Compositions and methods for modifying bile acids to regulate lipid and steroid metabolism - Patents.com

Abstract

Provided herein are methods and compositions for the treatment or prevention of metabolic disorders.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 288,980, filed December 13, 2021, U.S. Provisional Patent Application No. 63 / 289,412, filed December 14, 2021, and U.S. Provisional Patent Application No. 63 / 355,381, filed June 24, 2022, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Metabolic disorders are becoming an increasingly large global health problem due to their dramatic increase in prevalence. Metabolic disorders are associated with alterations in the composition and function of the gut microbiota. The gut microbiota can interact with the host by generating a diverse reservoir of metabolites from exogenous food substrates or endogenous host compounds. Certain classes of microbiota-derived metabolites, particularly bile acids, short-chain fatty acids, branched-chain amino acids, trimethylamine N-oxide, tryptophan, and indole derivatives, are involved in the development of metabolic disorders. Considerable efforts have been made to understand the mechanisms of metabolic disorders. Currently, it remains unclear which bacterial strains and by what mechanisms regulate bile acids and bile salts in the gut. Summary of the Invention [Problem to be solved by the invention]

[0003] There is an unmet need to identify gut bacterial strains that regulate bile acids and bile salts and to develop microbial therapeutics to treat metabolic disorders. [Means for solving the problem]

[0004] (Abstract) Methods and compositions are provided herein for regulating bile salts or bile acids by administering a composition (e.g., a composition disclosed herein) to a subject. In certain embodiments, the methods and compositions are for treating or preventing metabolic disorders in a subject (e.g., a subject with a lipid metabolism disorder, such as hyperlipidemia, hypercholesterolemia, acid lipase disease, Barth syndrome, Fabry disease, Farber disease, Gaucher disease, Niemann-Pick disease, or Tay-Sachs disease; or a steroid metabolism disorder, such as cytochrome p450 oxidoreductase deficiency, apparent mineralocorticoid excess, lipoid congenital adrenal hyperplasia, congenital bile acid synthesis deficiency, 3-beta-hydroxysteroid dehydrogenase deficiency, aldosterone-producing adenoma, polyendocrinopathy, or adrenal hyperplasia).

[0005] In some aspects, provided herein are methods for preventing or treating a metabolic disorder (e.g., a lipid metabolism disorder and / or a steroid metabolism disorder) in a subject, the method comprising administering to the subject a composition comprising a bacterial strain (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) that expresses a bile salt regulatory gene or a bile acid regulatory gene.

[0006] Also provided herein are methods of treating or preventing a metabolic disorder in a subject by depleting the subject's gut microbiota (e.g., by administering an antibiotic to the subject) and administering a composition comprising a bacterial strain that expresses a bile salt regulatory gene or a bile acid regulatory gene (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron).

[0007] Provided herein are bacterial strains (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) that contain a bile salt regulatory gene or a bile acid regulatory gene, which have been genetically engineered to express the bile salt regulatory gene or the bile acid regulatory gene. Similarly, provided herein are bacterial strains (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) that express a bile salt regulatory gene or a bile acid regulatory gene encoded by an exogenous nucleic acid, e.g., a plasmid or other vector, where the bile salt regulatory gene or the bile acid regulatory gene is operably linked to a promoter that drives expression (e.g., constitutive or inducible) of the bile salt regulatory gene or the bile acid regulatory gene in the bacterial strain.

[0008] Also provided herein are compositions, e.g., compositions comprising a bacterial strain described herein and a pharma- ceutically acceptable carrier; or compositions comprising a bacterial strain (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) expressing a bile salt regulatory gene or a bile acid regulatory gene (e.g., as described herein) and a pharma- ceutically acceptable carrier.

[0009] The composition may be formulated for oral or rectal delivery. The composition may be self-administered. The composition may be a food or beverage product. In some embodiments, the food is a dairy product (e.g., yogurt or kefir). In some embodiments, the composition comprises a probiotic. In some embodiments, the composition comprises a fecal sample (e.g., a fecal sample from a fecal bank) that includes a bacterial strain (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) that expresses a bile salt regulatory gene or a bile acid regulatory gene.

[0010] Provided herein are methods of making the bacterial strains described herein, comprising transforming the bacterial strain with a gene expression construct encoding a bile salt- or bile acid-regulated gene operably linked to a promoter that drives expression (e.g., constitutive or inducible) of the bile salt- or bile acid-regulated gene in the bacterial strain. In certain embodiments, the method further comprises formulating the bacterial strain for administration to a subject, e.g., in a pharmaceutical composition or in a food or beverage product. In some embodiments, the method further comprises culturing the bacterial strain to allow expression of the bile salt- or bile acid-regulated gene. [Brief description of the drawings]

