Methods and compositions for treating obesity using microorganisms

Abstract

The present invention provides compositions and methods for treating or reducing the risk of developing obesity and related diseases and disorders.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 356,945, filed on June 29, 2022, the contents of which are hereby incorporated herein in their entirety for all purposes. Background of the Invention

[0004] With the continuous improvement of living standards worldwide, the number of individuals suffering from unhealthy physical conditions such as obesity and related metabolic diseases (especially type 2 diabetes) is also increasing rapidly. The World Health Organization (WHO) estimates that by 2030, the number of people suffering from diabetes worldwide will exceed 350 million. Due to the rising incidence of obesity, diabetes and other metabolic diseases, their serious impact on health and their profound impact on the economy, new and effective methods are urgently needed to treat high-risk individuals, especially those individuals who have shown early signs of high probability of developing diseases in the future, such as obesity, higher than normal blood cholesterol or triglyceride levels, including higher than normal blood levels of low-density lipoprotein cholesterol (LDL-C) and / or lower than normal blood levels of high-density lipoprotein cholesterol (HDL-C), so as to reduce or eliminate the risk of them suffering from type 2 diabetes and other related conditions (for example, hypertension and cardiovascular disease) in the future.

[0005] There are a large number of microorganisms living in the human gastrointestinal tract, collectively referred to as the intestinal fungal microbiome (mycobiome). The composition of various microorganisms in the intestinal fungal microbiome can be used as an indicator of a person's general health status, as well as a person's risk or tendency to develop certain diseases such as metabolic diseases. Therefore, the largest variety of microorganisms can be used as biomarkers and / or therapeutic agents for treating certain diseases.

[0006] There is an urgent need for new and effective methods to treat or prevent conditions in individuals who already have or are likely to later develop diabetes and / or other metabolic diseases, so that preventive or therapeutic measures can be taken to delay the onset of these diseases in these individuals, reverse or eliminate the symptoms of these diseases in these individuals, or reduce the severity of related symptoms associated with these diseases / risk associated with these diseases. The present invention meets this and other related needs by identifying beneficial intestinal microorganisms in an effort to formulate new compositions and design new methods that are effective in improving metabolic health in individuals, especially those at risk of suffering from symptoms of various metabolic diseases associated with obesity or diabetes or individuals who already suffer from symptoms of various metabolic diseases associated with obesity or diabetes. SUMMARY OF THE INVENTION

[0008] The inventors of the present application have found in their research that certain intestinal microbial species and their relative abundance can affect the metabolism of the host individual, resulting in different effects on individual body weight and metabolic health. Their discovery provides important utility in preventing and treating diseases and conditions related to obesity and metabolic syndrome. The microorganisms identified in this way are now used to provide new methods and compositions for treating or preventing obesity and related conditions.

[0009] In a first aspect, the present invention provides a composition for preventing or treating obesity and related conditions in human subjects, such as metabolic syndrome, type 2 diabetes, fatty liver, hypertension, cardiovascular disease, etc. The composition contains an effective amount of (1) an inhibitor of one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisae; or one or more of Lachancea thermotolerans, Saccharomyces paradoxus, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthorathermophila, and (2) one or more physiologically acceptable excipients. In some embodiments, the composition consists essentially of or consists of the components specified above, such as: (1) one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae; or one or more inhibitors of Kluyveromyces thermotolerans, Saccharomyces paradoxica, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthora thermophila, and (2) one or more physiologically acceptable excipients. In some embodiments, the composition contains an effective amount of (1) Schizosaccharomyces pombe and / or Cryptococcus neoformans; or (2) one or more inhibitors of Kluyveromyces thermotolerans, Saccharomyces paradoxica, and Thielavia terrestris. In some embodiments, the composition comprises an effective amount of Schizosaccharomyces pombe and / or Cryptococcus neoformans, plus one or more physiologically acceptable excipients. In some embodiments, the composition further comprises at least one prebiotic, such as inulin and / or psyllium husk. In some embodiments, the composition is in the form of a food item or beverage that can be taken before, with, or after a meal. In some embodiments, the composition is introduced directly into the gastrointestinal tract of a subject, such as by colonoscopy.

[0010] In a second aspect, the present invention provides a method for treating obesity in an obese subject or a subject at risk of obesity by introducing into the gastrointestinal tract of the subject an effective amount of an inhibitor of one or more of (1) Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae; or (2) Kluyveromyces thermotolerans, Saccharomyces paradoxae, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthora thermophila. In some embodiments, introduced into the gastrointestinal tract of the subject is an effective amount of an inhibitor of one or more of (1) Schizosaccharomyces pombe and Cryptococcus neoformans; or (2) one or more of Kluyveromyces thermotolerans, Saccharomyces paradoxae, and Thielavia terrestris. In some embodiments, an effective amount of one or both of Schizosaccharomyces pombe and Cryptococcus neoformans is introduced into the gastrointestinal tract of the subject, for example, by colonoscopy.

[0011] In some embodiments, after the introduction step, the level of Schizosaccharomyces pombe, Cryptococcus neoformans and / or Saccharomyces cerevisiae increases by about 4-10 times. In some embodiments, after the introduction step, in the gastrointestinal tract of the subject, the relative level of Schizosaccharomyces pombe is greater than about 3.99% of the total intestinal fungi. In some embodiments, after the introduction step, in the gastrointestinal tract of the subject, the relative level of Kluyveromyces thermotolerans, Saccharomyces paradoxus, Thielavia terrestris, Parastagonospora nodorum and / or Myceliophthora thermophila is reduced to less than about 0.1% of the total intestinal fungi. In some embodiments, after the introduction step, the fungal alpha diversity index increases by about 1.2-6 times.

[0012] In some embodiments, the claimed method comprises introducing a composition into the gastrointestinal tract of a subject, the composition comprising: A. (i) one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae; or (ii) an inhibitor of one or more of Kluyveromyces thermotolerans, Saccharomyces paradoxus, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthora thermophila, and B. one or more physiologically acceptable excipients. In some embodiments, the composition comprises one or both of Schizosaccharomyces pombe and Cryptococcus neoformans. In some embodiments, the composition is delivered directly to the small intestine, ileum, or large intestine of the subject, for example, by colonoscopy. In some embodiments, the composition is formulated for oral administration to the subject. In some embodiments, the composition is formulated in a daily dose administered daily. In some embodiments, the composition is in the form of a food or beverage article. In some embodiments, the composition further comprises a prebiotic, such as inulin or psyllium husk. In some embodiments, the introduction step is performed by fecal microbiota transplantation (FMT). In some embodiments, the Schizosaccharomyces pombe, Cryptococcus neoformans, and / or Saccharomyces cerevisiae are derived from and partially purified from fecal material of a donor, or obtained from a culture. In some embodiments, the subject has reduced body weight, reduced serum low-density lipoprotein-cholesterol (LDL-C) levels, reduced serum glucose levels, increased glucose tolerance, and / or increased insulin responsiveness.

[0013] In a related aspect, the present invention provides a composition for treating obesity and related conditions in a subject, such as metabolic syndrome, type 2 diabetes (T2DM), fatty liver, hypertension, cardiovascular disease, etc., or for preventing these conditions in a subject. The composition contains an effective amount of (1) one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae; or an inhibitor of one or more of Kluyveromyces thermotolerans, Saccharomyces paradoxica, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthora thermophila, and (2) one or more physiologically acceptable excipients. In some embodiments, the composition consists essentially of or consists of a specified component, such as (1) one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae; or an inhibitor of one or more of Kluyveromyces thermotolerans, Saccharomyces paradoxica, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthora thermophila, and (2) one or more physiologically acceptable excipients. In some embodiments, the composition contains an effective amount of (1) Schizosaccharomyces pombe and / or Cryptococcus neoformans; or (2) an inhibitor of one or more of Kluyveromyces thermotolerans, Saccharomyces paradoxus, and Thielavia terrestris. In some embodiments, the composition comprises an effective amount of Schizosaccharomyces pombe and / or Cryptococcus neoformans, plus one or more physiologically acceptable excipients. In some embodiments, the composition further comprises at least one prebiotic, for example, inulin and / or psyllium husk. In some embodiments, the composition is in the form of a food item or beverage that can be taken before, with, or after a meal. In some embodiments, the composition is introduced directly into the gastrointestinal tract of a subject, for example by colonoscopy.

