Composition and use of turicimonas muris for the treatment of metabolic diseases

Abstract

The present invention relates to the prevention and / or treatment of metabolic diseases such as overweight and obesity as well as complications related thereto. The present invention more specifically relates to the bacterium Turicimonas muris or fragments thereof for preventing and / or treating metabolic diseases and complications related thereto.

Description

[0001] Composition and use of ripened Turicimonas for the treatment of metabolic diseases

[0002] The present invention relates to the prevention and / or treatment of metabolic diseases, such as, for example, overweight and obesity, and their complications. More specifically, the present invention relates to the bacterium Turicimonas matures or fragments thereof for preventing and / or treating metabolic diseases and their complications.

[0003] Obesity is defined as an excessive accumulation of fat mass or adipose tissue (AT) that can affect health and lead to the development of numerous pathologies (type 2 diabetes: T2D, cardiovascular diseases, dyslipidemia, cancers, etc.). This excessive gain of fat mass results from the interweaving of genetic, epigenetic, dietary and environmental factors that contribute to the imbalance of the energy balance.

[0004] The clinical diagnosis of obesity is traditionally based on the calculation of the body mass index (BMI, weight in kilograms divided by height in meters squared). In adults, a BMI between 18.5 and 24.99 kg / m 2 is considered normal. Overweight is defined as a BMI between 25 and 29.99, while a BMI > 30 defines obesity. The severity of obesity is classified into 3 grades: grades I (moderate obesity; 30 < BMI < 35), II (severe obesity; 35 < BMI < 40) and III (morbid obesity; BMI > 40). According to the World Health Organization, 39% of adults worldwide are overweight and 13% are obese.

[0005] Several studies report a strong and positive relationship between a high BMI and an increased risk of developing T2D. In this sense, individuals suffering from obesity are up to 80 times more likely to develop T2D compared to those with a BMI below 25 kg / m 2T2DM is characterized by chronic hyperglycemia due to insulin resistance in peripheral tissues (liver, skeletal muscle, adipose tissue) combined with insufficient insulin secretion by pancreatic P cells. Its clinical diagnosis is based on the demonstration of fasting blood glucose > 1.26 g / L on two occasions or > 2 g / L at any time of the day. T2DM significantly impairs quality and length of life, particularly through its vascular complications such as microangiopathies (nephropathy, retinopathy and neuropathy) and macroangiopathies (stroke and myocardial infarction). According to Sanjay Basu et al., nearly 406 million individuals worldwide suffered from T2D in 2018 (BASU et al. The Lancet. Diabetes & Endocrinology. January 2019, Vol. 7, no. 1, pp. 25-33).

[0006] These chronic diseases worsen over time and significantly reduce life expectancy. They therefore constitute a major public health problem with a significant economic and societal burden. The medical cost of obesity represents 1 to 3% of health expenditure in most countries (OECD. Health Goal. October 2010. https: / / www.oecd.org / fr / els / systemes-sante / 46044602.pdf.).

[0007] For patients with obesity and associated metabolic pathologies, a drug-based approach may be proposed in addition to dietary rebalancing and the promotion of regular physical activity. These drug-based approaches are non-curative and aim to control symptoms, slow the progression of the disease, and limit the risk of complications. To date, five obesity treatments—orlistat, phentermine-topiramate, naltrexonebupropion, liraglutide, and semaglutide—have been approved by the Food and Drug Administration in the United States for long-term use (BASU et al. The Lancet. Diabetes & Endocrinology. January 2019, Vol. 7, No. 1, pp. 25-33). In France, only orlistat and, more recently, liraglutide have received authorization for use in the treatment of obesity.

[0008] Treatments for T2D mainly aim to improve insulin sensitivity (metformin) or to potentiate insulin secretion by pancreatic B cells (sulfonylureas, glinide, semaglutide). More recently, gliflozins, or SGLT2 inhibitors which cause an increase in glycosuria, may be proposed.

[0009] Insulin therapy should be initiated when lifestyle and dietary approaches and non-insulin treatments do not allow the glycemic target to be reached (glycated hemoglobin level < 7%) (HAS. Drug strategy for glycemic control of type 2 diabetes, January 2013. https: / / www.has-

[0010] Current lifestyle, dietary, and drug approaches are insufficient to effectively and sustainably treat most patients with obesity and associated diseases. New therapeutic targets and treatments must therefore be developed to improve patient care, slow the progression of these diseases, and prevent the development of complications.

[0011] Over the past two decades, a growing 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 protozoa) living in a given ecological niche, in this case the digestive system. The latest studies estimate that the digestive tract (DT) of an adult would harbor approximately 100 trillion bacteria, themselves belonging to approximately 4,600 species (ALMEIDA et al. Nature Biotechnology. January 2021, Vol. 39, no. 1, pp. 105-114).

[0012] MI bacteria perform essential biological functions: they participate in the metabolism of xenobiotics and indigestible foods, they 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. A myriad of studies carried out in humans and mice report a strong and positive correlation between high bacterial richness (or alpha diversity) of MI, metabolic homeostasis and the absence of inflammation. Conversely, obesity and T2D are pathologies associated with a depletion of microbial diversity and MI dysbiosis, i.e. a persistent imbalance in the composition and functions of the bacterial community.