[0011] [Figure 1] Genomic comparison reveals distinct subgroups of Turicibacter sanguinis. A: Phylogenetic tree comparing full-length 16S rRNA sequences from the indicated T. sanguinis isolates. Circles indicate human isolates, triangles indicate mouse isolates, and squares indicate contaminating mouse isolates. B: Complete shotgun-assembled genome comparison of the T. sanguinis isolates listed in A. [Diagram 2] Figure 1: T. sanguinis isolates differ in their bile modifying capabilities. A: Liquid chromatograms of individual T. sanguinis isolates grown for 24 hours in medium with sub-inhibitory concentrations of five bile salts / acids: taurocholic acid (TCA), cholic acid (CA), glycochenodeoxycholic acid (GCDCA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA). Shaded areas indicate the expected retention time of each bile species. B: Relative amounts of conjugated bile salts (tauricholic acid, taurochenodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid) remaining after 24 hours of growth of the indicated T. sanguinis isolates. Magenta = glycine conjugated bile salts, blue = taurine conjugated bile salts. [Diagram 3]Figure 1. T. sanguinis isolates differ in their genetic ability to modify bile species. A: Liquid chromatograms of medium after 24 h incubation of E. coli expressing individual predicted bile salt hydrolases (BSH) from each sequence grouping, isolated, and grown with TCA and TCDCA. Control is E. coli with the same expression vector but expressing non-bile modifying genes. B: Same as A, but with GCA and GCDCA instead of taurine-conjugated bile salts. C. Amounts of TCA, TCDCA, GCA, GCDCA remaining after 24 h growth of E. coli expressing the indicated BSH homologs. D: Phylogenetic tree of each predicted BSH gene from T. sanguinis isolates showing bile salt specificity in boxes. No bile salt hydrolase activity was detected in unboxed sequences in this study. [Figure 4] Figure 1: Expression of T. sanguinis BSH is sufficient to alter the host lipidome and health-related lipid markers. A: Heatmap of plasma lipid species significantly altered by expression of at least one T. sanguinis BSH in Bacteroides thetaiotaomicron. Colors on the left correspond to lipid classes, and the cyan-magenta color scale represents Z-scores. B. Relative white adipose tissue weight in mice monocolonized with BSH-expressing B. thetatiotaomicron. C. Relative mixed plasma triglycerides (TG) in mice monocolonized with BSH-expressing B. thetatiotaomicron. D. Relative cholesterol esters (CE) in mice monocolonized with BSH-expressing B. thetatiotaomicron. All values ​​normalized to sex-matched littermates. *=p<0.05 two-tailed t-test. BSH3 corresponds to BSH-IV-MOL361, BSH4 corresponds to BSH-I-MOL361, BSH5 corresponds to BSH-III-1E2, and BSH7 corresponds to BSH-II-H121. [Diagram 5]Figure 1: T. sanguinis isolates differ in their effects on host lipid biology and bile acids. A: Relative abdominal fat pad mass from gender- and litter-matched mice monocolonized with individual T. sanguinis isolates. B: Top: Example images of histological sections of adipose tissue. Bottom: Relative adipocyte size from gender- and litter-matched mice monocolonized with individual T. sanguinis isolates. [Figure 6-1] Figure 2 shows circulating serum bile acid and cholesterol levels in mice monocolonized with individual T. sanguinis isolates. #=p<0.1, *=p<0.05. [Figure 6-2] Figure 2 shows circulating serum bile acid and cholesterol levels in mice monocolonized with individual T. sanguinis isolates. #=p<0.1, *=p<0.05. [Figure 7] Genome comparison reveals distinct subgroups of Turicibacter. a, Phylogenetic tree comparing full-length 16S rRNA gene sequences from the indicated Turicibacter isolates. Circles indicate isolates of human origin, triangles indicate isolates of mouse origin, and squares indicate contaminating isolates of mouse origin. b, Association between guanine-cytosine % (GC%) and calculated genome size in megabases (Mb) for shotgun assembled genomes of Turicibacter isolates from a. c. Whole-genome sequence comparison across Turicibacter strains. Positions of predicted bile modifying gene homologs are shown outside the rings, and the color of the gene name indicates the genome family in which the gene is found. Each ring represents one genome's sequence block. d, Average nucleotide identity (ANI) between the indicated Turicibacter genomes. Numbers indicate ANI, with the white to blue scale representing an ANI scale of 100% to 75%. [Figure 8]Figure 2: Turicibacter colonization alters host lipids in a strain-dependent manner. a, Heatmap of relative abundance of serum lipids from gnotobiotic mice monocolonized with the indicated Turicibacter strains. Heatmap values ​​represent the mean abundance of each detected lipid species from the labeled lipid category scaled across all of its individual lipid species' mean values. Black (p<0.05) and grey (p<0.1) rectangles indicate statistically significant differences of that metabolite between i) GF and MOL361 monocolonized mice, ii) CONV and MOL361 monocolonized mice, and iii) mice colonized with different Turicibacter strains. b, Serum cholesterol concentrations of mice colonized with the indicated Turicibacter strains. c, Relative epididymal / gonadal white adipose tissue (e / g WAT) mass of sex- and litter-matched mice monocolonized with the indicated Turicibacter strains. Figures show values ​​for individual mice, dotted bars represent pooled ANOVA statistics for each group relative to the experimental mean. Metabolite and cholesterol analyses n=6-10; WAT analyses n=6-26; Mann-Whitney test for comparisons of MOL361-GF and MOL361-CONV, Kruskal-Wallis for comparisons within Turicibacter. *p<0.05, ***p<0.0005. [Figure 9]Figure 2: Turicibacter colonization alters circulating host bile species in a strain-specific manner. a-c, Serum concentrations of: a-d) primary unconjugated bile acids, e-f) secondary unconjugated bile acids, or g-l) primary conjugated bile acids; serum levels of individual bile species from mice colonized with the indicated Turicibacter strains. Points show log-transformed values ​​for individual mice, shapes and colors match those in Figure 1, error bars represent mean + / - SEM. For a-f, Kruskal-Wallis test across all indicated colonizations with Dunn's multiple comparisons against GF. For g-l, Kruskal-Wallis test between indicated Turicibacter strains and multiple comparisons against H121. For g-l, Mann-Whitney test was used to compare GF and MOL361, p values ​​are shown above GF data points. n=6-10 for each group. Dotted bars represent ANOVA statistics for each group against pooled experimental means. *p<0.05, **p<0.005, ***p<0.0005. [Figure 10] Figure 7: Turicibacter isolates differ in their bile modification capabilities. a, Schematic of the types of bile transformations found to be performed by Turicibacter isolates. b, Inset: 16S rRNA-based phylogenetic tree from Figure 7a. Liquid chromatograms of individual Turicibacter isolates grown for 24 h in medium with subinhibitory concentrations of five bile acids: taurocholic acid (TCA), cholic acid (CA), glycochenodeoxycholic acid (GCDCA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA). Shaded areas indicate the expected retention time of each bile species. c, Percentage of remaining conjugated bile acids (TCA, taurochenodeoxycholic acid [TCDCA], glycocholic acid [GCA], GCDCA) after 24 h of growth with the indicated Turicibacter isolates (compared to cultures at time = 0). Yellow = glycine-conjugated bile acids, orange = taurine-conjugated bile acids. n=4 independent cultures. Values ​​not shown were <0.1% survival. Statistical analysis was performed by one-sample t-test and legend annotations indicate strains that differed significantly from 100% survival for each bile acid. [Figure 11]Turicibacter isolates differ in their genetic ability to modify bile species. a, Phylogenetic tree of amino acid sequences for each predicted bile salt hydrolase (BSH) sequence from Turicibacter isolates, with the observed bile species specificity shown in boxes. We did not detect bile salt hydrolase activity in unboxed sequences, representing groups V-VIII. b, Presence or absence of sequence homologs with potential BSH activity in Turicibacter strains. c, Liquid chromatograms of medium after 24 h incubation of E. coli expressing individual bsh genes predicted from each sequence grouping and grown with TCA and TCDCA. Control is E. coli with the same expression vector but expressing non-bile modifying genes. d, Same as c, but with GCA and GCDCA instead of tauro-bile acids. e, Quantification of survival (relative to medium control) of conjugated bile acids (TCA, TCDCA, GCA, GCDCA) after 24 h growth with E. coli expressing the indicated Turicibacter bsh genes. n=3 independent cultures, *p<0.05, **p<0.005, ***p<0.0005 using one-sample t-test comparisons with 100% survival. BSH nomenclature indicates homology group (e.g., III) and original isolate (e.g., MOL361). [Figure 12]Figure 1: Expression of Turicibacter bsh is sufficient to alter the host lipidome and health-related lipid markers. a, Percent survival of the indicated bile acids after 24 h growth with Bacteroides thetaiotaomicron expressing the indicated bsh genes; n=4 cultures per strain. b, Same as a but grown for 48 h with the indicated B. thetaiotaomicron strains; n=3 cultures per strain. For a and b, points represent individual comparisons with media control and legend annotations indicate strains with statistical significance for each bile acid using one-sample t-test comparisons at 100% survival. c, Quantification of colonic bile acids (BA) from mice colonized with bsh-expressing B. thetaiotaomicron. Values ​​were normalized to sex-matched littermates colonized with wild-type B. thetaiotaomicron. Statistical analysis was performed by Kruskal-Wallis test with Dunn's multiple comparison test; n=3-4. d, Heatmap of circulating lipid species significantly altered by expression of at least one Turicibacter bsh in B. thetaiotaomicron. Colors on the left correspond to lipid classes, the cyan-magenta color scale represents Z-scores, and each column represents one animal. e–i, Relative integrated circulating concentrations of e, triglycerides (TG), f, cholesterol esters (CE), g, diacylglycerides (DG), h, phosphotidylglycine (PG), or i, phosphotidylserine (PS) in mice monocolonized with bsh-expressing B. thetaiotaomicron. j, Relative white adipose tissue weight in mice monocolonized with bsh-expressing B. thetaiotaomicron. All values ​​were normalized to sex-matched littermates. Animals n = 4–6 colonization-1. Statistical analysis was performed by Welch's ANOVA with Dunnett's multiple comparisons against GF, and dotted bars in c-j represent ANOVA statistics for each group against the pooled experimental mean. *p<0.05, **p<0.005, ***p<0.0005. [Figure 13]Figure 1: Strain-dependent changes in colonization and adipocyte size. a-e, Representative images of adipose tissue from mice monocolonized with Turicibacter. f, Relative adipocyte area of ​​sex- and litter-matched mice monocolonized with individual Turicibacter strains. Each dot represents the average of 10 images of adipocyte area per animal. g, h, Colony-forming unit (CFU) equivalents per gram of distal small intestine (g) or proximal colon (h) contents. Each dot represents a sample from one animal, n=10-15 for qPCR and n=10-14 for adipocyte area calculations per colonization. Statistics for adipocyte area were performed by Welch's ANOVA with Dunnett's multiple comparisons, and dotted bars represent ANOVA statistics for each group relative to the pooled experimental mean. [Figure 14] Figure 2. Host effects of Turicibacter colonization vary by sex. a-c, Heatmaps of mean relative a) bile species, b) sterols or c) lipids. Column labels represent colonization status and sex. Blue = male, red = female. n = 3-6 / group, except for female T129 (n = 1). [Figure 15] Figure 10b shows a table summarizing the bile transformations performed by Turicibacter isolates. Table showing the presence / absence of the indicated bile species after growth in the mixtures of bile acids listed in Figure 10b. [Figure 16]Figure 2: Several strains of Turicibacter encode functional 7alpha-HSDH genes. a, Table showing amino acid similarity and sequence coverage between the closest predicted 7alpha-HSDH homologs in the genomes of the indicated isolates and the 7alpha-HSDH gene from Clostridium absonum. b, Chromatograms of cholic acid (CA) or cholic acid with two hydrogens removed (CA-2H) from E. coli cultures (cell control) expressing the MOL361 HSDH homolog (7alpha-HSDH) or non-bile modified gene sequences on the same plasmid. Chromatograms from triplicate independent cultures are shown, with dotted boxes indicating each bile species. c, Quantification of CA / CA-2H ratios determined from the reconstructed area under the curve in b. Statistical comparisons performed by Welch's t-test, n=3 cultures, bars indicate mean ± SEM. d, Same as b but with CDCA instead of CA. e, Same as b, but with DCA instead of CA. Triplicate chromatograms in d and e are stacked on the same axis. [Figure 17] Figure 11c-e: Bile transformation performed by B. thetaiotaomicron expressing the Turicibacter bsh gene. Table showing the presence / absence of the indicated bile species after growth in the mixtures of bile acids listed in Figure 11c-e. [Figure 18] FIG. 1 shows that expression of the bsh gene from Turicibacter does not significantly impair the in vitro growth of B. thetaiotaomicron. OD600 readings of the indicated B. thetaiotaomicron strains in BHI-S medium. Each point represents the mean + / - SEM for six independent cultures. [Figure 19] Figure 12 shows sex differences in lipidomic response to Turicibacter bsh expression in B. thetaiotaomicron. Similar to Figure 12d, but lipidomic analysis of animals is split into a) males and b) females. Note that the lipid species presented were found to be significantly altered by expression of at least one bsh in males and females combined (i.e., all lipid species shown across the three analyses are the same). Each row represents one animal. [Figure 20] Figure 12E-J: Expression of Turicibacter bsh by B. thetaiotaomicron is sufficient to cause widespread changes in circulating host lipids in male and female mice. Related to Figure 12E-J: Circulating concentrations of specific lipid categories in mice colonized with B. thetaiotaomicron expressing the indicated Turicibacter bsh homologs. Each dot represents one animal, red dots indicate females and blue indicates males. n=4-5 per colonization, error bars represent mean + / - SEM. Statistical comparisons were performed by Welch's ANOVA with Dunnett's multiple comparisons, dotted bars represent pooled ANOVA statistics for each group relative to the experimental mean. *p<0.05. [Figure 21] Figure 1: Turicibacter strains MOL361 and 1E2 can deconjugate at least six taurine-conjugated bile acids. Chromatograms (left) and unconjugated / conjugated bile acid ratios (right) of Turicibacter MOL361 or 1E2 grown for 24 h in YCFA + 0.5 mM for individual taurine-conjugated bile acids: a, TCA; b, TCDCA; c, tauroursodeoxycholic acid (TUDCA); d, taurolithocholic acid (TLCA); e, taurohyodeoxycholic acid (THDCA); f, taurodeoxycholic acid (TDCA). Chromatograms are concatenated reconstructed chromatograms for conjugated and unconjugated bile acids. Each trace and each point represents one biological replicate. n=3 cultures. Statistics were performed by Mann-Whitney test. #=p≦0.1 (Note: this p-value is the minimum for this test and our experimental parameters). [Figure 22] Figure 1 shows that bsh expression does not alter B. thetaiotaomicron colonization of the mouse intestine. Quantification of CFU / mL of the indicated B. thetaiotaomicron strains in a, distal small intestine; b, cecum; and c, proximal colon of gnotobiotic mice. [Figure 23]Figure 1: Turicibacter colonization and bsh expression induce similar gene expression patterns in the liver. qRT-PCR analysis of hepatic transcript levels of a, Fxr; b, Cyp7a1; and c, G6pase following colonization of B. thetaiotaomicron with the indicated Turicibacter strains expressing the indicated bsh genes. Data are presented as fold change relative to the appropriate control (GF for Turicibacter colonization, Bt-WT for bsh colonization). Each point represents data from one animal and statistics were performed by Kruskal-Wallis test with Dunn's multiple comparisons. The dashed horizontal line represents the sum of the ANOVA statistics for that comparison. *=p<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Provided herein are methods and compositions for modulating bile salts and / or bile acids by administering the compositions provided herein.

[0013] In some aspects, provided herein are methods for preventing or treating a metabolic disorder (e.g., a lipid metabolism disorder and / or a steroid metabolism disorder) in a subject, the method comprising administering to the subject a composition comprising a bacterial strain (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) that expresses a bile salt regulatory gene or a bile acid regulatory gene.

[0014] Also provided herein are methods of treating or preventing a metabolic disorder in a subject by depleting the subject's gut microbiota (e.g., by administering an antibiotic to the subject) and administering a composition comprising a bacterial strain that expresses a bile salt regulatory gene or a bile acid regulatory gene (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron).

[0015] Provided herein are bacterial strains (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) that contain a bile salt regulatory gene or a bile acid regulatory gene, which have been genetically engineered to express the bile salt regulatory gene or the bile acid regulatory gene. Similarly, provided herein are bacterial strains (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) that express a bile salt regulatory gene or a bile acid regulatory gene encoded by an exogenous nucleic acid, e.g., a plasmid or other vector, where the bile salt regulatory gene or the bile acid regulatory gene is operably linked to a promoter that drives expression (e.g., constitutive or inducible) of the bile salt regulatory gene or the bile acid regulatory gene in the bacterial strain.

[0016] Also provided herein are compositions, e.g., compositions comprising a bacterial strain described herein and a pharma- ceutically acceptable carrier; or compositions comprising a bacterial strain expressing a bile salt- or bile acid-regulating gene (e.g., as described herein) (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) and a pharma- ceutically acceptable carrier.

[0017] Provided herein are methods of making the bacterial strains described herein, comprising transforming the bacterial strain with a gene expression construct encoding a bile salt- or bile acid-regulated gene operably linked to a promoter that drives expression (e.g., constitutive or inducible) of the bile salt- or bile acid-regulated gene in the bacterial strain. In certain embodiments, the method further comprises formulating the bacterial strain for administration to a subject, e.g., in a pharmaceutical composition or in a food or beverage product. In some embodiments, the method further comprises culturing the bacterial strain to allow expression of the bile salt- or bile acid-regulated gene.

[0018] In certain embodiments, the methods and compositions are for the treatment or prevention of a metabolic disorder in a subject (e.g., a subject having a lipid metabolism disorder such as hyperlipidemia, hypercholesterolemia, acid lipase disease, Barth syndrome, Fabry disease, Farber disease, Gaucher disease, Niemann-Pick disease, or Tay-Sachs disease; or a steroid metabolism disorder such as cytochrome p450 oxidoreductase deficiency, apparent mineralocorticoid excess, lipoid congenital adrenal hyperplasia, congenital bile acid synthesis deficiency, 3-beta-hydroxysteroid dehydrogenase deficiency, aldosterone-producing adenoma, polyendocrinopathy, adrenal hyperplasia).

[0019] definition As used herein, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein, "another" may mean at least a second or more.

[0020] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and corn oil. (10) oils, such as soybean oil; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffers; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0021] The term "preventing" is art-recognized and, when used in reference to a condition such as local recurrence, is well understood in the art and includes administration of a composition to reduce the frequency or delay the onset of symptoms of a medical condition in a subject compared to a subject not receiving the composition.

[0022] The terms "prophylactic" or "therapeutic" treatment are art-recognized and include administration of one or more of the subject compositions to a host. If it is administered prior to the clinical manifestation of an undesirable condition (e.g., a disease or other undesirable condition of a host animal), the treatment is prophylactic (i.e., it protects the host against the onset of the undesirable condition), whereas if it is administered after the manifestation of the undesirable condition, the treatment is therapeutic (i.e., it is intended to reduce, ameliorate, or stabilize an existing undesirable condition or its side effects).

[0023] The term "subject" refers to a mammal, including, but not limited to, a human or a non-human mammal, such as a cow, horse, dog, sheep, or cat.

