and export of heparosan from EcN at 21 mg L-1 OD-1 . The average copy number of kps locus increased from 1 to 24 copies per cell, which produced up to 104 mg L-1 OD-1 of heparosan in the shaking flask cultures of engineered strains. The following in-frame deletion of recA stabilized the recombinant duplicates of chromosomal kps locus and the productivity of heparosan in continuous culture for at least 56 generations. Fed-batch fermentation of the engineered strain EcN8 was carried out to bring the yield of heparosan up to 9.1 g/L. Heparosan from the fermentation culture was further purified at a 75% overall recovery. The structure of purified heparosan was characterized and further modified by N-sulfotransferase with 3'-phosphoadenosine-5'-phosphosulfate as the sulfo-donor. The analysis of element composition showed that heparosan was N-sulfated by over 80%. These results indicated that duplicating large DNA cassettes up to 19-kb, followed by high-cell-density fermentation, was promising in the large-scale preparation of chemicals and could be adapted to engineer other industrial-interest bacteria metabolically.

Protective effects of E. coli Nissle 1917 on chickens infected with Salmonella pullorum.

The probiotic E. coli Nissle 1917 (EcN) plays an important role in regulating the microbial components of the gut and preventing inflammation of the gastrointestinal tract. Currently, the long-term use of antibiotics for the treatment of lethal white diarrhea in chicks caused by Salmonella has led to increased morbidity and mutation rates. Therefore, we want to use EcN as an antibiotic alternative as an alternative approach to prevent Salmonella-induced white diarrhea in chickens. To date, there are no reports of EcN being used for the prevention and control of Salmonella pullorum (S. pullorum) in chickens. In vitro, pretreatment with EcN significantly decreased the cellular invasion of S. pullorum CVCC533 in a chicken fibroblast (DF-1) cell model. Then, 0-day-old egg-laying chickens were orally inoculated with EcN at a dose of 109 CFU/100 µL at either Day 1 (EcN1) or both Day 1 and Day 4 (EcN2). Then, S. pullorum CVCC533 was used to challenge the cells at a dose of 1.0 × 107 CFU/100 µL on Day 8. Next, the body weights and survival rates were recorded for 14 consecutive days, and the colonization of S. pullorum in the spleen and liver at 7 days post-challenge (dpc) was determined. Chicken feces were also collected at 2, 4, 6 and 8 dpc to evaluate the excretion of pathogenic bacteria in feces. The liver, duodenum and rectum samples were collected and analyzed by pathological histology at 7 dpc to evaluate the protective effect of EcN on the mucosa, villi and crypts of the small intestine. The spleen and bursa were collected, and the immune organ index was calculated. In addition, the contents of the cecum of chicks were collected at 7 dpc for 16S rRNA sequencing to detect the distribution of microbial communities in the intestine. The results showed that EcN was able to protect against CVCC533 challenge, as shown by decreased body weight loss, mortality and shedding of pathogenic bacteria in fecal samples in the EcN1 plus Salmonella challenge group (EcN1S) but not the EcN2 plus Salmonella challenge group (EcN2S). The pathogenic changes in the liver, duodenum and rectum also demonstrated that one dose but not two doses of EcN effectively prolonged the length of the pilus with decreased crypt depth, indicating its protective effects against S. pullorum. In addition, the 16S rRNA sequencing results suggested that EcN could enlarge the diversity of intestinal flora, decrease the abundance of pathogenic bacteria and increase the abundance of beneficial bacteria, such as Lactobacillus. In conclusion, EcN has shown moderate protection against S. pullorum challenge in chickens.

High density fermentation of probiotic E. coli Nissle 1917 towards heparosan production, characterization, and modification.

