Compositions and uses of Turicimonas muris for treating metabolic disorders

Abstract

The present invention relates to the prevention and / or treatment of metabolic disorders such as overweight and obesity, and complications associated therewith. More specifically, the present invention relates to the bacterium Turicimonas muris or a fragment thereof for preventing and / or treating metabolic disorders and complications associated therewith.

Description

[Technical Field]

[0001] The present invention relates to the prevention and / or treatment of metabolic disorders, such as overweight and obesity, and complications associated therewith. More specifically, the present invention relates to the bacterium Turicimonas muris or fragments thereof for the prevention and / or treatment of metabolic disorders and complications associated therewith. [Background technology]

[0002] Obesity is defined as the excessive accumulation of fat mass or adipose tissue (AT) that impairs health and can lead to the development of numerous pathologies (e.g., type 2 diabetes (T2D), cardiovascular disease, dyslipidemia, cancer, etc.) This excessive adiposity results from the interplay of genetic, epigenetic, dietary, and environmental factors that contribute to an imbalance in energy balance.

[0003] The clinical diagnosis of obesity has traditionally been based on calculating the body mass index (BMI, weight in kilograms divided by height in meters squared). In adults, a BMI of 18.5 kg / m 2 from 24.99 kg / m 2 A BMI between 25 and 29.99 is considered normal. Overweight is defined by a BMI between 25 and 29.99, while obesity is defined by a BMI of 30 or greater. The severity of obesity is classified into three grades: Grade I (moderate obesity; 30≦BMI≦35), Grade II (severe obesity; 35≦BMI≦40), and Grade III (morbid obesity; BMI≧40). According to the World Health Organization, 39% of adults worldwide are overweight and 13% are obese.

[0004] Several studies have reported a strong positive relationship between high BMI and an increased risk of developing T2D. In this sense, obese individuals are defined as those with a BMI of 25 kg / m 2Individuals with a BMI lower than 1.25 are up to 80 times more likely to develop T2D than those with a BMI lower than 1.25. T2D is characterized by chronic hyperglycemia due to a combination of insulin resistance in peripheral tissues (liver, skeletal muscle, and adipose tissue) and insufficient insulin secretion by pancreatic beta cells. Clinical diagnosis is based on two fasting blood glucose levels of 1.26 g / L or higher or a blood glucose level of 2 g / L or higher at any time during the day. T2D significantly impairs quality of life and lifespan, particularly due to its vascular complications, such as microangiopathy (nephropathy, retinopathy, and neuropathy) and macroangiopathy (stroke and myocardial infarction). According to Sanjay Basu et al., nearly 406 million people worldwide suffered from T2D in 2018 (Non-Patent Document 1).

[0005] These chronic diseases worsen over time and significantly reduce life expectancy. Therefore, they represent a major public health problem with significant economic and social burdens. The medical costs of obesity account for 1% to 3% of healthcare expenditures in most countries (Non-Patent Document 2).

[0006] In addition to dietary rebalancing and the promotion of regular physical activity, pharmacological approaches may be offered to patients with obesity and associated metabolic pathologies. These drug approaches are non-curative and aim to suppress symptoms, slow disease progression, and limit the risk of complications. To date, five anti-obesity drugs have been approved for long-term use by the US Food and Drug Administration: orlistat, phentermine-topiramate, naltrexone-bupropion, liraglutide, and semaglutide (Non-Patent Document 1). In France, only orlistat, and more recently liraglutide, have been approved for use in the treatment of obesity.

[0007] On the other hand, treatments for T2D are mainly aimed at improving insulin sensitivity (metformin) or enhancing insulin secretion by pancreatic β-cells (sulfonylureas, glinides, semaglutide). More recently, gliflozins, i.e. SGLT2 inhibitors, which cause increased glycosuria, may be proposed.

[0008] If lifestyle and dietary approaches and non-insulin treatments fail to achieve glycemic control goals (glycated hemoglobin levels of 7% or less), insulin therapy should be initiated (Non-Patent Document 3).

[0009] Current lifestyle-, diet-, and drug-based approaches are unable to effectively and sustainably treat most patients with obesity and its associated diseases. Therefore, new therapeutic targets and treatments must be developed to improve patient care, slow the progression of these diseases, and prevent the development of complications.

[0010] Over the past 20 years, an increasing number of studies have identified the gut microbiota (GM) as a new player in the pathophysiology of obesity and associated metabolic disorders. The microbiota is an ecosystem consisting of a set of microorganisms (viruses, bacteria, archaea, fungi, and protists) that inhabit a given ecological niche (in this case, the digestive system). Recent studies have estimated that the adult gastrointestinal tract (DT) is home to approximately 100 trillion bacteria belonging to approximately 4,600 species (Non-Patent Document 4).

[0011] GM bacteria perform essential biological functions: they are involved in the metabolism of xenobiotics and indigestible food, synthesize vitamins, produce certain bioactive metabolites (e.g., short-chain fatty acids (SCFAs)), and are essential for the maturation and activation of the immune system. Numerous studies in humans and mice have reported a strong positive correlation between high bacterial richness (i.e., alpha diversity) in the GM, metabolic homeostasis, and the absence of inflammation. In contrast, obesity and T2D are pathologies associated with a reduction in GM microbial diversity and dysbiosis, i.e., a persistent imbalance in the composition and function of the bacterial community.

[0012] The advent of culturomics, i.e., high-throughput culturing to characterize the microbial composition of GM, and the recent optimization of bacterial culture media, have enabled an increasing number of strains to be isolated, cultivated in pure conditions, and characterized. These advances have paved the way for in vitro studies of the metabolic capabilities of these bacteria and for in vivo studies of target bacterial supplementation. This has therefore made it possible to study the causal relationship between certain bacterial species in GM and the regulation of the host's physiological state and to identify the cellular and molecular mechanisms involved.