[0024] A "therapeutically effective amount" of a compound in relation to a method of treating a subject refers to the amount of compound in a preparation that, when administered (to a mammal, preferably a human) as part of a desired dosing regimen, alleviates the symptoms, improves the condition, or delays the onset of a disease state in accordance with clinically accepted criteria for the disorder or condition to be treated or for cosmetic purposes, e.g., with a reasonable benefit / risk ratio applicable to any medical treatment.

[0025] As used herein, the term "treating" or "treatment" includes reversing, reducing, or preventing the symptoms, clinical signs, and underlying pathology of a condition so as to improve or stabilize the subject's condition.

[0026] As used herein, the terms "modulate" or "modulation", or "regulate" or "regulation" and "differentially regulated", unless otherwise specified or clear from the context of a particular usage, can refer to both upregulation (i.e., activating or stimulating, e.g., by agonizing or enhancing) and downregulation (i.e., inhibiting or suppressing, e.g., by antagonizing, decreasing or inhibiting).

[0027] Treatment method The disclosure herein relates in part to the discovery that different strains of Turicibacter sanguinis differentially deconjugate and dehydrogenate bile acids, differentially affecting host metabolic products, including adipose tissue mass, lipid composition, bile acid and tryptophan-related metabolites. The entire genomes of different strains of Turicibacter have been sequenced, and genes potentially involved in the different bile acid modifications (e.g., genes encoding bile salt hydrolases or 7-alpha hydroxysteroid dehydrogenases) have been identified. Additionally, strains of Escherichia coli and Bacteroides thetaiotaomicron have been developed to express Turicibacter sanguinis bile modifying genes (e.g., genes encoding bile salt hydrolases or 7-alpha hydroxysteroid dehydrogenases).

[0028] In some aspects, provided herein are methods for preventing or treating a metabolic disorder (e.g., a lipid metabolism disorder and / or a steroid metabolism disorder) comprising administering to a subject a composition comprising a bacterial strain (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) that expresses a bile salt regulatory gene or a bile acid regulatory gene.

[0029] Also provided herein are methods of treating or preventing a metabolic disorder in a subject by depleting the subject's gut microbiota and administering a composition comprising a bacterial strain that expresses a bile salt regulatory gene or a bile acid regulatory gene (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron).

[0030] In another aspect, provided herein are methods of treating or preventing a metabolic disorder in a subject by depleting the subject's gut microbiota and administering a composition comprising a bacterial strain expressing a bile salt regulatory gene or a bile acid regulatory gene (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron).

[0031] In some aspects, the methods include depleting the subject's gut microbiota (e.g., by administering an antibiotic to the subject) prior to administering a composition described herein.

[0032] In some embodiments, the bacterial strain expresses a bile salt regulatory gene or a bile acid regulatory gene encoded by an exogenous nucleic acid, e.g., a plasmid or other vector, and the bile salt regulatory gene or bile acid regulatory gene is operably linked to a promoter that drives expression (e.g., constitutive or inducible) of the bile salt regulatory gene or bile acid regulatory gene in the bacterial strain.

[0033] Provided herein are methods of making the bacterial strains described herein, comprising transforming the bacterial strain with a gene expression construct encoding a bile salt regulatory gene or a bile acid regulatory gene operably linked to a promoter that drives expression (e.g., constitutive or inducible) of the bile salt regulatory gene or the bile acid regulatory gene in the bacterial strain. In certain embodiments, the method further comprises formulating the bacterial strain for administration to a subject, e.g., in a pharmaceutical composition or in a food or beverage product. In some embodiments, the method further comprises culturing the bacterial strain to allow expression of the bile salt regulatory gene or the bile acid regulatory gene. In some embodiments, the bacterial strain (e.g., Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron) regulates bile salts by conjugation with glycine or conjugation with taurine. In some embodiments, the bile salt regulatory gene encodes a bile salt hydrolase (BSH), such as any one of the bile salt hydrolases listed in Table 1. In some embodiments, BSH is encoded by a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any one of the nucleic acid sequences in Table 1.

[0034] [Table 1] TIFF2025503418000002.tif229162TIFF2025503418000003.tif228162TIFF2025503418000004.tif229161TIFF20255034180 00005.tif229161TIFF2025503418000006.tif228161TIFF2025503418000007.tif230161TIFF2025503418000008.tif230162

[0035] In certain embodiments, the bile acid regulatory gene encodes a 7-alpha hydroxysteroid dehydrogenase, such as the 7-alpha hydroxysteroid dehydrogenase encoded by the exemplary nucleic acid sequences set forth below: In some embodiments, the 7-alpha hydroxysteroid dehydrogenase is encoded by a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the exemplary 7-alpha hydroxysteroid dehydrogenase nucleic acid sequences set forth below:

[0036] Exemplary 7-alpha hydroxysteroid dehydrogenase nucleic acid sequences:

[0037] [ka]

[0038] In some embodiments, the 7-alpha hydroxysteroid dehydrogenase is at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69% or at least 70% homologous to the amino acid sequence of Clostridium absonum 7-alpha hydroxysteroid dehydrogenase.

[0039] In certain embodiments, the bile salt or bile acid is any one of the bile salts or bile acids listed in Table 3. In some embodiments, administration of the composition increases bile acids in the subject.

[0040] [Table 2] TIFF2025503418000011.tif239163TIFF2025503418000012.tif218162TIFF2025503418000013.tif53162

[0041] In certain embodiments, administration of the composition alters the lipidome of the subject.

[0042] In some embodiments, administration of the composition reduces white adipose tissue mass in the subject.

[0043] In certain embodiments, administration of the composition alters health-related lipid biomarkers in the subject (e.g., reduces triglyceride (TG) levels in the subject, reduces cholesterol levels and / or cholesterol ester (CE) levels in the subject).

[0044] In some embodiments, administration of the composition reduces abdominal fat pad mass in the subject.

[0045] In some embodiments, the bacterial strain is Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron.

[0046] In certain embodiments, the T. sanguinis bacterial strain is any one of the T. sanguinis bacterial strains listed in Table 4.

[0047] [Table 3]

[0048] In some embodiments, the T. sanguinis bacterial strain comprises a 16S nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the 16S nucleic acid sequences of the exemplary Turicibacter sanguinis strains shown below.

[0049] 16S nucleic acid sequence of an exemplary Turicibacter sanguinis strain:

[0050] [ka] TIFF2025503418000016.tif249162TIFF2025503418000017.tif248161TIFF2025503418 000018.tif251162TIFF2025503418000019.tif248162TIFF2025503418000020.tif25116 3TIFF2025503418000021.tif249165TIFF2025503418000022.tif245163TIFF2025503418 000023.tif248164TIFF2025503418000024.tif247165TIFF2025503418000025.tif31163

[0051] In some embodiments, the subject has a metabolic disorder (e.g., a lipid metabolism disorder and / or a steroid metabolism disorder). The lipid metabolism disorder can be hyperlipidemia, hypercholesterolemia, acid lipase disease, Barth syndrome, Fabry disease, Farber disease, Gaucher disease, Niemann-Pick disease, or Tay-Sachs disease. The steroid metabolism disorder can be cytochrome p450 oxidoreductase deficiency, apparent mineralocorticoid excess, lipoid congenital adrenal hyperplasia, congenital bile acid synthesis deficiency, 3-beta-hydroxysteroid dehydrogenase deficiency, aldosterone-producing adenoma, polyendocrinopathy, adrenal hyperplasia.

[0052] The composition may be formulated for oral delivery. In some embodiments, the composition may include probiotics. In some embodiments, the compositions disclosed herein are food products. The composition may be in the form of a pill, tablet, or capsule. In some embodiments, the subject may be a mammal (e.g., a human). In some embodiments, the composition is self-administered. Although a single composition preferably includes all bacteria to be administered, it will be appreciated that for any of the various embodiments described herein, combinations of bacteria may similarly be administered in multiple compositions that include the combinations of bacteria together. For example, the present invention further provides kits that include multiple compositions that include Turicibacter sanguinis bacterial strains (e.g., the bacterial strains listed in Table 4) that modulate bile salts and / or bile acids together.

[0053] In some embodiments, the composition is formulated for rectal delivery (e.g., fecal sample). In some embodiments, the subject undergoes a fecal microbiota transplant, the transplant comprising a composition disclosed herein. Fecal microbiota transplantation (FMT), also commonly known as "fecal bacteriotherapy", represents a treatment protocol that allows for the reconstitution of the colonic microbial community. This process involves the transplantation of fecal bacteria from a healthy individual to a recipient. FMT restores the colonic microbiota by introducing healthy bacterial flora through injection of a fecal sample obtained from a healthy donor, for example, by enema, oral gavage, or by mouth in the form of a capsule containing a freeze-dried material. In some embodiments, the fecal sample is from a fecal bank.

[0054] In some embodiments, the bacterial DNA in the gut microbiota of a subject is sequenced.The gut bacterial DNA of a subject can be sequenced before administering the composition.For example, a sample containing bacterial DNA can be obtained from a subject, and then the bacterial DNA is sequenced for any one of the bacteria listed in Table 4, thus measuring the presence or level of any one of such bacteria (e.g., one or more, two or more, five or more, or ten or more of the bacteria of interest) in the gut microbiota of the subject.The composition disclosed herein can then be administered to the subject when the level of bacteria is low. In some embodiments, a subject is considered to have low levels of any one of the bacteria listed in Table 4 if less than 0.0001%, less than 0.001%, less than 0.01%, less than 0.02%, less than 0.03%, less than 0.04%, less than 0.05%, less than 0.06%, less than 0.07%, less than 0.08%, less than 0.09%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 5%, less than 7%, less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% of the bacteria in the sample are the bacteria of interest. The bacterial DNA to be sequenced can be obtained by any means known in the art, including, but not limited to, obtaining a fecal sample from the subject and isolating the bacterial DNA. Sequencing of bacterial DNA by any known art technique includes, but is not limited to, Maxam-Gilbert sequencing, Sanger sequencing, shotgun sequencing, bridge PCR or next generation sequencing methods such as massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing or nanopore DNA sequencing.

[0055] In some embodiments, the methods described above act directly to reduce the amount of pathogenic bacteria in a subject (i.e., in the subject's gastrointestinal tract). In some embodiments, this includes any such therapy that achieves the same goal of reducing the number of pathogens, which, when used in combination with the compositions described herein, will lead to replacing the pathogenic microflora involved in a disease state with less pathogenic species that occupy the same ecological niche as the microflora associated with a non-disease state or the type that causes the disease state. For example, a subject may be treated with an antibiotic (e.g., an antibacterial compound) or a composition that includes an antibiotic to target and reduce the spread of pathogens, and then treated with a composition described herein. The treatment may also include an antifungal or antiviral compound.

[0056] Suitable antibacterial compounds include capreomycin, including capreomycin IA, capreomycin IB, capreomycin IIA, and capreomycin IIB; carbomycin, including carbomycin A; carumonam; cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefbuperazone, cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefime, ceftamet, cefmenoxime, cefmetzole, cefminox, cefmetazolin ... cefotaxime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefpimizole, cefpiramide, cefpirome, cefprozil, cefroxadine, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephalexin, cephalogycin, cephaloridine, cephalosporin C, cephalothin, cephapirin, cephamycin, e.g., cephamycin C, cepharomycin ... doxycycline, cyclosporine, chlortetracycline, clarithromycin, clindamycin, clometocillin, clomocycline, cloxacillin, cyclacillin, danofloxacin, demeclocycline, destomycin A, dicloxacillin, dirithromycin, doxycycline, epicillin, erythromycin A, ethambutol, fenbenicillin, flomoxef, florfenicol, floxacillin, flumequine, fortimicin A, fortimicin B, forfomycin n), foraltadone, fusidic acid, gentamicin, glyconiazid, guamecycline, hetacillin, idarubicin, imipenem, isepamicin, josamycin, kanamycin, leumycin, e.g. leumycin A1, lincomycin, lomefloxacin, loracarbef, lymecycline, meropenam, methampicillin, methacycline, methicillin, mezlocillin, micronomycin, midecamycin, e.g. midecamycin A1,Mikamycin, Minocycline, Mitomycins such as Mitomycin C, Moxalactam, Mupirocin, Nafcillin, Netilicin, Norcardian, such as Norcardian A, Oleandomycin, Oxytetracycline, Panipenam, Pazufloxacin, Penamecillin, Penicillins such as Penicillin G, Penicillin N and Penicillin O, Penicillic acid, Pentylpenicillin, Peplomycin, Phenethicillin, Pipacyclin, Piperacillin, Pirrimycin, Pivampicillin, Pivocephalexin, Porfiromycin, Propiallin, Quinacillin, Ribo Stamycin, rifabutin, rifamide, rifampin, rifamycin SV, rifapentine, rifaximin, ritipenem, rekitamycin, rolitetracycline, rosaramycin, roxithromycin, sancycline, sisomicin, sparfloxacin, spectinomycin, streptozocin, sulbenicillin, sultamicillin, talampicillin, teicoplanin, temocillin, tetracycline, tostrepton, tiamulin, ticarcillin, tigemonam, tilmicosin, tobramycin, tropospectromycin, trovafloxacin, tylosin and vancomycin, as well as analogs, derivatives, pharmaceutically acceptable salts, esters, prodrugs and protected forms thereof.