[0013] With the emergence of culturomics, i.e. high-throughput culture to characterize the microbial composition of MI, and with the recent optimization of bacterial culture media, an increasing number of strains have been isolated, cultivated in pure conditions and characterized. These advances open the way to in vitro studies of the metabolic potential of these bacteria and to in vivo studies of supplementation with bacteria of interest. They thus make it possible to study the causal relationships between certain bacterial species of MI and the modulation of the physiological state of the host and to identify the cellular and molecular mechanisms involved.

[0014] To date, a limited number of isolated species and strains (less than ten) have been characterized and identified as microorganisms with a direct causal effect on metabolic health. The mechanisms involved, even partial, have only been identified for an even smaller number of bacteria. These results, which are based on studies made possible only by pure bacterial culture, demonstrate the causal and undeniable role of certain MI bacteria in the regulation of the host's energy metabolism. The isolation and functional analysis of new bacterial strains potentially beneficial to the host are therefore necessary in order to consider effective and non-invasive therapeutic strategies aimed at treating, or even preventing, obesity and associated metabolic pathologies.

[0015] In this context, the inventors have demonstrated the beneficial effect of the bacterium Turicimonas matures on energy metabolism and physiology in mice; more particularly, the administration of Turicimonas matures reduces the weight gain and fat mass induced by an obesogenic diet. The beneficial effects of this bacterium on the body composition of mice on a high-fat diet (HFHS) are associated with improved glucose tolerance compared to control mice, fed under the same conditions. It appears that the administration of Turicimonas matures has a beneficial effect on the body composition and carbohydrate metabolism of mice without affecting their food intake or the quantity of stools excreted. The inventors have also demonstrated a correlation between the relative abundance of Turicimonas matures measured in stools in human subjects and the good metabolic state of these subjects (in particular a decrease in blood sugar).

[0016] The present invention thus relates to ripened Turicimonas for its use in the prevention and / or treatment of metabolic diseases and / or their complications.

[0017] It also relates to the use of ripened Turicimonas to improve glucose and fat metabolism, increase energy expenditure and / or promote weight loss, particularly in an overweight or obese individual. The species Turicimonas ripen (Taxonomy ID: 1796652) was isolated from the caecal contents of mice by Lagkouvardos et al. during the establishment of the “Mouse Intestinal Bacterial Collection”, a public repository consisting of 100 bacterial strains isolated from the caecal contents of mice with conventional microbiota (SPF status for Specific Pathogen Free) (LAGKOUVARDOS et al. Nature Microbiology. October 2016, Vol. 1, no. 10, p. 16131). This Gram-negative bacterium grows optimally at temperatures ranging from 20 to 45°C (mesophilic) and is also present in human MI. It belongs to the Sutterellaceae family and the Proteobacteria phylum (now Pseudomonadota).

[0018] The present invention also relates to bacterial strains belonging to the same family as Turicimonas matures or to strains of Turicimonas matures genetically modified but retaining the same properties as Turicimonas matures.

[0019] The mature Turicimonas bacteria can be cultured as described in the experimental section.

[0020] For the purposes of the present invention, it can be used in viable or non-viable form, but also in pasteurized or freeze-dried form. It can also be used in the form of fragments. The term "fragment" refers to cellular components, metabolites, secreted molecules and compounds resulting from the metabolism of matured Turicimonas. The fragments can be obtained, for example, by recovering the supernatant of a culture of matured Turicimonas or by extracting cellular components or cell fractions, metabolites or secreted compounds from a culture of matured Turicimonas. The term "fragment" can also refer to a degradation product, in particular metabolites.

[0021] According to one embodiment, matured Turicimonas or its fragments are substantially purified. As used herein, the term "substantially purified" means that matured Turicimonas or its fragments are included in a sample where it represents at least about 50%, preferably at least about 60, 70, 80, 85, 90, 95, 99% or more of the bacterial strains or their fragment by weight of said sample.

[0022] The term "metabolic disease" refers to disorders, diseases, and conditions caused by or characterized by weight gain, abnormal energy utilization or consumption, 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 a combination thereof. A metabolic disease may be associated with a deficiency or excess in a metabolic pathway resulting in an imbalance in the metabolism of carbohydrates, lipids, proteins, and / or nucleic acids.

[0023] Examples of metabolic diseases include, but are not limited to, metabolic syndrome, disorders related to insulin deficiency or resistance, diabetes mellitus (such as, for example, type 2 diabetes), impaired glucose tolerance, abnormal lipid metabolism (or dyslipidemia), overweight and obesity; Complications of metabolic diseases (also referred to as comorbidities) include atherosclerosis, hypertension, preeclampsia, cardiac disease, stroke, non-alcoholic fatty liver disease, hyperglycemia, fatty liver disease of various etiologies, immune system dysfunction associated with overweight and obesity, cardiovascular disease, hypercholesterolemia, elevated triglycerides, asthma, sleep apnea, osteoarthritis, neurodegeneration, gallbladder disease, syndrome X, inflammatory and immune disorders,atherogenic dyslipidemia and cancer, in particular, esophageal cancer, gastric cardia cancer (a type of stomach cancer), bowel 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.

[0024] The quantities of matured Turicimonas to be administered are preferably chosen to restore a normal bacterial population in the intestine, that is to say a population such as that found in a healthy subject (not overweight or obese).