Heparosan is a naturally occurring non-sulfated glycosaminoglycan. Heparosan serves as the substrate for chemoenzymatic synthesis of biopharmaceutically important heparan sulfate and heparin. Heparosan is biologically inert molecule, non-toxic, and non-immunogenic and these qualities of heparosan make it an ideal drug delivery vehicle. The critical-to-quality (CTQ) attributes for heparosan applications include composition of heparosan, absence of any unnatural moieties, and heparosan molecular weight size and unimodal distribution. Probiotic bacteria E. coli Nissle 1917 (EcN) is a natural producer of heparosan. The current work explores production of EcN heparosan and process parameters that may impact the heparosan CTQ attributes. Results show that EcN could be grown to high cell densities (OD600 160-180) in a chemically defined media. The fermentation process is successfully scaled from 5-L to 100-L bioreactor. The chemical composition of heparosan from EcN was confirmed using nuclear magnetic resonance. Results demonstrate that heparosan molecular weight distribution may be influenced by fermentation and purification conditions. Size exclusion chromatography analysis shows that the heparosan purified from fermentation broth results in bimodal distribution, and cell-free supernatant results in unimodal distribution (average molecular weight 68,000 Da). The yield of EcN-derived heparosan was 3 g/L of cell free supernatant. We further evaluated the application of Nissle 1917 heparosan for chemical modification to prepare N-sulfo heparosan (NSH), the first intermediate precursor for heparin and heparan sulfate. KEY POINTS: • High cell density fermentation, using a chemically defined fermentation media for the growth of probiotic bacteria EcN (E. coli Nissle 1917, a natural producer of heparosan) is reported. • Process parameters towards the production of monodispersed heparosan using probiotic bacteria EcN (Nissle 1917) has been explored and discussed. • The media composition and the protocol (SOPs and batch records) have been successfully transferred to contract manufacturing facilities and industrial partners.

Transcriptional Profiling of the Probiotic Escherichia coli Nissle 1917 Strain under Simulated Microgravity.

Long-term space missions affect the gut microbiome of astronauts, especially the viability of some pathogens. Probiotics may be an effective solution for the management of gut microbiomes, but there is a lack of studies regarding the physiology of probiotics in microgravity. Here, we investigated the effects of microgravity on the probiotic Escherichia coli Nissle 1917 (EcN) by comparing transcriptomic data during exponential and stationary growth phases under simulated microgravity and normal gravity. Microgravity conditions affected several physiological features of EcN, including its growth profile, biofilm formation, stress responses, metal ion transport/utilization, and response to carbon starvation. We found that some changes, such as decreased adhesion ability and acid resistance, may be disadvantageous to EcN relative to gut pathogens under microgravity, indicating the need to develop probiotics optimized for space flight.

Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK-cell responses.

Lactobacillus rhamnosus GG (LGG), a gram-positive lactic acid bacterium, is one of the most widely used probiotics; while fewer gram-negative probiotics including Escherichia coli Nissle 1917 (EcN) are characterized. A mechanistic understanding of their individual and interactive effects on human rotavirus (HRV) and immunity is lacking. In this study, noncolonized, EcN-, LGG-, and EcN + LGG-colonized neonatal gnotobiotic (Gn) pigs were challenged with HRV. EcN colonization is associated with a greater protection against HRV, and induces the highest frequencies of plasmacytoid dendritic cells (pDCs), significantly increased NK-cell function and decreased frequencies of apoptotic and TLR4+ mononuclear cells (MNCs). Consistent with the highest NK-cell activity, splenic CD172+ MNCs (DC enriched fraction) of EcN-colonized pigs produced the highest levels of IL-12 in vitro. LGG colonization has little effect on the above parameters, which are intermediate in EcN + LGG-colonized pigs, suggesting that probiotics modulate each other's effects. Additionally, in vitro EcN-treated splenic or intestinal MNCs produce higher levels of innate, immunoregulatory and immunostimulatory cytokines, IFN-α, IL-12, and IL-10, compared to MNCs of pigs treated with LGG. These results indicate that the EcN-mediated greater protection against HRV is associated with potent stimulation of the innate immune system and activation of the DC-IL-12-NK immune axis.