[0057] Suitable antifungal compounds include ketoconazole, miconazole, fluconazole, clotrimazole, undecylenic acid, sertaconazole, terbinafine, butenafine, clioquinol, haloprogin, nystatin, naftifine, tolnaftate, ciclopirox, amphotericin B or tea tree oil, and analogues, derivatives, pharmaceutically acceptable salts, esters, prodrugs and protected forms thereof.

[0058] composition In some aspects, the invention relates to compositions (e.g., food or pharmaceutical compositions). Provided herein are compositions (e.g., compositions comprising a Turicibacter sanguinis bacterial strain, such as any of the bacteria listed in Table 4, and a pharma- ceutically acceptable carrier; or compositions comprising a Turicibacter sanguinis bacterial strain that modulates bile salts and / or bile acids, such as any of the bacteria listed in Table 4, and a pharma- ceutically acceptable carrier). The compositions may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 bacteria that modulate bile salts and / or bile acids, such as any of the bacteria listed in Table 4. Any combination of the bacteria listed in Table 4 may be included in the composition.

[0059] The composition may include a pharma- ceutically acceptable carrier. The composition may include a probiotic. The pharmaceutical compositions disclosed herein may be delivered by any suitable route of administration, including oral, buccal, sublingual, parenteral, and rectal, such as by powder, ointment, drop, liquid, gel, tablet, capsule, pill, or cream. In certain embodiments, the pharmaceutical compositions are delivered generally (e.g., by oral administration). In certain other embodiments, the compositions disclosed herein are delivered rectally.

[0060] In certain embodiments, the present invention provides kits that include a plurality of compositions (e.g., a composition comprising a Turicibacter sanguinis bacterial strain, such as any of the bacteria listed in Table 4, and a pharma- ceutically acceptable carrier; or a composition comprising a Turicibacter sanguinis bacterial strain that modulates bile salts and / or bile acids, such as any of the bacteria listed in Table 4, and a pharma- ceutically acceptable carrier). The kits disclosed herein may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 of the compounds listed in Table 1. The kits provided herein may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 of the bacteria that modulate bile salts and / or bile acids, such as any of the bacteria listed in Table 4. Any combination of the bacteria listed in Table 4 may be included in the composition.

[0061] In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the bacteria in the composition that modulate bile salts and / or bile acids, such as any of the bacteria listed in Table 4.

[0062] The compositions described herein may be used for oral administration to the gastrointestinal tract for the purpose of introducing bacteria (e.g., bacteria disclosed herein) into the tissues of the gastrointestinal tract. Formulations for compositions of the present invention (e.g., probiotic compositions) may also include other probiotic agents or nutrients that promote spore germination and / or bacterial growth. An exemplary material is bifidobacterium oligosaccharides that promote the growth of beneficial probiotic bacteria. In some embodiments, the probiotic bacterial composition is administered with a therapeutically effective amount of a (preferably broad-spectrum) antibiotic or antifungal agent. In some embodiments, the compositions described herein are encapsulated in an enteric-coated, sustained-release capsule or tablet. The enteric coating allows the capsule / tablet to remain intact (i.e., undissolved) as it passes through the gastrointestinal tract and / or until it reaches a certain portion of the GI tract (e.g., the small intestine). The sustained-release component prevents the "release" of the probiotic bacterial strains in the compositions described herein for a predetermined period of time.

[0063] The composition may be a food product, such as, but not limited to, a dairy product. The dairy product may be a cultured dairy product or a non-cultured (e.g., milk) dairy product. Non-limiting examples of cultured dairy products include yogurt, cottage cheese, sour cream, kefir, buttermilk, and the like. Dairy products often also contain various specialty dairy ingredients, such as whey, skim milk powder, whey protein concentrate solids, and the like. Dairy products may be processed in any manner known in the art to achieve desirable qualities, such as flavor, thickening power, nutrition, specific microorganisms, and other properties, such as mold growth control. The compositions of the present invention may also include other agents, such as known antioxidants, buffers, and colorants, flavors, vitamins, or minerals.

[0064] In some embodiments, the compositions of the present invention are mixed with a carrier (e.g., a pharma- ceutically acceptable carrier) that is physiologically compatible with the gastrointestinal tissues of the subject to which they are administered. The carrier may be composed of a solid-based dry material for formulation into a tablet, capsule, or powder form; or the carrier may be composed of a liquid or gel-based material for formulation into a liquid or gel form. The specific type of carrier and the final formulation will depend in part on the route of administration selected. The therapeutic compositions of the present invention may also include various carriers and / or binders. In some embodiments, the carrier is microcrystalline cellulose (MCC) added in an amount sufficient to complete one gram of total dosage weight. The carrier may be a solid-based dry material for formulation into a tablet, capsule, or powder form, or a liquid or gel-based material for formulation into a liquid or gel form, which form will depend in part on the route of administration. Typical carriers for dry formulation include, but are not limited to, trehalose, malto-dextrin, rice flour, microcrystalline cellulose (MCC), magnesium stearate, inositol, FOS, GOS, dextrose, sucrose and similar carriers. Suitable liquid or gel-based carriers include, but are not limited to, water and saline; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol, propylene glycol and the like). Preferably, aqueous carriers have a neutral pH value (i.e., pH 7.0). Other carriers or agents for administering the compositions described herein are known in the art, for example, U.S. Pat. No. 6,461,607.

[0065] In some embodiments, the composition further comprises other bacteria or microorganisms known to colonize the gastrointestinal tract. For example, the composition can include species belonging to the phyla Firmicutes, Proteobacteria, Tenericutes, Actinobacteria, or combinations thereof. Examples of additional bacteria and microorganisms that may be included in the subject compositions include, but are not limited to, Saccharomyces, Bacteroides, Eubacterium, Clostridium, Lactobacillus, Fusobacterium, Propionibacterium, Streptococcus, Enteroccus, Lactococcus, and Staphylococcus, Peptostreptococcus. In certain embodiments, the composition is substantially free of bacteria that increase the risk of metabolic disorders. Such bacteria include Bifidobacterium bacteria. Thus, in some embodiments, the composition is substantially free of Bacteroides bacteria. A composition is substantially free of a bacterial type when that bacterial type constitutes less than 10% of the bacteria in the composition, preferably less than 5%, even more preferably less than 1%, and most preferably less than 0.5% or even 0% of the bacteria in the composition.

[0066] In some embodiments, the composition comprises a fecal sample comprising at least one bacterium that modulates bile salts and / or bile acids, such as any of the bacteria listed in Table 4. In some embodiments, the fecal sample is from a stool bank. In some embodiments, the composition may be added to the fecal sample prior to administration to the subject.

[0067] In some embodiments, provided herein are methods of treating or preventing a metabolic condition by administering a bile salt and / or bile acid modulating composition (e.g., a fecal sample), such as any of the bacteria listed in Table 4. A fecal sample is enriched if at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1% or at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the bacteria in the fecal sample are bile salt and / or bile acid modulating bacteria, such as any of the bacteria listed in Table 4. In some embodiments, the fecal sample is from a stool bank. In some embodiments, the fecal sample is from a donor.

[0068] The composition may further comprise nutrients. In some embodiments, the nutrients support the growth of bacteria (e.g., bacteria disclosed herein). In some embodiments, the nutrients are lipids (e.g., linoleic acid, stearic acid, or palmitic acid). In some embodiments, the nutrients may be administered in conjunction with the compositions disclosed herein. As used herein, the phrase "administered together" refers to any form of administration of two or more different agents (e.g., a composition disclosed herein and a nutrient disclosed herein) such that the second agent is administered while the previously administered agent is still effective in the body. For example, the compositions disclosed herein and the nutrients disclosed herein may be administered either concomitantly or sequentially, either in the same formulation or in separate formulations.

[0069] Actual dosage levels of the active ingredients in pharmaceutical compositions can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without toxicity to the patient.

[0070] The selected dosage level will depend on a variety of factors, including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0071] A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can prescribe and / or administer doses of the compounds used in the pharmaceutical composition at levels lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. EXAMPLES

[0072] Example 1: Genomic comparison reveals distinct subgroups of Turicibacter sanguinis Figures 1A and 1B show genomic comparisons reveal distinct subgroups of Turicibacter sanguinis. A: Phylogenetic tree comparing full-length 16S rRNA sequences from the indicated T. sanguinis isolates. Circles indicate human isolates, triangles indicate mouse isolates, and squares indicate contaminating mouse isolates. B: Comparison of complete shotgun assembled genomes of the T. sanguinis isolates listed in A.

[0073] Example 2: T. sanguinis isolates differ in their bile-modifying abilities Figures 2A and 2B show that T. sanguinis isolates differ in their bile modifying abilities. A: Liquid chromatograms of individual T. sanguinis isolates grown for 24 hours in medium with sub-inhibitory concentrations of five bile salts / acids: taurocholic acid (TCA), cholic acid (CA), glycochenodeoxycholic acid (GCDCA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA). Shaded areas indicate the expected retention time of each bile species. B: Relative amounts of conjugated bile salts (tauricholic acid, taurochenodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid) remaining after 24 hours of growth of the indicated T. sanguinis isolates. Magenta = glycine conjugated bile salts, blue = taurine conjugated bile salts.

[0074] Example 3: T. sanguinis isolates differ in their genetic ability to modify bile species Table 2 shows the presence (+) or absence (-) of sequence homologs with potential bile-modifying activity in T. sanguinis isolates. For the 7-alpha hydroxysteroid dehydrogenase homologs (right-most column), the percent amino acid identity with Clostridium absonum is also shown.

[0075] [Table 4]

[0076] Figures 3A-D show that T. sanguinis isolates differ in their genetic ability to modify bile species. A: Liquid chromatograms of medium after 24 h of incubation of E. coli expressing individual predicted bile salt hydrolases (BSH) from each sequence grouping, isolated, and grown with TCA and TCDCA. Control is E. coli with the same expression vector but expressing non-bile modifying genes. B: Same as A, but with GCA and GCDCA instead of taurine-conjugated bile salts. C. Amounts of TCA, TCDCA, GCA, and GCDCA remaining after 24 h of growth of E. coli expressing the indicated BSH homologs. D: Phylogenetic tree of each predicted BSH gene from T. sanguinis isolates, showing bile salt specificity in boxes. We did not detect bile salt hydrolase activity in unboxed sequences.

[0077] Example 4: Expression of BSH in T. sanguinis is sufficient to alter the host lipidome and health-related lipid markers Figures 4A-D show that T. sanguinis BSH expression is sufficient to alter the host lipidome and health-related lipid markers. A: Heatmap of plasma lipid species significantly altered by expression of at least one T. sanguinis BSH in Bacteroides thetaiotaomicron. Colors on the left correspond to lipid classes, and the cyan-magenta color scale represents Z-scores. B. Relative white adipose tissue weight in mice monocolonized with BSH-expressing B. thetaiotaomicron. C. Relative mixed plasma triglycerides (TG) in mice monocolonized with BSH-expressing B. thetaiotaomicron. D. Relative cholesterol esters (CE) in mice monocolonized with BSH-expressing B. thetaiotaomicron. All values ​​normalized to sex-matched littermates. *=p<0.05 two-tailed t-test.