[0013] To date, only a limited number of species and strains (fewer than 10) have been isolated and characterized as microorganisms with direct causal effects on metabolic health. The mechanisms involved have been identified, even partially, for even fewer bacteria. These results, based on studies made possible only by pure bacterial culture, demonstrate an undeniable causal role for certain GM bacteria in regulating host energy metabolism. Therefore, the investigation of effective, non-invasive therapeutic strategies aimed at treating, or even preventing, obesity and its associated metabolic pathologies requires the isolation and functional analysis of new bacterial strains potentially beneficial to the host. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Basu et al. The Lancet. Diabetes & Endocrinology. January 2019, Vol. 7, No. 1, pp. 25-33 [Non-patent document 2] OECD. Health Goals. October 2010. https: / / www.oecd.org / fr / els / systemes-sante / 46044602.pdf [Non-patent document 3] HAS. Drug strategy for glycemic control in Type 2 diabetes, January 2013. https: / / www.hassante.fr / uploa / docs / application / pdf / 2023-02 / 10irp04_synth_diabete_type_2_objectif_glycemique_messages_cles.pdf [Non-patent document 4] Almeida et al. Nature Biotechnology. January 2021, Vol. 39, No. 1, pp. 105-114 Summary of the Invention

[0015] In this context, we demonstrated the beneficial effects of the bacterium Turicimonas muris on energy metabolism and physiology in mice. More specifically, administration of Turicimonas muris reduced weight gain and fat mass induced by an obesogenic diet. The beneficial effects of this bacterium on body composition in mice fed a high-fat diet (HFHS) were associated with improved glucose tolerance compared with control mice fed under the same conditions. Administration of Turicimonas muris appears to have beneficial effects on body composition and carbohydrate metabolism in mice without affecting their food intake or fecal volume. We also demonstrated a correlation between the relative abundance of Turicimonas muris measured in the feces of human subjects and their favorable metabolic status (particularly reduced blood glucose).

[0016] Thus, the present invention relates to Turicimonas muris for use in the prevention and / or treatment of metabolic diseases and / or complications associated therewith.

[0017] The present invention also relates to the use of Turicimonas muris to improve glucose and fat metabolism, increase energy expenditure and / or promote weight loss, particularly in overweight or obese individuals.

[0018] The species Turicimonas muris (Taxonomic ID: 1796652) was isolated from the cecal contents of mice by Lagkouvardos et al. during the establishment of the Mouse Intestinal Bacterial Collection, a public repository of 100 bacterial strains isolated from the cecal contents of mice with a conventional microbiota (specific pathogen-free (SPF) status) (LAGKOUVARDOS et al. Nature Microbiology. October 2016, Vol. 1, No. 10, p. 16131). This Gram-negative bacterium grows optimally at temperatures ranging from 20°C to 45°C (mesophilic) and is present in the human GM. Turicimonas muris belongs to the family Sutterellaceae and the phylum Proteobacteria (hereafter referred to as Pseudomonadota).

[0019] The present invention also relates to bacterial strains belonging to the same family as Turicimonas muris, or genetically modified Turicimonas muris strains that retain the same properties as Turicimonas muris.

[0020] Turicimonas muris bacteria can be cultured as described in the experimental section.

[0021] For purposes of the present invention, Turicimonas muris can be used in viable or non-viable form, as well as in pasteurized or lyophilized form. Turicimonas muris can also be used in fragmented form. The term "fragment" refers to cellular components, metabolites, secreted molecules, and compounds resulting from the metabolism of Turicimonas muris. Fragments can be obtained, for example, by collecting the supernatant of a Turicimonas muris culture or by extracting cellular components or cell fractions, metabolites, or secreted compounds from a Turicimonas muris culture. The term "fragment" can also refer to degradation products, particularly metabolic products.

[0022] According to one embodiment, the Turicimonas muris or fragments thereof are substantially purified. As used herein, the term "substantially purified" means that the Turicimonas muris or fragments thereof comprise at least about 50%, preferably at least about 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more of the bacterial strain or fragments thereof by weight in the sample.

[0023] The term "metabolic disease" refers to disorders, diseases, and conditions caused by or characterized by weight gain, abnormal energy utilization or expenditure, altered responses to ingested or endogenous nutrients, energy sources, hormones, or other signaling molecules in the body, or altered metabolism of carbohydrates, lipids, proteins, nucleic acids, or combinations thereof. Metabolic diseases can be associated with deficiencies or excesses in metabolic pathways that result in imbalances in carbohydrate, lipid, protein, and / or nucleic acid metabolism.

[0024] Examples of metabolic disorders include, but are not limited to, metabolic syndrome, disorders of insulin deficiency or insulin resistance, diabetes (e.g., type 2 diabetes), impaired glucose tolerance, abnormal lipid metabolism (or dyslipidemia), overweight, and obesity; complications of metabolic disorders (also called comorbidities) include, among others, atherosclerosis, hypertension, preeclampsia, heart disease, stroke, non-alcoholic fatty liver disease, hyperglycemia, and fatty liver disease of various etiologies. These include immune system dysfunction associated with overweight and adiposity, cardiovascular disease, hypercholesterolemia, elevated triglycerides, asthma, sleep apnea, osteoarthritis, neurodegeneration, gallbladder disease, syndrome X, inflammatory and immune disorders, atherogenic dyslipidemia, and cancer, particularly esophageal cancer, gastric cardia (a type of stomach cancer), colon cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer in postmenopausal women, breast cancer, ovarian cancer, kidney cancer, meningioma (brain tumor), thyroid cancer, and multiple myeloma.