[0025] As an example, the administered quantity of ripened Turicimonas can be chosen between approximately 1.10 2 and 1.10 15 cfu (colony forming unit), especially between about 1.10 4 and 1.10 12 cfu or about 1.10 6 and 1.1010 cfu, preferably this amount is administered daily. When matured Turicimonas is administered in non-viable form or fragments, the above amounts are used for the preparation of this form. The administered amount of matured Turicimonas may also be in the order of about 1.10 6 at about 1.10 12 cells, preferably between about 1.10 8 and about 1.1O 10 cells or between approximately 1.10 9 and about 1.1O 10 cells, preferably this amount is administered daily.

[0026] The present invention further relates to a composition comprising ripened Turicimonas and a physiologically acceptable carrier.

[0027] By physiologically acceptable vehicle is meant a vehicle that can be administered and well tolerated by an individual, in particular a mammal, preferably a human.

[0028] 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 capsule of plant origin; the composition may also include anti-caking agents such as magnesium stearate or silicon dioxide.

[0029] The composition according to the invention comprises matured Turicimonas in viable, non-viable or fragment form, in an amount of between about 1.10 2 and 1.10 15 cfu, especially between about 1.10 4 and 1.10 12 cfu or about 1.10 6 and 1.1O 10 cfu or between approximately 1.10 6 and about 1.10 12 cells, preferably between about 1.10 8 and about 1.1O 10 cells or between approximately 1.10 9 and about 1.1O 10 cells.

[0030] Ripened Turicimonas or a fragment thereof or the composition according to the invention can be administered by several routes of administration.

[0031] 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.

[0032] According to one embodiment, ripened Turicimonas or a fragment thereof or the composition of the invention is in a form suitable for oral administration. According to this embodiment, the form suitable for oral administration may be:

[0033] - a solid form selected from the group comprising tablets, pills, capsules, soft gelatin capsules, sugar-coated pills, orodispersible tablets, effervescent tablets or other solids; according to a particular embodiment, the oral solid form is gastro-resistant so that its contents are not degraded during its stay in the stomach.

[0034] - a liquid form, such as, for example, an oral solution, liposomal forms and others.

[0035] When the administration is carried out orally, the composition of the invention may be a nutritional composition or a food product.

[0036] According to one embodiment, ripened Turicimonas or a fragment thereof or the composition of the invention is in a form suitable for rectal administration. According to this embodiment, the composition may be a suppository or a rectal capsule.

[0037] According to one embodiment, the composition according to the invention further comprises a probiotic and / or a prebiotic. According to this embodiment, the composition of the invention is preferably administered orally.

[0038] The term "probiotic" refers to microbial cell preparations (such as, for example, live microbial cells) that, when administered in an effective amount, have a beneficial effect on the health or well-being of a subject. By definition, all probiotics have a proven non-pathogenic character. In one embodiment, these health benefits are associated with improving the balance of the human or animal microbiota in the gastrointestinal tract, and / or restoring normal microbiota.

[0039] For example, the probiotic can be chosen from:

[0040] - les bactéries : Lactobacillus, Lactococcus, Bifidobacterium, Veillonella, Desemzia, Christensenella, Allobaculum, Coprococcus, Collinsella, Citrobacter, Turicibacter, Sutterella, Subdoligranulum, Streptococcus, Sporobacter, Sporacetigenium, Ruminococcus, Roseburia, Proteus, Propionobacterium, Leuconostoc, Weissella, Pediococcus, Streptococcus, Prevotella, Parabacteroides, Papillibacter, Oscillospira, Melissococcus, Dorea, Dialister, Clostridium, Cedecea, Catenibacterium, Butyrivibrio, Buttiauxella, Bulleidia, Bilophila, Bacteroides, Anaerovorax, Anaerostopes, Anaerofilum, Enterobacteriaceae, Fermicutes, Atopobium, Alistipes, Acinetobacter, Slackie, Shigella, Shewanella, Serratia, Mahella, Lachnospira, Klebsiella, Idiomarina, Fusobacterium, Faecalibacterium, Eubacterium, Enterococcus, Enterobacter, Eggerthella ;

[0041] In particular, Bifidobacterium animalis, in particular Bifidobacterium animalis spp. lactis, Bifidobacterium lactis, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Akkermansia muciniphila;

[0042] - les microorganismes procaryotes -. Archaea, Firmicutes, Verrucomicrobia, Christensenella, Bacteroidetes (for example, Allistipes, Bacteroides ovatus, Bacteroides splachnicus, Bacteroides stercoris, Parabacteroides, Prevotella ruminicola, Porphyromondaceae), Proteobacteria, Betaproteobacteria (for example, Aguabacterium and Burkholderia), Gammaproteobacteria (for example, Dorea, Bulleidia, Anaerofustis, Gemella, Roseburia, Dialister, Anaerotruncus, Staphylococcus, Micrococcus, Propionobacteria, Enterobacteriaceae, Faecalibacterium, Bacteroides, Parabacteroides, Prevotella, Eubacterium, Bacilli (for example, Lactobacillus salivarius, Aerococcus, Granulicatella,Streptococcus bovis and Streptococcus intermedius), Clostridium (such as, for example, Eubacterium hallii, Eubacterium limosum) and Butyrivibrio.,

[0043] - yeasts: Ascomycètes, Zygomycetes and Deuteromycetes, notably from the groups Aspergillus, Torulopsis, Zygosaccharomyces, Hansenula, Candida, Saccharomyces, Clavispora, Bretanomyces, Pichia, Amylomyces, Zygosaccharomyces, Endomyces, Hyphopichia, Zygosaccharomyces, Kluyveromyces, Mucor, Rhizopus, Yarrowia, Endomyces, Debaryomyces, and Penicillium.