[0078] Example 5: T. sanguinis isolates differ in their effects on host lipid biology and bile acids 5A-5B show that T. sanguinis isolates differ in their effects on host lipid biology and bile acids. A: Relative abdominal fat pad mass from gender- and litter-matched mice monocolonized with individual T. sanguinis isolates. B: Top: Example images of histological sections of adipose tissue. Bottom: Relative adipocyte size from gender- and litter-matched mice monocolonized with individual T. sanguinis isolates.

[0079] Example 6: T. sanguinis isolates differ in their effects on host lipid biology and bile acids Figure 6 shows circulating serum bile acid and cholesterol levels in mice monocolonized with individual T. sanguinis isolates. #=p<0.1, *=p<0.05.

[0080] Example 7: Turicibacter alters host bile acids and lipids in a strain-specific manner Bacteria of the genus Turicibacter are important members of the mammalian gut microbiota and have been associated with changes in dietary fat and body weight, but the specific links between these commensals and host physiology are poorly understood. We genomically and phenotypically characterized diverse Turicibacter strains from mice and humans and found them to fall into three clades that differ in bile acid transformation. We identified Turicibacter bile salt hydrolases that confer strain-specific differences in bile deconjugation. Colonization with individual Turicibacter strains caused changes in the host bile acid profile that were generally consistent with those produced in vitro. Furthermore, colonization of mice with additional bacteria expressing bile-modifying genes from these strains reduced serum cholesterol and triglycerides, as well as adipose tissue mass. This study identified genes by which diverse Turicibacter strains can differentially modify host bile acid and lipid metabolism, positioning multiple Turicibacter strains as candidates for altering host lipid biology.

[0081] introduction The gut microbiota forms a complex relationship with its host organism and regulates metabolism. 1、2 and Neurobiology 3、4In many cases, the link between the gut microbiota and host physiology is most easily interpreted by the presence / absence of large sectors of the microbial community, but 5~7 (e.g., microbial communities), in some cases, specific microbial characteristics and / or taxa play important roles in the physiology of the host. 8~10 .

[0082] The mammalian gut microbiota has long been associated with obesity, 25、26 ,In many cases, studies have shown strong correlations rather than mechanistic determinants of these relationships, indicating the need for further fundamental investigation of the link between the microbiota and host fat. 27 Numerous microbiota community profiling studies have revealed correlations between Turicibacter and features of host fat metabolism, such as obesity and dietary lipids. 28~33 ,The nature of these correlations varies. 34、35 Recently, the type strain of T. sanguinis, MOL361 36、37 was observed to reduce serum cholesterol and triglycerides in mice while extensively altering the host serum lipidome. 38 This same strain has also been reported to modify biliary species by deconjugation and dehydrogenation in vitro. 21 , suggesting at least one means by which Turicibacter may affect the lipid status of the host. Based on these findings, it is hypothesized that there may be variation in the functional activities of Turicibacter strains that accounts for differences in host bile and lipid biology, providing a mechanism linking this taxon to aspects of host physiology.

[0083] result Turicibacter isolates are divided into genetically distinct strains To better understand the diversity within the genus Turicibacter, we collected nine isolates from mouse and human fecal microbiota that were identified as T. sanguinis based on 16S rRNA gene sequences (97% full-length 16S rRNA gene sequence similarity cutoff, Table 5). Two of these isolates had been previously identified (human-derived reference strains MOL361 and H121, which were derived from mice free of contaminating bacteria). 36、39 five were isolated but not published (human isolates 18F6, T46, and T129, and mouse isolates 1E2 and TA25); two were isolated from human fecal samples specifically for this study (GALT-E2 and GALT-G1) using an array-based isolation and culture platform (see Methods). Shotgun short-read sequencing was performed and draft assemblies of each isolate genome were generated. Comparison of 16S rRNA gene phylogenies (Fig. 7a), general genomic characteristics (Fig. 7b) or specific genomic sequences (Fig. 7c, d) revealed the presence of at least three distinct subgroups even in this rather small sample of 16S rRNA gene-based "T. sanguinis" species designations: two from humans (typological isolates MOL361 and H121, with 99.3% full-length 16S rRNA gene similarity) and one from mice (typological strain 1E2, with 97.5% and 97.8% 16S rRNA gene similarity to MOL361 and H121, respectively). Genome alignments showed a significant amount of shared DNA sequences among members of the same subgroup (all average nucleotide identity [ANI] within the group >98.3%), while the remaining amount of genetic variation indicated smaller genetic differences between related isolates. The shared sequences within these subgroups were distinct from members of the other two subgroups (intergroup ANI: MOL361-H121 = 76.80%, MOL361-1E2 = 74.95%, H121-1E2 = 77.43%). The H121 group is currently home to the newly described species Turicibacter bilis, the only other named species from this genus. 13It is important to note that the genomes of these strains are genomically similar (98.8% ANI) to those of the host origin. Overall, these genomic divergences suggest distinct evolutionary histories that correspond, at least in part, to host origins.

[0084] [Table 5] TIFF2025503418000028.tif74161

[0085] Turicibacter isolates differ in their impact on host lipid biology and circulating metabolome Previous studies have demonstrated that monocolonization of mice with T. sanguinis MOL361 alters host adipose tissue and circulating lipids. 38 Due to the large genomic variations among our Turicibacter strains, it was predicted that they would have different effects on the lipid biology of the host. We selected representative isolates from each of the different phylogenetic groups (MOL361, H121 and T129, and 1E2) and measured their effects on circulating metabolites and adipose tissue in monocolonized mice compared to germ-free mice (GF) and conventional controls (i.e., gavaged with the complete microbiota, CONV). Compared to GF littermates, CONV mice had decreased levels of several dicarboxylate fatty acids, long-chain fatty acids and long-chain acylcarnitines, and widespread increases in short- and medium-chain acylcarnitines (Figure 8a). In line with previous reports, 38Consistent with this, colonization with individual Turicibacter strains also caused widespread changes in host serum lipids, with many strain-level differences in host lipid changes (Fig. 8a, Table 6). Compared to GF controls, MOL361 increased a subset of long-chain acylcarnitines and decreased many long-chain saturated fatty acids and dicarboxylic acids. Furthermore, MOL361 colonization caused a significant reduction in host cholesterol to levels below those seen in both GF and CONV animals (Fig. 8b). Compared to GF, H121 colonization significantly increased serum levels of several medium-chain fatty acids, dicarboxylic acids, and short-, medium-, and long-chain acylcarnitines. Colonization with 1E2 had a smaller overall effect on host lipids, but did lead to a decrease in some dicarboxylate fatty acids (Fig. 8a), and in addition to the differences between GF or CONV mice and animals mono-colonized with Turicibacter, there were also extensive changes in host lipids in response to colonization with different Turicibacter strains, with large differences in dicarboxylate and long-chain saturated fatty acids (Fig. 8a). At the tissue level, two of the four Turicibacter strains promoted a statistically significant increase in epididymal / gonadal white adipose tissue (e / gWAT) mass compared to GF controls, and the third strain caused a similar increase, but not statistically significant. In contrast, there was no notable effect of H121 (Fig. 8c). Consistent with this, H121 showed the smallest e / gWAT adipocyte size within the fat body (Fig. 13a-f). This may be due to less colonization of H121 in both the small intestine and colon (Fig. 13g, h). These results indicate that changes in specific lipid species are not necessarily associated with adipose tissue mass.

[0086] [Table 6] TIFF2025503418000030.tif230161TIFF2025503418000031.tif232161TIFF2025503418000032.tif225161TIFF2025503418000033.tif221161TIFF2025503418000034.tif232161TIFF2025503418000035.tif225161TIFF2025503418000036.tif232161TIFF2025503418000037.tif225161TIFF2025503418000038.tif225161TIFF2025503418000039.tif230161TIFF2025503418000040.tif225161TIFF2025503418000041.tif225161TIFF2025503418000042.tif232161TIFF2025503418000043.tif230161TIFF2025503418000044.tif232161TIFF2025503418000045.tif225161TIFF2025503418000046.tif229161TIFF2025503418000047.tif229161TIFF2025503418000048.tif232161TIFF2025503418000049.tif220161TIFF2025503418000050.tif232161TIFF2025503418000051.tif226161TIFF2025503418000052.tif221161TIFF2025503418000053.tif225161TIFF2025503418000054.tif230161TIFF2025503418000055.tif224161TIFF2025503418000056.tif225161TIFF2025503418000057.tif220161TIFF2025503418000058.tif225161TIFF2025503418000059.tif225161TIFF2025503418000060.tif232161TIFF2025503418000061.tif232161TIFF2025503418000062.tif225161TIFF2025503418000063.tif233161TIFF2025503418000064.tif220161T IFF2025503418000065.tif229161TIFF2025503418000066.tif232161TIFF2025503 418000067.tif225161TIFF2025503418000068.tif230161TIFF2025503418000069. tif224160TIFF2025503418000070.tif232161TIFF2025503418000071.tif185161.

[0087] Bile acids may affect circulating host lipids by altering fat digestion and systemic hormonal signaling 40 They are produced by the host and released into the small intestine, where they enhance fat digestion by promoting micelle formation and the farnesoid X receptor (FXR). 40 and GPBAR1 / TGR5 41 Intestinal bacteria can modify bile acids mainly through transformation such as deconjugation. Previous reports have shown that MOL361 can modify a wide range of bile acids in vitro. 21To determine whether MOL361 and other Turicibacter strains could modify host bile acids in vivo, this study profiled serum bile acids from mice monocolonized with Turicibacter. Although each strain had unique effects on host serum metabolites, this study found several patterns in bile acids across monocolonized mice compared to either their GF or CONV controls. (Note: bile species with amino acid conjugates are commonly referred to as "bile salts," but for brevity, this study refers to conjugated and unconjugated bile species as "bile acids" herein.) Colonization with all Turicibacter strains led to an overall increase in serum levels of unconjugated primary bile acids such as cholic acid (CA), chenodeoxycholic acid (CDCA) and β-muricholate (βMCA) (Fig. 9a-d), as well as the unconjugated secondary bile acids 3-dehydrocholate and 7-ketodeoxycholate (Fig. 9e, f). These responses were highly variable in the case of T129 colonization, so we decided not to emphasize this strain in further experiments. Overall, these results suggest that these Turicibacter strains may affect host bile acids by potentially deconjugating them in the gut.

[0088] To determine the possible cause of the increase in unconjugated bile species, it was observed that the levels of conjugated bile acids differed between animals colonized by the different Turicibacter strains, most clearly distinguishing H121 from MOL361 and 1E2. Animals colonized with MOL361 and 1E2 strains had overall lower levels of taurine-conjugated primary bile acids compared to animals colonized with H121 (Fig. 9g-j), whereas H121 colonization resulted in an increase in glyco-beta-muricholic acid (GβMCA) (Fig. 9k). Female mice showed the most pronounced Turicibacter-associated changes in circulating bile species, lipids and cholesterol, indicating a sex-dependent response to Turicibacter colonization (Fig. 14a-c). This sex difference was also reported in C57BL / 6 mice monocolonized with T. sanguinis MOL361, although there was a directional change in adipocyte size. 38 Overall, these data demonstrate that Turicibacter colonization alters serum bile acids, lipids and cholesterol, as well as the lipid content of the host. Furthermore, while some changes, such as an increase in unconjugated bile acids, were conserved across Turicibacter colonization, changes to specific conjugated bile acids differed between Turicibacter strains, with MOL361 and 1E2 resulting in lower levels of taurine-conjugated bile acids than H121 colonization.