[0014] In a third aspect, the present invention provides a kit for promoting weight loss in a subject or reducing the risk of obesity in a subject, comprising a plurality of compositions, each of which comprises an effective amount of one or more of the following substances: Schizosaccharomyces pombe; Cryptococcus neoformans; Saccharomyces cerevisiae; inhibitors of thermotolerant Kluyveromyces; inhibitors of Saccharomyces paradisi; inhibitors of Thielavia terrestris; inhibitors of Parastagonospora nodorum and inhibitors of Myceliophthora thermophila. In some embodiments, the kit comprises a first composition and a second composition, the first composition comprising an effective amount of Schizosaccharomyces pombe and / or Cryptococcus neoformans, and the second composition comprising an inhibitor of thermotolerant Kluyveromyces, Saccharomyces paradisi and / or Thielavia terrestris. In some embodiments, the kit comprises a first composition and a second composition, the first composition comprising an effective amount of Schizosaccharomyces pombe, and the second composition comprising an effective amount of Cryptococcus neoformans. In some embodiments, the compositions contained in the claimed kits can be formulated for oral administration or for direct delivery to the gastrointestinal tract of a subject, for example, by colonoscopy.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Fecal fungal community structure in the Hong Kong and Kunming cohorts. (A) Compositional changes at the genus level in the gut fungal microbiome associated with obesity in the two regions. (B) Effect size of metadata variables in changes in the human gut fungal microbiome. *P<0.05, ***P<0.001. (CE) Changes in α-diversity of the human gut fungal microbiome associated with obesity in the two regions. Fecal fungal diversity (C), uniformity (D), and richness (E) were compared between the lean and obese groups in the two regions. HK, Hong Kong, China; KM, Kunming. Statistical significance was determined by t-test, and the numbers on the bars represent p-values. For box plots, the vertical line extends 1.5 times the median value depicted by the interquartile range (top and bottom boundaries of the box) and the horizontal line within the box.

[0017] Figure 2. Changes in gut fungal microbiome composition at the species level associated with obesity in the Hong Kong and Kunming cohorts (A) and differences in the relative abundance of major fungal taxa (B). Differences in abundance were detected using linear discriminant analysis effect size (LEfSe) analysis. Only dominant species (relative abundance ≥ 1.0%) were shown, and when analyzed with LefSE analysis, the FDR correction had an adjusted p value of <.05 and an effect size of >2. The logarithmic transformation of relative abundance is shown in the box plot. HK, Hong Kong, China; KM, Kunming. *P<0.05, **P<0.01. P values ​​were determined by Wilcoxon rank-sum test.

[0018] Figure 3 . Putative fungal functional characteristics in fecal samples of lean and obese subjects from the Hong Kong and Kunming cohorts. (A) Pie chart of 12 functional groups (guilds) in fecal samples. Lean inHK: lean individuals in Hong Kong; Obesity in HK: obese individuals in Hong Kong; Lean inKM: lean individuals in Kunming; Obesity in KM: obese individuals in Kunming. (BE) Relative abundance of endosymbiotic fungi (B), epiphytic fungi (C), plant pathogenic fungi (D), and plant saprophytic fungi (E) in fecal samples. All data are expressed as mean ± SEM. Bars with numbers indicate statistical differences. p values ​​are from t-tests. HK, Hong Kong; KM, Kunming.

[0019] Figure 4 .Co-occurrence network between fungal taxa in feces of lean and obese subjects from the Hong Kong and Kunming cohorts. (A) Lean in HK; (B) Obese in HK; (C) Lean in KM; (D) Obese in KM. Colored nodes represent operational taxonomic units (OTUs) assigned to major genera. Edges between nodes represent positive (blue) or negative (red) co-occurrence relationships inferred from OTU abundance features using the SparCC method (pseudo p < 0.05, correlation value < -0.6 or > 0.6). HK, Hong Kong; KM, Kunming.

[0020] Figure 5 .Correlation between alpha diversity (diversity, evenness, and richness) of fecal bacteria and fungi in lean and obese subjects from the Hong Kong and Kunming cohorts. (A) Lean in HK; (B) Obese in HK; (C) Lean in KM; (D) Obese in KM. Spearman's correlation coefficient was calculated, and statistical significance was determined for all pairwise comparisons. Significant correlations (p<0.05) are indicated with asterisks. *p<0.05, **p<0.01, ***p<0.001. Blue circles indicate positive correlations, and red circles indicate negative correlations. Size and shading indicate the magnitude of the correlation, where darker shading indicates stronger correlations than lighter shading. HK, Hong Kong; KM, Kunming.

[0021] Figure 6. Effects of Schizosaccharomyces pombe on obesity and serological parameters. (A) Schematic, (B) Weight gain, (C) Fat index, (D) Subcutaneous adipose tissue weight, (E) Epididymal adipose tissue weight, (F) Perirenal fat weight, (G) Serum glucose profile of insulin tolerance test (ITT), (H) Area under the curve (AUC) measured during ITT, (I) Serum glucose profile of oral glucose tolerance test (OGTT), (J) AUC measured during OGTT. Data are shown as mean ± SEM (n = 4 or 5). p values ​​were corrected with false discovery rate (FDR). *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significant difference. p values ​​are from one-way ANOVA. ND: normal diet; HF: high-fat diet; SE: group treated with S. cerevisiae; SP: group treated with S. pombe; OGTT: oral glucose tolerance test; ITT: insulin tolerance test.

[0022] Figure 7. Changes in fungal microbiome composition with respect to region and obesity. (A) Beta-diversity of fecal fungi in obese and lean subjects from Hong Kong and Kunming, China. NMDS (non-metric multidimensional scaling) plot based on Bray-Curtis dissimilarity at the fungal species level. Differences in fecal fungal community structure between lean and obese subjects in Hong Kong, China (B) and Kunming, China (C).

[0023] Figure 8. Changes in α-diversity of the human gut fungal microbiome according to medication and type 2 diabetes mellitus (T2DM). Effects of mediation on the richness (A) and diversity (B) of the gut fungal microbiome. Effects of type 2 diabetes on the richness (C) and diversity (D) of the gut fungal microbiome (as measured in Chao1 richness and Simpson diversity index, respectively). Bars with numbers indicate statistical differences. Statistical significance between Y and N was determined by Wilcoxon. HK, Hong Kong, China; KM, Kunming. Y, yes; N, no.

[0024] Figure 9. Negative correlation between Schizosaccharomyces pombe and BMI. (A) Negative correlation between Schizosaccharomyces pombe and BMI in the Hong Kong and Kunming cohorts, respectively. (B) Association between major fungal species and BMI. p values ​​are from t-tests. HK, Hong Kong; KM, Kunming.

[0025] Fig.10 Differences in relative abundance of putative fungal functional traits in fecal samples between the Hong Kong and Kunming cohorts. All data are presented as mean ± SEM. Bars with numbers indicate statistical differences. p values ​​are from t-tests.

[0026] Fig.11.Liver mass index and blood lipid levels in mice. (A) Liver mass index; (B) High-density lipoprotein-cholesterol (HDL-C); (C) Low-density lipoprotein-cholesterol (LDL-C); (D) Total cholesterol (TC); (E) Total triglycerides (TG). Data are shown as mean ± SEM (n = 4 or 5). *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significant difference. p values ​​are from one-way analysis of variance.

[0027] definition

[0028] The term "fecal microbiota transplant (FMT)" or "fecal transplant" refers to a medical procedure in which fecal material containing live fecal microorganisms (bacteria, fungi, etc.) obtained from a healthy individual is transferred to the gastrointestinal tract of a recipient to restore healthy intestinal microflora that has been disrupted or destroyed by various medical conditions. Typically, fecal material from a healthy donor is first processed into a suitable form for transplantation, which can be achieved by direct deposition into the lower gastrointestinal tract (such as by colonoscopy or by nasal cannula) or by oral ingestion of packaged materials containing dried and frozen fecal material.

[0029] The term "antifungal agent" refers to any substance capable of inhibiting, suppressing or preventing the growth or proliferation of fungal species, either generally or specifically against certain fungal species, e.g., Kluyveromyces thermotolerans, Saccharomyces paradoxus, Thielavia terrestris, Parastagonospora nodorum or Myceliophthora thermophila. Known agents with general fungicidal activity include amphotericin B, echinocandins, fluconazole, nystatin and clotrimazole.