[0044] En particulier, on peut citer les souches suivantes :

[0045] Type Lactobacillus: L. acidophilus, L. amylovorus, L. casei, L. gasseri, L. helveticus, L. johnsonii, L. pentosus, L. plantarum, L. reuteri and L. rhamnosus

[0046] Type Bifidobacterium B. adolescentis, B. animalis, B. bifidum, B. breve, B. infantis and B. longum.

[0047] Other lactic acid bacteria: Enterococcus faecium, Lactococcus lactis and Streptococcus thermophilus; Other microorganisms: Bacillus clausii, Escherichia coli Nissle 1917 and Saccharomyces cerevisiae (boula rdi)

[0048] Preferably, the probiotic (or LBP) is chosen from: Escherichia coli K12, Christensenella minuta, Anaerobutyricum soehngenii, Dysosmobacter welbionis, and Akkermansia muciniphila, Faecalibacterium prausnitzii, Afnia alvei 4597, P. goldsteinii.

[0049] For the purposes of the present invention, the probiotic may be used in viable or non-viable form; or in the form of fragments. It is administered in the same quantities as those indicated for matured Turicimonas.

[0050] The term "prebiotic" refers to a substance, such as, for example, a substance that cannot be digested by humans, but which modulates the composition and / or activity of the intestinal microbiota through its metabolism by microorganisms in the intestine, thus conferring a beneficial physiological effect on the host.

[0051] Prebiotics may be dietary fiber. Dietary fiber may be selected from the group consisting of fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), xylo-oligosaccharides, isomalto-saccharides, soy oligosaccharides, pyrodextrins, transgalactosylated oligosaccharides, lactulose, beta-glucan, inulin, raffinose, stachyose. Dietary fiber also has the advantage of withstanding a number of conditions, including heating and long periods of storage. It may further contribute to a treatment within the scope of the present invention by improving gastrointestinal health and increasing satiety.

[0052] Other non-limiting examples of prebiotics include water-soluble cellulose derivatives, water-insoluble cellulose derivatives, unprocessed oat flakes, psyllium, wheat bran, and a mixture thereof. Examples of water-soluble cellulose derivatives include, but are not limited to, methylcellulose, methylethylcellulose, also hydroxyethylcellulose, ethylhydroxyethylcellulose, cationic hydroxyethylcellulose, as well as hydroxypropylcellulose, hydroxyethylmethylcellulose, but also hydroxypropylmethylcellulose and carboxymethylcellulose.

[0053] The composition according to the invention can be administered in combination with a drug intended for the treatment of obesity, such as orlistat, phentermine-topiramate, naltrexone-bupropion, liraglutide, semaglutide and tirzepatide and / or in combination with a drug intended for the treatment of type 2 diabetes such as metformin, sodium-glucose co-transporter type 2 inhibitors, gliptins, acarbose, glinides, sulfonamides or sulfonylureas such as Glimeperide.

[0054] The present invention also relates to the (non-therapeutic) use of ripened Turicimonas to promote weight loss in an individual as well as to a method of preventing and / or treating metabolic diseases and their complications comprising the administration of ripened Turicimonas or fragments thereof or a composition according to the invention.

[0055] FIGURES

[0056] Figure 1 - T. mature is predominantly present in the ileum of non-obese mice.

[0057] qPCRs were performed with specific (T. matures) and non-specific (total bacteria) primers on samples of jejunal (Figures a to c) and ileal (Figures d to f) contents of mice fed a control diet (Chow diet) or HFD supplemented or not with FOS and / or 5-ASA.

[0058] (a, d) Bacterial load for each group of mice.

[0059] (b, e) Concentration of T. matures.

[0060] (c, f) Relative abundance of T. matures.

[0061] Data were analyzed using the Kruskal Wallis test followed by Dunn's pairwise multiple comparisons test and are represented as scatter plots with mean ± SEM. Number of mice per group: n=5 to 12. *q < 0.05; **q < 0.01; ***q < 0.001; ****q < 0.0001.

[0062] Figure 2 - T. matures prevents diet-induced obesity.

[0063] (a) Schematic diagram of the experimental design. Animals in two of the four groups were fed a HFHS diet, and those in the remaining two groups were fed a control diet (chow diet). Mice in one of the HFHS and chow diet groups were supplemented once daily with T. matures accompanied by its freeze-dried culture supernatant or with the uninoculated T. matures culture medium.

[0064] (b, c) Body weight and body weight gain.

[0065] (d, e) Fat mass and fat mass gain. Weight (in mg) of different fat depots after sacrifice: (f) TAS: subcutaneous (inguinal) TAS,

[0066] (g) TAE: epididymal BP,

[0067] (h) TAB: TA brown.

[0068] Data in figures b and d were analyzed using 2-way ANOVA followed by Tukey's post hoc test. Data in figures c, e, f, g, and h were analyzed using the Kruskal Wallis test followed by Dunn's pairwise multiple comparisons test. Results are represented 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 comparisons. matured and <i q < 0.01 ; q < 0.001 ; q < 0.0001 for chow diet vs HFHS comparisons.

[0069] Figure 3 - T. matures prevents diet-induced alteration of carbohydrate metabolism.

[0070] (a) Fasting blood glucose measured during week 3.

[0071] (b) Glycemic profile and

[0072] (c) area under the curve measured during an oral glucose tolerance test (OGTT) performed during week 10.