[0025] The amount of Turicimonas muris administered is preferably selected to restore the normal bacterial population in the intestine, i.e., the bacterial population found in a healthy subject (not overweight or obese).

[0026] For example, the dose of Turicimonas muris administered is approximately 1 × 10 2 cfu (colony forming units) to 1 x 10 15 Among cfu, especially approximately 1 × 10 4 cfu to 1 × 10 12 Between cfu, or approximately 1 x 10 6 cfu to 1 × 10 10 cfu, and this amount is preferably administered daily. If Turicimonas muris is administered in a non-viable form or as fragments, the above-mentioned amounts are used to prepare this form. The amount of Turicimonas muris administered is also approximately 1 x 10 6 cells ~ approximately 1 x 10 12 in the range of 1 x 10 cells, preferably approximately 1 x 10 8 cells to approximately 1 × 10 10 between cells, or approximately 1 x 10 9 cells to approximately 1 × 10 10 The amount may be between 100 and 100 cells, and this amount is preferably administered daily.

[0027] The present invention also relates to a composition comprising Turicimonas muris and a physiologically acceptable carrier.

[0028] The term "physiologically acceptable carrier" means a carrier that can be administered and well tolerated by an individual, particularly a mammal, preferably a human.

[0029] Examples include, but are not limited to, bulking agents such as corn starch, and carrier agents such as potato starch. The composition may be incorporated into a vegetable-derived capsule, and may also include an anti-caking agent such as magnesium stearate or silicon dioxide.

[0030] The composition of the present invention can be used to kill approximately 1 x 10 Turicimonas muris in viable, non-viable, or fragmented form. 2 cfu to 1 × 10 15 Among cfu, especially approximately 1 × 10 4 cfu to 1 × 10 12 Between cfu, or approximately 1 x 10 6 cfu to approximately 1 x 10 10 cfu, or approximately 1 x 10 6 cells to approximately 1 × 10 12 Between 1 x 10 cells, preferably approximately 1 x 10 8 cells to approximately 1 × 10 10 between cells, or approximately 1 x 10 9 cells to approximately 1 × 10 10 It contains the amount contained between cells.

[0031] Turicimonas muris or a fragment thereof, or a composition according to the invention, can be administered by several routes of administration.

[0032] Examples of suitable routes of administration include, but are not limited to, oral administration, rectal administration, administration by esophagogastroduodenoscopy, administration by colonoscopy, administration using a nasogastric or orogastric tube, etc., preferably administration is by oral or rectal route.

[0033] According to one embodiment, Turicimonas muris or a fragment thereof, or a composition of the present invention, is in a form suitable for oral administration. According to this embodiment, the form suitable for oral administration comprises: a solid dosage form selected from the group consisting of a tablet, pill, capsule, soft gelatin capsule, dragee, orodispersible tablet, effervescent tablet, or other solid form (according to certain embodiments, the oral solid dosage form is gastroresistant, so that its contents do not decompose while remaining in the stomach); Liquid dosage forms, such as oral liquids, liposomal dosage forms, etc. It could be.

[0034] When administered orally, the composition of the present invention may be a nutritional composition or a foodstuff.

[0035] According to one embodiment, Turicimonas muris or a fragment thereof, or a composition of the present invention, is in a form suitable for rectal administration. According to this embodiment, the composition may be a suppository or a rectal capsule.

[0036] According to one embodiment, the composition of the invention further comprises probiotics and / or prebiotics. According to this embodiment, the composition of the invention is preferably administered orally.

[0037] The term "probiotic" refers to a microbial cell preparation (e.g., live microbial cells) that, when administered in an effective amount, has a beneficial effect on the health or well-being of a subject. By definition, all probiotics are proven to be non-pathogenic. In one embodiment, these health benefits are related to improving the balance of, and / or restoring normal, the human or animal microflora in the gastrointestinal tract.

[0038] For example, probiotics Bacteria: Lactobacillus, Lactococcus, Bifidobacterium, Veillonella, Desemzia, Christensenella, Allobaculum, Coprococcus, Collinsella, Citrobacter, Turicibacter ), Sutterella, Subdoligranulum, Streptococcus, Sporobacter, Sporacetigenium, Ruminococcus, Roseburia, Proteus, Propionobacterium, Leuconostoc, Weissell a), Pediococcus, Streptococcus, Prevotella, Parabacteroides, Papillibacter, Oscillospira, Melissococcus, Dorea, Dialister, Clostridium, Cedecea, Catenibacterium Genus (Catenibacterium), genus Butyrivibrio, genus Buttiauxella, genus Bulleidia, genus Bilophila, genus Bacteroides, genus Anaerovorax, genus Anaerostopes, genus Anaerofilum, family Enterobacteriaceae, phylum Firmicutes,Atopobium, Alistipes, Acinetobacter, Slackie, Shigella, Shewanella, Serratia, Mahella, Lachnospira, Klebsiella, Idiomarina, Fusobacterium, Faecalibacterium, Eubacterium, Enterococcus, Enterobacter, Eggerthella, In particular, Bifidobacterium animalis, especially Bifidobacterium animalis spp. lactis, Bifidobacterium lactis, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Akkermansia muciniphila, Prokaryotic microorganisms: Archaea, Firmicutes, Verrucomicrobia, Christensenella, Bacteroidetes (e.g., Allistipes, Bacteroides ovatus, Bacteroides splachnicus, Bacteroides stercoris, Parabacteroides, Prevotella ruminicola) ruminicola, Porphyromondaceae, etc.), Proteobacteria, Betaproteobacteria (e.g., Aguabacterium and Burkholderia, etc.), Gammaproteobacteria (e.g., Xanthomonadaceae, etc.), Actinobacteria (e.g., Actinomycetaceae and Atopobium, etc.), Fusobacteria, Mesophyllaceae, Methanobacteria, Spirochaetes, Fibrobacteres, Deferribacteres, Deinococcus, Thermus, Cyanobacteria, Methanobrevibacteria, Peptostreptococcus, Ruminococcus, Coprococcus, Subdoligranulum, Dorea, Bulleidia,The genera Anaerofustis, Gemella, Roseburia, Dialister, Anaeroturuncus, Staphylococcus, Micrococcus, Propionobacteria, Enterobacteriaceae, Faecalibacterium, Bacteroides, Parabacteroides, Prevotella, Eubacterium, Bacilli (e.g., Lactobacillus salivarius), salivarius, Aerococcus, Granulicatella, Streptococcus bovis, and Streptococcus intermedius, etc.), Clostridium (e.g., Eubacterium hallii, Eubacterium limosum, etc.), and Butyrivibrio, Yeasts: Ascomycetes, Zygomycetes, and Deuteromycetes, especially Aspergillus, Torulopsis, Zygosaccharomyces, Hansenula, Candida, Saccharomyces, Clavispora, Bretanomyces, Pichia, and Amylomyces the genera Amylomyces, Zygosaccharomyces, Endomyces, Hyphopichia, Zygosaccharomyces, Kluyveromyces, Mucor, Rhizopus, Yarrowia, Endomyces, Debaryomyces and the group of the genera Penicillium, may be selected from:

[0039] In particular, the following strains can be mentioned: Lactobacillus species: L. acidophilus, L. amylovorus, L. casei, L. gasseri, L. helveticus, L. johnsonii, L. pentosus, L. plantarum, L. reuteri, and L. rhamnosus; Bifidobacterium species: B. adolescentis, B. animalis, B. bifidum, B. breve, B. infantis, and B. longum; Other lactic acid bacteria: Enterococcus faecium, Lactococcus lactis, and Streptococcus thermophilus, Other microorganisms: Bacillus clausii, Escherichia coli Nissle 1917, and Saccharomyces cerevisiae (boulardi).

[0040] Preferably, the probiotic (or LBP) is selected from Escherichia coli K12, Christensenella minuta, Anaerobutyricum soehngenii, Dysosmobacter welbionis, and Akkermansia muciniphila, Faecalibacterium prausnitzii, Hafnia alvei 4597, and P. goldsteinii.

[0041] For purposes of the present invention, probiotics can be used in viable or non-viable form, or in fragmented form. The probiotics are administered in the same amounts as those indicated for Turicimonas muris.

[0042] The term "prebiotic" refers to substances, e.g., substances that cannot be digested by humans but that, through their metabolism by intestinal microorganisms, modulate the composition and / or activity of the intestinal microbiota and therefore confer a beneficial physiological effect on the host.

[0043] The prebiotic may be dietary fiber. The dietary fiber may be selected from the group consisting of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides, isomaltose, soybean oligosaccharides, pyrodextrin, transgalactosylated oligosaccharides, lactulose, beta-glucan, inulin, raffinose, and stachyose. Dietary fiber also has the advantage of being resistant to some conditions, particularly heat and long-term storage. Dietary fiber may also contribute to the treatment within the scope of the present invention by improving gastrointestinal health and increasing satiety.

[0044] Other non-limiting examples of prebiotics include water-soluble cellulose derivatives, water-insoluble cellulose derivatives, raw oat flakes, psyllium, wheat bran, and mixtures thereof. Examples of water-soluble cellulose derivatives include, in particular, methylcellulose, methylethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, cationic hydroxyethylcellulose, as well as hydroxypropylcellulose, hydroxyethylmethylcellulose, but also hydroxypropylmethylcellulose and carboxymethylcellulose.

[0045] The compositions according to the invention may be administered in combination with drugs intended for the treatment of obesity, such as orlistat, phentermine-topiramate, naltrexone-bupropion, liraglutide, semaglutide, and tirzepatide, and / or in combination with drugs intended for the treatment of type 2 diabetes, such as metformin, sodium-glucose cotransporter type 2 inhibitors, gliptins, acarbose, glinides, sulfonamides, or even sulfonylureas, such as glimepiride.

[0046] The present invention also relates to the (non-therapeutic) use of Turicimonas muris to promote weight loss in an individual, as well as to a method for preventing and / or treating metabolic diseases and their complications, comprising the administration of Turicimonas muris or a fragment thereof or a composition according to the invention. [Brief explanation of the drawings]