[0030] "Relative abundance percentage," when used in the context of describing the presence of a particular fungal species (e.g., Schizosaccharomyces pombe or Cryptococcus neoformans) relative to all fungal species present in the same environment, refers to the amount of the particular fungal species relative to the amount of all fungal species expressed as a percentage. For example, the relative abundance percentage of the fungal species Schizosaccharomyces pombe can be determined by comparing the amount of Schizosaccharomyces pombe-specific DNA in a given sample (e.g., as determined by quantitative polymerase chain reaction) to the amount of all fungal DNA in the same sample (e.g., as determined by quantitative PCR and sequencing based on the internal transcribed spacer 2, or ITS2, sequence).

[0031] "Absolute abundance," when used in the context of describing the presence of a particular fungal species (e.g., Schizosaccharomyces pombe or Cryptococcus neoformans) in feces, refers to the amount of DNA derived from the fungal species relative to the amount of all DNA in a fecal sample. For example, the absolute abundance of Schizosaccharomyces pombe can be determined by comparing the amount of Schizosaccharomyces pombe-specific DNA in a given sample (e.g., as determined by quantitative PCR) to the amount of all fecal DNA in the same sample.

[0032] As used herein, the "total fungal load" of a stool sample refers to the amount of all fungal DNA in the total amount of DNA in the stool sample. For example, the absolute abundance of fungi can be determined by comparing the amount of fungal-specific DNA (e.g., 18S rDNA measured by quantitative polymerase chain reaction) in a given sample with the amount of all fecal DNA in the same sample.

[0033] The term "fungal alpha diversity index" used with respect to fungal species describes the diversity of the fungal community in a predetermined sample or site, for example, a fecal sample taken from the human gastrointestinal tract. In the present disclosure, "alpha diversity" means the complexity of the community in each sample when the species and the relative abundance of each species are considered separately: a higher alpha diversity indicates that the sample contains a more complex community, including a higher number of different species (kinds) and a higher frequency of different species (relative abundance of each species). The definition of alpha diversity is usually based on the values ​​given by one or more diversity indices, such as species richness (a count of the number of different species present in a sample), species evenness (a measure of the degree of separation of individuals between species, where low values ​​indicate that one or a few species are dominant and high values ​​indicate that a relatively equal number of individuals belong to each species), and the Shannon index or Simpson index (which takes into account the proportional abundance of species in the community).

[0034] As used herein, the term "obese" is used to describe non-Asians with a body mass index (BMI) of 30 or higher and Asians with a BMI of 25 or higher, while the term "overweight" is used to describe non-Asians with a BMI of 25 or higher and Asians with a BMI of 23 or higher.

[0035] As used herein, the term "inhibiting" or "inhibition" refers to any detectable negative effect on a target biological process, such as RNA / protein expression of a target gene, biological activity of a target protein, cell signal transduction, cell proliferation, the presence / level of an organism, especially a microorganism, any measurable biomarker, biological parameter, or symptom in a subject, etc. In general, inhibition is reflected in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in any of the target process (e.g., weight or blood glucose / cholesterol levels of a subject, or any measurable symptom or biomarker in a subject, such as infection rate of a pathogenic infectious agent among subjects) or any of the above downstream parameters when compared to a control. "Inhibition" also includes a 100% reduction, i.e., complete elimination, prevention, or abrogation of a target biological process or signal. Other related terms such as "suppressing," "suppression," "reducing," and "reduction" are used in a similar manner in the present disclosure to refer to a decrease to varying levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater decrease compared to a control level) up to complete elimination of a target biological process or signal. On the other hand, terms such as "activate," "activating," "activation," "increase," "increasing," "promote," "promoting," "enhance," "enhancing," or "enhancement" are used in the present disclosure to encompass various levels of positive changes (e.g., at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more, such as a 3, 5, 8, 10, 20-fold increase, compared to a control level in a process, signal or parameter of interest).

[0036] As used herein, the term "treatment" or "treating" includes therapeutic and preventive measures taken to address the presence of a disease or condition or the risk of developing such a disease or condition in the future. It covers treatment or preventive measures used to alleviate ongoing symptoms, inhibit or slow disease progression, delay the onset of symptoms, or eliminate or reduce side effects caused by such diseases or conditions. Preventive measures and variations thereof in this context do not require 100% elimination of the occurrence of an event; rather, they refer to suppressing or reducing the likelihood or severity of such an occurrence or delaying such an occurrence.

[0037] A "patient" or "subject" receiving a composition or treatment method of the present invention is a person of any age, gender, and ethnic background, including adults and adolescents, who may not have been diagnosed with any specific disease or condition (e.g., no blood glucose test results for diabetes diagnosis), but who needs to improve metabolic health (e.g., reduce their weight or eliminate their risk of developing obesity or diabetes). Typically, patients or subjects who receive treatment according to the methods of the present invention to improve metabolic health such as weight loss or reduce the risk of becoming overweight or obese do not need to be treated with the same therapeutic agent in addition. For example, if a subject is receiving a composition according to the claimed method, the subject does not suffer from any disease known to be treated with the same therapeutic agent. Although the patient can be of any gender and any age, in some cases, the patient is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years old; in some cases, the patient can be 40-45 years old, or 50-65 years old, or 65-85 years old. A "child" subject is a subject under the age of 18 years, e.g., about 5-17 years, 9 years, or 10-17 years, or 12-17 years, and includes "infants," which are less than about 12 months, e.g., less than about 10, 8, 6, 4, or 2 months, while an "adult" subject is a subject 18 years or older.

[0038] The term "effective amount" as used herein refers to the amount of the expected (e.g., treatment or prevention) effect of the administered substance. The effect includes preventing, correcting or inhibiting the symptoms of a particular disease / condition and the progression of related complications to any detectable extent, for example, the incidence of obesity, type 2 diabetes, hypertension, heart disease, etc., one or more symptoms of these conditions and related disorders. The exact "effective" amount of any therapeutic agent will depend on the properties of the agent and the purpose of treatment, and will be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Volume 1-Volume 3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0039] As used herein, the term "about" refers to a range of values ​​of + / - 10% of the specified value. For example, "about 10" refers to a range of values ​​of 10 + / - 10 x 10%, i.e., 9-11.

[0040] A "pharmaceutically acceptable" or "pharmacologically acceptable" excipient is a substance that is not biologically harmful or otherwise undesirable, i.e., the excipient can be administered to an individual with a biologically active agent without causing any undesirable biological effect. The excipient also will not interact in a deleterious manner with any component of the composition in which it is contained.

[0041] The term "excipient" refers to any substantially auxiliary material that may be present in the final dosage form of the composition of the present invention. For example, the term "excipient" may include, but is not limited to, vehicles, buffers, binders, disintegrants, fillers (diluents), lubricants, glidants (flow enhancers), compression aids, colorants, sweeteners, preservatives, suspending agents / dispersants, film formers / coating agents, flavoring agents, and printing inks.

[0042] When used in the context of describing a composition containing an active ingredient or multiple active ingredients, the term "consisting essentially of" refers to the fact that the composition does not contain other ingredients that have any similar or related biological activity of the active ingredient or that can enhance or suppress the activity, although one or more inactive ingredients such as physiologically or pharmaceutically acceptable excipients may be present in the composition. For example, a composition consisting essentially of an active agent effective to reduce the weight of a subject or improve metabolic health in a subject (e.g., a composition comprising Schizosaccharomyces pombe or Cryptococcus neoformans) is a composition that does not contain any other agent that may have any detectable positive or negative effect on the same target process (e.g., weight loss, reduction in blood sugar, lipid or cholesterol levels, improvement in glucose tolerance or improvement in insulin sensitivity) or that may increase a disease or symptom in a recipient subject or that may reduce a disease or symptom in a recipient subject to any measurable degree. DETAILED DESCRIPTION OF THE INVENTION

[0044] I. Introduction

[0045] The purpose of the present invention relates to new methods and compositions for preventing, improving and treating obesity and obesity-related conditions involving specific fungal species. In the present invention, the inventors of the present application have found that certain fungal species are at a changed level in objects suffering from obesity. Health benefits can be achieved by regulating the level of these fungal species in the intestinal tract of the patient, such as by oral administration of beneficial species or agents that can suppress undesirable species, such as preventing and alleviating obesity and obesity-related conditions. Fecal microbiota transplantation (FMT) technology is a preferred method for implementing the present invention.

