Amuc-1100 polypeptide variants for inducing immune signaling, and / or influencing gut barrier function, and / or regulating metabolic status.

Abstract

Polypeptide variants of Akkermansia muciniphila extracellular polypeptides are provided that are capable of regulating and / or promoting intestinal mucosal immune system function, maintaining and / or restoring metabolic status, and / or enhancing the physical integrity of the intestinal mucosal barrier in a mammal. The polypeptide variants or host cells containing such polypeptide variants may be utilized to prevent and / or treat a variety of conditions that benefit from increased physical integrity of the intestinal mucosal barrier and / or improved intestinal mucosal immune system function and metabolic status.

Description

[Technical Field]

[0001] The present invention relates to the field of pharmaceutical, food or feed compositions comprising the intestinal mucosal immune system, the intestinal mucosal barrier, polypeptides and / or host cells, which can regulate and / or promote the function of the intestinal mucosal immune system and / or maintain and / or restore and / or enhance the physical integrity of the intestinal mucosal barrier, and / or maintain, restore or improve glucose and / or cholesterol and / or triglyceride homeostasis in mammals (e.g., humans). [Background technology]

[0002] Increased or hyperpermeability of the intestinal mucosal barrier is thought to be involved in several disorders and conditions, including gut-related diseases, autoimmune diseases, allergies, cancer, type 2 diabetes, obesity, depression, anxiety, and many others. Therefore, there is growing interest in understanding the contributing factors to intestinal mucosal barrier dysfunction in the pathogenesis of many conditions affecting the mammalian gastrointestinal tract (GI).

[0003] Under normal conditions, the intestinal mucosal barrier acts as a selective barrier that allows for the absorption of nutrients, electrolytes, and water, while preventing exposure to harmful macromolecules, microorganisms, food-derived antigens, and microbial antigens (e.g., food allergens). The intestinal mucosal barrier is basically composed of a layer of mucus and underlying epithelial cells (referred to herein as “intestinal epithelial cells”). Intestinal epithelial cells are tightly bound to one another by so-called “tight junctions,” which are essentially “physical connections” between the membranes of two intestinal epithelial cells. Maintaining the intestinal mucosal barrier, in particular maintaining the physical integrity of the intestinal epithelial cell layer (i.e., maintaining tight connections between cells), is crucial for protecting the host from the translocation of pathogenic microorganisms, antigens, and other undesirable agents from the intestines into the bloodstream.

[0004] The intestinal mucosal barrier has approximately 10 12 ~10 14The intestinal mucosa is home to numerous symbiotic microorganisms, primarily anaerobic or microaerophilic bacteria, most of which live in symbiosis with the host. These bacteria are beneficial to the host in several ways. They provide protection against pathogenic bacteria and play a nutritional role in the host by synthesizing vitamin K and some components of the vitamin B complex. Furthermore, the intestinal mucosa barrier has evolved a complex “intestinal mucosal immune system” to distinguish symbiotic bacteria (i.e., beneficial bacteria) from pathogenic bacteria and other harmful agents. The intestinal mucosal immune system is an essential part of the intestinal mucosa barrier and includes lymphoid tissue and specialized immune cells (i.e., lymphocytes and plasma cells), which are widely distributed throughout the intestinal mucosa barrier. One of the microorganisms naturally present on the mucous membranes of healthy subjects is the mucin-degrading Akkermansia muciniphila, which has been shown to enhance intestinal barrier function (Everard et al., PNAS 110 (2013), pp. 9066-9071; Reunanen et al., Appl Environ Microbiol, March 20, 2015), thereby influencing diseases associated with impaired intestinal barrier function.

[0005] Under certain circumstances, the intestinal mucosal barrier may be vulnerable to a wide variety of infectious organisms or agents that, while normally unable to penetrate it, cleverly pass through it (for example, through gaps resulting from loose, tight junctions between intestinal epithelial cells). Organisms or other agents that pass through the intestinal mucosal barrier may cause disease or other undesirable conditions in the host (e.g., allergies). Examples of such conditions include obesity, metabolic syndrome, insulin deficiency or insulin resistance-related disorders, type 2 diabetes, type 1 diabetes, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), impaired glucose tolerance, abnormal lipid metabolism, atherosclerosis, hypertension, heart conditions, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, fatty liver, lipid metabolism disorders, immune system dysfunction associated with obesity (weight gain), allergies, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other disorders associated with impaired barrier function, wound healing, behavioral disorders, alcoholism, cardiovascular disease, high cholesterol, elevated triglycerides, atherosclerosis, sleep apnea, osteoarthritis, gallbladder disease, and cancer.

[0006] Conversely, diseases such as those mentioned above, as well as other conditions such as food allergies, including underdeveloped intestines due to premature birth, exposure to radiation, chemotherapy and / or toxins, autoimmune disorders, malnutrition, and sepsis, can alter the physical integrity of the intestinal mucosal barrier (i.e., cause loosening of tight junctions between intestinal epithelial cells), which may in turn allow undesirable microorganisms or other agents to pass through the host's intestinal mucosal barrier.

[0007] Several vaccines and / or antibodies targeting such microorganisms or agents have been developed over many years. However, such approaches have not been very successful because some microorganisms or agents cannot be effectively targeted or eradicated even with vaccines or antibodies.

[0008] Other approaches have also been investigated that aim to prevent harmful microorganisms and other agents from passing through the host's intestinal mucosal barrier at the initial stage, and / or to prevent increased permeability of the intestinal mucosal barrier. For example, compositions containing glutamic acid have been developed to prevent and / or treat conditions associated with increased permeability of the intestinal mucosal barrier (WO01 / 58283). Other substances, including spermine and spermidine and their precursors, have also been used for the same purpose (Dorhout et al., (1997), British J. Nutrition, pp. 639-654). Preparations containing arabinoxylan to promote beneficial effects against GI bacteria residing around the intestinal mucosal barrier have also been developed to modulate the intestinal mucosal barrier (US2012 / 0230955).

[0009] WO2016177797 discloses a polypeptide derived from Ackermansia muciniphila, namely polypeptide Amuc-1100, which can maintain, restore or enhance the physical integrity of the intestinal mucosal barrier and / or maintain, restore or improve glucose and / or cholesterol and / or triglyceride homeostasis in mammals, and / or, in particular, can improve the metabolic or immune status of mammals by interacting with Toll-like receptor 2 (TLR2) and / or modulating TLR2 and / or NFk-B-dependent signaling pathways and / or promoting cytokine release (e.g., IL-6, IL-8, and IL-10) from immune cells surrounding the mucointestinal barrier of mammals (e.g., humans).

[0010] It is an object of the present invention to provide novel or improved agents and / or compositions comprising such agents that are suitable for maintaining, / or restoring and / or enhancing the physical integrity of the intestinal mucosal barrier, and / or preventing increased intestinal mucosal barrier permeability in mammals (e.g., humans), and / or suitable for maintaining, restoring and / or improving glucose and / or cholesterol and / or triglyceride homeostasis in mammals, preferably thereby preventing or treating a disease or condition associated with suboptimal intestinal mucosal barrier permeability and / or an imbalance in glucose and / or cholesterol and / or triglyceride homeostasis in said mammals. Alternatively or additionally, it is an object of the present invention to provide further or improved agents and / or compositions comprising such agents suitable for modulating and / or promoting intestinal mucosal immune system function in mammals. [Prior Art Literature] [Patent Literature]

[0011] [Patent Literature 1] WO01 / 58283 [Patent Literature 2] US2012 / 0230955 [Patent Literature 3] WO2016177797 [Patent Literature 4] U.S. Patent No. 4,554,101 [Non-Patent Literature]

[0012] [Non-Patent Literature 1] Everard et al., PNAS 110 (2013) pp. 9066-9071 [Non-Patent Literature 2] Reunanen et al., Appl Environ Microbiol, March 20, 2015 [Non-Patent Literature 3] Dorhout et al., (1997), British J. Nutrition, pp. 639-654 [Non-Patent Document 4] Xing et al., (2019); Genes & Genomics 41: pp. 1253-1264 [Non-Patent Document 5] Lee H and Ko G, Appl Environ Microbiol, October 2014; 80(19): pp. 5935-5943 [Non-Patent Document 6] Syngelaki et al., N Engl J Med, February 4, 2016; 374(5): pp. 434-443 [Non-Patent Document 7] Rajilic-Stojanovic and de Vos, The first 1000 cultured species of the human gastrointestinal microbiota, FEMS Microbiol Rev. 38: pp. 996-1047 [Non-Patent Document 8] Derrien et al. (2004, Int. J. Syst. Evol. Microbiol. 54: pp. 1469-1476) [Non-Patent Document 9] Lukovac et al. (2014, mBio 01438-14) [Non-Patent Document 10] Visweswaran GR et al., 2014, Appl Microbiol Biotechnol. 98: pp. 4331-4345 [Non-Patent Document 11] Solanki et al., BioMed Res. Int. 2013, Article ID 620719 [Non-Patent Document 12] Schulz, G. E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1979 [Non-Patent Document 13] Creighton, TE, Proteins: Structure and Molecular Principles, WH Freeman & Co, San Francisco, 1984. [Non-Patent Document 14] Kyte et al., J. Mol. Biol. 157, pp. 105-132 (1982) [Non-Patent Document 15] Henikoff and Henikoff, 1992, PNAS 89, pp. 915-919. [Non-Patent Document 16] Reunanen J et al. 2012, Appl Environ Microbiol 78:2337–44 pages [Non-Patent Document 17] Tailford LE et al., 2015, Nat Commun. 6:7624. [Overview of the project] [Means for solving the problem]

[0013] The inventors have identified a distant variant of polypeptide Amuc-1100 in Ackermansia glycanipira that can modulate and / or promote intestinal immune system function and / or maintain and / or restore and / or enhance the physical integrity of the intestinal mucosal barrier and / or maintain and / or restore and / or improve glucose and / or cholesterol and / or triglyceride homeostasis in mammals (e.g., humans). This is surprising, as previous studies have reported that Ackermansia glycanipira has no homolog of Amuc-1100 (see Xing et al., (2019); Genes & Genomics 41: pp. 1253-1264).