[0047] [Figure 1] This figure shows that T. muris is primarily present in the ileum of non-obese mice. qPCR was performed using specific (T. muris) primers and nonspecific (total bacterial) primers on jejunal (a-c) and ileal (d-f) content samples from mice fed a control chow diet or an HFD supplemented with or without FOS and / or 5-ASA. (a, d) Bacterial load for each mouse group. (b, e) T. muris concentration. (c, f) Relative abundance of T. muris. Data were analyzed using a Kruskal-Wallis test followed by Dunn's pairwise multiple comparison test and are presented as scatter plots with mean ± SEM. Number of mice per group: n = 5–12. *q<0.05, **q<0.01, ***q<0.001, ****q<0.0001. [Figure 2]Figure 1 shows that T. muris prevents diet-induced obesity. (a) Diagram of experimental procedures. Animals in two of the four groups were fed a HFHS diet, while animals in the remaining two groups were fed a control diet (chow diet). Mice in the HFHS diet group and one of the chow diet groups were supplemented once daily with T. muris, its lyophilized culture supernatant, or culture medium uninoculated with T. muris. (b, c) Body weight and weight gain. (d, e) Fat mass and fat mass gain. Weights (mg) of various fat depots after sacrifice: (f) SAT: subcutaneous (inguinal) AT, (g) EAT: epididymal AT, (h) BAT: brown AT. Data in (b) and (d) were analyzed using two-way ANOVA followed by Tukey's post-hoc test. Data in (c), (e), (f), (g), and (h) were analyzed using a Kruskal-Wallis test followed by Dunn's pairwise multiple comparison test. Results are presented as histograms with mean ± SEM. Number of mice per group: n = 9. *q<0.05, **q<0.01, ***q<0.001, ****q<0.0001 for HFHS vs. HFHS-T. muris; ◇◇q<0.01, ◇◇◇q<0.001, ◇◇◇◇q<0.0001 for chow vs. HFHS. [Figure 3] Figure 1: T. muris prevents diet-induced changes in carbohydrate metabolism. (a) Fasting blood glucose measured during week 3. (b) Blood glucose profile, and (c) Area under the curve measured during an oral glucose tolerance test (OGTT) performed during week 10. Results in panels a and c were analyzed using a Kruskal-Wallis test followed by Dunn's post-hoc test. Results in panel b were analyzed using a two-way ANOVA followed by Tukey's post-hoc test. Number of mice per group: n = 9. *q < 0.05, **q < 0.01, ***q < 0.001 for the HFHS vs. HFHS-T. muris comparison; ◇◇q < 0.01, ◇◇◇◇q < 0.0001 for the chow vs. HFHS comparison. [Figure 4]A figure measuring the relative abundance of Turicimonas muris by quantitative PCR in the feces of individuals having either a normal body mass index (18 < BMI < 25) or an obese body mass index (BMI > 30), and individuals who initially had obesity and type 2 diabetes but either had or did not have remission of type 2 diabetes five years after undergoing bariatric surgery (Roux-en-Y bypass). (A) Relative abundance of T. muris (% of total bacteria) according to body mass index. Mann-Whitney test. (B) Proportion of individuals having a detectable level of T. muris by quantitative PCR (i.e., a level exceeding 0.00009% of total bacteria) according to body mass index. Chi-square test. (C) Relative abundance of T. muris according to fasting blood glucose in obese patients (BMI > 30). A threshold (1.1 g / L) for moderate fasting hyperglycemia is shown. Overall, T. muris is not detected in patients with moderate fasting hyperglycemia, but is detected in some patients with normal fasting blood glucose values. (D) Relative abundance of T. muris in patients who initially had obesity and type 2 diabetes but either had or did not have remission of type 2 diabetes. T. muris is not detected in patients still having diabetes, but is detected in some patients with remission. Mann-Whitney test.

Example

[0048] I. Materials and Methods Bacterial culture of Turicimonas muris: Culture medium: The powder (Table 1) was dissolved in distilled water, followed by the addition of formic acid, resazurin solution (a colored redox indicator), and hemin stock solution. The pH was adjusted to 7.8. The resulting medium was transferred into glass vials and boiled in a microwave oven to remove dissolved oxygen. The vials were then capped and autoclaved. At the same time, temperature-sensitive components (Table 2) were transferred into empty vials. The vials were capped, and a mixture of CO2, H2, and N2 was injected using a needle. A second needle was then inserted into the septum to expel the O2 present in the air. Anaerobic water, FCS, and vitamin stock solution were added to the powder. 3 ml of the resulting solution and filter-sterilized menadione solution (final 80 ng / ml) were added to each medium vial. Finally, these medium vials were pressurized by injecting a mixture of filter-sterilized CO2, H2, and N2 and stored at 4°C.

[0049] [Table 1]

[0050] [Table 2]

[0051] Inoculation and administration: Turicimonas muris (DSM 22575) was purchased from the German Public Collection of Microbial Cell Cultures (DSMZ) and cultivated for 48 h at 37°C under sterile anaerobic conditions. For administration to mice, bacterial cultures were centrifuged at 4000g for 20 min at 4°C. The bacterial pellet was resuspended in culture supernatant, which had been concentrated 5-fold by lyophilization. 5 × 10 9 These preparations, measured as cfu (colony forming units) / ml, were finally stored in glycerol at -80°C until force-feeding of the mice.

[0052] Animal Model: Thirty-six 8-week-old male C57BL / 6J SPF mice (Charles River Laboratories, France) were housed in a controlled environment (temperature 22 ± 2°C, 12-h light / dark cycle) with food and water available ad libitum. Mice were allowed to acclimate for 7 days without handling while being fed a control diet (chow diet, 3.91 kcal / g, 4% fat, Research Diet, Reference No. 98121701). After acclimatization, mice were identified by ear tag and divided into four groups of nine mice with similar weight and body composition. Two of the four groups were fed the control diet for 12 weeks, while both groups were fed a diet enriched in lipids and sucrose (high-fat, high-sucrose diet: HFHS, 4.68 kcal / g, 21% fat, and 50% carbohydrate, Research Diet, Reference No. D12079B).

[0053] To demonstrate the effect of Turicimonas muris on host physiology, bacteria were administered once daily by oral gavage in the form of viable cells resuspended in culture supernatant (200 μL of a solution containing Turicimonas muris, i.e., 1 × 10 9 cfu). Control mice received uninoculated culture medium, which was also concentrated five-fold by freeze-drying. Force-feeding of the animals began 1 week after the animals arrived at the animal facility, coinciding with the diet change for the HFHS group.