[0046] II. Pharmaceutical Compositions and Administration

[0047] The present invention provides a pharmaceutical composition comprising an effective amount of one or more of Schizosaccharomyces pombe, Cryptococcus neoformans and Saccharomyces cerevisiae, for reducing the weight of a person, to reduce the risk of obesity or conditions associated with obesity such as diabetes, hypertension or cardiovascular disease. The pharmaceutical composition of the present invention is suitable for a variety of drug delivery systems. Suitable formulations for the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th edition (1985). For a brief review of drug delivery methods, see Langer, Science 249: 1527-1533 (1990).

[0048] In another aspect of the invention, an alternative composition for improving metabolic health in humans, such as for weight loss or for reducing the risk of developing obesity in humans, contains an inhibitor or specific antifungal agent capable of suppressing the proliferation of at least one, possibly two or more (e.g., 3, 4, or 5) of the following fungal species: Kluyveromyces thermotolerans, Saccharomyces paradoxus, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthora thermophila. This inhibitor or antifungal agent is preferably not a broad-spectrum fungicide; instead, it should be a specific antifungal agent that specifically targets one or more of the above-named species. For example, it can be a short polynucleotide such as a small inhibitory RNA, microRNA, miniRNA, lncRNA, or an antisense oligonucleotide that is capable of disrupting the expression of at least one key gene in the life cycle of the target fungal species, so that the agent can specifically target only the species without significantly affecting other closely related fungal species.

[0049] The pharmaceutical compositions of the present invention can be administered by various routes, such as systemic administration via oral ingestion or local delivery using rectal suppositories. For example, a preferred route of administering the pharmaceutical compositions is in the form of about 10 6 to about 10 12 One or more of Schizosaccharomyces pombe, Cryptococcus neoformans and Saccharomyces cerevisiae are orally administered at a daily dose of CFU. Optionally, the composition also contains at least one prebiotic, such as inulin or psyllium husk. In addition, the composition can be formulated in a daily dosage form. Suitable dosages can be administered in a single daily dose or in divided doses presented at appropriate intervals, such as twice, three times, four times or more sub-doses per day. The duration of administration can range from about 2 weeks to about 4 weeks, such as about 1 week to about 2 weeks.

[0050] To prepare the pharmaceutical compositions of the present invention, one or more inert and pharmaceutically acceptable carriers are used. Pharmaceutical carriers can be solid or liquid. Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. A solid carrier can be one or more substances that can also act as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, or tablet disintegrating agent; it can also be an encapsulating material.

[0051] In powders, the carrier is generally a finely divided solid which is mixed with the finely divided active ingredient, e.g., one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0052] To prepare pharmaceutical compositions in suppository form, a low melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into convenient sized molds and allowed to cool and solidify.

[0053] Powders and tablets preferably contain from about 5% to about 100% by weight of the active ingredient (e.g., one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae). Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like.

[0054] The pharmaceutical composition may include an active ingredient, such as a preparation of one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae, wherein the encapsulating material is used as a carrier to provide a capsule in which the active ingredient (with or without other carriers) is surrounded by the carrier so that the carrier is combined with the active ingredient. In a similar manner, a sachet may also be included. Tablets, powders, sachets, and capsules may be used as solid dosage forms suitable for oral administration.

[0055] Liquid pharmaceutical compositions include, for example, solutions suitable for oral administration or topical delivery, suspensions suitable for oral administration and emulsions. Water-based solutions prepared by adding active ingredients (e.g., one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae) to a previously sterilized aqueous solution in a solvent comprising water, buffered water, saline, PBS, ethanol, or propylene glycol are examples of liquid or semi-liquid compositions suitable for oral administration or topical delivery (e.g., via rectal suppositories). The compositions may contain one or more pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting agents and buffers, tension adjusting agents, wetting agents, detergents, and the like.

[0056] A sterile solution (e.g., aqueous solution) may be prepared first, and then an active component (e.g., one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae, which may be obtained from a culture or may have been inactivated, for example, by heating or by chemical treatment) may be added to a desired solvent system. Depending on the nature of the active ingredient / component, different methods may be used to prepare a suitable solvent-based delivery system. For example, one or more active compounds may be first dissolved in a solution, and then the resulting solution may be passed through a membrane filter to sterilize it, or alternatively, one or more previously sterilized active compounds may be dissolved in a previously sterilized solvent under aseptic conditions. In the case where the active component is one or more microorganisms, they may optionally be first purified to remove unrelated microbial species to a substantially undetectable level (e.g., not greater than 110%, 120%, or 150% of the background signal), and then placed in a sterile solution to produce a solution-based delivery system. The resulting aqueous solution may be packaged for use as is or lyophilized, and the lyophilized preparation may be combined with a sterile aqueous carrier prior to administration. The pH of the preparation is typically 3-11, more preferably 5-9, and most preferably 7-8.

[0057] Single or multiple administrations of the composition may be performed using a dosage level and pattern selected by the treating physician. In any event, the pharmaceutical preparation should provide an amount of active agent sufficient to effectively enhance the efficacy of a preventive or therapeutic regimen (such as a plan of diet, physical exercise and other lifestyle changes) and / or reduce or eliminate the undesirable effects of weight-related conditions (such as type 2 diabetes, hypertension, heart disease, etc.).

[0058] III. Kit

[0059] The present invention also provides a kit for improving the metabolic health of an individual or for preventing a disease associated with obesity according to the methods disclosed herein. The kit generally includes a plurality of containers, each of which contains a composition comprising: (1) one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae; (2) one or more inhibitors of Kluyveromyces thermotolerans, Saccharomyces paradoxus, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthora thermophila. Optionally, additional ingredients such as one or more prebiotics (e.g., inulin or psyllium husk) may be included in the composition. In addition, other agents or drugs known to be effective in preventing and / or treating related conditions, especially metabolic diseases associated with obesity, including improving the symptoms of these diseases and reducing the severity of these diseases, and for promoting recovery from diseases (such as those described in the last section or those known in the relevant art) may be included in the kit. The multiple containers in the kit may each contain different active agents / drugs or different combinations of two or more active agents or drugs.

[0060] The kit may also include informational material providing instructions on how to dispense the pharmaceutical composition, including a description of the types of patients that can be treated (e.g., human patients who are overweight or obese or have been identified as being at high risk for developing metabolic diseases associated with overweight or obesity and are therefore seeking to improve metabolic health, as well as patient subpopulations not included in the claimed methods, such as those who have been diagnosed with a pre-existing condition that already requires administration of an active ingredient such as an inhibitor of one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae, or one or more of Kluyveromyces thermotolerans, Saccharomyces paradoxica, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthora thermophila), the dosage, frequency, and mode of administration, etc. Example

[0061] The following examples are provided by way of illustration only, and not by way of limitation. Those skilled in the art will readily recognize that a variety of noncritical parameters can be changed or modified to produce essentially the same or similar results.

[0062] method

[0063] Study subjects

[0064] Recruited from Hong Kong and Kunming, China, with a body mass index (BMI) ≥ 28 kg / m 2 Obese subjects aged 18-70 years and with a BMI of 18.5 to 23 kg / m 2 Lean subjects aged 18-70 years. Subjects were excluded if they had current severe gastrointestinal diseases, including inflammatory bowel disease (IBD), cancer, advanced adenoma or gastroenteritis; or if they often had digestive system symptoms, including blood in the stool, diarrhea, abdominal pain or vomiting; or if they had current rheumatic immune diseases, active infections or acquired immunodeficiency syndrome. Subjects were excluded if they had a known history of organ dysfunction or failure, including the cardiopulmonary system or urinary system, etc.; or if they had a known history of abdominal surgery, radio-chemotherapy or immunotherapy; or if they had current incurable cancer. Subjects who were pregnant or planned to become pregnant within 1 year were also excluded.

[0065] Written informed consent was obtained from all subjects. Their information on age, sex, BMI, alcohol intake, smoking, type 2 diabetes mellitus (T2DM), and medications was recorded. Fecal samples from study subjects were collected and then stored at −80 °C for downstream gut fungal microbiome analysis. This study was approved by The Joint Chinese University of Hong Kong—New TerritoriesEast Cluster Clinical Research Ethics Committee (No.: 2014.026) and The Clinical Research Ethics Committee of Kunming Medical University (No.: 2019L23).