[0014] While we do not wish to be bound by any theory, we believe that the beneficial effects of the polypeptides of this disclosure result from their ability to interact with the TLR2 signaling pathway present on the surface of immune cells surrounding the mammalian intestinal mucosal barrier. More specifically, we have found that the polypeptides taught herein can modulate and / or stimulate the TLR2 signaling pathway in said immune cells surrounding the intestinal mucosal barrier to interact with TLR2 present on the surface of the immune cells and / or stimulate the secretion of cytokines (e.g., IL-6, IL-8, and IL-10) from the immune cells.

[0015] Furthermore, the inventors have discovered that the polypeptides taught herein can modulate and / or enhance the transepithelial resistance of the mammalian intestinal mucosal barrier. Since increased transepithelial resistance measures serve as an indicator of reduced permeability of the intestinal mucosal barrier, it is believed that the polypeptides taught herein, including variants, can modulate the physical integrity of the intestinal mucosal barrier, particularly at the level of tight junctions between epithelial cells.

[0016] Combined, these effects are thought to result in improved or enhanced intestinal mucosal immune system function (e.g., more release of cytokines from the intestinal mucosal barrier) and improved or enhanced physical integrity of the intestinal mucosal barrier, particularly at the level of intercellular connections (i.e., through tighter intercellular junctions).

[0017] Furthermore, treatment of mice given HFD with the polypeptide according to this disclosure significantly reduced weight and fat mass gain without affecting food intake. Treatment with this polypeptide also resulted in a significant decrease in serum HDL cholesterol and a similar trend in LDL cholesterol, and may also cure HFD-induced hypercholesterolemia. In addition, administration of this polypeptide may alleviate impaired glucose tolerance with the same or better efficacy as the Amuc-1100 polypeptide of Ackermansia muciniphila.

[0018] Finally, metformin is known to stimulate the proliferation of Akkermansia (Lee H and Ko G, A ppl Environ Microbiol, October 2014; 80(19): 5935-43), and therefore, Akkermansia as a polypeptide and its extracellular peptides with similar functionality may have similar effects to metformin on gestational diabetes and pre-eclampsia (Syngelaki et al., N Engl J Med, February 4, 2016; 374(5): 434-43).

[0019] Polypeptide This disclosure is, a) Having at least 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with sequence number 9 (over its entire length); b) comprising at least 1, 2, 3, 4, 5, 6, or 7 amino acid residues from the following set i. R, S, I, S, A, and / or P (or their conservative substitutions) at positions 1, 2, 8, 20, 23, and / or 27 of sequence number 9; ii. C, K, K, I, and / or T (or their conservative substitutions) at positions 92, 93, 95, 97, and / or 100 of sequence number 9; iii. W, L, G, and / or F (or their conservative substitutions) at positions 105, 106, 107, and / or 108 of sequence number 9; iv. F and / or E (or their conservative substitutions) at positions 126 and / or 127, respectively, of sequence number 9; v. V, Y, and / or R (or their conservative substitutions) at positions 149, 150, and / or 151, respectively, of sequence number 9; vi. P, E, I, F, Q, R, S, and / or V (or their conservative substitutions) at positions 179, 181, 182, 184, 185, 188, 190, and / or 191 of sequence number 9; vii. P, P, P, A, A, P, G, T, A, E, A, P, Q, K, G, and / or E (or its conservative substitution) at positions 220, 222, 229, 230, 231, 234, 248, 258, 260, 262, 264, 172, 175, 279, 283, and / or 285 of sequence number 9, This document provides instructions for an isolated polypeptide characterized by the following features.

[0020] The polypeptides defined above can induce immune signaling and / or affect intestinal barrier function and / or affect glucose and / or cholesterol and / or triglyceride homeostasis. Preferably, the isolated polypeptide does not contain SEQ ID NO: 1 or an amino acid sequence having more than 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 1. The polypeptides taught herein may also be able to bind to Toll-like receptor 2 (TLR2).

[0021] In one embodiment, the polypeptide defined above is preferably included in a composition further comprising a carrier, for example, a physiologically acceptable carrier, a pharmaceutically acceptable carrier, a digestively acceptable carrier, or a nutritionally acceptable carrier. The carrier may be any inert carrier. For example, non-limiting examples of suitable physiologically or pharmaceutically acceptable carriers include any well-known physiological or pharmaceutically acceptable carrier, buffers, diluents, and excipients.

[0022] In one embodiment, polypeptides and their variants taught herein can stimulate the TLR2 signaling pathway in cells, stimulate the release of cytokines (e.g., IL-6, IL-8, IL-10, etc.) from cells, and / or increase the epithelial membrane resistance (TER) of mammalian cells, such as human cells, and / or improve the metabolic or immune status of mammals, such as mice or humans.

[0023] a) As described below, the polypeptides taught herein may also include variants of the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of the variant having more than 25% sequence identity with the amino acid sequence of SEQ ID NO: 9. Variants of the polypeptide also include polypeptides derived from the polypeptide having the amino acid sequence of SEQ ID NO: 9 by one or more amino acid substitutions, deletions, or insertions. Preferably, such polypeptides contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions, or insertions compared to the polypeptide having the amino acid sequence of SEQ ID NO: 9, up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, or 15 amino acid substitutions, deletions, or insertions. As described, the polypeptide may have, for example, at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 9 throughout its entire length, for example, at least 50% sequence identity with SEQ ID NO: 9. The polypeptide according to this disclosure may or may not include a leader sequence.

[0024] In one embodiment, the polypeptide according to this disclosure is - i) at least five amino acid residues (or their conservative substitutions) as defined below; - ii) at least four amino acid residues (or their conservative substitutions) as defined below; - iii) at least three amino acid residues (or their conservative substitutions) as defined below; - iv) at least one amino acid residue (or a conservative substitution thereof) as defined below; - v) at least two amino acid residues (or their conservative substitutions) as defined below; - vi) at least seven amino acid residues (or their conservative substitutions) as defined below; and / or vii) comprising at least 15 (or at least 12) amino acid residues (or their conserved substitutions) as defined below. Alternatively, or simultaneously, the polypeptides taught herein may, in particular, comprise the following set of amino acid residues as defined above. - i); - i) and vii); - i), ii), vi), and vii); - i), iii), iv), and vii); - i), ii), iii), iv), v), vi), vii).

[0025] Alternatively, or simultaneously, the polypeptides taught herein may, for example, have at least 75% sequence identity with SEQ ID NO: 9 throughout their entire length.

[0026] In a preferred embodiment, the isolated polypeptide according to the present disclosure further comprises amino acid residues S, N, E, N, (A,) P, Q, L, and / or L (or their conserved substitutions) at positions corresponding to positions 28, 29, 35, 37, (40,) 71, 78, 81, and / or 88 of SEQ ID NO: 9, respectively. Preferably, at least eight of these listed amino acid residues are included.

[0027] In yet another preferred embodiment, the isolated polypeptide according to the Disclosure further comprises amino acid residues P, L, N, G, K, W, I, Y, R, I, V, L, F, and / or P (or their conserved substitutions) at positions corresponding to positions 116, 124, 136, 142, 148, 175, 198, 204, 212, 213, 289, 295, 298, and / or 301 of SEQ ID NO: 9. Preferably, at least 13 (or at least 11) of these listed amino acid residues are included.

[0028] The isolated polypeptides according to this disclosure may be natural variants of polypeptides according to Sequence ID No. 9, for example, naturally occurring polypeptides having the same functionality or synthetic polypeptides having the same functionality, i.e., polypeptides that can induce immune signaling and / or affect intestinal barrier function and / or affect glucose and / or cholesterol and / or triglyceride homeostasis. The polypeptides may also be able to bind to Toll-like receptor 2 (TLR2).