[0054] Mouse Sacrifice: Immediately prior to euthanasia by cervical dislocation, 36 mice were anesthetized with isoflurane (3%) and intracardiac blood samples were collected. Tissues and organs of interest were collected, weighed, and frozen in liquid nitrogen (liver, spleen, inguinal, epididymal, and interscapular AT, ileum and ileal contents, jejunum and jejunal contents, colon, and gastrocnemius muscle). Liver and jejunum were fixed in paraformaldehyde (4%) for histological analysis before various tissues / organs were embedded in paraffin using the HISTOMICS (ICM-Pitié-Salpêtrière) platform.

[0055] All experiments were approved and conducted in accordance with the guidelines of the Charles Darwin Ethics Committee.

[0056] Body composition analysis: To monitor the development of obesity, the body composition (fat mass, lean mass, and fluid) of the mice was determined by nuclear magnetic resonance (NMR-LF90 Minispec+ scanner) at the time of feeding, then every 3 weeks, and the day before sacrifice.

[0057] Oral glucose tolerance test (OGTT): Mice were fasted for 6 hours and given an oral glucose load (2 g glucose / kg body weight). Blood glucose levels were measured using a glucose meter (ROCHE AccuCheck) from a drop of blood collected from the lateral tail with a 23G needle at -30, 0.15, 30, 60, and 90 minutes after the carbohydrate load. At 0 and 15 minutes, 30 μL of blood was collected from the tail using an EDTA-coated capillary tube, and insulin assays were performed.

[0058] Transit time measurement: Mice were given 200 μL of carmine red solution (concentration 10 mg / ml) by oral gavage. Transit time corresponds to the period elapsed from gavage to the appearance of the first red stool.

[0059] Food Intake: Food intake of mice was determined throughout the experiment by calculating the difference in weight between the food placed in the cage and the food remaining after one week. The following formula was used to determine daily food intake per mouse per cage:

number

[0060] Results are expressed in grams of feed / day / animal and kilocalories / day / animal.

[0061] Calories excreted: The calorie excretion is determined by fecal calorimetry.To do this, mice are placed in cages with mesh bottoms that allow feces to be collected for 3 days during the 11th week.After collecting all the feces from the mice for 3 days, they are placed in an incubator at 60°C to dehydrate them.The collected feces are then weighed, and the amount of energy contained in 1g of feces is measured using a bomb calorimeter.The percentage of absorbed calories, i.e., the proportion of ingested energy that is not excreted in feces, is calculated using the following formula:

number

[0062] Calories per gram of food x mass of food ingested. Feed efficiency is determined by calculating the ratio of weight gain / calories absorbed.

[0063] DNA extraction from mouse feces and digesta - adapted GODON protocol: Bacterial lysis: Samples of jejunal and ileal contents were weighed and placed on ice. To each tube, 250 μL of 4 M guanidine thiocyanate, 350 μL of 6% sarcosine, and 30 μL of DTT were added. After vortexing, the contents were transferred to Precellys-compatible tubes and incubated at 95°C for 15 minutes. To enhance cell lysis, silica beads with diameters of 0.1 mm and 2 mm were added to the samples. The tubes were then shaken at 10,000 Hz for six 30-second cycles with a 30-second pause between each cycle, and then placed on ice for 2 minutes.

[0064] Nucleic acid isolation and protein removal Next, 20 mg of PVPP (polyvinylpolypyrrolidone) was homogenized, and 600 μL of TEN-PVPP buffer (a solution containing PVPP, Tris-HCl, EDTA, and NaCl) was added to each tube. The samples were centrifuged, and the supernatant was collected. The steps of TEN-PVPP, centrifugation, and supernatant collection were repeated three times. Under a fume hood, phenol, chloroform, and isoamyl alcohol (25:24:1) were added to the supernatant in a 1:1 ratio. The tubes were vortexed, centrifuged, and the aqueous phase was collected. The samples were then incubated with 15 μL of proteinase K (19 mg / mL) at 60°C for 1 hour. After centrifugation, nucleic acids present in the supernatant were precipitated with isopropanol and left on ice for 20 minutes. The tubes were then mixed by inversion, centrifuged, and the supernatant was removed. The previously air-dried pellet was gently resuspended in phosphate buffer and potassium acetate. After centrifugation, the supernatant was collected.

[0065] RNA digestion and purification The samples were incubated with RNase at 37°C for 45 minutes. DNA was precipitated using absolute ethanol and sodium acetate. The samples were then centrifuged three times. Between each centrifugation, the supernatant was removed and the pellet was washed with 500 μL of 70% ethanol. Finally, the pellet was air-dried and resuspended in 60 μL of TE.

[0066] The OD ratios of 260 nm / 230 nm and 260 nm / 280 nm (indicators of nucleic acid purity) and DNA concentration were determined using a NanoDrop1000 (Thermo Fisher Scientific, USA). Finally, the samples were stored at -20°C.

[0067] DNA extraction from human stool Fecal DNA was extracted from stool using the PureLink™ Microbiome DNA Purification Kit (Invitrogen, Paris, France) according to the protocol provided by the manufacturer.

[0068] Quantification of T. muris by quantitative polymerase chain reaction (qPCR): The extracted bacterial DNA was diluted to a concentration of 10 ng / μL, and 2.5 μL of the diluted DNA and 7.5 μL of the master mix were added to the bottom of a 96-well ABI FAST plate.

[0069] The master mix for one reaction consisted of: Fast Sybr Green (Applied Biosystems 4385612) (5 μL), forward and reverse primers (200 nM), BSA (2.5 μg), and water (qs to 10 μL).