[0066] DNA extraction, library preparation, and metagenomic sequencing

[0067] According to Zuo et al. 1 DNA was extracted using the method reported above (with minor modifications). 2 O pre-washed 100 mg fecal sample and pelleted by centrifugation at 13,000 × g for 1 min. The fecal pellet was resuspended in 800 μL TE buffer (pH 7.5) supplemented with 16 μL β-mercaptoethanol and 250 U lysing enzyme (Sigma) and incubated at 37°C for 90 min. The sample was then centrifuged at 13,000 × g for 3 min and then lysed using Fecal DNA was extracted from the pellet using the RSC PureFood GMO and Authentication Kit (Promega, Madison, Wisconsin). Briefly, 800 mL of cetrimonium bromide (CTAB) buffer was added to the fecal pellet and vortexed for 30 seconds, and the mixture was then incubated at 95°C for 5 minutes. The sample was then vortexed thoroughly with the beads for 15 minutes. Next, 40 μL of proteinase K and 20 μL of RNase A were added to the sample, and the mixture was incubated at 70°C for 10 minutes. The supernatant was then obtained by centrifugation at 13,000 × g for 5 minutes and added to RSC machine for DNA extraction. DNA library was constructed through end repair, purification and PCR amplification. Whole genome shotgun sequencing of samples was performed on Illumina NovaSeq 6000 (Illumina, San Diego, California, USA) using the paired end 150 bp sequencing strategy of Novogene, Beijing, China.

[0068] Bioinformatics and statistical analysis

[0069] Adaptor contamination and low-quality reads were discarded from the raw data using Kneaddata (website: huttenhower.sph.harvard.edu / kneaddata). 2 Fungal sequence annotation was performed and the MetaPhlAn2 classifier was used 3 Perform bacterial taxonomy analysis. Use the FUNGuild database (website: github.com / UMNFuN / FUNGuild) to annotate fungal functions 4 Fungal microbiome and bacterial group abundance tables were imported into R (version 3.6.1) for statistical analysis.

[0070] The R package Vegan 2.5-6 was used to calculate and visualize alpha diversity indices, including observed species, chao1, Simpson and Shannon diversity indices. Non-metric multidimensional scaling analysis (NMDS) was also performed using the Vegan package. The R package Vegan2.5-6 was used to examine the factors of region, BMI, gender, age, lifestyle (alcohol intake and smoking), and type 2 diabetes (T2DM) to explore which factors were associated with the established fungal microbiome by permutation multivariate analysis of variance (PERMANOVA). The linear discriminant analysis effect size (LEfSe) was used to identify biomarkers associated with obesity and thinness in Hong Kong and Kunming, China. Those with a linear discriminant analysis (LDA) score ≥ 2.0 were considered to be important biomarkers associated with obesity and thinness.

[0071] To identify positive and negative correlations between individual operational taxonomic units (OTUs), sparse correlations of compositional data (SparCC) were developed to quantify intra- and inter-kingdom relationships. Co-occurrence and mutual exclusion networks within fungi were then visualized using Cytoscape 3.7.2. Correlation matrices within and between kingdoms were created using the R package Corrplot.

[0072] Statistically significant differences between groups were determined using Student's t-test and Wilcoxon rank-sum test where appropriate and adjusted for multiple comparisons with Bonferroni correction.

[0073] Animal experiments

[0074] 29 male C57BL / 6J mice (6-8 weeks old, n=4 / group or 5 / group) were obtained from the Laboratory Animal Services Centre of the Chinese University of Hong Kong. All animals were housed in standard plastic cages (four or five mice per cage) under a 12 h light-dark cycle at a constant temperature and humidity (23±1°C and 55%±5%, respectively), with free access to food and water. A schematic diagram is shown in FIG6A . After acclimation for 1 week, the mice were divided into six groups (n=4 / group or 5 / group). Three groups received a normal diet (ND) (Labdiet Picolab Select Mouse diet 50IF / 9F Irradiated, containing 18 wt% protein, 8.0 wt% fat and 5 wt% fiber; POBOX 19788, BRENTWOOD, MO 63144), and at the same time, 0.2 mL of phosphate buffered saline (PBS) (ND), 1×10 7 CFU / mouse) in 0.2 mL PBS (ND+SC) or containing Schizosaccharomyces pombe (ATCC 16979, 1×10 7 CFU / mouse) in 0.2 mL PBS (ND+SP) for 11 weeks. The other three groups received a high sucrose cholesterol diet (HF) (SF11-078, 23 wt% fat, 42.4 wt% sucrose, and 19.4 wt% protein; Specialty Feeds, Perth, Australia) and were simultaneously gavaged with 0.2 mL phosphate buffered saline (PBS) (HF), 1×10 CFU / mouse containing Saccharomyces cerevisiae (ATCC 36375, 1×10 7 CFU / mouse) in 0.2 mL PBS (HF+SC) or containing Schizosaccharomyces pombe (ATCC 16979, 1×10 7 CFU / mouse) in 0.2 mL PBS (HF+SP) for 11 weeks. The body weight of each individual mouse was measured weekly.

[0075] At the 10th week (Fig. 6A), glucose intolerance was evaluated using a standard oral glucose tolerance test (OGTT). In short, after fasting for 14h, 20% (w / v) glucose solution was administered to mice at a dose of 1g / kg body weight by oral gavage. Blood samples were collected from a tail incision (by removing 2mm from the tail end) before (0) and 15, 30, 60 and 90min after glucose administration. Glucose levels were analyzed with a handheld blood glucose meter (Sannuo; Changsha Sinocare, Inc., Changsha, China). One week later, an insulin tolerance test (ITT) was performed. Under non-fasting conditions, diluted regular human insulin (Sigma-Aldrich) was injected intraperitoneally into mice at a dose of 0.75 units / kg body weight. Blood samples were collected from a tail incision (by removing 2mm from the tail end) before (0) and 15, 30, 60 and 90min after insulin injection, and glucose levels were also analyzed with a handheld blood glucose meter. At the end of the 12-week experiment (Figure 6A), mice were euthanized by cervical dislocation. The liver and adipose tissue (subcutaneous, epididymal and perirenal) were precisely dissected and weighed. The liver index was defined as the ratio of liver weight divided by body weight. The fat index was defined as the ratio of fat weight divided by body weight. Total cholesterol (TC), total triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels in serum were measured using commercial enzyme kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's instructions. All animal experiments were approved by the Animal Ethics Committee (AEEC) of the Chinese University of Hong Kong and performed in accordance with the requirements of the Animal Ethics Committee (AEEC) of the Chinese University of Hong Kong.

[0076] Schizosaccharomyces pombe and Saccharomyces cerevisiae cultures

[0077] Schizosaccharomyces pombe was grown in yeast and mold agar medium (3 g / L yeast extract, 3 g / L malt extract, 5 g / L peptone, 10 g / L dextrose, 20 g / L agar for solid medium, pH 6.2 ± 0.2) at 37°C, 200 rpm. The culture was then frozen and stored at -80°C with glycerol. The glycerol stock was thawed and cultured overnight under the same conditions one day before administration by oral gavage, and then diluted to the final concentration on the day of oral gavage.

[0078] Similarly, Saccharomyces cerevisiae was grown in yeast peptone dextrose (YPD) medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, 20 g / L agar for solid medium) at 37°C, 200 rpm. The culture was then frozen and stored in glycerol at -80°C. The glycerol stock was thawed one day before administration by oral gavage and cultured overnight under the same conditions, then diluted to the final concentration on the day of oral gavage.

[0079] result

[0080] Clinical characteristics of the study subjects

[0081] A total of 217 subjects from Hong Kong and Kunming were included in this study. 118 subjects (59 lean subjects and 59 obese subjects) were recruited in Hong Kong, China, and 99 subjects (49 lean subjects and 50 obese subjects) were recruited in Kunming. In the Hong Kong group, the median age of the lean and obese participants was 45.88 years and 47.85 years, respectively. In the Kunming group, the median age of the lean and obese participants was 48.14 years and 40.56 years, respectively. There were significant differences in smoking status, T2DM, and medication use between the lean and obese subjects in the Hong Kong group, and significant differences in age and gender between the lean and obese subjects in the Kunming group. Other clinical characteristics were comparable (Table 1).

[0082] Table 1 Clinical characteristics of the subjects

[0083]

[0084]

[0085] aFor continuous variables, from the Wilcoxon rank-sum test; for categorical variables, from Fisher’s exact test; bBMI, body mass index.

[0086] cExpressed as mean ± SD or median (25th to 75th percentiles, P25–P75) where appropriate dAny medication, including metformin, sulfonylureas, statins, proton pump inhibitors and nonsteroidal anti-inflammatory drugs, antibiotics, angiotensin-converting enzyme inhibitors, and calcium channel blockers.