Genetically engineered Escherichia coli Nissle 1917 synbiotic counters fructose-induced metabolic syndrome and iron deficiency.

Consumption of fructose leads to metabolic syndrome, but it is also known to increase iron absorption. Present study investigates the effect of genetically modified Escherichia coli Nissle 1917 (EcN) synbiotic along with fructose on non-heme iron absorption. Charles foster rats weighing 150-200 g were fed with iron-deficient diet for 2 months. Probiotic treatment of EcN (pqq) and EcN (pqq-glf-mtlK) was given once per week, 109 cells after 2 months with fructose in drinking water. Iron levels, blood, and liver parameters for oxidative stress, hyperglycemia, and dyslipidemia were estimated. Transferrin-bound iron levels in the blood decreased significantly after 10 weeks of giving iron-deficient diet. Probiotic treatment of EcN (pqq-glf-mtlK) and fructose together led to the restoration of normal transferrin-bound iron levels and blood and hepatic antioxidant levels as compared to iron-deficient control group. The probiotic also led to the restoration of body weight along with levels of serum and hepatic lipid, blood glucose, and antioxidant in the blood and liver as compared to iron-deficient control group. Restoration of liver injury marker enzymes was also seen. Administration of EcN-producing PQQ and mannitol dehydrogenase enzyme together with fructose led to increase in the transferrin-bound iron levels in the blood and amelioration of consequences of metabolic syndrome caused due to fructose consumption.

Pyrroloquinoline quinone-secreting probiotic Escherichia coli Nissle 1917 ameliorates ethanol-induced oxidative damage and hyperlipidemia in rats.

BACKGROUND: Chronic ethanol (EtOH) consumption is associated with oxidative tissue damage, decrease in antioxidant enzyme activities, and increase in hepatic and plasma lipids. This study investigates the effect of modified probiotic Escherichia coli Nissle 1917 (EcN) secreting pyrroloquinoline quinone (PQQ) against EtOH-induced metabolic disorder in rats. METHODS: Male Charles Foster rats were gavaged with EtOH (5 g/kg body weight [acute study] and 3 g/kg body weight per day for 10 weeks [chronic study]). RESULTS: Pretreatment of PQQ, vitamin C, and PQQ-secreting EcN prevented acute EtOH-induced oxidative damage in rats reflected by reduced lipid peroxidation in blood and liver and increased hepatic reduced glutathione. However, PQQ given externally was found to be most effective against acute EtOH toxicity. In the chronic study, rats treated with PQQ-secreting EcN showed remarkable reduction in oxidative tissue damage (liver, colon, blood, and kidney) with significant increase in antioxidant enzyme activities as compared to only EtOH-treated rats. Additionally, these rats had significantly lowered hepatic and plasma lipid levels with concomitant reduction in mRNA expression of fatty acid synthase (0.5-fold) and increase in mRNA expression of acyl coenzyme A oxidase (2.4-fold) in hepatic tissue. Antioxidant and hyperlipidemic effects of PQQ-secreting EcN are correlated with increased colonic short chain fatty acids (SCFAs; i.e., acetate, propionate, and butyrate) levels, and PQQ concentration in fecal samples (2-fold) and liver (4-fold). Extracted PQQ and vitamin C were given once a week, but they did not exhibit any ameliorative effect against chronic EtOH toxicity. CONCLUSIONS: Accumulated PQQ in tissues prevents hepatic and systemic oxidative damage. PQQ along with SCFAs reduced hyperlipidemia, which can be correlated with changes in mRNA expression of hepatic lipid metabolizing genes. Our study suggests that endogenous generation of PQQ by EcN could be an effective strategy in preventing alcoholic liver disease.

Deciphering the Crucial Roles of the Quorum-Sensing Transcription Factor SdiA in NADPH Metabolism and (S)-Equol Production in Escherichia coli Nissle 1917.