[0070] The plates were then centrifuged (800 rpm for 1 minute), placed in the qPCR instrument (StepOnePlus system), and the program run (StepOne v2.3 software) was initiated. To determine absolute bacterial concentrations, a standard range was applied to each PCR plate by diluting genomic DNA from a pure Turicimonas muris culture of known concentration. This range was 4 x 10 per well. 5 The relative Turicimonas muris concentrations were determined by dividing the absolute Turicimonas muris concentrations by the absolute total bacterial concentration.

[0071] Thermal cycler program: First, one cycle of 5 min at 95°C for initial denaturation, followed by a second 30 s denaturation step at 95°C, a 30 s annealing step at 60°C, and a 30 s extension step at 72°C, all for 40 cycles.

[0072] [Table 3]

[0073] statistical analysis Statistical analysis was performed using GraphPad Prism software (version 7.00 for Windows) (GraphPad Software, San Diego, CA, USA). Data distribution was verified using the Shapiro-Wilk test. Comparisons between groups at a given time point were performed using one-way ANOVA followed by Tukey's post-hoc test for parametric data or Kruskal-Wallis test followed by Dunn's correction for nonparametric data. Comparisons between groups at various time points were performed using two-way ANOVA followed by Tukey's post-hoc test. In all graphs, data are presented as mean ± SEM (standard error of the mean). A corrected q value of ≤0.05 was considered statistically significant.

[0074] II. Results II.1. Study of intestinal localization of Turicimonas muris in mice with complex microbiota To study the localization of Turicimonas muris throughout the gut, its abundance was quantified by qPCR using samples of ileal and jejunal contents from mice with a complex microbiota. Mice were divided into five groups: A chow diet group, equivalent to the control group, in which mice were fed a control diet for 15 weeks (the same period for all groups); The HFD group, in which mice were fed a high-fat diet (60% of calories came from fat), induced obesity. Finally, mice in the last three groups (HFD / FOS, HFD / 5-ASA (5-aminosalicylic acid), and HFD / FOS / 5-ASA) were fed an HFD diet supplemented with FOS and / or 5-ASA in the drinking water.

[0075] As expected, FOS treatment increased the bacterial load in the intestinal contents of mice fed an HFD diet (treated or untreated) compared with untreated mice fed under the same conditions (Figure 1a and d).

[0076] In both ileal and jejunal content samples, Turicimonas muris was detected primarily in the chow, HFD / FOS, and HFD / FOS / 5-ASA groups (Figure 1b and e). A similar trend was observed in the relative abundance of T. muris (Figure 1c and f). Thus, unlike 5-ASA, which has no effect on the growth of Turicimonas muris in the intestine, FOS is a prebiotic that partially restores its abundance in the intestinal contents of mice with diet-induced obesity (DIO).

[0077] In mice fed the chow diet, the relative abundance of Turicimonas muris was higher in the ileal contents (7.6%) than in the jejunal contents (4.3%) (Figure 1c and f). These results support the findings published by Andrew J. Macpherson et al., which showed that Turicimonas muris is primarily present in the ileum of mice with the simplified Oligo-MM12 microbiota.

[0078] II.2. Administration of Turicimonas muris prevents obesity induced by an obesogenic diet To induce obesity and impaired carbohydrate metabolism, mice in two of the four groups (18 of 36 mice) were fed a high-fat, high-sucrose (HFHS) diet for 12 weeks, while mice in the remaining two groups were fed a control diet (chow).

[0079] To demonstrate the effects of T. muris on host physiology, regardless of the mechanism involved, T. muris was administered by oral gavage to half of the mice in each group in the form of cells (for cellular components) resuspended in culture supernatant (for secreted metabolites). Control mice received uninoculated culture medium, also concentrated by lyophilization (Fig. 2a).

[0080] Data from a 6-week follow-up study showed that mice fed the HFHS diet gained significantly more weight than mice fed the control diet. This difference increased throughout the experiment (Figure 2b). NMR analysis indicated that the weight gain in these HFHS mice was primarily due to an increase in fat mass, rather than lean mass (Figure 2d and e). Overall, these results support the validity of the DIO model.

[0081] Interestingly, administration of Turicimonas muris limited the gains in body weight and fat mass induced by the HFHS diet (Figure 2b and d). Compared with untreated HFHS mice, the gains in body weight and fat mass of HFHS-Turicimonas muris mice were significantly lower by 3.4 g and 3.8 g, respectively (Figure 2c and e). When mice were fed a control diet, the differences between groups in weight gain and fat mass remained virtually unchanged. This indicates that the effects of Turicimonas muris are observable only in the context of obesity induced by a diet enriched in fat and sucrose. It should be noted that the lean mass of the mice was not affected by either the diet or Turicimonas muris administration (data not shown).

[0082] The observations made regarding mouse body weight and composition were substantiated by tissue weights measured during animal dissection. Indeed, the mass of subcutaneous AT (SAT) and epididymal AT (EAT) depots in mice fed the HFHS diet was significantly greater than that in mice fed the control diet (Figure 2, f and g). A similar trend, although not significant, was observed for BAT (Figure 2, h). Administration of Turicimonas muris to mice limited the increase in the weight of these various depots. Again, the positive effect of this bacterium was limited to the AT in HFHS-fed mice.

[0083] Finally, neither Turicimonas muris administration nor the diet had any effect on the mass of the gastrocnemius muscle, liver, or spleen in mice (data not shown). These results indicate that Turicimonas muris administration prevents the development of obesity induced by an obesogenic diet by limiting the increase in body weight and fat mass in mice.

[0084] II.3. Administration of Turicimonas muris prevents changes in carbohydrate metabolism induced by an obesogenic diet The reduced adiposity of HFHS-fed and T. muris-treated mice was associated with reduced fasting blood glucose (Figure 3A) and improved glucose tolerance, as demonstrated by the OGTT performed during week 10 and the resulting area under the curve (Figure 3B and C). Indeed, hyperglycemia induced by a glucose load (15-90 min duration) was lower in HFHS-T. muris mice compared to untreated HFHS mice (Figure 3B).