[0087] Variations in the composition of the gut fungal microbiome in lean and obese individuals between regions

[0088] At the phylum level, Ascomycota was the most prevalent fungus detected in all samples, while at the genus level, Saccharomyces was the most prevalent fungus detected in individuals from Hong Kong (average relative abundance: 48.53%) and Kunming (average relative abundance: 34.85%). Figure 1 The most abundant species were Saccharomyces cerevisiae (detected in 90.68% of Hong Kong subjects and 86.87% of Kunming subjects) and Sporisorium reilianum (detected in 84.75% of Hong Kong subjects and 95.96% of Kunming subjects) ( Figure 2A ). The prevalence of other fungal species is shown in Table 2.

[0089] Table 2 Prevalence of selected gut fungal species detected in the Hong Kong and Kunming cohorts

[0090] Species Hong Kong Kunming Candida dubliniensis 36.44％ 55.56％ Cryptococcus neoformans 54.24％ 67.68％ Kluyveromyces thermotolerant 18.64％ 30.30％ Myceliophthora thermophila 23.73％ 37.37％ Parastagonospora nodorum 29.66％ 32.32％ Schizosaccharomyces pombe 52.54％ 68.69％ Thielavia terrestris 30.51％ 51.52％

[0091] Among all clinical variables (region, sex, alcohol intake, obesity, smoking, T2DM, age, and medications), region (effect size R 2 =0.015, p<0.001) and obesity (effect size R 2 =0.010, p<0.05) showed the largest effect size for changes in the fecal fungal microbiome ( Figure 1 B, Table 3). By nonmetric multidimensional scaling (NMDS), Kunming subjects (yellow dots) clustered separately from Hong Kong subjects (blue dots), reinforcing the finding of significant differences in fungal microbiome composition between Hong Kong and Kunming subjects (PERMANOVA test, p < 0.001, Fig. 7A ).

[0092] Table 3 Effects of obesity, region, gender, diabetes, smoking and alcohol on intestinal fungal flora analyzed by PERMANOVA

[0093]

[0094] The fecal fungal microbiome diversity was then evaluated between regions and between obese subjects relative to lean subjects. The fecal fungal microbiome diversity and richness in Kunming subjects were significantly higher than those in Hong Kong subjects (t-test, p<0.01 and p<0.001, respectively, Figure 1 C and 1E). Regarding obesity, lower fecal fungal evenness, but not diversity, was observed in obese subjects compared with lean subjects in the Hong Kong cohort (t-test, p<0.05 and p=0.079, respectively, Figure 1 C and 1D). However, lean and obese individuals in the Kunming cohort showed a more homogeneous fecal fungal composition (t-test, p = 0.763 in diversity; p = 0.664 in evenness; p = 0.61 in richness, respectively). Figure 1 C-1E). Overall, the pattern of disruption of the gut mycoflora in obese subjects compared with lean individuals was regionally distinct.

[0095] In addition, the association between medications and the composition of the gut fungal microbiome was further explored. Beta blockers, nonsteroidal anti-inflammatory drugs (NSAIDs), proton pump inhibitors (PPIs), statins, and sulfonylureas (SUs) were recorded in the medical history of the included participants (Figure 8). The results showed that there was no significant correlation between the use of these five drugs and fungal diversity or richness (Figures 8A and 8B). In addition, T2DM did not show a significant effect on the composition of fecal fungi (Figures 8C and 8D).

[0096] Prediction of fecal fungal trophic patterns and functional groups in lean and obese individuals across regions

[0097] To further explore how obesity and region affect the function of the gut fungal microbiome, the fecal fungal microbiome nutritional patterns and ecological functional group predictions were performed in obese and lean subjects in relation to region ( Figure 3 According to the classification based on nutrient substrate preference, fungi are divided into twelve functional groups (fecal saprophytes, wood saprophytes, soil saprophytes, animal pathogens, plant pathogens, epiphytes, endophytes, animal endosymbionts, fungal parasites, and undetermined saprophytes) and three trophic modes (pathotroph, saprotroph, and symbiotroph) ( Figure 3 A) The majority of fungi detected in the fecal fungal microbiome were saprotrophic, accounting for more than 79% of the community and mainly including the taxa Saccharomycetales.

[0098] Fungal functions vary widely among geographic regions ( Figure 3 Compared with Kunming subjects, the fecal fungal microbiota of Hong Kong subjects was more abundant in animal endosymbiotic fungi (t test, p < 0.05), animal pathogenic fungi (t test, p < 0.05), endophytic fungi (t test, p < 0.01), parasitic fungi (t test, p < 0.05), soil saprophytic fungi (t test, p < 0.05), and wood saprophytic fungi (t test, p < 0.01) ( Fig.10 ). The nutritional pattern of the fecal fungal microbiome also showed significant associations with obesity, which was also region-specific ( Figure 3In Hong Kong, China, the feces of the lean group had significantly higher levels of animal endosymbiotic fungi (t-test, p<0.05) and less phytopathogenic fungi (t-test, p<0.05) ( Figure 3 B and 3D). In Kunming, epiphytic fungi and plant saprophytic fungi were relatively more abundant in the feces of the obese group than in the lean group, showing a higher relative abundance of about 74.48% (Wilcoxon rank sum test, p < 0.05) and 79.24% (Wilcoxon rank sum test, p = 0.01), respectively. Figure 3 C and 3E). These data suggest that region and obesity have a combined effect on the functional landscape of gut fungi.

[0099] Reduced fungal-intra-kingdom ecological interactions in the fecal fungal microbiome in obesity across regions

[0100] In the host, fungi interact with each other through hyphae or hitchhiking to promote colonization in the intestine. To investigate whether obesity affects fungal-fungal interactions, network analysis was performed to depict the relationships between fungal species in the two groups ( Figure 4 ).

[0101] When correlated with co-abundance in obesity ( Figure 4 B and 4D) compared with the Figure 4 A) and Kunming ( Figure 4 In the two lean groups of the C) group, more complex within-kingdom co-abundance relationships within the fecal fungal microbiome were observed. Interestingly, a negative correlation between Ustilago serrata and Saccharomyces cerevisiae was identified in all four groups (lean subjects in Hong Kong, China, r = -0.18, p < 0.001; obese subjects in Hong Kong, China, r = -0.08, p = 0.14; lean subjects in Kunming, r = -0.05, p = 0.32; obese subjects in Kunming, r = -0.13, p = 0.32, Figure 4 ), although not all significant, may suggest an antagonistic relationship between these two species in the human gut. In addition, a robust correlation between S. pombe and C. neoformans was observed in both lean groups from Hong Kong, China (SparCC correlation, r = 0.26, p < 0.001) and Kunming, China (SparCC correlation, r = 0.33, p < 0.001). Figure 4 A and 4C), however, this correlation was not observed in their obese groups.

[0102] The fungus-fungus interaction network between the lean and obese groups was also region-dependent ( Figure 4). Verticillium dahliae and Alternaria solani were uniquely identified in the co-occurrence network of the Hong Kong group, and in the obese group, Alternaria solani was closely associated with Verticillium dahliae (r = 0.15, p = 0.01) and Kluyveromyces marxianus (SparCC correlation, r = -0.13, p < 0.05), implying that Alternaria solani may be a key player in the gut of obese subjects in Hong Kong ( Figure 4 A and 4B). Meanwhile, Kunming subjects were specifically distinguished from Hong Kong subjects by Talaromyces pinophilus and Torulaspora delbruckli in the intestine, and Talaromyces pinophilus was positively correlated with Candida dubliniensis in the feces of lean subjects (SparCC correlation, r = 0.16, p < 0.05), whereas this symbiotic or synergistic relationship was clearly absent in the intestine of obese individuals ( Figure 4 C and 4D).

[0103] In summary, the healthy gut fungal microbiome ecological network is characterized by complex gut fungal-fungal interactions, with different fungal species competing or cooperating with each in the host. However, in obese subjects, while no significant fungal diversity reduction was shown in obesity, the ecological network became much less diverse. There were reduced interactions, and their characteristics depended on the region.

[0104] Reduced fungal-bacterial ecological interactions in the fecal fungal microbiome in obesity across regions

[0105] Previous studies have provided evidence that inter-kingdom interactions play a potential role in human metabolic health. We further explored the relationship between gut fungi and bacteria in feces by investigating the correlation between the alpha diversity (diversity and richness) of the fungal microbiome and the bacterial microbiome in obese subjects and lean controls.