[0029] The polypeptides taught herein may be preceded by an N-terminal signal sequence that stimulates the secretion of the polypeptide from a cell. In one embodiment, the N-terminal signal sequence may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, which is the predicted naturally occurring N-terminal signal sequence of the Amuc-1100 polypeptide. However, other N-terminal signal sequences that enable the secretion of Amuc-1100 from a cell are also available. For example, such an N-terminal signal sequence may be a cleaved or elongated version of the predicted naturally occurring N-terminal signal sequence of the Amuc-1100 polypeptide, as long as it enables the secretion of Amuc-1100 from a cell. Alternatively, a non-naturally occurring N-terminal signal sequence may be used. Those skilled in the art will be able to identify N-terminal signal sequences suitable for use in this disclosure. Thus, the polypeptides of this disclosure may have the amino acid sequence of SEQ ID NO: 3 at the N-terminus of the amino acid sequence.

[0030] Amino acid sequence identity can be determined by any suitable means available in the art. For example, amino acid sequence identity may be determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as defined above. Naturally, many methods such as Western blotting, immunohistochemistry, ELISA, and amino acid synthesis can be used to identify, synthesize, or isolate variants of the polypeptides taught herein.

[0031] Naturally, any variant of the polypeptide taught herein will perform the same functions and / or have the same activity as the polypeptide taught herein. The functionality or activity of any variant may be confirmed by any method known in the art that a person skilled in the art would consider suitable for these purposes.

[0032] Polynucleotides This disclosure also teaches nucleic acid molecules, such as isolated, synthesized, or recombinant nucleic acid molecules, that include nucleic acid sequences encoding polypeptides taught herein, for example, nucleic acid sequences as shown in SEQ ID NO: 29 or SEQ ID NO: 33, or nucleic acid sequences having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 29 or SEQ ID NO: 33.

[0033] The term “isolated nucleic acid molecule” (e.g., cDNA, genomic DNA, or RNA) includes naturally occurring nucleic acid molecules, artificial nucleic acid molecules, or synthetic nucleic acid molecules. A nucleic acid molecule may encode any polypeptide taught herein. Such nucleic acid molecules may be used to produce the polypeptides taught herein. Due to the degeneracy of genetic coding, various nucleic acid molecules may encode the same polypeptide (e.g., a polypeptide containing the amino acid sequence of SEQ ID NO: 9).

[0034] Naturally, many methods, including nucleic acid hybridization, PCR techniques, computer analysis, and nucleic acid synthesis, can be used to identify, synthesize, or isolate the polynucleotide variants taught herein.

[0035] The nucleic acid molecules taught herein may include nucleic acid molecules encoding an N-terminal signal sequence suitable for stimulating the secretion of the polypeptide taught herein from a host cell. The N-terminal signal sequence encoding the nucleic acid molecule may include the nucleic acid sequence described in Sequence ID No. 4.

[0036] In one embodiment, the nucleic acid molecule taught herein may be included in a chimeric gene, in which case the nucleic acid molecule is operably linked to a promoter. Thus, this disclosure also relates to a chimeric gene containing the nucleic acid molecule taught herein.

[0037] Any promoter known in the art and suitable for binding to the nucleic acid molecules taught herein may be used. Non-limiting examples of suitable promoters include those enabling constitutive or controlled expression, weak expression, and strong expression. Any method known in the art may be used to include the nucleic acid molecules taught herein in the chimeric gene.

[0038] In some cases, it may be advantageous to operably bind the nucleic acid molecules taught herein to a so-called "constitutive promoter."

[0039] Alternatively, it may be advantageous to operably conjugate the polynucleotides and their variants taught herein to a so-called “inducible promoter.” The inducible promoter may be a physiologically controlled promoter (for example, by external application of a particular compound).

[0040] The chimeric genes taught herein may be included in a “vector” or a “nucleic acid construct.” Therefore, this disclosure also relates to vectors containing the chimeric genes taught herein or the nucleic acid molecules taught herein.

[0041] In one embodiment, the disclosure relates to a host cell genetically modified to include, for example, a nucleic acid molecule, a chimeric gene, or a vector as taught herein in its genome.

[0042] Genetically modified host cells taught herein may be used to produce the polypeptides and their variants taught herein ex vivo and / or in vitro, either within the cytoplasm of the host cell or released from the cell by any means. The polypeptides taught herein may, in particular, be expressed as soluble or secreted molecules. The genetically modified host cells taught herein may be any host cell suitable for a transformation or genetic engineering procedure. Non-limiting examples of suitable host cells include any cultureable cell, such as any prokaryotic or eukaryotic cell. In one embodiment, the polypeptides according to this disclosure are expressed in bacteria such as Escherichia coli.

[0043] In some embodiments, the host cells taught herein may be any cells that originally express the polypeptide or its variants taught herein. In such cases, the host cells may overexpress the polypeptide or its variants taught herein.

[0044] In another embodiment, the host cells taught herein may be any cells that do not originally express the polypeptide or its variants taught herein.

[0045] In one embodiment, the host cells taught herein do not belong to the species Ackermansia muciniphylla or Ackermansia glycanipira.

[0046] In another embodiment, the host cells may belong to the species Ackermansia muciniphila or Ackermansia glycanipira and are genetically modified to contain additional copies of the nucleic acid molecules taught herein, or to contain the chimeric genes or vectors taught herein. Such Ackermansia muciniphila or Ackermansia glycanipira cells may also overexpress the polypeptides or variants thereof taught herein.

[0047] The host cells taught herein may be genetically modified using any method known in the art. For example, the host cells or organisms taught herein may be a) a step of transforming a host cell with a nucleic acid molecule taught herein, such as a nucleic acid sequence capable of encoding a polypeptide and its variants as taught herein; b) A step of culturing the host cells under conditions suitable for enabling the expression of nucleic acid molecules taught herein and / or the production of polypeptides or variants thereof taught herein; c) Optionally, a step of screening host cells that can express nucleic acid molecules taught herein and / or produce polypeptides or variants thereof taught herein. Genetic modification may be performed by methods including the following:

[0048] In some embodiments, the genetically modified host cells taught herein may belong to bacterial species that naturally occur or inhabit the periphery or within the mammalian intestinal mucosal barrier. Such bacterial species are often referred to as “intestinal mucosal-associated bacterial species.” Non-limiting examples of “intestinal mucosal-associated bacterial species” include Akkermansia muciniphylla (ATTC BAA-835), Faecalibacterium prausnitzii (A2-165), Lactobacillus rhamnosus (ATCC 53103), and Bifidobacterium breve (DSM-20213).

[0049] In certain embodiments, it may be advantageous to genetically modify intestinal mucosa-related bacteria having any of the polynucleotides and their variants taught herein to express or overexpress the polynucleotides taught herein, or to produce or overproduce the polypeptides taught herein, for example, directly around or within the intestinal mucosal barrier of mammals (e.g., humans). In preferred embodiments, the intestinal mucosa-related bacteria may be any bacterium of the species Ackermansia muciniphila or Ackermansia glycanipira. Such overproduction may be achieved by recombinant DNA technology, gene modification tools including genome editing, such as by using CRISPR / cas-like system-based tools, or by classical mutaselection systems.

[0050] In one embodiment, the genetically modified host cell may be any bacterium that is not originally present or inhabits the periphery or within the mammalian intestinal mucosal barrier. Non-limiting examples of such bacteria include any beneficial isolated enterobacteria strain, e.g., probiotic bacteria, particularly strains selected from the genera Lactococcus, Lactobacillus, or Bifidobacterium. Furthermore, obligate anaerobic enterobacteria, such as those belonging to genera known to occur in the human intestinal tract, may be used (Rajilic-Stojanovic and de Vos, The first 1000 cultured species of the human gastrointestinal microbiota, FEMS Microbiol Rev. 38: pp. 996-1047).

[0051] Methods for producing polypeptides In another embodiment, the present disclosure provides a method for producing a polypeptide taught herein, including a variant, (a) a step of culturing host cells as taught herein under conditions that enable the production of polypeptides or variants thereof as taught herein; (b) Optionally, a step to isolate the polypeptide produced in step (a) and Regarding methods including

[0052] In step (a), the host cells taught herein may be cultured in any known culture medium according to any known culture method. Those skilled in the art will be able to select suitable host cells and establish suitable conditions that enable polypeptide production.

[0053] Alternatively, this polypeptide is (a) A step of culturing the bacteria species Akkermansia muciniphila or Akkermansia glycanipira in a suitable culture medium; (b) Optionally, a step to isolate the polypeptide produced in step (a) and It may be produced by a method that includes [a certain element].

[0054] The polypeptide produced in step (a) of the above method may be isolated by any method known in the art. Those skilled in the art will be able to isolate polypeptides produced from such culture media.