[0073] Results in Figures a and c were analyzed using the Kruskal Wallis test followed by Dunn's post hoc test. Those in Figure b were analyzed using a 2-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 HFHS vs. HFHS-T comparisons. matured and <i q < 0.01; oooo q < 0.0001 for chow diet vs HFHS comparisons.

[0074] Figure 4: The relative abundance of matured Turicimonas was measured by quantitative PCR in the stools of individuals with either normal (18 < BMI < 25) or obese (BMI > 30) body mass index, as well as in individuals initially suffering from obesity and type 2 diabetes before undergoing bariatric surgery (Roux-en-Y bypass) and having, or not, entered into remission of their type 2 diabetes on average 5 years after surgery.

[0075] (A) Relative abundance of T. matures (% of total bacteria) as a function of body mass index. Mann Whitney test.

[0076] (B) Proportion of individuals with detectable levels of T. matures by quantitative PCR (i.e., greater than 0.00009% of total bacteria) as a function of body mass index. Chi-square test. (C) Relative abundance of T. matures as a function of fasting glucose in patients with obesity (BMI > 30). The threshold for moderate fasting hyperglycemia (1.1 g / L) is indicated. Overall, T. matures is not detected in patients with moderate fasting hyperglycemia, while it is detected in a proportion of patients with normal fasting glucose.

[0077] (D) Relative abundance of T. matures in patients initially suffering from obesity and type 2 diabetes who have entered, or not, remission from their type 2 diabetes. T. matures is not detected in patients still suffering from diabetes while it is detected in a proportion of patients who have entered remission. Mann Whitney test.

[0078] EXAMPLE

[0079] I. MATERIALS AND METHODS

[0080] Bacterial culture of ripened Turicimonas:

[0081] Culture medium: The powders (Table 1) were dissolved in distilled water, then formic acid, rezasurin solution (redox indicator color), and hemin stock solution were added. The pH was adjusted to 7.8. The resulting medium was transferred to glass vials and boiled in a microwave to remove dissolved oxygen. The vials were crimped and autoclaved. In parallel, the heat-sensitive ingredients (Table 2) were transferred to empty vials. The vials were crimped, a mixture of CO2, H2, N2 was injected using a needle, and then a second needle was inserted into the septum to evacuate the CH present in the air. Anaerobic water, FCS, and the vitamin stock solution were added to the powders. Three ml of the resulting solution and a filter-sterilized menadione solution (80 ng / ml final) were added to each of the medium flasks.These were finally pressurized by injection of the CO2, H2, N2 mixture sterilized by filtration and stored at 4°C.

[0082] Table 1: Composition of the culture medium

[0083] Ingredients for 11 of culture medium Quantity

[0084] Yeast extract 5 g

[0085] Soy peptone 10 g

[0086] NaCl 2.5 g

[0087] Na2CO3 1.5 g Formic acid 80% 1.5 ml

[0088] Resazurin solution (1 g / L) 1 ml

[0089] KH2PO4 1.33 g

[0090] Na2CO3 1.5 g

[0091] NH4CI 1.5 g

[0092] Hemin solution (20 mg / ml) 66.5 pl

[0093] H2O 900 ml

[0094] Table 2: Culture medium composition (ingredients added sterilely by filtration after thermal sterilization of the medium)

[0095] Autoclave-sensitive ingredients for 11 medium quantity

[0096] Galactose 3 g

[0097] Ascorbic acid 0.5 g

[0098] Reduced glutathione lg

[0099] Vitamin mix solution 0.2 ml

[0100] Cysteine ​​1 g

[0101] Na2CO3 1.2 g

[0102] Anaerobic water 60 ml

[0103] SVF 20 ml

[0104] Inoculation and administration: Matured Turicimonas (DSM 22575) was purchased from the German Public Collection of Microorganisms and Cell Cultures (DSMZ) and then cultured under sterile, anaerobic conditions for 48 hours at 37°C. For administration to mice, the bacterial culture was centrifuged at 4000 g for

[0105] 20 min at 4°C. Bacterial pellets were resuspended in culture supernatant concentrated by a factor of 5 by lyophilization. These preparations, measured by qPCR at 5.10 9cfu (colony forming units) / ml, were finally stored at -80 °C in glycerol until gavage of the mice.

[0106] Animal models: 36 8-week-old male C57BL / 6J SPF mice (Charles River Laboratories, France) were housed in a controlled environment (temperature 22 ± 2°C, 12-h day / night cycle) with free access to food and water. The mice were acclimatized for 7 days while being fed a control diet (chow diet, 3.91 kcal / g, 4% fat, Research Diet, ref. 98121701) and without being handled. After this acclimatization, the mice were identified by ear tag and 4 groups of 9 mice of similar weight and body composition were formed. Mice in two of the four groups were fed the control diet for 12 weeks, while those in 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% carbohydrates, Research diet, ref. D12079B).

[0107] In order to highlight the impact of matured Turicimonas on host physiology, the bacteria were administered by oral gavage once daily in the form of live cells resuspended in culture supernatant (200 μl of the solution containing matured Turicimonas, i.e. 1.10 9 cfu). Control mice received the uninoculated culture medium also concentrated by a factor of 5 by lyophilization. Force-feeding of the animals began 1 week after their arrival at the animal facility, at the same time as the diet change for the HFHS groups.