[0106] like Figure 5 As shown, in the Figure 5 A) and Kunming ( Figure 5 C) showed a positive correlation between fungal-bacterial alpha diversity measures in both lean groups. However, compared with Hong Kong ( Figure 5 A, Bacterial group diversity and fungal microbiome diversity, SparCC correlation, r = 0.23, p < 0.01) and Kunming group ( Figure 5 C, Bacterial microbiome diversity and fungal microbiome diversity, SparCC correlation, r = 0.40, p < 0.05) compared with the lean group in Hong Kong, China ( Figure 5 B, Bacterial group diversity and fungal microbiome diversity, SparCC correlation, r = -0.007, p < 0.01) and Kunming group ( Figure 5 D, Correlation between bacterial group diversity and fungal microbiome diversity, SparCC, r = -0.01, p < 0.01), the correlation between fungal-bacterial diversity was weak or even lost in the obese group (Figure 5A), indicating that the fungal-bacterial network in obesity was altered compared with the lean control. In addition, the correlation in the obese group became opposite to that observed in the lean group ( Figure 5 For example, in Hong Kong, China, the evenness of the gut fungal microbiome of lean subjects was similar to their bacterial microbiome diversity ( Figure 5 A, SparCC correlation, r = 0.19, p < 0.05), bacterial group uniformity ( Figure 5 A, r = 0.13, p < 0.05) and bacterial group richness ( Figure 5 A, SparCC correlation, r = 0.24, p = 0.06) showed a positive correlation, while there was a significant negative correlation in the obese group ( Figure 5 B, SparCC correlation, respectively

[0107] r=-0.04, p<0.01; r=-0.06, p<0.01; r=-0.18, p<0.001).

[0108] Altered gut fungal microbiome composition in obese and lean subjects from Hong Kong and Kunming, China

[0109] To avoid geographical effects, fungal species associated with obesity were identified by LEfSe analysis comparing obese and lean individuals from Hong Kong and Kunming, China, respectively. Differences in relative abundance were confirmed by the Wilcoxon rank-sum test. The relative abundance of Candida dubliniensis was significantly higher in lean subjects compared with obese subjects from Hong Kong (mean: 1.26% vs 0.66%, p < 0.05, Wilcoxon rank-sum test) and Kunming (mean: 5.41% vs 0.83%, p < 0.05, Wilcoxon rank-sum test).

[0110] In the Hong Kong cohort, Cryptococcus neoformans and Schizosaccharomyces pombe were enriched in lean subjects (Cryptococcus neoformans: mean relative abundance 2.41% vs 1.36%, p<0.05; Schizosaccharomyces pombe: mean relative abundance 3.99% vs 0.92%, p<0.05; Wilcoxon rank-sum test). Kluyveromyces thermotolerans, Saccharomyces paradoxus, and Thielavia terrestris were enriched in obese subjects from Hong Kong, China (Kluyveromyces thermotolerans: mean relative abundance 0.01% vs 0.05%, p<0.05; Schizosaccharomyces pombe: mean relative abundance 0.18% vs 0.26%, p<0.05; Thielavia terrestris: mean relative abundance 0.57% vs 0.84%, Wilcoxon rank-sum test). In the Kunming cohort, Parastagonospora nodorum and Myceliophthora thermophila were associated with obesity ( Figure 2B ).

[0111] Anti-obesity effects of Schizosaccharomyces pombe in high-fat diet-induced obese mice

[0112] Among the differentially enriched fungal species identified by LEfSe analysis, the difference in Schizosaccharomyces pombe in the Hong Kong cohort was the most statistically significant (log relative abundance in lean vs. obese: 3.99 ± 7.49 vs 0.92 ± 2.46, p < 0.01, Figure 2B ). Further correlation studies showed that it was negatively correlated with body mass index (BMI) in the Hong Kong group (r 2 =0.08, p=0.002, Fig. 9B ). This suggests that S. pombe may protect against obesity and potential obesity-related conditions, such as diabetes and dyslipidemia.

[0113] To determine the causal effect of S. pombe on obesity, S. pombe (ATCC 16979) was simultaneously administered to mice fed a high-fat diet (HF). In addition to the PBS control, S. cerevisiae (ATCC 36375) was also included as a control (ND+SC group and HF+SC group, Figure 6A). After 11 weeks of treatment, the HF standardized diet supplemented with S. pombe, but not S. cerevisiae, induced weight gain. Compared with mice treated with HF and PBS, the body weight of mice treated with HF and S. pombe decreased by 20.86% (Figure 6B, p<0.001), and the body weight of mice treated with HF and S. pombe decreased by 13.87% (Figure 6B, p<0.05) compared with mice treated with HF and S. cerevisiae. Interestingly, the body weight of mice fed with ND supplemented with S. pombe remained similar to that of mice fed with ND and PBS (p=0.101). This suggests that S. pombe is well tolerated.

[0114] The risks associated with being overweight or obese are primarily related to the deposition of adipose tissue. Although both subcutaneous and visceral fat stores are associated with progressive deterioration in the metabolic risk profile, visceral fat is more detrimental. 5 . The effects of S. pombe on subcutaneous and visceral (epididymal and perirenal) adipose tissue were next evaluated. As expected, HF resulted in a significant increase in fat weight relative to ND (fat mass index for HF vs ND, p<0.001, t-test; Fig. 6C). Oral supplementation with S. pombe, but not S. cerevisiae, attenuated HDF-induced adiposity, as shown by reduced subcutaneous (p<0.001; Fig. 6D) and perirenal (p<0.01; Fig. 6F) adipose tissue weight compared to mice fed HF and PBS.

[0115] Dyslipidemia is a common metabolic disorder associated with obesity. In the present study, it was observed that HF ​​significantly increased the serum level of low-density lipoprotein-cholesterol (LDL-C) (p<0.01) and decreased the serum level of high-density lipoprotein-cholesterol (HDL-C) (p<0.001) compared with mice fed with ND ( Fig.11 B and 11C). Oral supplementation with S. pombe maintained serum LDL-C levels similar to those of mice fed ND ( Fig.11 B). Serum HDL-C levels were also significantly increased in mice fed HF supplemented with S. pombe compared to mice fed HF and PBS (p<0.05, Fig.11 C). Since fatty liver is often associated with dyslipidemia, and elevated LDL-C levels were detected in mice fed HF, lipid accumulation in liver tissue was next evaluated by assessing liver mass index. It was observed that HF ​​significantly increased liver mass index relative to mice fed ND, and oral supplementation with S. pombe prevented this increase in liver mass index in mice fed HF (p<0.001, Fig.11 A).

[0116] It is known that nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM) often coexist and act synergistically. The presence of NAFLD increases the risk of concomitant T2DM, with a relative risk of 1.86 (95% CI, 1.76–1.95). 6 , and improvements in NAFLD are generally associated with a reduced risk of T2DM 7. Therefore, the effect of S. pombe on glucose tolerance and insulin resistance was evaluated. Mice fed with HF developed glucose intolerance, as shown by a significant increase in the area under the curve (AUC) obtained from the oral glucose tolerance test (OGTT) compared to mice fed with ND (p<0.001, Figures 6I-6J). Although supplementation with S. cerevisiae did not result in any significant change in the AUC of the OGTT, supplementation with S. pombe significantly reduced the AUC (p<0.001, Figures 6I-6J). Insulin responsiveness was then assessed by the insulin tolerance test (ITT). Mice fed with HF showed a significant increase in the AUC of the ITT compared to mice fed with ND (p<0.001, Figures 6G-6H). Although supplementation with S. cerevisiae (p<0.05, Figures 6G-6H) resulted in a decrease in the AUC of the ITT, the effect of supplementation with S. pombe was much more significant (p<0.001, Figures 6G-6H).

[0117] In conclusion, these results indicate that supplementation with S. pombe improves HF-induced obesity, adiposity, dyslipidemia, fatty liver, and T2DM (glucose tolerance and insulin sensitivity). Importantly, S. pombe was well tolerated in both ND- and HF-fed mice. These findings provide support for the use of S. pombe as a therapeutic option targeting obesity and obesity-related conditions such as adiposity, dyslipidemia, fatty liver, and T2DM.