[0055] Suitable culture media are taught, for example, by Derrien et al. (2004, Int. J. Syst. Evol. Microbiol. 54: pp. 1469-1476). Derrien et al. used the A. muciniphila strain Muc T The authors teach that A. muciniphila was isolated and grown in a basic anaerobic medium containing porcine stomach mucin as the sole carbon and nitrogen source. They also teach that A. muciniphila can be grown in nutrient-rich media, such as Columbia culture medium (CB) and Brain Heart Infusion (BHI) culture medium, or in basic media containing glucose and high concentrations of casiton and yeast extract. Similarly, Lukovac et al. (2014, mBio 01438-14) teach the growth of A. muciniphila in a basic medium containing glucose and fucose, as well as large amounts of casiton. A similar method can be used for Ackermansia glycanipira.

[0056] composition In another embodiment, this disclosure relates to compositions comprising any polypeptide taught herein.

[0057] In yet another embodiment, this disclosure relates to a composition comprising a host cell taught herein. The host cell comprises about 10 4 From about 10 15 It may be present in amounts ranging from colony-forming units (CFUs). For example, the effective amount in a host cell is approximately 10 5 From CFU 14 CFU, preferably about 10 6 From CFU13 CFU, preferably about 10 7 CFU to about 10 12 CFU, more preferably about 10 8 CFU to about 10 12 CFU. The host cell may be viable or non-viable. The efficacy of the host cell correlates with the presence of the polypeptide taught herein.

[0058] In one embodiment, the composition taught herein further comprises a carrier, for example, a physiologically acceptable carrier, a pharmaceutically acceptable carrier, a digestively acceptable carrier, or a nutritionally acceptable carrier. The carrier may be any inert carrier. For example, non-limiting examples of suitable physiologically or pharmaceutically acceptable carriers include any known physiologically or pharmaceutical carriers, buffers, diluents, and excipients. It will be understood that the selection of a suitable physiologically or pharmaceutical carrier, digestively acceptable carrier, or nutritionally acceptable carrier will depend on the intended route of administration (e.g., oral) of the composition taught herein and the intended form of the composition (e.g., beverage, yogurt, powder, capsule, etc.). A person skilled in the art would know how to select a suitable carrier appropriate or compatible for the composition taught herein, for example, a physiologically acceptable carrier, a nutritionally acceptable carrier, or a pharmaceutically acceptable carrier.

[0059] In one embodiment, the composition taught herein may be a nutritional or digestive composition. For example, the composition taught herein may be a food, a nutritional supplement for food, a feed, or a nutritional supplement for feed, for example, a dairy product, for example, a fermented dairy product such as yogurt or a yogurt drink. In this case, the composition may comprise a nutritionally acceptable carrier or a digestively acceptable carrier, which may be a suitable food base.

[0060] In some embodiments, the composition taught herein may be a pharmaceutical composition. The pharmaceutical composition may also be intended for use as a nutritional supplement (e.g., a dietary supplement). The pharmaceutical composition taught herein may include, in addition to the polypeptide and / or host cell taught herein, a pharmaceutically, nutritionally, digestively, or physiologically acceptable carrier. The preferred form will depend on the intended mode of administration and (therapeutic) use. The carrier may be any suitable, physiologically acceptable, and non-toxic substance suitable for delivering the polypeptide and / or host cell taught herein to the gastrointestinal tract of a mammal (e.g., a human), preferably around or within the mammalian intestinal mucosal barrier (more preferably the colonic mucosal barrier). For example, sterile water or an inert solid may be used as the carrier, usually supplemented with pharmaceutically acceptable adjuvants, buffers, dispersants, etc.

[0061] The compositions taught herein may be in liquid form, for example, a stabilized suspension of the polypeptide or host cell taught herein, or in solid form, for example, a lyophilized powder of the host cell taught herein. When the host cell taught herein is lyophilized, cryoprotective substances such as lactose, trehalose, or glycogen may be used. With regard to oral administration, the polypeptide or lyophilized host cell taught herein may be administered in solid dosage forms such as capsules, tablets, and powders, or in liquid dosage forms such as elixirs, syrups, and suspensions. The polypeptide or host cell taught herein may be encapsulated in a capsule such as a gelatin capsule together with an inert component and a powder carrier, for example, glucose, lactose, sucrose, mannitol, starch, cellulose, or a cellulose derivative, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate, etc.

[0062] In some embodiments, the compositions taught herein may comprise one or more components suitable for promoting survival and / or viability during storage and / or exposure to bile and / or passage through the gastrointestinal tract of mammals (e.g., humans), and / or for maintaining the integrity of the polypeptides and / or host cells taught herein. Non-limiting examples of such components include enteric coatings that allow passage through the stomach, and controlled-release agents. Those skilled in the art will know how to select components suitable for receiving the intended destination (whether polypeptides or host cells) where the active ingredient is to exert its action.

[0063] In some embodiments, the compositions taught herein may further comprise a mucosal binding agent or mucosal binding polypeptide. As used herein, the terms “mucosal binding agent” or “mucosal binding polypeptide” refer to an agent or polypeptide capable of adhering the composition to the intestinal mucosal surface of the intestinal mucosal barrier of a mammal (e.g., human).

[0064] Alternatively, a specific docking system may be used to attach the polypeptides or cells that produce them as taught herein, or to attach living or dead non-producing cells. Binding may be at either the C-terminus or the N-terminus, whichever is deemed most efficient, while the use of spacer peptides has also been demonstrated. An example is the use of a LysM-based peptidoglycan docking system (Visweswaran GR et al., 2014, Appl Microbiol Biotechnol. 98:4331-45). Furthermore, a variety of mucosal-binding polypeptides have been disclosed in the art. Non-limiting examples of mucosal-binding polypeptides include bacterial toxin membrane-binding subunits, such as the B subunit of cholera toxin, the B subunit of Escherichia coli thermolabile enterotoxin, Bordetella pertussis toxin subunits S2, S3, S4 and / or S5, the B fragment of diphtheria toxin, and the membrane-binding subunit of Shiga toxin or Shiga-like toxin. Other suitable mucosal binding polypeptides include bacterial pili proteins, such as Escherichia coli pili (K88, K99, 987P, F41, FAIL, CFAIII ICES1, CS2 and / or CS3, CFAIIV ICS4, CS5 and / or CS6), and P pili. Other non-limiting examples of pili include Bordetella pertussis filamentous hemagglutinin, Vibrio cholerae toxin-coregulate pilus (TCP), mannose-sensitive hemagglutinin (MSHA), and fucose-sensitive hemagglutinin (PSHA). Further mucosal binding agents include viral adhesion proteins containing influenza and Sendai virus hemagglutinins, as well as animal lectins or lectin-like molecules containing immunoglobulin molecules or their fragments, calcium-dependent (type C) lectins, selectins, collectins, or apple snail hemagglutinins. Plant lectins with mucosal binding subunits include concanavalin A, wheat germ agglutinin, phytohemagglutinin, abrin, and lysine. The advantage of this delivery method is that it eliminates the need for the use of live recombinant organisms.

[0065] Although not required, it may be advantageous to add one or more mucosal binding agents or mucosal binding polypeptides to the composition taught herein so as to direct the polypeptides or host cells taught herein to the intestinal mucosal barrier.

[0066] The compositions taught herein may further include components selected from the group consisting of prebiotics, probiotics, carbohydrates, polypeptides, lipids, vitamins, minerals, pharmaceutical agents, preservatives, antibiotics, or any combination thereof.

[0067] In one embodiment, the compositions taught herein may further contain one or more components that further enhance the nutritional and / or therapeutic value of the compositions taught herein. For example, it may be advantageous to add one or more components selected from proteins, amino acids, enzymes, inorganic salts, vitamins (e.g., thiamine HCl, riboflavin, pyridoxine HCl, niacin, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, vitamin B12, p-aminobenzoic acid, vitamin A acetate, vitamin K, vitamin D, vitamin E, etc.), sugars and complex carbohydrates (e.g., water-soluble and water-insoluble monosaccharides, disaccharides, and polysaccharides), pharmaceutical compounds (e.g., antibiotics), antioxidants, and trace element components (e.g., compounds such as cobalt, copper, manganese, iron, zinc, tin, nickel, chromium, molybdenum, iodine, chlorine, silicon, vanadium, selenium, calcium, magnesium, sodium, and potassium) (e.g., nutritional components, veterinary or pharmaceutical agents, etc.). Those skilled in the art will be familiar with suitable methods and ingredients for improving the nutritional value and / or therapeutic value / pharmacological effects of the compositions taught herein.

[0068] In one embodiment, the host cells may be incorporated in a lyophilized form, a microencapsulated form (e.g., as investigated by Solanki et al., BioMed Res. Int. 2013, Article ID 620719), or any other form that preserves the activity and / or viability of the host cells (e.g., bacterial strain).