[0108] Sacrifice of mice:

[0109] An intracardiac blood sample was taken under isofluorane anesthesia (3%) immediately before euthanasia by cervical dislocation of the 36 mice. The tissues and organs of interest were collected, weighed and frozen in liquid nitrogen (liver, spleen, inguinal, epididymal and interscapular TA, ileum and ileal contents, jejunum and jejunal contents, colon, gastrocnemius muscle). Before the inclusion of the different tissues / organs in paraffin by the HISTOMICS ICM - Pitié Salpêtrière platform, the liver and jejunum were fixed in paraformaldehyde (4%) for histological analyses.

[0110] All experiments were approved and performed in accordance with the guidelines of the Charles Darwin Ethics Committee.

[0111] Body composition analysis:

[0112] To monitor the development of obesity, the body composition of the mice (fat mass, lean mass and fluids) was determined by nuclear magnetic resonance (NMR - LF90 Minispec+ scanner) at the time the animals were put on the diet, then every 3 weeks and the day before sacrifice. Oral glucose tolerance test (OGTT):

[0113] Mice fasted for 6 hours received an oral glucose load (2 g of glucose per kg of body weight). Blood glucose levels were measured using a glucometer (Accu Check ROCHE) from a drop of blood collected with a 23G needle from the side of the tail at times -30, 0, 15, 30, 60, and 90 min post-carbohydrate load. At times 0 and 15 min, 30 μl of blood was collected from the tail using an EDTA-coated capillary for insulin assay.

[0114] Transit time measurement:

[0115] Mice were given 200 μl of carmine red solution (concentration 10 mg / ml) by oral gavage. Transit time is the period between gavage and the appearance of the first red stool.

[0116] Food intake:

[0117] The food intake of the 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. To determine the daily food intake per cage and per mouse, the following formula was used:

[0118] Weight of food put in the cage — Weight remaining after one week.

[0119] Number of days between the 2 measurements ' number of animals per cage

[0120] The results are expressed in grams of food / day / animal and in kilocalories / day / animal.

[0121] Calories excreted:

[0122] The calories excreted will be determined by fecal calorimetry. For this, the mice were placed for 3 days during week 11 in cages equipped with a mesh bottom to collect the stools. All the mice's feces were collected for 3 days and then placed in an incubator at 60°C to dehydrate them. The collected feces were then weighed and the amount of energy contained per gram of stool with a calorimetric bomb is measured. The percentage of calories absorbed, that is to say the proportion of ingested energy that has not been evacuated in the feces, is calculated with the following formula:

[0123] % of calories absorbed = 100 Feed efficiency is determined by calculating the ratio: weight gain / calories absorbed.

[0124] DNA extraction from mouse feces and digestive contents - GODON protocol o Bacterial lysis

[0125] Jejunal and ileal content samples were weighed and placed on ice. In each tube, 250 μl of 4M guanidine thiocyanate, 350 μl of 6% sarcosine and 30 μl of DTT were added. Once vortexed, the contents were transferred into Precellys compatible tubes and incubated for 15 minutes at 95°C. To improve cell lysis, silica beads of 0.1 and 2 mm diameter were added to the samples. The tubes were then shaken 6 times for 30s interspersed with 30s of rest at a frequency of 10,000 Hz, then left on ice for 2 minutes. o Nucleic acid isolation and protein removal

[0126] 20 mg of PVPP (polyvinylpolypyrrolidone) and, after homogenization, 600 μl of TEN-PVPP buffer (solution containing PVPP, Tris HCl, EDTA and NaCl) were added to each tube. The samples were centrifuged and the supernatant was collected. The steps: TEN-PVPP, centrifugation and supernatant collection were repeated 3 times. Under a fume hood, a volume (1:1) of phenol, chloroform, isoamyl alcohol (25:24:1) was added to the supernatants. The tubes were vortexed and then centrifuged; the aqueous phase was collected. The samples were then incubated for 1 h at 60°C with 15 μl of proteinase K (19 mg / mL). After centrifugation, the nucleic acids present in the supernatant were precipitated with isopropanol and left for 20 minutes on ice. The tubes were then mixed by inversion, centrifuged and the supernatant was discarded.The pellets, previously air-dried, were gently resuspended in phosphate buffer and potassium acetate. After centrifugation, the supernatants were collected. o RNA digestion and purification.

[0127] Samples were incubated for 45 min at 37°C with RNase. DNA was precipitated with absolute ethanol and sodium acetate. Samples were then centrifuged 3 times. Between each centrifugation, the supernatant was removed and the pellet was washed with 500 μl of 70% ethanol. Pellets were finally air-dried and resuspended in 60 μl of TE.

[0128] The ratios of OD 260 / 230 nm and 260 / 280 nm (indicators of nucleic acid purity) and DNA concentration were determined using a NanoDroplOOO (Thermo Fisher Scientific, USA). The samples were finally stored at -20°C. DNA extraction from human stool

[0129] Fecal DNA was extracted from stool using the PureLink™Microbiome DNA Purification Kit (Invitrogen, Paris, France) according to the protocol provided by the manufacturer.

[0130] Quantification of matured T. by quantitative Polymerase Chain Reaction (qPCR):

[0131] The extracted bacterial DNA was diluted to a concentration of 10 ng / µl. 2.5 µl of diluted DNA and 7.5 µl of master mix were placed in the bottom of an ABI FAST 96-well plate.

[0132] Master MIX composition for one reaction: Fast Sybr Green Applied Biosystems 4385612 (5 pl), forward and reverse primer (200 nM), BSA (2.5 pg) and water (gsp 10 pl).