[0089] Turicibacter strains differ in their ability to modify host bile acids Based on the large genetic variation among Turicibacter strains (Figure 7) and the differences in serum lipid and bile acid profiles observed in response to colonization with the different bacteria (Figure 8), we hypothesized in this study that these strains differ in their ability to modify bile acids. To test this, we used subinhibitory concentrations of five bile acid species. 21Each of the nine isolates was grown to stationary phase in rich medium supplemented with: cholic acid, chenodeoxycholic acid deoxycholic acid (DCA), taurocholic acid (TCA) and glycochenodeoxycholic acid (GCDCA). The study then used liquid chromatography-mass spectrometry (LC-MS) to characterize the resulting bile transformations performed by each isolate (Figure 10a). This study discovered that not only did the strains differ in their ability to modify this combination of bile species, but these abilities largely mirrored the groupings identified in the genome comparison (Figure 10b). MOL361, 18F6 and GALT-E2 deconjugated both tauro- and glyco-bile acids and also dehydrogenated CA and CDCA (Figure 10b). 1E2 and TA25 deconjugated tauro bile acids but did not detectably deconjugate or dehydrogenate glycobile acids (Figure 10b). H121 and T129 deconjugated glycobile acids but did not readily deconjugate or detectably dehydrogenate tauro bile acids (Figure 10b). T46 and GALT-G1 did not have bile modification capabilities that reflected their genetic phylogeny; T46, which is genomically similar to the MOL361 group, performed modifications similar to the H121 group (i.e., glycodeconjugation but not taurodeconjugation, minimal dehydrogenation), whereas GALT-G1, which is genomically similar to the H121 group, performed transformations more similar to MOL361 (i.e., glycodeconjugation and taurodeconjugation, dehydrogenation of CDCA) (Figures 7, 10b, 15). Overall, each strain performed at least one of the three bile transformations, with some exhibiting capabilities in all three (Figures 10b, 15).

[0090] To confirm such intragroup differences, in this study, we selected one isolate from each of the subgroups (MOL361, H121, 1E2) and grew them in the presence of four primary conjugated bile acids: TCA, taurochenodeoxycholic acid (TCDCA), glycocholic acid (GCA) and GCDCA. This supported the same pattern seen above; MOL361 deconjugated both groups of bile acids, 1E2 deconjugated tauroconjugates, and H121 preferentially deconjugated glycoconjugates (Figure 10c). MOL361 and 1E2 showed extensive deconjugation of tauroconjugates and could process at least six taurine-conjugated bile acids (Figure 21). These data revealed that all strains tested were efficient modifiers of bile species, but their specific transformation differed in a strain-dependent manner, likely reflecting features that influence their differential effects on host lipid biology.

[0091] Turicibacter genomes possess distinct repertoires of bile salt hydrolases The differing bile-modifying capabilities across the strain subgroups suggested that each contained a unique repertoire of bile-modifying genes.

[0092] Certain bacteria from the gut microbiota dehydrogenate the hydroxyl group from the steroid core of bile acids. 42 , increasing their polarity and modulating their affinity for host bile acid receptors 43 This study aimed to characterize 7α-hydroxysteroid dehydrogenase (7α-HSDH) from Clostridium absolum. 21 ) were surveyed in the Turicibacter genome for homologues. 44This revealed a gene with 57% amino acid identity in MOL361, 18F6, T46 and GALT-E2, and a homolog with 59% amino acid identity in H121 and T129 (Figure 16a). The putative homolog from H121 had higher overall sequence identity than the homolog from MOL361, but it lacked certain features predicted to be important for dehydrogenase activity, such as the similar Asp38, which is catalytically important for this reaction. 44 Since in vitro experiments showed that only isolates from the MOL361 group performed bile dehydrogenation, this study cloned the putative 7α-HSDH homolog from MOL361 into E. coli C41-pLys and then grew these cells in the individual unconjugated bile acids that could be dehydrogenated: CA, CDCA, and DCA. Indeed, the protein encoded by this gene removed a mass equivalent to two hydrogens from CA (Fig. 16b, c). Although background transformation with E. coli prevented clear evidence of CDCA dehydrogenation by this putative 7α-HSDH homolog (Fig. 16d), this homolog did not act on DCA (Fig. 16e), supporting its annotation as a 7α-HSDH.

[0093] Conjugation increases the solubility and emulsifying ability of bile acids, but 45, deconjugation reverses these effects, resulting in reduced dietary lipid absorption. To identify Turicibacter bile salt hydrolase (bsh) genes responsible for strain-specific differences in bile deconjugation, this study first surveyed our assembled genome for annotations of "choloylglycine hydrolase," a broad category that includes these genes. Of these annotated genes, this study identified eight groups of potential homologous sequences and found that each Turicibacter strain encoded putative choloylglycine hydrolases from at least two of the eight groups (Fig. 11a, b). Isolates within the same phylogenetic and phenotypic subgroups shared mostly similar sequences (Fig. 11a, b). To assay the function of strain-specific putative bsh genes, this study cloned and individually expressed one representative sequence from each of the eight putative choloylglycine hydrolase groups in E. coli C41-pLys and measured the ability of these engineered bacteria to perform deconjugation, a study observed in native Turicibacter. This study cultured individual E. coli strains to stationary phase in the presence of two tauro (TCA, TCDCA) or glyco (GCA, GCDCA) bile acids and then measured their ability to deconjugate these bile acid pools. This study found that E. coli expressing four of the eight putative bsh gene groups exhibited deconjugation activity for at least one of the bile acids (Figure 11c-e). From MOL361, one BSH (group IV) is tauro-specific and one (group I) deconjugates both glycoconjugates and tauroconjugates (Fig. 11c, d). 1E2 shares a tauro-specific BSH with MOL361 (group IV) and has another BSH (group III) with moderate activity in TCDCA (Fig. 11c, d). H121 has a BSH (group II) with activity in TCDCA and GCDCA (Fig. 11c, d), but this was blunted when the four bile acids were presented in combination (Fig. 11e), possibly due to competitive inhibition.Taken together, these results reveal that Turicibacter strains contain various bile salt hydrolases with deconjugation preferences for different bile acids.

[0094] Strain- and substrate-specific bile salt hydrolases from Turicibacter differentially alter host lipid composition Given that Turicibacter colonization extensively modifies the host lipid and bile pools ( Fig. 8 ) and that bile transformation has previously been shown to alter host lipids. 46、47 In this study, we predicted that expressing Turicibacter bile-modifying genes outside of the context of Turicibacter colonization would be sufficient to affect host lipid biology. To measure the individual effects of these bile acid transformations, this study used genomically integrated high-expression vectors in the common gut bacterium Bacteroides thetaiotaomicron. 48 We expressed the Turicibacter bsh gene from T. sanguinis MOL361, which stably colonized the mouse intestine and, unlike E. coli C41-pLys, was able to express the Turicibacter bsh gene from T. sanguinis MOL361. 49、50These strains were selected because they contain a similarly characterized homolog of 7α-HSDH to that of E. coli, allowing the engineered bacteria to more completely mimic bile transformation in Turicibacter. These bacteria generally transformed tauro- and glycoconjugated bile acids, as predicted based on the characterization of BSH in E. coli, including preferential TCDCA transformation, indicating that they are capable of bile transformation similar to Turicibacter (Figure 12a, Figure 17). However, B. thetaiotaomicron strains expressing bsh (group III) from strain 1E2 transformed glycoconjugated bile acids more completely than strains expressing bsh (group II) from H121, the opposite of the results in E. coli. This observation led us to discontinue further use of the bsh (group II)-expressing strains. Furthermore, although no significant in vitro growth defects were observed in the engineered B. thetaiotaomicron strains (Figure 22), we observed a delay in bile transformation in B. thetaiotaomicron expressing bsh (group I) from MOL361, a study that could be complemented by extending the growth period (Figure 12b).

[0095] In this study, we monocolonized mice with bsh-expressing B. thetaiotaomicron strains and assessed their circulating lipid profile and abdominal WAT mass. We found that the engineered strains colonized the intestines of gnotobiotic mice at least as well as the parental strain (Supplementary Fig. 8), and that expressing individual Turicibacter bsh genes in a B. thetaiotaomicron background was sufficient to significantly alter host colonic bile levels (Fig. 12c) and absolute abundance of 346 circulating lipid species (Fig. 12d, Table 7). Notably, expression of either group I or group IV bsh led to a reduction in triglycerides (Fig. 12d, e). Expressing the tauro-specific bsh (group IV) from MOL361 also reduced diacylglycerides (Fig. 12g). Expression of the broader specificity bsh (group I) from MOL361 also resulted in a reduction in phosphatidylglycine, phosphatidylserine and cholesterol (Fig. 12d-f, h, i). Despite having broad transformation potential, B. thetaiotaomicron expressing bsh (group III) from 1E2 did not alter the host lipid profile as much as the other strains (Fig. 12d-i). At the tissue level, bsh expression also altered WAT accumulation in colonized mice, with broad tauro-deconjugated BSH (groups I and IV) significantly reducing WAT mass (Fig. 12j). Similar to our results with Turicibacter monocolonization, we also observed sex differences in the BSH response, with male mice showing a more consistent triglyceride reduction in response to tauro-specific BSH from MOL361 and females showing a more consistent triglyceride response in response to extensively deconjugated BSH (Supplementary Figure 9).

[0096] To further explore possible factors that may drive cholesterol and WAT changes in response to Turicibacter colonization and bsh expression, we measured hepatic transcript levels of farnesoid X receptor, (Fxr), the major nuclear receptor for bile acids; cytochrome P450 family A subfamily A member 1 / cholesterol 7α-hydroxylase (Cyp7a1), the rate-limiting enzyme for cholesterol to bile acid conversion; and glucose-6-phosphatase (G6pase), the key enzyme for gluconeogenesis. There were no differences in Fxr transcript levels across either native or engineered bacterial colonization conditions (Figure 23a). However, a similar increase in Cyp7a1 expression (Figure 23b) and decrease in G6pase expression (Figure 23c) were found between Turicibacter and bsh colonization. Overall, these results demonstrate that expression of Turicibacter -derived strain-specific bsh genes, particularly those capable of processing the abundant taurine-conjugated bile acids present in the mouse intestine, is sufficient to dramatically alter the cholesterol, bile, and lipid biology of the host.

[0097] Consideration Results from this study indicate that Turicibacter bacteria from mammalian gut microbiota modulate host bile and lipid composition in a strain-dependent manner. In this study, we identified and characterized five novel Turicibacter genes (four bsh, one 7α-HSDH) capable of bile transformation and revealed that expression of individual bsh genes is sufficient to extensively and differentially alter the host lipid profile. Furthermore, this study found that although bile transformation genes are present in all our examined Turicibacter strains, the specific transforming ability of BSH variants differs between strains in a manner consistent with coevolution of the host environment: whereas bile acids in the human intestine are a mixture of taurine and glycine conjugates, bile acids in mice are primarily taurine conjugates. 51、52These results suggest that the bile-modifying bacteria MOL361 and Turicibacter spp., which are isolated from the gastrointestinal tract, provide distinct bile environments that are preferentially processed by Turicibacter strains isolated from their respective gastrointestinal tracts. This close relationship between host-specific bile composition and bacterial modification may be due in part to the bile sensitivity previously demonstrated by MOL361 and / or the high abundance of Turicibacter in the small intestine, which leads to stronger associations with host genes for bile reabsorption and luminal bile levels than other bile-modifying enterobacteria. 21、53~57 .

[0098] This study provides metabolic consequences of colonization by specific gut bacteria and improves our understanding of the association of specific taxa (in this case at the strain level) with host physiology. In rodent and human studies, the relative abundance of Turicibacter is often correlated with dietary fat. 29、31、58~62 and host obesity 28、33、63 negatively correlated with 30、64、65 This may be a result of the phenotypic diversity identified herein among Turicibacter isolates, where hosts may experience different lipid outcomes depending on their own particular Turicibacter strain, but may also vary depending on other characteristics such as host genetics and sex. 21、38 Importantly, the efficacy of Turicibacter colonization may also be influenced by the biogeographic composition of an individual's microbiota; in addition to specific taxonomic membership, Turicibacter location in the small and large intestine may affect the host outcome from their respective bile modifications by transforming the bile pool in either part of the intestinal tract. Despite the genomic and localization differences between them, this study found that MOL361 and H121 induced changes in lipid metabolites indicative of increased fatty acid oxidation, suggesting that these strains share at least some features that alter the host's fatty acid metabolism.