The active metabolite (S)-equol, derived from daidzein by gut microbiota, exhibits superior antioxidative activity compared with its precursor and plays a vital role in human health. As only 25% to 50% of individuals can naturally produce equol when supplied with isoflavone, we engineered probiotic E. coli Nissle 1917 (EcN) to convert dietary isoflavones into (S)-equol, thus offering a strategy to mimic the gut phenotype of natural (S)-equol producers. However, co-fermentation of EcN-eq with fecal bacteria revealed that gut microbial metabolites decreased NADPH levels, hindering (S)-equol production. Transcriptome analysis showed that the quorum-sensing (QS) transcription factor SdiA negatively regulates NADPH levels and (S)-equol biosynthesis in EcN-eq. Screening AHLs showed that SdiA binding to C10-HSL negatively regulates the pentose phosphate pathway, reducing intracellular NADPH levels in EcN-eq. Molecular docking and dynamics simulations investigated the structural disparities in complexes formed by C10-HSL with SdiA from EcN or E. coli K12. Substituting sdiA_EcN in EcN-eq with sdiA_K12 increased the intracellular NADPH/NADP+ ratio, enhancing (S)-equol production by 47%. These findings elucidate the impact of AHL-QS in the gut microbiota on EcN NADPH metabolism, offering insights for developing (S)-equol-producing EcN probiotics tailored to the gut environment.

Minimizing endogenous cryptic plasmids to construct antibiotic-free expression systems for Escherichia coli Nissle 1917.

The probiotic bacterium Escherichia coli Nissle 1917 (EcN) holds significant promise for use in clinical and biological industries. However, the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its benefits. In this study, we addressed this issue by engineering the endogenous cryptic plasmids pMUT1 and pMUT2. The non-essential elements were removed to create more stable derivatives pMUT1NR△ and pMUT2HBC△. Synthetic promoters by integrating binding motifs on sigma factors were further constructed and applied for expression of Bacteroides thetaiotaomicron heparinase III and the biosynthesis of ectoine. Compared to traditional antibiotic-dependent expression systems, our newly constructed antibiotic-free expression systems offer considerable advantages for clinical and synthetic biology applications.

Engineering a Novel Probiotic Toolkit in Escherichia coli Nissle 1917 for Sensing and Mitigating Gut Inflammatory Diseases.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation with no cure and limited treatment options that often have systemic side effects. In this study, we developed a target-specific system to potentially treat IBD by engineering the probiotic bacterium Escherichia coli Nissle 1917 (EcN). Our modular system comprises three components: a transcription factor-based sensor (NorR) capable of detecting the inflammation biomarker nitric oxide (NO), a type 1 hemolysin secretion system, and a therapeutic cargo consisting of a library of humanized anti-TNFα nanobodies. Despite a reduction in sensitivity, our system demonstrated a concentration-dependent response to NO, successfully secreting functional nanobodies with binding affinities comparable to the commonly used drug Adalimumab, as confirmed by enzyme-linked immunosorbent assay and in vitro assays. This newly validated nanobody library expands EcN therapeutic capabilities. The adopted secretion system, also characterized for the first time in EcN, can be further adapted as a platform for screening and purifying proteins of interest. Additionally, we provided a mathematical framework to assess critical parameters in engineering probiotic systems, including the production and diffusion of relevant molecules, bacterial colonization rates, and particle interactions. This integrated approach expands the synthetic biology toolbox for EcN-based therapies, providing novel parts, circuits, and a model for tunable responses at inflammatory hotspots.

Production of Reactive Oxygen (ROS) and Nitrogen (RNS) Species in Macrophages J774A.1 Activated by the Interaction between Two Escherichia coli Pathotypes and Two Probiotic Commercial Strains.