[0069] Treatment method In another aspect, the disclosure relates to obesity, metabolic syndrome, insulin deficiency or insulin resistance-related disorders, type 2 diabetes, type 1 diabetes, gestational diabetes, pre-eclampsia, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), impaired glucose tolerance, abnormal lipid metabolism, atherosclerosis, hypertension, cardiac conditions, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, fatty liver, lipid metabolism disorders, immune system dysfunction associated with obesity (weight gain), allergies, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other diseases associated with impaired barrier function, wound healing, behavioral disorders, alcoholism, cardiovascular disease, high cholesterol, elevated triglycerides, atherosclerosis, and sleep apnea in mammals. Methods for treating and / or preventing disorders or conditions selected from the group including inhalation, osteoarthritis, gallbladder disease, cancer, and conditions that alter the physical integrity of the intestinal mucosal barrier, such as food allergies, intestinal underdevelopment due to premature birth of an infant, exposure to radiation, chemotherapy and / or toxins, autoimmune disorders, malnutrition, sepsis, etc.; methods for promoting weight loss in mammals; methods for promoting anti-inflammatory activity in the intestines of mammals; methods for promoting intestinal mucosal immune system function in mammals; methods for maintaining, restoring and / or improving glucose and / or cholesterol and / or triglyceride homeostasis; and methods for maintaining, restoring and / or enhancing the physical integrity of the mucointestinal barrier in mammals. The method comprises the step of administering an effective amount of a polypeptide taught herein, a host cell taught herein, or a composition taught herein to a mammal in need thereof.

[0070] In one embodiment, the polypeptides, host cells, or compositions taught herein may be administered by any known method of administration. For example, the compositions taught herein may be administered orally, intravenously, topically, enterally, or parenterally. Naturally, the mode or route of administration will depend on the current case (e.g., the age of the subject, the desired site of effect, the disease state, etc.) and the intended form of the composition (e.g., pills, liquid, powder, etc.).

[0071] In preferred embodiments, the polypeptides, host cells, or compositions taught herein are administered orally.

[0072] use In another embodiment, this disclosure relates to the use of nucleic acid molecules, chimeric genes, and / or vectors taught herein for producing polypeptides taught herein and / or forming host cells taught herein. Polynucleotides and / or host cells taught herein may have an enhanced ability to interact with TLR2 receptors on cells and / or an enhanced ability to stimulate the TLR2 signaling pathway in cells and / or an enhanced ability to stimulate the production of cytokines from cells, particularly IL-1β, IL-6, IL-8, IL-10, and TNF-α, and / or an enhanced ability to increase TER in mammalian cells, such as human cells, compared to host cells (e.g., bacteria) that have not been genetically modified with polynucleotides, chimeric genes, or vectors taught herein.

[0073] In another embodiment, the Disclosure relates to a pharmaceutically acceptable use; in particular, to use to promote intestinal mucosal immune system function, or to maintain, restore, and / or enhance the physical integrity of the intestinal mucosal barrier in mammals; to maintain, restore, and / or improve glucose and / or cholesterol and / or triglyceride homeostasis in mammals; and in mammals, obesity, e.g., dietary obesity, metabolic syndrome, insulin deficiency or insulin resistance-related disorders, type 2 diabetes, type 1 diabetes, gestational diabetes, pre-eclampsia, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), impaired glucose tolerance, abnormal lipid metabolism, atherosclerosis, hypertension, cardiac conditions, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, fatty liver, lipid metabolism disorders, immune system dysfunction associated with obesity (weight gain), allergies, The present invention relates to polypeptides, host cells, or compositions taught herein for use in preventing and / or treating disorders or conditions selected from the group consisting of asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other diseases associated with impaired barrier function, wound healing, behavioral disorders, alcoholism, cardiovascular diseases, high cholesterol, elevated triglycerides, atherosclerosis, sleep apnea, osteoarthritis, gallbladder disease, cancer, and conditions that alter the physical integrity of the intestinal mucosal barrier, such as food allergies, for example, underdeveloped intestines due to premature birth of an infant, exposure to radiation, chemotherapy and / or toxins, autoimmune disorders, malnutrition, sepsis, etc.; for use in promoting anti-inflammatory activity in the intestines of mammals; or for use in promoting weight loss in mammals.

[0074] In one embodiment, the mammal, for example, a human, may be of any age group (e.g., infant, adult, elderly) and of any sex (male and female). In one embodiment, the mammal may be an infant (e.g., newborn, baby, toddler, etc.), particularly a premature infant.

[0075] The mammal may be any mammal, such as a human, a non-human primate, a rodent, a cat, a dog, a cow, a horse, etc. In a preferred embodiment, the mammal is a human.

[0076] The isolated polypeptides in this disclosure also instead a) Having at least 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with sequence number 5 (over its entire length); b) comprising at least 1, 2, 3, 4, 5, 6, or 7 amino acid residues from the following set i. R, S, I, S, A, and / or P (or their conservative substitutions) at positions 6, 7, 13, 22, 25, and / or 30 of sequence number 5; ii. C, K, K, I, and / or T (or their conservative substitutions) at positions 88, 89, 91, 93, and / or 96 of sequence number 5; iii. W, L, G, and / or F (or their conservative substitutions) at positions 101, 102, 103, and / or 104 of sequence number 5; iv. F and / or E (or their conservative substitutions) at positions 122 and / or 123, respectively, of sequence number 5; v. V, Y, and / or R (or their conservative substitutions) at positions 145, 146, and / or 147, respectively, of sequence number 5; vi. P, E, I, F, Q, R, S, and / or V (or their conservative substitutions) at positions 174, 176, 177, 179, 180, 183, 185, and / or 186 of sequence number 5; vii. P, P, P, A, A, P, G, T, A, E, A, P, Q, K, G, and / or E (or its conservative substitution) at positions 215, 217, 221, 222, 223, 226, 234, 239, 241, 243, 245, 150, 153, 257, 261, and / or 263, respectively. It may also be characterized by the following:

[0077] The polypeptides defined above can induce immune signaling and / or affect intestinal barrier function and / or affect glucose and / or cholesterol and / or triglyceride homeostasis. Preferably, the isolated polypeptide does not contain SEQ ID NO: 1 or an amino acid sequence having more than 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO: 1. The polypeptide may also be able to bind to Toll-like receptor 2 (TLR2).

[0078] In one embodiment, the polypeptide defined above is preferably included in a composition further comprising a carrier, for example, a physiologically acceptable carrier, a pharmaceutically acceptable carrier, a digestively acceptable carrier, or a nutritionally acceptable carrier. The carrier may be any inert carrier. For example, non-limiting examples of suitable physiologically or pharmaceutically acceptable carriers include any well-known physiological or pharmaceutically acceptable carrier, buffers, diluents, and excipients.

[0079] a) As described below, the polypeptide may also include variants of the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of the variant having more than 25% sequence identity with the amino acid sequence of SEQ ID NO: 5. Variants of the polypeptide also include polypeptides derived from the polypeptide having the amino acid sequence of SEQ ID NO: 5 by one or more amino acid substitutions, deletions, or insertions. Preferably, such polypeptides contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions, or insertions, up to a maximum of about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, or 15 amino acid substitutions, deletions, or insertions compared to the polypeptide having the amino acid sequence of SEQ ID NO: 5. As described, the polypeptide may have, for example, at least 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 5, or at least 50% sequence identity with SEQ ID NO: 5, over its entire length. The polypeptide according to this disclosure may or may not include a leader sequence.

[0080] In one embodiment, the polypeptide according to this disclosure is - i) at least five amino acid residues (or their conservative substitutions) as defined below; - ii) at least four amino acid residues (or their conservative substitutions) as defined below; - iii) at least three amino acid residues (or their conservative substitutions) as defined below; - iv) at least one amino acid residue (or a conservative substitution thereof) as defined below; - v) at least two amino acid residues (or their conservative substitutions) as defined below; - vi) at least seven amino acid residues (or their conservative substitutions) as defined below; and / or - vii) at least 15 (or at least 12) amino acid residues (or their conservative substitutions) as defined below Includes.

[0081] Alternatively, or simultaneously, the polypeptides taught herein may, in particular, comprise the following set of amino acid residues as defined above. - i); - i) and vii); - i), ii), vi), and vii); - i), iii), iv), and vii); - i), ii), iii), iv), v), vi), vii).

[0082] Alternatively, or simultaneously, the polypeptides taught herein may, for example, have at least 75% sequence identity with SEQ ID NO: 5 throughout their entire length.

[0083] In a preferred embodiment, the isolated polypeptide according to the Disclosure further comprises amino acid residues S, N, E, N, (A,) P, Q, L, and / or L (or their conserved substitutions) at positions corresponding to positions 34, 35, 41, 43, (46,) 67, 74, 77, and / or 84 of SEQ ID NO: 5. Preferably, at least eight of these listed amino acid residues are included.