[0099] Further investigations into bsh gene regulation in Turicibacter, the relationship between bile and Turicibacter colonization and infection behavior, and the native functionality of the putative BSH and 7α-HSDH homologs examined in this study will further elucidate how these bacteria perform bile modification in the gut. Interestingly, while some of the in vitro bile transformations, particularly the increase in unconjugated bile acids, were generalizable between our in vitro characterization and Turicibacter colonization, the concomitant in vivo increase in certain conjugated bile acids seen during 1E2 and H121 colonization indicates the possibility of a more complex interplay between host bile production and Turicibacter colonization. The fact that the specificity and activity of individual BSH homologs differ when expressed in different bacterial backgrounds indicates that other unknown cellular or environmental factors influence the means by which individual BSHs act in vivo. This may also include mechanisms to modify the functionality of proteins encoded by other putative BSH homologs, studies identified from Turicibacter that did not deconjugate the specific tauro- or glycoconjugated bile acids used in our experiments. These results may be used to guide how bile-modifying genes can be used to shape the host lipid profile through microbiota manipulation, positioning certain microbial species as more suitable vectors for conferring specific host effects. It would also be informative to determine what other activities performed by Turicibacter result in increased WAT in colonized animals, which contrasts with results from mice colonized with a specific bsh-overexpressing strain of B. thetaiotaomicron. Given this result, Turicibacter may also be affecting host lipids through other mechanisms in addition to bile transformation, studies characterized.

[0100] Our study also links specific Turicibacter members and BSH activity to specific host outcomes. Although some host responses, such as broadly reduced triglycerides, were consistent across our BSH recipient mice, the exact lipid and cholesterol responses differed, indicating that the type of deconjugation may have different associations with host physiology. Further studies may provide insight into the mechanism by which deconjugation may play a role, as has been proposed for other bacteria. 54、68 , the exciting prospect of using Turicibacter and / or its bile modifications to purposefully alter the host's lipid biology to improve the host's metabolic and lipid-related health will continue to be strengthened. 66、67 Besides lipid biology, Turicibacter abundance is associated with Parkinson's disease. 69 and depression 70 It has been found that selective serotonin reuptake inhibitors (SSRIs) have a negative effect on the growth and colonization of Turicibacter. 71 This is because selective serotonin reuptake inhibitors (SSRIs) may inhibit the activity of Turicibacter's specific serotonin transporter. 38 SSRI use may lead to weight gain 72、73 SSRI use is frequently associated with metabolic side effects such as inflammatory bowel disease, and our results suggest a hypothesis linking these side effects to SSRI use: SSRI use may reduce gut colonization of bacteria such as Turicibacter, thus unintentionally altering their impact on host physiology. Future studies may develop strategies to reduce the interaction of SSRIs with the activity of the microbiota, minimizing the side effects of these drugs and improving host outcomes. Overall, these associations further highlight the importance of understanding the mechanisms linking diverse Turicibacter members to host physiology.

[0101] method Mouse husbandry Adult (6–8 weeks old) germ-free Swiss Webster mice were used for all animal experiments, following UCLA Animal Care and Use Committee-approved protocols. Mice were housed in flexible gnotobiotic isolators on a 12 h:12 h light:dark schedule on standard chow (Labdiet 5K52, 22.1%:16.6%:61.3% protein:fat:carbohydrate by calories) and then transferred to autoclaved filter-top cages with autoclaved chow (Labdiet 5010, 28.7%:13.1%:58.2% protein:fat:carbohydrate by calories) and water. After 1 day of acclimation to the cages, the indicated Turicibacter or Bacteroides thetaiotaomicron strains were grown overnight in YCFA medium (see below), pelleted by centrifugation, and resuspended in 1x PBS. Approximately 10 6 of colony forming units (CFU) of Turicibacter or approximately 10 8 Mice were colonized by oral gavage of 200 μL containing 100 CFU of B. thetaiotaomicron. Alternatively, mice were gavaged with the same volume of PBS alone (referred to as germ-free [GF]) or PBS-suspended fecal slurry from specific pathogen-free adult mice (referred to as conventional [CONV]). Colonization was quantified using strain-specific TuriSERT primers (Table 7) and quantitative PCR (qPCR) from weight-normalized contents from distal small intestine and proximal colon after addition of Low Abundance Microbiota Standard (Zymo) and extraction using the Zymo DNA Mini kit (Zymo).

[0102] bacterial culture Turicibacter isolates and Bacteroides thetaiotaomicron strains (Table 5) were cultured in an anaerobic 85% / 10% / 5% nitrogen / carbon dioxide / hydrogen mixture (Airgas) in flexible vinyl chambers (Coy). Turicibacter were grown on Schaedler's agar (BD Biosciences) or modified YCFA. 74Grown at 37°C on 100 mM MOPS, 10 g casitone, 2.5 g yeast extract, 2 g glucose, 2 g maltose monohydrate, 2 g cellobiose, 44 mg MgSO4, 68 mg CaCl2, 0.9 g NaCl, 10 mg hemin, 0.45 g K2HPO4, 0.45 g KH2PO4, 4 g NaHCO3, 1 g cysteine, 1 mg resazurin, 1.9 mL glacial acetic acid, 0.7 mL propionic acid, 90 μL isobutyric acid, 100 uL isovaleric acid, 100 uL valeric acid, 10 mL ATCC vitamin mix, 0.2% Tween-80). Cells were typically grown without shaking, but where appropriate, Turicibacter cultures were anaerobically transferred to sealed Hungate tubes or 1.7 mL microcentrifuge tubes and shaken at 37°C and 225 RPM.

[0103] For growth curves of B. thetaiotaomicron, overnight cultures were grown anaerobically in BHI-S at 37°C for approximately 48 hours to ensure culture saturation and then subcultured 1:50 for 6 hours at 37°C (final OD 600 = 0.41–0.51). All subcultures were then cultured at OD 600 = 0.1 and then six replicates were further diluted 1:10 in 100 μL of BHI-S in a 96-well plate. The plates were anaerobically sealed with parafilm and incubated at 37°C. OD 600 Readings were taken every 15 min in a Biotek Synergy H1 microplate reader (Agilent).

[0104] Escherichia coli C41-pLys (Lucigen) was used to characterize putative bile-modifying genes expressed from the pET21+ plasmid. E. coli was cultured at 100 μg mL -1 The strains were grown in Luria Broth (LB, 1% NaCl, 1% tryptone, 0.5% yeast extract) supplemented with 100 μM ampicillin with shaking aerobically at 37° C. Gene expression was induced by the addition of 100 μM IPTG.

[0105] Bacterial isolation and identification Frozen fecal samples were thawed on ice and diluted 1:10 with PRAS anaerobic dilution blank medium (Anaerobe Systems). 100 μL of diluted feces was further diluted 1:1000 with modified YCFA medium containing 0.05% bovine bile, 0.2% Tween-80, and 50 mM resorufin and loaded onto a Prospector® system array (Isolation Bio, San Carlos, CA, USA) according to the manufacturer's instructions. The fluorescent green signal of the array at time 0 was read on a Prospector® instrument in a Coy anaerobic chamber, and the array was incubated at 37° C. in an Anaerobic Systems AS-580 anaerobic chamber (Anaerobe Systems). At 17 and 41 hours of incubation, the array was scanned again, and the decrease in green fluorescence from time 0 was used as an indicator of bacterial growth in the array nanowells. Bacteria from the arrays were transferred to a 96-well transfer plate containing 200 μL of modified YCFA medium per well without the addition of 50 mM resorufin. The transfer plate was sealed with gas-permeable film and incubated at 37° C. for 7 days in a Mitsubishi AnaeroPack jar with a gassing bag (Remel). After incubation, the contents of 538 wells from transfer plates with visible turbidity were pooled into a secondary 96-well plate, preserved in reduced glycerol, and stored at −80° C. until needed. All manipulations of feces and isolates were performed anaerobically in a 5% CO2 / 5% H2 / 90% N2 atmosphere unless otherwise stated.

[0106] Genomic DNA was extracted from assembled Prospector® culture plates in 96-well format using the Extract All Kit (Applied Biosystems). 20 μL of culture was combined with 20 μL of lysis solution and incubated at 95° C. for 10 min followed by room temperature for 3 min. 20 μL of DNA stabilization solution was added to stabilize the DNA and the resulting DNA lysate was stored at −20° C. until required.

[0107] qPCR screening of novel Turicibacter isolates Genomic DNA from 538 isolates was analyzed using multiplex primer sets for Turicibacter 16S and Turicibacter TuriSERT. 38 Genes were screened for (Table 7). Each 25 μL qPCR reaction mixture had 1 μL Extract All lysate, 10 μL SYBR Power master mix (Applied Biosystems), 0.5 μL of each 10 μM primer, and 12 μL molecular grade water. Reactions were run in a QuantStudio 6 Flex (Thermo Fisher) with a 95°C hold followed by 40 cycles of 95°C for 15 s, 50°C for 30 s, and 72°C for 30 s. Turicibacter sanguinis MOL361 gDNA and water were used as positive and negative controls, respectively.

[0108] Molecular cloning Turicibacter genes were amplified from template culture lysates using Phusion or Q5 DNA polymerase (NEB) and primers designed to amplify the indicated Turicibacter genes. 48 The derived expression plasmids were assembled using Gibson assembly (see Table 7 for oligos) for expression in E. coli or Bacteroides thetaiotaomicron, respectively. Cloned constructs were verified by Sanger sequencing prior to functional characterization. pWW3837-derived constructs were synthesized as previously described 48 , and cloned into B. thetaiotaomicron VPI-5482. B. thetaiotaomicron expressing bsh was compared to B. thetaiotaomicron containing the original pWW3837 construct (referred to as wild-type B. thetaiotaomicron).

[0109] Genome assembly Each strain was streaked onto Schaedler agar plates and incubated anaerobically, then individual colonies from each isolate were picked onto YCFA medium and grown overnight at 37 °C. DNA was extracted using Zymo DNA mini kit (Zymo) and cells were lysed using bead beating. Purified genomic DNA was sequenced by MiGS (migscenter.org) and 151 bp paired-end sequences were assembled using CLC Genomics Workbench (Qiagen). The genome assembly has been deposited at NCBI under BioProject PRJNA846348.

[0110] Whole genome and gene comparisons Thank you 75 was used to profile and visualize DNA sequences of different Turicibacter strains, to identify the location of putative bile salt hydrolases and 7α-HSDH homologs within contig groups, to generate variability profiles, and to measure gene coverage and detection statistics. Average nucleotide identity (ANI) was calculated using the OrthoANIu 76 (available at https: / / www.ezbiocloud.net / tools / ani) was used to calculate the

[0111] Sequence comparison between 16S rRNA and bsh gene / BSH amino acid sequences was performed in CLC Genomics Workbench (Qiagen). 44 tblastn using the translated amino acid sequence from 77 This was done using

[0112] Assessment of biliary transformation In vitro characterization of bile transformation by engineered E. coli or B. thetaiotaomicron strains was performed by growing cells in each of the media conditions listed above supplemented with 0.5 mM (total concentration combined) of the indicated bile species. Cells were grown to stationary phase (shaking at 37 °C) and then frozen at -80 °C until further processing. Cells were then thawed, pelleted (5 min at 16,000 × g), and the supernatant removed to a new microcentrifuge tube. Three volumes of methanol were added, and the mixture was then mixed vigorously for 30-60 seconds and incubated (room temperature, 15 min). The mixture was centrifuged (5 min, 16,000 × g), and the supernatant removed to a sterile microcentrifuge tube and dried in a vacuum concentrator. The dried residue was treated with methanol / water / formic acid (50 / 50 / 0.1, all by volume), then mixed vigorously and centrifuged as above. The supernatant was transferred to a polypropylene HPLC vial, capped, and kept at 4 °C while an aliquot (usually 5 μL) was injected onto a reverse-phase HPLC column (Cadenza CD-C18, 3.0 μm, 250 × 2 mm, Imtakt), equilibrated with solution A (water / formic acid, 100 / 0.1, vol. / vol.), and eluted (0.2 mL min ) with increasing concentrations of solution B (acetonitrile / formic acid, 100 / 0.1, vol. / vol.). -1 ); min / %B: 0 / 30, 45 / 70, 48 / 100, 50 / 30, 67 / 30). The effluent from the column was analyzed by electrospray ionization using an electrospray ion source (capillary voltage 42 V, capillary temperature 275 °C, sheath gas flow rate 15 L min) connected to a linear ion trap mass spectrometer (Thermo LTQ). -1 Spectra were recorded and analyzed using software supplied by the instrument manufacturer. Confirmation of the proposed elemental composition was achieved by scanning with an Orbitrap mass spectrometer (Thermo LTQ XL) using the same chromatographic and ion source configuration as the spectra recorded.