Probiotics play an important role against infectious pathogens, such as Escherichia coli (E. coli), mainly through the production of antimicrobial compounds and their immunomodulatory effect. This protection can be detected both on the live probiotic microorganisms and in their inactive forms (paraprobiotics). Probiotics may affect different cells involved in immunity, such as macrophages. Macrophages are activated through contact with microorganisms or their products (lipopolysaccharides, endotoxins or cell walls). The aim of this work was the evaluation of the effect of two probiotic bacteria (Escherichia coli Nissle 1917 and Bifidobacterium animalis subsp. lactis HN019 on macrophage cell line J774A.1 when challenged with two pathogenic strains of E. coli. Macrophage activation was revealed through the detection of reactive oxygen (ROS) and nitrogen (RNS) species by flow cytometry. The effect varied depending on the kind of probiotic preparation (immunobiotic, paraprobiotic or postbiotic) and on the strain of E. coli (enterohemorrhagic or enteropathogenic). A clear immunomodulatory effect was observed in all cases. A higher production of ROS compared with RNS was also observed.

Neuroprotective effects of an engineered Escherichia coli Nissle 1917 on Parkinson's disease in mice by delivering GLP-1 and modulating gut microbiota.

Considerable evidence suggests that insulin resistance is closely linked to Parkinson's disease (PD), leading to agents aiming at treating diabetes can be regarded as new neuroprotective strategies in PD, notably glucagon-like peptide-1 (GLP-1). However, the extremely short half-life of GLP-1 due to degradation by the ubiquitous proteolytic enzyme limits its clinical application. In this study, we engineered the recombinant integrant probiotic strain Escherichia coli Nissle 1917 (EcN) to create a strain EcN-GLP-1 that effectively delivers the heterologous GLP-1 molecule. Subsequently, we assessed its neuroprotective effects on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD mice. We demonstrated that EcN-GLP-1 treatment could improve motor deficits, increase tyrosine hydroxylase-positive neurons, suppress microglia and astrocyte activation, reduce brain and colon inflammation, and ameliorate colonic barrier function damaged by MPTP induction. Meanwhile, we confirmed that the oral administration of EcN-GLP-1 could restore the disturbance of gut microbiota in the MPTP-induced PD mice, by reducing the relative abundances of Akkermansia and Oscillospira, and increasing the level of Prevotella in the gut. These results support further development of an engineered probiotic platform in which production of GLP-1 for gut-brain disorders, such as PD.

Engineered living materials for the conversion of a low-cost food-grade precursor to a high-value flavonoid.

Microbial biofactories allow the upscaled production of high-value compounds in biotechnological processes. This is particularly advantageous for compounds like flavonoids that promote better health through their antioxidant, anti-bacterial, anti-cancer and other beneficial effects but are produced in small quantities in their natural plant-based hosts. Bacteria like E. coli have been genetically modified with enzyme cascades to produce flavonoids like naringenin and pinocembrin from coumaric or cinnamic acid. Despite advancements in yield optimization, the production of these compounds still involves high costs associated with their biosynthesis, purification, storage and transport. An alternative production strategy could involve the direct delivery of the microbial biofactories to the body. In such a strategy, ensuring biocontainment of the engineered microbes in the body and controlling production rates are major challenges. In this study, these two aspects are addressed by developing engineered living materials (ELMs) consisting of probiotic microbial biofactories encapsulated in biocompatible hydrogels. Engineered probiotic E. coli Nissle 1917 able to efficiently convert cinnamic acid into pinocembrin were encapsulated in poly(vinyl alcohol)-based hydrogels. The biofactories are contained in the hydrogels for a month and remain metabolically active during this time. Control over production levels is achieved by the containment inside the material, which regulates bacteria growth, and by the amount of cinnamic acid in the medium.

Recombinant production of a diffusible signal factor inhibits Salmonella invasion and animal carriage.