[0118] The use of this species, Schizosaccharomyces pombe (NCBI: txid4896), a fission yeast, is suitable for preventing or ameliorating and treating the above-mentioned diseases. This can be achieved by regulating or adjusting the level of Schizosaccharomyces pombe in the GI tract of a subject in need thereof. Preferably, the level of Schizosaccharomyces pombe should be increased to a relative abundance of 3.99%, which is the average relative abundance in lean subjects from the Hong Kong group, China.

[0119] Other Beneficial Species for Weight Loss

[0120] Comparison of the fungal microbiome between lean and obese subjects from Hong Kong, China, revealed that, in addition to Schizosaccharomyces pombe, Cryptococcus neoformans (NCBI: txid5207) was also enriched in lean subjects compared to obese subjects. Thus, in summary, in order to reduce the weight of a subject in need thereof, the level of one or more fungal species shown in Table 4 in the GI tract can be regulated or adjusted by delivering an effective amount of these species to the GI tract of the subject. Preferably, the level of these species should be increased at least to the average relative abundance in lean subjects (Table 4).

[0121] Table 4 Fungal species used for weight loss

[0122]

[0123] Administration of fungal species for the prevention or improvement of obesity and obesity-related conditions or for the maintenance of intestinal health against pathogenic Candida

[0124] Schizosaccharomyces pombe, Saccharomyces cerevisiae, Debaryomyces hansenii, and the fungal species listed in Table 4 can be obtained from a sufficient amount of fungal culture and then formulated into a suitable composition for introduction into a subject by oral, nasal, or rectal administration. The amount of each fungus or yeast in the composition is about 1×10 6 -1×10 12 CFU or 50-1000mg. Ideally, the relative abundance should reach the median relative abundance (cutoff), or be within the range of the lower and upper quartiles listed in Table 4. Although some of these species are present only in low relative abundance (<0.005%), they still play an important role in preventing or improving obesity and obesity-related conditions. For those species with a median relative abundance of <0.005%, a detectable level (e.g., at least 0.005%) can be used as a cutoff.

[0125] All patents, patent applications, and other publications cited in this application, including GenBank Accession Numbers and equivalents, are incorporated herein by reference in their entirety for all purposes.

[0126] References

[0127] 1. Zuo T, Wong SH, Cheung CP, et al. Gut fungal dysbiosis correlates with reduced efficacy of fecal microbiota transplantation in Clostridium difficileinfection. Nature communications 2018; 9:1-11.

[0128] 2.Soverini M, Turroni S, Biagi E, et al. HumanMycobiomeScan: a new bioinformatics tool for the characterization of the fungal fractioninmetagenomic samples. BMC genomics 2019;20:496.

[0129] 3.Truong DT,Franzosa EA,Tickle TL,et al.MetaPhlAn2 forenhancedmetagenomic taxonomic profiling.Nature methods 2015;12:902-903.

[0130] 4.Nguyen NH,Song Z,Bates ST,et al.FUNGuild:an open annotation toolforparsing fungal community datasets by ecological guild.Fungal Ecology2016;20:241-248.

[0132] 5.Cornier MA,Despres JP,Davis N,et al.Assessing adiposity:ascientificstatement from the American Heart Association.Circulation 2011;124:1996-2019.

[0133] 6.Ballestri S,Zona S,Targher G,et al.Nonalcoholic fatty liver diseaseisassociated with an almost twofold increased risk of incident type2diabetesand metabolic syndrome.Evidence from a systematic review and meta-analysis.J Gastroenterol Hepatol 2016;31:936-44.

[0135] 7.Targher G,Corey KE,Byrne CD,et al.The complex link between NAFLDandtype 2diabetes mellitus-mechanisms and treatments.Nat RevGastroenterolHepatol 2021;18:599-612.

[0136] 8.Breuer U,Harms H.Debaryomyces hansenii—an extremophilic yeastwithbiotechnological potential.Yeast 2006;23:415-437.

[0137] 9.Capece A,Romano P.“Pecorino di Filiano”cheese as a selectivehabitat forthe yeast species,Debaryomyces hansenii.International journal offoodmicrobiology 2009;132:180-184.

[0138] 10.Ferreira A,Viljoen B.Yeasts as adjunct starters in matured Cheddarcheese.International journal of food microbiology 2003;86:131-140.

[0140] 11.Buzzini P,Martini A.Large-scale screening of selected Candidamaltosa,Debaryomyces hansenii and Pichia anomala killer toxin activityagainstpathogenic yeasts.Sabouraudia 2001;39:479-482.

Claims

1. A method for treating obesity in an obese subject or a subject at risk of obesity, comprising introducing into the gastrointestinal tract of the subject an effective amount of an inhibitor of (1) one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisae; or (2) one or more of Lachancea thermotolerans, Saccharomyces paradoxus, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthorathermophila.

2. The method of claim 1, comprising introducing into the gastrointestinal tract of the subject an effective amount of an inhibitor of (1) one or more of Schizosaccharomyces pombe and Cryptococcus neoformans; or (2) one or more of Kluyveromyces thermotolerans, Saccharomyces paradoxus, and Thielavia terrestris.

3. The method of claim 1, wherein the level of Schizosaccharomyces pombe, Cryptococcus neoformans and / or Saccharomyces cerevisiae increases by about 4-10 fold after the introducing step.

4. The method of claim 1, wherein after said introducing step, the relative level of Schizosaccharomyces pombe in the gastrointestinal tract of said subject is greater than about 3.99% of the total intestinal fungi.

5. The method of claim 1, wherein after the introducing step, the relative level of Kluyveromyces thermotolerans, Saccharomyces paradoxus, Thielavia terrestris, Parastagonospora nodorum and / or Myceliophthora thermophila in the gastrointestinal tract of the subject is less than about 0.1% of the total intestinal fungi.

6. The method of claim 1, wherein after the introducing step, the fungal alpha diversity index increases by about 1.2-6 times.

7. The method of claim 1, comprising introducing into the gastrointestinal tract of the subject a composition comprising: (1) one or more of Schizosaccharomyces pombe, Cryptococcus neoformans, and Saccharomyces cerevisiae; or (2) one or more of Kluyveromyces thermotolerans, Saccharomyces paradoxus, Thielavia terrestris, Parastagonospora nodorum, and Myceliophthora thermophila, and physiologically acceptable excipients.

8. The method of claim 7, wherein the composition comprises Schizosaccharomyces pombe and / or Cryptococcus neoformans.

9. The method of claim 7 or 8, wherein the composition is delivered directly to the small intestine, ileum, or large intestine of the subject.

10. The method of claim 7 or 8, wherein the composition is formulated for oral ingestion.

11. The method of claim 10, wherein the composition is formulated as a daily dose.

12. The method of claim 10, wherein the composition is in the form of a food or beverage article.

13. The method of claim 10, wherein the composition further comprises a prebiotic.

14. The method of any one of claims 1-13, wherein the introducing step is performed by fecal microbiota transplantation (FMT).

15. The method of claim 14, wherein the Schizosaccharomyces pombe, Cryptococcus neoformans and / or Saccharomyces cerevisiae are derived from fecal material of a donor.

16. The method of any one of claims 1-15, wherein the subject has reduced body weight, reduced serum low-density lipoprotein-cholesterol (LDL-C) levels, reduced serum glucose levels, improved glucose tolerance and / or increased insulin responsiveness.

17. A kit for promoting weight loss in a subject or reducing the risk of obesity in a subject, comprising a plurality of compositions, each of the compositions comprising an effective amount of one or more of the following: Schizosaccharomyces pombe; Cryptococcus neoformans; Saccharomyces cerevisiae; an inhibitor of Kluyveromyces thermotolerans; an inhibitor of Saccharomyces paradoxalis; an inhibitor of Thielavia terrestris; an inhibitor of Parastagonospora nodorum, and an inhibitor of Myceliophthora thermophila.

18. The kit of claim 17, comprising a first composition and a second composition, wherein the first composition comprises an effective amount of Schizosaccharomyces pombe and / or Cryptococcus neoformans, and the second composition comprises an inhibitor of Kluyveromyces thermotolerans, Saccharomyces paradoxica and / or Thielavia terrestris.

19. The kit of claim 17, comprising a first composition comprising an effective amount of Schizosaccharomyces pombe and a second composition comprising an effective amount of Cryptococcus neoformans.

20. The kit of claim 18 or 19, wherein the composition is formulated for oral administration or direct delivery to the gastrointestinal tract of the subject.

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