[0133] The plates were then centrifuged (1 min at 800 rpm), placed in the qPCR apparatus (StepOnePlus System) and the program execution (StepOne software v2.3) was started.

[0134] To determine the absolute concentration of bacteria, a standard range was applied to each PCR plate by diluting the genomic DNA of a pure culture of matured Turicimonas of known concentration. This range varied from 4.10 5 at 26 cfu per well. The relative concentration of matured Turicimonas was determined by dividing the absolute concentration of matured Turicimonas by the absolute concentration of total bacteria.

[0135] Thermocycler program: first 1 cycle of 5 min at 95°C for initial denaturation, followed by a second denaturation step at 95°C for 30s, an annealing step at 60°C for 30s and an elongation step at 72°C for 30s. All in all, 40 cycles were carried out. ues

[0136] Statistical analyses were performed using GraphPad Prism software version 7.00 for Windows (GraphPad Software, San Diego, CA, USA). Data distribution was checked for normality or non-normality using the Shapiro-Wilk test. Comparison between groups at a given time point was 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. Comparison between groups at different time points was performed using two-way ANOVA followed by Tukey's post hoc test. For all graphs, data are represented as mean plus or minus SEM (standard error mean). A corrected q-value < 0.05 was considered statistically significant.

[0137] II. RESULTS ll.l. Study of the intestinal localization of Turicimonas matured in mice with complex microbiota

[0138] To study the localization of matured Turicimonas throughout the intestine, its abundance was quantified by qPCR from ileal and jejunal content samples of Ml complex mice. For this experiment, the mice were divided into 5 groups:

[0139] The chow diet group, corresponding to the control group in which the mice were fed for 15 weeks (same duration for all groups) with a control diet.

[0140] The HFD group in which mice were fed a fat-enriched diet to induce obesity (60% of calories from fat).

[0141] Finally, mice in the last 3 groups HFD / FOS, HFD / 5-ASA (5-aminosalicylic acid) and HFD / FOS / 5-ASA were fed with HFD diet supplemented with FOS and / or 5-ASA in the drinking water.

[0142] As expected, FOS treatment increased the bacterial load in the intestinal contents of mice fed the HFD diet (treated or not with 5-ASA) compared to that of untreated mice fed under the same conditions (Figures 1a and d). Whether from ileal or jejunal content samples, Turicimonas matures was predominantly detected in the chow diet, HFD / FOS and HFD / FOS / 5-ASA groups (Figures 1b and e). In terms of relative abundance of T. matures, similar trends were obtained (Figures 1c and f). Thus, in contrast to 5-ASA which has no influence on the proliferation of Turicimonas matures in the intestine, FOS is a prebiotic that partially restores its abundance in the intestinal contents of mice with diet-induced obesity (DIO).

[0143] The relative abundance of matured Turicimonas was higher in the ileal contents (7.6%) than in the jejunal contents (4.3%) of chow-fed mice (Figures 1e and f). These results confirm those published by Andrew J. Macpherson et al. showing that matured Turicimonas is predominantly present in the ileum of mice with a simplified Oligo-MM12 microbiota.

[0144] 11.2. Administration of ripened Turicimonas prevents obesity induced by obesogenic diet

[0145] 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. Mice in the remaining two groups received a control diet (chow diet).

[0146] To highlight the impact of matured Turicimonas on host physiology regardless of the mechanisms involved, it 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 the uninoculated culture medium also concentrated by lyophilization (Figure 2 a).

[0147] As early as 6 weeks of follow-up, the data show that mice fed a HFHS diet gained significantly more weight than those fed a control diet. This difference increased throughout the experiment (Figure 2 b). NMR analyses indicate that the increase in body weight in these HFHS mice was mainly due to an increase in fat mass rather than lean mass (Figures 2 d and e). Overall, these results confirm the efficacy of the DIO model. Interestingly, the administration of ripened Turicimonas limited the body weight and fat mass gain induced by the HFHS diet (Figures 2 b and d). Compared with untreated HFHS mice, the body weight and fat mass gain of HFHS-ripened Turicimonas mice was significantly lower, by 3.4 and 3.8 g, respectively (Figures 2 c and e).The between-group difference in body weight gain and fat mass remained substantially unchanged when mice were fed the control diet, showing that the effect of ripened Turicimonas is observable only in the context of obesity induced by the fat- and sucrose-enriched diet. It should be noted that lean mass of the mice was not affected by either the diet or the administration of ripened Turicimonas (data not shown).

[0148] The observations made on the weight and body composition of the mice were corroborated by the tissue weight measured during dissection of the animals. Indeed, the mass of subcutaneous (SAT) and epididymal (EAT) AT of mice fed with the HFHS diet was significantly greater than that of mice fed with the control diet (Figures 2 f and g). Although not significant, similar trends were obtained for the BAT (Figure 2 h). The administration of matured Turicimonas to the mice limited the increase in the weight of these different adipose depots. Here again, the positive effect of this bacterium was limited to the AT of mice under HFHS.

[0149] Finally, neither the administration of ripened Turicimonas nor the diet had any effect on the mass of the gastrocnemius muscle, liver, and spleen of the mice (data not shown). These results show that the administration of ripened Turicimonas opposes the development of obesity induced by the obesogenic diet by limiting the gain of body weight and fat mass in the mice.