The complex chemical environment of the intestine is defined largely by the metabolic products of the resident microbiota. Enteric pathogens, elegantly evolved to thrive in the gut, use these chemical products as signals to recognize specific niches and to promote their survival and virulence. Our previous work has shown that a specific class of quorum-sensing molecules found within the gut, termed diffusible signal factors (DSF), signals the repression of Salmonella tissue invasion, thus defining a means by which this pathogen recognizes its location and modulates virulence to optimize its survival. Here, we determined whether the recombinant production of a DSF could reduce Salmonella virulence in vitro and in vivo. We found that the most potent repressor of Salmonella invasion, cis-2-hexadecenoic acid (c2-HDA), could be recombinantly produced in E. coli by the addition of a single exogenous gene encoding a fatty acid enoyl-CoA dehydratase/thioesterase and that co-culture of the recombinant strain with Salmonella potently inhibited tissue invasion by repressing Salmonella genes required for this essential virulence function. Using the well characterized E. coli Nissle 1917 strain and a chicken infection model, we found that the recombinant DSF-producing strain could be stably maintained in the large intestine. Further, challenge studies demonstrated that this recombinant organism could significantly reduce Salmonella colonization of the cecum, the site of carriage in this animal species. These findings thus describe a plausible means by which Salmonella virulence may be affected in animals by in situ chemical manipulation of functions essential for colonization and virulence.

Despite our best efforts, infections of agricultural animals with Salmonella persist, posing threats to food safety. Few, if any, measures have proven effective in reducing Salmonella carriage in animals used for food, a major source of this pathogen. Antibiotics are ineffective at curtailing infection and have served only to exacerbate the global crisis of antimicrobial resistance. The alternative then is to seek novel means to reduce Salmonella disease and carriage by preventing its colonization of livestock and poultry. Here we describe an approach targeting invasion, a function essential for Salmonella carriage and disease in animals. We show that a potent chemical inhibitor of invasion, the diffusible signal factor cis-2 hexadecenoic acid, can be produced by recombinant E. coli strains capable of stably colonizing the animal intestine, providing a means to directly affect the virulence of Salmonella within an animal host. These studies may thus provide a route to reduce the carriage of this pathogen in production animals and thus the spread of disease to humans.

E. coli Nissle 1917 ameliorates mitochondrial injury of granulosa cells in polycystic ovary syndrome through promoting gut immune factor IL-22 via gut microbiota and microbial metabolism.

Objective: Gut microbiota and its metabolites have regulatory effects on PCOS related ovarian dysfunction and insulin resistance. Escherichia coli Nissle 1917 (EcN) is a genetically controlled probiotic with an excellent human safety record for improving gut microbiome metabolic disorders and immune system disorders. Here we focused to explore the application and effect of probiotic EcN on the gut microbiota-metabolism-IL-22-mitochondrial damage axis in PCOS. Methods: PCOS mice were constructed with dehydroepiandrosterone (DHEA) and treated with EcN, FMT or IL-22 inhibitors. Clinically control and PCOS subjects were included for further analysis. Serum and follicular fluid supernatant levels of sex hormones, insulin, glucose, cholesterol, and inflammatory factors were detected by ELISA and biochemical reagents. The pathological changes of ovarian tissues were observed by HE staining. The JC-1 level and COX4 gene expression in granulosa cells was detected by ELISA and RT-qPCR. The expressions of progesterone receptor A (PR-A), LC3II/I, Beclin1, p62 and CytC were detected by western blot. The number of autophagosomes in granulosa cells was observed by electron microscopy. 16S rRNA and LC-MS/MS were used to analyze the changes of gut microbiota and metabolism. Results: EcN promoted the recovery of sex hormone levels and ovarian tissue morphology, promoted the expression of IL-22, COX4 and PR-A in granulosa cells, and inhibited mitophagy in PCOS mice. EcN decreased the number of gut microbiota, and significantly increased the abundance of Adlercreutzia, Allobaculum, Escherichia-Shigella and Ileibacterium in PCOS mice. EcN improved metabolic disorders in PCOS mice by improving Amino sugar and nucleotide sugar metabolism pathways. IL-22 was positively associated with Ileibacterium, Adlercreutzia and Progesterone, negatively associated with RF39, Luteinizing hormone, Testosterone, N-Acetylglucosamin, L-Fucose and N-Acetylmannosamin. FMT reconfirmed that EcN ameliorated mitochondrial damage in granulosa cells of PCOS mice by gut microbiota, but this process was blocked by IL-22 inhibitor. Clinical trials have further demonstrated reduced IL-22 levels and mitochondrial damage in granulosa cells in PCOS patients. Conclusion: EcN improved IL-22 level and mitochondrial damage of granulosa cells in PCOS mice by promoting the recovery of sex hormone levels and ovarian tissue morphology, inhibiting the amount of gut microbiota, and promoting amino sugar and nucleotide sugar metabolism.