[0084] In yet another preferred embodiment, the isolated polypeptide according to the Disclosure further comprises amino acid residues P, L, N, G, K, W, I, Y, R, I, V, L, F, and / or P (or their conserved substitutions) at positions corresponding to positions 112, 120, 132, 138, 144, 170, 193, 199, 207, 208, 297, 273, 276, and / or 279 of SEQ ID NO: 5. Preferably, at least 13 (or at least 11) of these listed amino acid residues are included.

[0085] The isolated polypeptides according to this disclosure may be natural variants of polypeptides according to Sequence ID No. 5, for example, naturally occurring polypeptides having the same functionality or synthetic polypeptides having the same functionality, i.e., polypeptides that can induce immune signaling and / or affect intestinal barrier function and / or affect glucose and / or cholesterol and / or triglyceride homeostasis. The polypeptides may also be able to bind to Toll-like receptor 2 (TLR2).

[0086] The isolated polypeptides according to this disclosure are - Isolated polypeptides having at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with Sequence ID No. 5 (over its entire length); - Isolated polypeptides having at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 6 (over its entire length); - Isolated polypeptides having at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% sequence identity with Sequence ID No. 7 (over its entire length); - Isolated polypeptides having at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% sequence identity with SEQ ID NO: 8 (over its entire length); and - Isolated polypeptide having at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% sequence identity with SEQ ID NO: 9 (over its entire length) You may choose from the following: Preferably, it contains the set of conserved amino acid residues as taught herein.

[0087] general definition In the context of this disclosure, the term “polypeptide” is equivalent to the term “protein.” A polypeptide has a specific amino acid sequence. A “variant” of a polypeptide in this disclosure preferably has an amino acid sequence that has at least 25% sequence identity with a reference polypeptide. The polypeptides in this disclosure are isolated when they are no longer present in their natural environment, i.e., no longer present in association with pili, and / or no longer present in association with cells such as Ackermansia muciniphila or Ackermansia glycanipira cells. The leader sequence is the (encoded) region between the promoter and the coding region and is involved in the regulation of expression. The leader sequence (or a portion thereof) can be translated into a leader peptide, but, in contrast to signal peptides, the leader peptide does not become part of a structural protein.

[0088] Where used herein, the term “conserved substitution” may also refer to the substitution of one or more amino acids in a polypeptide without substantial impairment of functionality. It is common knowledge that certain amino acids can be substituted with other amino acids without loss of polypeptide activity. For example, the following amino acids are generally interchangeable: Ala, Ser, Thr, Gly (small aliphatic nonpolar or slightly polar residues) Asp, Asn, Glu, Gln (polar, negatively charged residues and their amides) His, Arg, Lys (polar residues with a positive charge) Met, Leu, Ile, Val(Cys) (large aliphatic nonpolar residues) Phe, Ty, Trp (large aromatic residues) (See, for example, Schulz, GE et al., Principles of Protein Structure, Springer-Verlag, New York, 1979, and Creighton, TE, Proteins: Structure and Molecular Principles, WH Freeman & Co, San Francisco, 1984)

[0089] A preferred “substitution” is a conservative substitution, i.e., a residue is replaced by another residue of the same general type. When making a modification, the hydropathic index of the amino acid may be taken into consideration (see, e.g., Kyte et al., J. Mol. Biol. 157, pp. 105-132 (1982)). It is known in the art that a particular amino acid can be substituted with another amino acid having a similar hydropathic index or score, and still result in a polypeptide with similar biological activity. When making such a modification, substitutions of amino acids with a hydropathic index of ±2 are preferred, substitutions of ±1 are more preferred, and substitutions of ±0.5 are even more preferred. Similarly, a selected amino acid may be substituted with another amino acid having similar hydrophilicity, as described in U.S. Patent No. 4,554,101. When making such a modification, as with the hydropathic index, substitutions of amino acids with a hydrophilic index of ±2 are preferred, substitutions of ±1 are more preferred, and substitutions of ±0.5 are even more preferred.

[0090] The terms “sequence identity” or “sequence similarity,” as used herein, refer to the situation in which an amino acid or nucleic acid sequence has sequence identity or sequence similarity with another reference amino acid or nucleic acid sequence. “Sequence identity” or “sequence similarity” can be determined by the alignment of two polypeptides or two nucleotide sequences using a global or local alignment algorithm. Sequences may also be referred to as “substantially identical” or “basically similar” if, as a result, they share at least a certain minimum percentage of sequence identity (as defined below) (for example, when optimally aligned by the program GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, GAP default parameters are used with a gap insertion penalty of 50 (nucleotides) / 8 (protein) and a gap elongation penalty of 3 (nucleotides) / 2 (protein). The default scoring matrix used for nucleotides is nwsgapdna, and the default scoring matrix for proteins is Blosum62 (Henikoff and Henikoff, 1992, PNAS 89, pp. 915-919). Sequence alignment and scores for sequence identity percentages can be obtained using computer programs such as the GCG Wisconsin package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or EmbossWin version 2.10.0 (using the program "needle"). Alternatively, similarity or identity percentages can be obtained by searching databases using algorithms such as FASTA or BLAST. Preferably, sequence identity refers to sequence identity over the entire length of the sequence.

[0091] "Epithelial membrane resistance" (abbreviated as TER) is a measure of the permeability of the epithelial cell layer in vitro. Increased epithelial permeability has been associated with weakened tight junctions and decreased TER.

[0092] When used herein, the term “chimeric gene” refers to any gene that does not exist in nature, i.e., a gene not typically found in nature within a species, in particular a gene in which one or more parts of the nucleic acid sequence are not bound to each other in nature. For example, a promoter is not bound to some or all of a transcription region or to another regulatory region in nature. The term “chimeric gene” is understood to include an expression construct in which a heterogeneous promoter or transcription regulatory sequence is operably linked to one or more coding sequences and optionally a 3'-untranslated region (3'-UTR). Alternatively, a chimeric gene may include a promoter, coding sequence, and optionally a 3'-UTR of the same species but in a combination that does not exist in nature.

[0093] Where used herein, the term “genetically modified host cell” refers to a cell that has been genetically modified, for example, by the introduction of an exogenous nucleic acid sequence or by specific alterations to an endogenous gene sequence. Such cells may be genetically modified by the introduction of, for example, one or more mutations, insertions and / or deletions in the endogenous gene and / or by the insertion of a gene construct (e.g., a vector or chimeric gene) into the genome. Genetically modified host cells may also refer to isolated cells or cells in a culture. Genetically modified cells may also be “transduced cells” in which the cells have been infected with, for example, a modified virus, for example, a retrovirus may be used, but other suitable viruses such as lentiviruses may also be considered. Non-viral methods such as transfection may also be used. Thus, genetically modified host cells may be “stablely transfected cells” or “transiently transfected cells.” Transfection refers to a non-viral method of introducing DNA (or RNA) into a cell so that a gene is expressed. Transfection methods such as calcium phosphate transfection of nucleic acids, PEG transfection, and liposome or lipoplex transfection are widely known in the art. Such transfections may be transient or stable, and cells into which the gene construct has been incorporated into the genome may be selected.

[0094] Where used herein, the term “effective dose” refers to the amount necessary to achieve the effects taught herein. For example, an effective dose of a polypeptide or genetically engineered host cell taught herein is necessary to modulate and / or promote intestinal mucosal immune system function, and / or maintain and / or restore and / or enhance the physical integrity of the intestinal mucosal barrier (e.g., to promote the formation of tighter junctions between intestinal epithelial cells), and / or modulate and / or stimulate the Toll-like receptor signaling pathway (i.e., the TLR2 pathway) in immune cells, and / or increase cytokine production (e.g., IL-6, IL-8, and IL-10) in immune cells, and / or treat obesity, metabolic syndrome, insulin deficiency or insulin resistance-related disorders, type 2 diabetes, type 1 diabetes, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), impaired glucose tolerance, and abnormal lipid metabolism. It is an effective amount for preventing and / or treating atherosclerosis, hypertension, heart conditions, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, fatty liver, lipid metabolism disorders, immune system dysfunction associated with obesity (weight gain), allergies, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other diseases associated with impaired barrier function, wound healing, behavioral disorders, alcoholism, cardiovascular disease, high cholesterol, elevated triglycerides, atherosclerosis, sleep apnea, osteoarthritis, gallbladder disease, cancer, and conditions that alter the physical integrity of the intestinal mucosal barrier, such as food allergies, underdeveloped intestines due to premature birth, exposure to radiation, chemotherapy and / or toxins, autoimmune disorders, malnutrition, sepsis, and other disorders or conditions.