[0085] II.4. Effects of Turicimonas muris administration on energy balance and digestive physiology Gain in weight and fat mass occurs when the calories provided by the diet and absorbed by the intestine exceed the calories excreted and expended by the body.

[0086] We then assessed whether the beneficial effects of Turicimonas muris administration on body composition and carbohydrate metabolism were the result of a decrease in food intake and / or intestinal nutrient absorption, and / or an increase in energy expenditure.

[0087] The mean daily food intake (g) of mice fed the chow diet was found to be significantly higher than that of mice fed the HFHS diet (Figure 2a). Considering the higher calorie content of the HFHS diet, the resulting absorbed energy was similar for all groups (Figure 2b). Neither food intake nor transit time of the mice was significantly affected by the administration of Turicimonas muris (Figure 2a, b, and c).

[0088] Finally, the daily fecal weight per mouse varied according to the diet but was not affected by the administration of bacteria. Indeed, the daily fecal weight was higher in mice fed chow than in mice fed a high-fat, high-sucrose (HFHS) diet (d in FIG. 3).

[0089] Overall, these data indicate that administration of Turicimonas muris has beneficial effects on mouse body composition and carbohydrate metabolism without affecting mouse food intake or fecal excretion. These latter observations suggest that Turicimonas muris is well tolerated by mice.

[0090] III. CONCLUSIONS The results obtained demonstrate the beneficial effects of Turicimonas muris on mouse energy metabolism and physiology.

[0091] More specifically, administration of Turicimonas muris reduces the increase in body weight and fat mass induced by a high-fat, high-sucrose (HFHS) diet. The beneficial effect of this bacterium on the body composition of HFHS-fed mice is associated with an improvement in glucose tolerance compared to control mice fed under the same conditions. Finally, administration of Turicimonas muris appears to have beneficial effects on mouse body composition and carbohydrate metabolism without affecting mouse food intake or fecal excretion.

[0092] The relative abundance of Turicimonas muris was measured by quantitative PCR in the feces of human subjects.

[0093] The relative abundance of Turicimonas muris was measured by quantitative PCR (see the "Materials and Methods" section above) in the feces of individuals having either a normal body mass index (18 < BMI < 25) or an obese body mass index (BMI > 30), and in the feces of individuals who initially had obesity and type 2 diabetes before undergoing bariatric surgery (Roux-en-Y bypass) and in whom remission of type 2 diabetes was or was not observed an average of 5 years after surgery.

[0094] The results obtained are shown in FIG.

[0095] In humans, detection of T. muris in stool has been associated with improved metabolic status in some situations.

[0096] First, the mean relative abundance of T. muris was significantly higher in stool samples from patients with a normal body mass index than in those from patients with obesity (Figure 4A). This also translated to a higher frequency of T. muris in the microbiota of patients with a normal body mass index, as quantitative PCR detected T. muris in 35.8% of these patients, compared with only 19.6% of patients with obesity (Figure 4B). The frequency of T. muris was also associated with better carbohydrate metabolism, as in the obese patient population, T. muris was detected only in patients with fasting blood glucose levels below approximately 1.1 g / L (Figure 4C). Finally, among patients who initially had obesity and type 2 diabetes, T. muris was detected only in a subset of patients who experienced remission of their type 2 diabetes after bariatric surgery. T. muris was not detected in any patients who still had type 2 diabetes after bariatric surgery (Figure 4D). This negative association between the abundance and presence of T. muris and the obesity and blood glucose levels of patients indicates a positive effect of T. muris on the metabolism of patients.

Claims

1. Turicimonas muris for the prevention and / or treatment of metabolic diseases and / or complications associated therewith.

2. 2. Turicimonas muris according to claim 1, characterized in that it exists in a viable, non-viable or fragmented form.

3. 1 x 10 ２ cfu to 1 x 10 １５ Turicimonas muris according to claim 1 or 2, characterized in that it is administered in an amount comprised between cfu.

4. A composition comprising Turicimonas muris and a physiologically acceptable carrier.

5. 1 x 10 ２ cfu to 1 x 10 １５ 5. The composition according to claim 4, characterized in that it contains between cfu of Turicimonas muris.

6. 6. The composition according to claim 4 or 5, characterized in that it further comprises at least one probiotic and / or at least one prebiotic.

7. 7. The composition according to claim 6, characterized in that it contains at least one probiotic selected from Escherichia coli K12, Christensenella minuta, Anaerobutyricum saenggenii, Zysosmobacter welbiornis, and Akkermansia muciniphila, Faecalibacterium prausnitzii, Hafnia alvei 4597, and P. goldsteinii.

8. The composition according to any one of claims 4 to 7, which is administered orally or rectally.

9. The composition according to any one of claims 4 to 8 for the prevention and / or treatment of metabolic diseases and / or complications associated therewith.

10. The composition according to any one of claims 4 to 8 of claim 9, characterized in that it is administered in combination with a drug intended for the treatment of obesity, such as orlistat, phentermine-topiramate, naltrexone-bupropion, liraglutide, semaglutide, and tirzepatide.

11. The composition according to any one of claims 4 to 8, characterized in that it is administered in combination with a medicine intended for the treatment of type 2 diabetes, such as metformin, a sodium-glucose cotransporter type 2 inhibitor, a gliptin, acarbose, a glinide, a sulfonamide, or a sulfonylurea such as glimepiride.

12. Use of Turicimonas muris to promote weight loss in an individual.

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