[0150] 11.3. Administration of ripened Turicimonas prevents the alteration of carbohydrate metabolism induced by the obesogenic diet

[0151] The lower adiposity of HFHS-fed mice treated with ripened Turicimonas was associated with decreased fasting blood glucose (Figure 3 a) and improved glucose tolerance as shown by the OGTT performed during week 10 and the resulting area under the curve (Figures 3 b and c). HFHS-T. ripened mice indeed had lower glucose-load-induced hyperglycemia (time 15 min to 90 min) compared to untreated HFHS mice (Figure 3 b). II.4. Impact of ripened Turicimonas administration on energy balance and digestive physiology

[0152] Weight gain and fat mass occur when the calories supplied by food and absorbed by the intestines are greater than those excreted and expended by the body.

[0153] It was then evaluated whether the beneficial effect of the administration of ripened Turicimonas on body composition and carbohydrate metabolism was the consequence of a decrease in food intake and / or nutrient absorption at the intestinal level and / or an increase in energy expenditure.

[0154] It was found that the average daily food intake (in g) of mice on the chow diet was significantly higher than that of mice fed the HFHS diet (Figure 2 a). Since the caloric content is higher for the HFHS diet, the resulting amount of energy intake was identical for all groups (Figure 2 b). This food intake, as well as the transit time of the mice, was not significantly impacted by the administration of ripened Turicimonas (Figures 2 a, b and c).

[0155] Finally, daily stool weight per mouse varied depending on the diet but was not affected by administration of the bacteria. It was indeed greater in mice on the chow diet than in those fed the obesogenic diet (Figure 3 d).

[0156] Overall, these data indicate that the administration of ripened Turicimonas beneficially impacts the body composition and carbohydrate metabolism of mice without affecting their food intake or the quantity of stool excreted. These latter observations suggest that ripened Turicimonas was well tolerated by the mice.

[0157] III. CONCLUSIONS

[0158] The results obtained show a beneficial effect of matured Turicimonas on energy metabolism and physiology of mice.

[0159] More specifically, the administration of ripened Turicimonas reduces the weight gain and fat mass induced by the obesogenic diet. The beneficial effects of this bacterium on the body composition of mice under HFHS are associated with improved glucose tolerance compared to control mice, fed under the same conditions. Finally, it appears that the administration of ripened Turicimonas acts beneficially on the body composition and carbohydrate metabolism of mice without affecting their food intake or the quantity of stool excreted.

[0160] Relative abundance of matured Turicimonas was measured by quantitative PCR in stools of human subjects

[0161] The relative abundance of matured Turicimonas was measured by quantitative PCR (see the "Materials and Methods" section above) in the stools of individuals with either normal (18 < BMI < 25) or obese (BMI > 30) body mass index, as well as in individuals initially suffering from obesity and type 2 diabetes before undergoing bariatric surgery (Roux-en-Y bypass) and having, or not, entered into remission of their type 2 diabetes on average 5 years after surgery.

[0162] The results obtained are presented in Figure 4.

[0163] In humans, the detection of T. matures in stool is associated with improved metabolic status in several contexts.

[0164] First, the mean relative abundance of T. mature is significantly higher in stools of patients with normal body mass index than in stools of patients with obesity (Fig 4A). This also translates into a higher prevalence of T. mature in the microbiota of patients with normal body mass index since T. mature is detected by quantitative PCR in 35.8% of these patients while it is only detected in 19.6% of patients with obesity (Figure 4B). The prevalence of T. mature is also associated with better carbohydrate metabolism since, in a population of patients with obesity, it is detected almost exclusively in patients with fasting blood glucose levels below 1.1 g / L (Figure 4C). Finally, in patients with initial obesity and type 2 diabetes, T.T. matures is detected only in a portion of patients who have entered remission from their type 2 diabetes following bariatric surgery. It is not detected in any patient still suffering from type 2 diabetes after bariatric surgery (Figure 4D). This negative association of the abundance and presence of T. matures with the body size and blood glucose levels of the patients demonstrates a positive effect of T. matures on the metabolism of the patients.

Claims

Claims 1. Turicimonas ripened for its use in the prevention and / or treatment of metabolic diseases and / or their complications.

2. Ripened Turicimonas for use according to claim 1, characterized in that it is in viable, non-viable or fragment form.

3. Ripened Turicimonas for use according to claim 1 or claim 2, characterized in that it is administered in an amount of between 1.10 2 and 1.10 15 cfu.

4. Composition comprising ripened Turicimonas and a physiologically acceptable vehicle.

5. Composition according to claim 4, characterized in that it comprises between 1.10 2 and 1.10 15 CFU of ripened Turicimonas.

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

7. Composition according to claim 6, characterized in that it comprises at least one probiotic chosen from Escherichia coli K12, Christensenella minuta, Anaerobutyricum soehngenii, Dysosmobacter welbionis, and Akkermansia muciniphila, Faecalibacterium prausnitzii, Afnia alvei 4597, P. goldsteinii.

8. Composition according to any one of claims 4 to 7, characterized in that it is administered orally or rectally.

9. Composition according to any one of claims 4 to 8 for its use for the prevention and / or treatment of metabolic diseases and / or their complications.

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

11. Composition according to any one of claims 4 to 8 for its use according to claim 8 characterized in that it is administered in combination with a medicament intended for the treatment of type 2 diabetes such as metformin, inhibitors of the sodium-glucose co-transporter type 2, gliptins, acarbose, glinides, sulfonamides or sulfonylureas such as Glimeperide.

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

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