[0113] For quantification of biliary species, an internal spike-in standard of 100 mM chenodeoxycholic acid-D4 (CDCA-D4, Sigma) was added to the initial culture supernatant as a normalization reference. Areas under the curve from reconstructed ion chromatograms were used to quantitate the abundance of each species.

[0114] Serum metabolite analysis Mice were euthanized with isoflurane and whole blood was collected by cardiac puncture. Blood was allowed to clot on ice in SST Vacutainer tubes (BD) and then centrifuged (4°C, 1 min, 1500 x g). Supernatants were removed and flash frozen in liquid nitrogen. Serum metabolites were analyzed using the Global Metabolomics Platform by Metabolon (Morrisville, NC, USA). Unless otherwise stated, values ​​presented are arbitrary units (au) for the specific metabolites, determined by log transformation of the volume-corrected quantitative values.

[0115] Circulating lipid analysis Mice were fasted for 4-6 h and then euthanized as described above. Blood was collected by cardiac puncture and placed on ice into anticoagulated K2EDTA Vacutainer tubes (BD). Blood was centrifuged (4°C, 15 min, 2000 × g) and then plasma was collected from the supernatant and flash frozen in liquid nitrogen. Shotgun lipidomics was performed by the UCLA Lipidomics Core (Los Angeles, CA, USA) using the following protocol: Thawed plasma was pipetted into glass tubes and a mixture of 70 internal standard lipids (Sciex and Avanti) was added and purified using a modified Bligh and Dyer extraction. 79Lipids were extracted using 100% ethanol. The pooled organic layers from the two extractions were dried in a vacuum concentrator and resuspended in 50 / 50 (vol. / vol.) methanol / dichloromethane and 10 mM ammonium acetate. After transfer to robovials, samples were analyzed using a Sciex 5500 equipped with a DMS Device (Lipidyzer Platform) using an acquisition list covering 1450 lipid species. The Lipidyzer Differential Mobility device was calibrated using EquiSPLASH LIPIDOMIX standard mixture (Avanti). Parameters previously described were used. 80 Data were analyzed using an in-house platform using and quantitative values ​​were normalized to input volume. Identification of statistical significance for species for inclusion in heatmaps was performed by two-tailed Welch's t-test (p-value cutoff <0.05).

[0116] Measurement of total colonic bile concentration Mice were colonized and fasted as above, then at the time of sacrifice, contents from approximately 1 cm of the proximal colon were collected into microcentrifuge tubes and flash frozen in liquid nitrogen. Thawed contents were weighed and suspended in water, then total bile levels were measured using a bile acid assay kit (Sigma). Total bile values ​​were normalized by sample mass, and each sample value was compared to gender-matched littermate controls. Values ​​marked "0" were below the limit of detection.

[0117] Calculation of fat cell area After sacrifice, the epididymal or gonadal white adipose tissue (e / g WAT) bodies of the mice were weighed and placed in 4% paraformaldehyde in 1× PBS at 4°C for 48 h. The fat bodies were washed twice with 70% ethanol and then submitted to the UCLA Translational Pathology Core Laboratory (Los Angeles, CA, USA) for paraffin embedding, sectioning and H&E staining. Ten adipocyte images from each animal (five from each fat body) were visualized by a 20x objective lens on an EVOS microscope (Thermo). Fiji 78 Adiposoft 79A plug-in (version 1.1.16) was used to automatically measure adipocyte area for all cells completely contained within the field of view.

[0118] qRT-PCR measurement of liver transcripts Gnotobiotic mice were monocolonized as above, and upon sacrifice, the median lobe of the liver was dissected and either directly flash frozen in liquid nitrogen (all Turicibacter colonized animals) or placed in Trizol, bead beat for 1 min, and then frozen in liquid nitrogen (all B. thetaiotaomicron colonized animals). All livers were then transferred to -80°C until further processing. Directly flash frozen livers were thawed overnight at -20°C in RNALater-ICE (ThermoFisher) and then bead beat in Trizol for 1 min, after which all samples were processed similarly. RNA was extracted from thawed Trizol samples using the Direct-Zol RNA Miniprep Kit (Zymo), and cDNA was then generated using the qScript cDNA Synthesis Kit (Quantabio). qPCR was performed using PowerUp SYBR Green Master Mix (ThermoFisher) on a QuantStudio5 Real-Time PCR System (ThermoFisher) (primers available in Table 7). 83、84 ) (Cycling conditions: 50°C for 2 min, 95°C for 2 min, 50 cycles of 95°C for 15 s, 55°C for 15 s, 72°C for 1 min, followed by a melting curve. Fold changes compared to gender-matched controls (GF for Turicibacter colonization and Bt-WT for B. thetaiotaomicron colonization) were calculated using the ΔΔCt method with autothreshold Ct values ​​with ppia as the housekeeping gene.

[0119] statistical analysis Statistical calculations were performed with Prism 9.3.1 (Graphpad). Unless otherwise stated, ***=p<0.0005, **=p<0.005, *=p<0.05, stated p-values=0.05<0.2. Heatmaps were generated using pheatmap in R. 80Package (version 3.6.3) 81 Created using.

[0120] Table 7 shows the list of oligos used in this study.

[0121] Table 8 shows the list of strains used in this study.

[0122] Table 9 shows serum metabolomics from mice monocolonized with GF, CONV, and Turicibacter. Volume-adjusted log-transformed levels of the listed serum metabolites from mice with different colonization states. Note that the statistics on the far right of the sheet are automatically generated as part of the analysis pipeline, but were not used in this study because the analyzed data did not have a normal distribution.

[0123] Table 10 shows absolute quantification of lipid species from the plasma of mice colonized with B. thetaiotaomicron engineered to express the Turicibacter bsh gene.

[0124] [Table 7] TIFF2025503418000073.tif252161TIFF2025503418000074.tif250160

[0125] [Table 8] TIFF2025503418000076.tif168161

[0126] [Table 9] TIFF2025503418000078.tif250161TIFF2025503418000079.tif250161TIFF2025503418000080.tif250161TIFF2025503418000081.tif251161TIFF2025503418000082.tif250162TIFF2025503418000083.tif250162TIFF2025503418000084.tif251161TIFF2025503418000085.tif251161TIFF2025503418000086.tif251161TIFF2025503418000087.tif249161TIFF2025503418000088.tif251161TIFF2025503418000089.tif250162TIFF2025503418000090.tif251161TIFF2025503418000091.tif251161TIFF2025503418000092.tif251161TIFF2025503418000093.tif252161TIFF2025503418000094.tif252162TIFF2025503418000095.tif250162TIFF2025503418000096.tif252161TIFF2025503418000097.tif252161TIFF2025503418000098.tif250162TIFF2025503418000099.tif252161TIFF2025503418000100.tif251162TIFF2025503418000101.tif252162TIFF2025503418000102.tif251161TIFF2025503418000103.tif250161TIFF2025503418000104.tif251161TIFF2025503418000105.tif251161TIFF2025503418000106.tif250161TIFF2025503418000107.tif251162TIFF2025503418000108.tif251161TIFF2025503418000109.tif251161TIFF2025503418000110.tif250161TIFF2025503418000111.tif250161TIFF2025503418000112.tif239161TIFF2025503418000113.tif239161TIFF2025503418000114.tif237162TIFF2025503418000115.tif237162TIFF2025503418000116.tif237160TIFF2025503418000117.tif237162TIFF2025503418000118.tif239161TIFF2025503418000119.tif237161TIFF2025503418000120.tif237161TIFF2025503418000121.tif236161TIFF2025503418000122.tif237161TIFF2025503418000123.tif238162TIFF2025503418000124.tif239161TIFF2025503418000125.tif238163TIFF2025503418000126.tif235158TIFF2025503418000127.tif237161TIFF2025503418000128.tif238161TIFF2025503418000129.tif237162TIFF2025503418000130.tif239161TIFF2025503418000131.tif237162TIFF2025503418000132.tif239161TIFF2025503418000133.tif238162TIFF2025503418000134.tif238162TIFF2025503418000135.tif237159TIFF2025503418000136.tif237161TIFF2025503418000137.tif237161TIFF2025503418000138.tif238162TIFF2025503418000139.tif238161TIFF2025503418000140.tif238162TIFF2025503418000141.tif238161TIFF2025503418000142.tif238162TIFF2025503418000143.tif237161TIFF2025503418000144.tif239162TIFF2025503418000145.tif238162TIFF2025503418000146.tif239161.

[0127]

Table 10

[0128] [Table 11] TIFF2025503418000261.tif245166TIFF2025503418000262.tif247166TIFF2025503418000263.tif245162TIFF20255034180 00264.tif250165TIFF2025503418000265.tif246166TIFF2025503418000266.tif244165TIFF2025503418000267.tif247164

[0129] Incorporation by Reference All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0130] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A composition comprising a bacterial strain that expresses a bile salt regulatory gene or a bile acid regulatory gene, and a pharmaceutically acceptable carrier.

2. 2. The composition of claim 1, wherein the bacterial strain is Turicibacter sanguinis, Escherichia coli, or Bacteroides thetaiotaomicron, and optionally the bacterial strain modulates bile salts by conjugation with glycine or conjugation with taurine.

3. 3. The composition of claim 2, wherein the T. sanguinis bacterial strain is any one of the T. sanguinis bacterial strains listed in Table 4.

4. 4. The composition of any one of claims 1 to 3, wherein the bile salt regulatory gene encodes a bile salt hydrolase (BSH), preferably wherein said BSH is any one of the bile salt hydrolases listed in Table 1.

5. 4. The composition of claim 1, wherein the bile acid regulatory gene encodes a 7-alpha hydroxysteroid dehydrogenase, and preferably the amino acid sequence of said 7-alpha hydroxysteroid dehydrogenase is at least 40% homologous to the amino acid sequence of the 7-alpha hydroxysteroid dehydrogenase of Clostridium absonum.

6. 4. The composition of any one of claims 1 to 3, wherein the bile salt or bile acid is any one of the bile salts or bile acids listed in Table 3.

7. The composition of any one of claims 1 to 3, wherein the composition is formulated for oral delivery.

8. The composition of claim 7, wherein the composition is a food product.

9. The composition of claim 8 wherein the food product is a dairy product.

10. 10. The composition of claim 9, wherein the food product is yogurt.

11. The composition of any one of claims 1 to 3, wherein the composition is formulated for rectal delivery.

12. A composition described in any one of claims 1 to 3 for preventing or treating a metabolic disorder in a subject.

13. The composition described in claim 12, wherein the metabolic disorder is a lipid metabolism disorder and / or a steroid metabolism disorder.

14. A composition comprising: a. a composition for administering to a subject a composition for administering to a subject a composition for administering to a subject; b. administration of the composition reduces white adipose tissue mass in the subject; c. administration of the composition alters health-related lipid biomarkers in the subject; d. administration of the composition reduces abdominal fat pad mass in the subject; or e. administration of the composition increases bile acids in the subject; The composition of claim 12.

15. The composition described in claim 12, wherein administration of the composition reduces plasma triglyceride (TG) levels, and / or cholesterol levels, and / or cholesterol ester (CE) levels.

16. Use of a composition according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of a metabolic disorder.

17. The use described in claim 16, wherein the metabolic disorder is a lipid metabolism disorder and / or a steroid metabolism disorder.

18. transforming the bacterial strain with a gene expression construct encoding a bile salt-regulated gene or a bile acid-regulated gene operably linked to a promoter that drives expression of the bile salt-regulated gene or the bile acid-regulated gene in the bacterial strain; and Culturing the bacterial strain to express a bile salt-regulated gene or a bile acid-regulated gene. A method of making the composition of any one of claims 1 to 3, comprising:

19. A bacterial strain comprising a bile salt regulatory gene or a bile acid regulatory gene, wherein the bacterial strain expresses the bile salt regulatory gene or the bile acid regulatory gene encoded by an exogenous nucleic acid.

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