Microbiota-Derived Extracellular Vesicles Promote Immunity and Intestinal Maturation in Suckling Rats.

Microbiota-host communication is primarily achieved by secreted factors that can penetrate the mucosal surface, such as extracellular membrane vesicles (EVs). The EVs released by the gut microbiota have been extensively studied in cellular and experimental models of human diseases. However, little is known about their in vivo effects in early life, specifically regarding immune and intestinal maturation. This study aimed to investigate the effects of daily administration of EVs from probiotic and commensal E. coli strains in healthy suckling rats during the first 16 days of life. On days 8 and 16, we assessed various intestinal and systemic variables in relation to animal growth, humoral and cellular immunity, epithelial barrier maturation, and intestinal architecture. On day 16, animals given probiotic/microbiota EVs exhibited higher levels of plasma IgG, IgA, and IgM and a greater proportion of Tc, NK, and NKT cells in the spleen. In the small intestine, EVs increased the villi area and modulated the expression of genes related to immune function, inflammation, and intestinal permeability, shifting towards an anti-inflammatory and barrier protective profile from day 8. In conclusion, interventions involving probiotic/microbiota EVs may represent a safe postbiotic strategy to stimulate immunity and intestinal maturation in early life.

Advances in Escherichia coli Nissle 1917 as a customizable drug delivery system for disease treatment and diagnosis strategies.

With the in-depth and comprehensive study of bacteria and their related ecosystems in the human body, bacterial-based drug delivery system has become an emerging biomimetic platform that can retain the innate biological functions. Benefiting from its good biocompatibility and ideal targeting ability as a biological carrier, Escherichia coli Nissle 1917 (ECN) has been focused on the treatment strategies of inflammatory bowel disease and tumor. The advantage of a bacterial carrier is that it can express exogenous protein while also acting as a natural capsule by releasing drug slowly as a result of its own colonization impact. In order to survive in harsh environments such as the digestive tract and tumor microenvironment, ECN can be modified or genetically engineered to enhance its function and host adaptability. The adoption of ECN carries or expresses drugs which are essential for accurate diagnosis and treatment. This review briefly describes the properties of ECN, the relationship between ECN and inflammation and tumor, and the strategy of using surface modification and genetic engineering to modify ECN as a delivery carrier for disease treatment.

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 5-HT producing gut probiotic improves gastrointestinal motility and behavior disorder.

Slow transit constipation is an intractable constipation with unknown aetiology and uncertain pathogenesis. The gut microbiota maintains a symbiotic relationship with the host and has an impact on host metabolism. Previous studies have reported that some gut microbes have the ability to produce 5-hydroxytryptamine (5-HT), an important neurotransmitter. However, there are scarce data exploiting the effects of gut microbiota-derived 5-HT in constipation-related disease. We genetically engineered the probiotic Escherichia coli Nissle 1917 (EcN-5-HT) for synthesizing 5-HT in situ. The ability of EcN-5-HT to secrete 5-HT in vitro and in vivo was confirmed. Then, we examined the effects of EcN-5-HT on intestinal motility in a loperamide-induced constipation mouse model. After two weeks of EcN-5-HT oral gavage, the constipation-related symptoms were relieved and gastrointestinal motility were enhanced. Meanwhile, administration of EcN-5-HT alleviated the constipation related depressive-like behaviors. We also observed improved microbiota composition during EcN-5-HT treatment. This work suggests that gut microbiota-derived 5-HT might promise a potential therapeutic strategy for constipation and related behavioral disorders.