[0095] The terms “physiologically acceptable carrier,” “digestively acceptable carrier,” “nutritionally acceptable carrier,” and “pharmaceutically acceptable carrier,” as used herein, refer to physiologically acceptable or digestively acceptable carriers, or nutritionally acceptable or pharmaceutically acceptable carrier materials, such as liquid or solid extenders, diluents, excipients, solvents, or encapsulating materials, involved in providing the polypeptide or host cell dosage forms of the herein disclosure. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the composition and is not harmful to the subject, i.e., suitable for ingestion or nutritionally acceptable. The terms “suitable for ingestion” or “nutritionally acceptable” refer to components or substances that are generally considered safe for ingestion by humans (and other mammals). Non-limiting examples of materials that can act as physiologically acceptable carriers or nutritionally or pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, e.g., cocoa butter and suppository waxes; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, and sesame oil. (10) Glycols, e.g., propylene glycol; (11) Polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; (12) Esters, e.g., ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, e.g., magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Pyrogen-free water; (17) Physiological saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) Phosphate buffer; (21) Other non-toxic and suitable substances used in pharmaceutical formulations.Furthermore, the terms “nutritionally acceptable” and “pharmaceutically acceptable,” as used herein, mean, within the bounds of appropriate medical judgment, a composition or drug, material, or combination of composition and / or dosage forms thereof that is suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that have a reasonable benefit-to-risk ratio.

[0096] The term "homeostasis" refers to the property of a system in which changes are regulated so that the internal state remains stable and relatively constant. All animals regulate blood glucose levels. Glucose regulation in the body is the process of maintaining "glucose homeostasis" in the body. Mammals regulate blood glucose through various hormones (e.g., insulin, glucagon, glucagon-like peptide-1, catecholamines, and many others) and various neural pathways (e.g., neural relay, gut-brain-peripheral organ axis). The human body maintains a nearly constant glucose level for almost the entire day, even after 24 hours of fasting. Glucose levels only decrease very slightly during prolonged fasting. Insulin, secreted by beta cells of the pancreas, effectively transports glucose to the body's cells by instructing them to hold more glucose for use by the cells themselves. When there is a lot of glucose in the cells, the cells convert glucose into insoluble glycogen to prevent soluble glucose from interfering with cellular metabolism. Ultimately, this lowers blood glucose levels, allowing insulin to help prevent hyperglycemia. Diabetes occurs when insulin is deficient or when cells become insulin-resistant. Glucagon, secreted by alpha cells in the pancreas, promotes the breakdown of stored glycogen or the conversion of non-carbohydrate carbon sources into glucose through gluconeogenesis, thereby preventing hypoglycemia. Numerous other factors and hormones (e.g., glucagon-like peptide-1, catecholamines, and many others) are involved in regulating glucose metabolism. Various mechanisms, including neural pathways, also contribute to this complex regulation.

[0097] Cholesterol homeostasis is a mechanism that contributes to the process of maintaining a balanced internal state of cholesterol within living organisms. Cholesterol, an essential biomolecule in the human body, performs various physiological functions, including acting as a precursor for the production of bile acids, vitamin D, and steroid hormones. It also functions as an important structural element in the cell membranes of all cells present in the body. Despite cholesterol's beneficial and essential functions, dysfunction of cholesterol homeostasis can lead to an increased risk of heart disease and disrupt other homeostatic feedback systems associated with cholesterol metabolism. The liver is the most prominent organ regulating cholesterol homeostasis because it not only biosynthesizes cholesterol released into the circulatory system but also breaks down potentially harmful free-floating cholesterol in the bloodstream. HDL is beneficial in maintaining cholesterol homeostasis because it picks up potentially dangerous cholesterol and sends it directly back to the liver where cholesterol is synthesized into harmless bile acids used by the digestive system. LDL has a less beneficial effect. This is because cholesterol tends to accumulate in somatic cells and artery walls. Excessive levels of LDL have been shown to increase the risk of cardiovascular disease. In healthy individuals, cholesterol homeostasis is tightly regulated by a complex feedback loop. In this case, even if a healthy individual consumes large amounts of dietary cholesterol, biosynthesis in the liver is significantly reduced to maintain balance. In healthy individuals with high baseline LDL levels due to long-term poor eating habits or other genetic or medical conditions, the feedback loop and systemic coping mechanisms may be impaired by the same large intake, leading to a dangerous homeostatic imbalance.

[0098] Triglyceride homeostasis is a mechanism that contributes to maintaining a balanced internal state of triglycerides in living organisms. Triglyceride metabolism has significant clinical relevance. Hypertriglyceridemia refers to high (hyper-) blood or serum levels (-emia) of triglycerides, the most abundant lipid molecules. Elevated levels of triglycerides are associated with atherosclerosis and increase the risk of cardiovascular disease, even without hypercholesterolemia (high cholesterol levels). High triglyceride levels also increase the risk of acute pancreatitis. Furthermore, elevated and increasing TG levels increase the risk of developing diabetes over time. Insulin resistance has been shown to be associated with high levels of triglycerides (TG).

[0099] When used herein, the term "approximately" refers to a range of tolerances that are normal in the art, for example, within two standard deviations of the mean. The term "approximately" can be understood to include values ​​that are up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the indicated value.

[0100] The terms “comprising” or “to comprise,” as used herein, refer to situations in which the terms are used in a non-restrictive sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. This also includes the more restrictive verbs “to consist essentially of” and “to consist of.”

[0101] The indefinite article "a" or "an" does not rule out the possibility of two or more elements being present unless the context clearly requires the presence of only one or one element. Therefore, the indefinite article "a" or "an" usually means "at least one." [Brief explanation of the drawing]

[0102] [Figure 1A] This graph shows the total body weight gain (g) (n=8-10). [Figure 1B] This graph shows the increase in total fat mass (g, fat mass gain) (n=8~10) measured by time-domain nuclear magnetic resonance. [Figure 1C] This is a graph showing the daily food intake. [Figure 1D] This graph shows plasma VLDL, LDL, and HDL cholesterol levels (n=8-10, Plasma cholesterol). [Figure 1E] This graph shows the plasma glucose (mg dl-1) profile. [Figure 1F] This graph shows the mean area under the curve (AUC) measured between -30 and 120 minutes after glucose loading (mg.dl-1.min-1; n=8~10). [Figure 1G] This graph shows the ratio (n=3-5) of insulin-stimulated p-IRβ to a loading control measured by densitometry. [Figure 1H] This graph shows the ratio of insulin-stimulated p-Aktthr308 to a loading control, as measured by densitometry. [Figure 1I] This graph shows the ratio (n=3-5) of insulin-stimulated p-Aktser473 to a loading control measured by densitometry. [Figure 2]This diagram shows the conserved residues in the natural variants of Amuc-1100 (SEQ ID NOs. 1, 5, 6, 7, 8, and 9). Viewed from top to bottom and left to right, the first box shows outward-facing conserved residues that may play a role in interactions. The second, third, and fourth boxes show hydrophobic residues that may be involved in structural integrity. The fifth box shows outward-facing conserved residues that may play a role in interactions. The sixth box shows loops. [Figure 3] This figure shows the sequence of Amuc-1100 (SEQ ID NO: 1). Conserved residues are circled, and deletions are shown in gray. [Figure 4] This figure shows a bicistronic design used in expression plasmids. Translation of short peptides driven by RBS1 ensures accessibility for RBS2, which drives translation of the target protein. Linearization of RBS2 ensures the elimination of potential inhibitory secondary structures in the 5'UTR, thereby improving translation efficiency (Mutalik et al., 2013; Nieuwkoop et al., 2019). [Figure 5] This graph shows the SEAP activity (in AU units) of a positive control (Pam3CSK4, 1 ug / ml), negative controls (PBS and DMEM), and various Amuc_1100 variants (all 50 ug / ml) purified after TEV cleavage. The pTH00×ID at the beginning of the native variant name refers to the plasmid name used to purify each protein.

[0103] Sequence ID 1: Amino acid sequence of Amuc-1100 polypeptide (conserved residues are underlined)

[0104] [ka]

[0105] Sequence ID 2: Nucleotide sequence encoding the Amuc-1100 polypeptide

[0106] [ka]

[0107] Sequence ID 3: Amino acid sequence of the predicted N-terminal signal sequence of the Amuc-1100 polypeptide

[0108] [ka]

[0109] Sequence ID 4: Nucleotide sequence of the predicted N-terminal signal sequence of the Amuc-1100 polypeptide

[0110] [ka]

[0111] Sequence ID 5: Amino acid sequence of Ackermansia muciniphila protein WP_094137363.1 (pTH008, conserved residues are underlined)

[0112] [ka]

[0113] Sequence ID 6: Amino acid sequence of Ackermansia muciniphila protein WP_022398192.1 (pTH009, conserved residues are underlined)

[0114] [ka]

[0115] Sequence ID 7: Amino acid sequence of Akkermansia muciniphila protein WP_102725837.1 (pTH010, conserved residues are underlined)

[0116] [ka]

[0117] Sequence ID 8: Amino acid sequence of Akkermansia species KLE1797 protein WP_067981703.1 (pTH011, conserved residues are underlined)

[0118] [ka]

[0119] Sequence ID 9: Amino acid sequence of Ackermansia glycanipira protein WP_067777749.1 (pTH012, conserved residues are underlined)

[0120] [ka]

[0121] Sequence ID 13

[0122] [ka]

[0123] Sequence ID 14

[0124] [ka]

[0125] Sequence ID 15

[0126] [ka]

[0127] Sequence ID 16

[0128] [ka]

[0129] Sequence ID 17

[0130] [ka]

[0131] Sequence ID 29 (pTH008)

[0132] [ka]

[0133] Sequence ID 30 (pTH009)

[0134] [ka]

[0135] Sequence ID 31 (pTH010)

[0136] [ka]

[0137] Sequence ID 32 (pTH011)

[0138] [ka]

[0139] Sequence ID 33 (pTH012)

[0140] [ka] [Examples]

[0141] Example 1: Formation of genetically modified bacteria to produce the Amuc-1100 protein. method: The polynucleotide encoding mature Amuc-1100 (nucleotide sequence of SEQ ID NO: 2) was cloned into E. coli TOP10 with a C-terminal His tag under the control of an inducible T7 promoter of a pET28 derivative, and introduced into E. coli BL21 (DE3) for overproduction. For this purpose, an ATG start codon was added to the nucleotide sequence of SEQ ID NO: 2 so that the resulting polypeptide would begin with the amino acid sequence MIVNS. All constructs were confirmed by Sanger sequence analysis. Constructs containing overexpressed Amuc-1100 resulted in overproduction of soluble Amuc-1100 protein, which was purified to appear homogeneous by Ni-column affinity chromatography and used at a concentration of 100-300 ug / ml. Antibodies were generated in rabbits using the purified Amuc-1100, essentially as previously described (Reunanen J et al., 2012, Appl Environ Microbiol 78: pp. 2337-44).

[0142] result: These results indicate that *E. coli* transformed with polynucleotide (SEQ ID NO: 2) were able to produce soluble Amuc-1100 protein that could be easily isolated using Ni-column chromatography, as shown (Tailford LE et al., 2015, Nat Commun. 6:7624). Similar results were obtained with polynucleotide SEQ ID NO: 29 or 33.

[0143] Example 2: Interaction and stimulation of the TLR2 signaling pathway method: To test the ability of Amuc-1100 to stimulate TLR2 and other TLR signaling pathways after binding to TLR2 and other TLR receptors, reporter cell lines expressing TLR2 and TLR4 receptors were created. After binding to cell lines expressing TLR2 or TLR4, the ability of Amuc-1100 to stimulate the TLR2 and / or TLR4 signaling pathways in these cells was tested in vitro by measuring NK-κB production from the reporter cells.

[0144] In short, hTLR2 and hTLR4 cell lines (Invivogen, CA, USA) were used. Stimulation of the receptors with the corresponding ligands activated NF-κB and AP-1, which induced the production of secreted embryonic alkaline phosphatase (SEAP), the level of which could be measured by a spectrophotometer (Spectramax). All cell lines were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 4.5 g / l D-glucose, 50 U / ml penicillin, 50 μg / ml streptomycin, 100 μg / ml Normocin, 2 mM L-glutamine, and 10% (v / v) heat-inactivated fetal bovine serum (FBS) as maintenance medium, and subcultured to a maximum concentration density of 70-80%. Immunotherapy experiments were performed by adding 20 μl of Amuc-1100 suspension to each cell line. Reporter cells were incubated with Amuc-1100 in a 5% CO2 incubator at 37°C for 20–24 hours. Receptor ligands Pam3CSK4 (10 ng / ml relative to hTLR2) and LPS-EB (50 ng / ml relative to hTLR4) were used as positive controls, and maintenance medium without any selective antibiotics was used as a negative control. SEAP secretion was detected by measuring OD600 at 15 minutes, 1 hour, 2 hours, and 3 hours after adding 180 μL of QUANTI-Blue (Invivogen, CA, USA) to 20 μL of induced hTLR2 and hTLR4 supernatants. The experiment was performed three times.

[0145] result: These results indicate that Amuc-1100 was able to interact with TLR2. Furthermore, these results show that Amuc-1100 exerted an immunostimulatory effect on reporter cells expressing TLR2, i.e., that Amuc-1100 was able to stimulate the release of NF-κB from reporter cells. Similar results were obtained with the polypeptides of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0146] Example 3. Stimulation of cytokine release from peripheral blood mononuclear cells. method: The ability of Amuc-1100 to stimulate cytokine production or release from peripheral blood mononuclear cells (PBMCs) was tested in vitro. In short, peripheral blood from three healthy donors was received from Sanquin Blood Bank, Nijmegen, The Netherlands. Peripheral blood mononuclear cells (PBMCs) were isolated from the healthy donor blood using Ficoll-Paque Plus gradient centrifugation according to the manufacturer's (Amersham biosciences, Uppsala, Sweden) procedure. After centrifugation, the mononuclear cells were collected, washed in Iskoff-modified Dulbecco's medium (IMDM) + Glutamax (Invitrogen, Breda, The Netherlands), and then refueled in IMDM + Glutamax with penicillin (100 U / ml) (Invitrogen), streptomycin (100 μg / ml) (Invitrogen), and 10% heat-inactivated FBS (Lonza, Basel, Switzerland) at 0.5 × 10⁶ times. 6 Adjusted to cells / ml. PBMC(0.5×10 6 Cells were seeded (per well) into 48-well tissue culture plates. A negative control (culture medium only) was used for each donor.

[0147] After 1 day of stimulation of PBMCs with either live or A. musciniphila cells heated at 99°C for 10 minutes (in a 1:10 ratio to PBMCs) or Amuc-1100, the production of cytokines IL-6, IL-8, IL-10, TNF-α, IL-1β, and IL-12p70 in the culture supernatant was measured using FACS CantoII (Becton Dickinson) and analyzed using BD FCAP software (Becton Dickinson). The manufacturer's detection limits were as follows: 3.6 pg / ml IL-8, 7.2 pg / ml IL-1β, 2.5 pg / ml IL-6, 3.3 pg / ml IL-10, 3.7 pg / ml TNF-α, and 1.9 pg / ml IL-12p70.

[0148] result These results indicate that Amuc-1100 was able to stimulate cytokine production compared to the control state (culture medium only), specifically, an increase in the levels of IL-1β, IL-6, IL-8, IL-10, and TNF-α was observed. The cytokine levels induced by 4.5 μg / ml Amuc-1100 were 5 × 10⁶ in either the live or heat-dead state. 6 The levels were similar to those of A. muciniphila cells (see Table 1 below).

[0149] [Table 1]

[0150] Similar results were obtained with polypeptides of sequence number 5, 6, 7, 8, or 9.

[0151] Example 4: Modification of epithelial membrane resistance (TER) method: The ability of Amuc-1100 to promote the integrity of the intestinal epithelial cell layer was evaluated by measuring its ability to stimulate or enhance TER in Caco-2 cells in vitro. In short, Caco-2 cells (5 × 10 4 Cells (insert) were seeded into Millicell cell culture inserts (3 μm pore size; Millipore) and grown for 8 days. Bacterial cells were washed once with RPMI1640 and 0.25 OD600 nm (approximately 10) of RPMI1640 were added. 8 The solution was applied to cell cultures. Purified Amuc-1100 was applied to the inserts at concentrations of 0.05, 0.5, and 5 μg / ml. Epithelial membrane resistance was measured from cell cultures at 0 and 24 hours after Amuc-1100 addition using a Millicell ERS-2 TER analyzer (Millipore).

[0152] result: This result indicates that after 24 hours of co-culture with Caco-2 cells, 0.05 μg / ml of Amuc-1100 was already present, approximately 10 8 This demonstrated that TER could be significantly increased to a similar level as in A. muciniphila cells. Similar results were obtained with polypeptides of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0153] Example 5: Regulation of diet-induced metabolic dysfunction As previously described by Everard et al. (2013, PNAS. Vol. 110(22):9066~9071), a cohort of 10-11 week old C57BL / 6J mice (n=10 per subpopulation) was fed either a control diet (ND) or an HF diet (HFD; 60% fat and 20% carbohydrates (kcal / 100g) D12492i, Research Diet, New Brunswick, NJ, USA). Synthetic medium (0.4g KH2PO4, 0.669g Na2HPO4.2H2O, 0.3g NH4Cl, 0.3g NaCl, 0.1g MgCl2.6H per liter of deionized water) was used as shown by Lukovac et al. (2014, mBio 01438-14). 2A. muciniphylla Muc (containing 10g of caditon, 1mM L-threonine, 1ml of trace mineral solution, 5mM L-fucose and 5mM D-glucose) T The cells were grown, concentrated as described above by Everard et al., and mixed in PBS containing 25% glycerol, and stored at -80°C. A subpopulation of HFD-treated mice was orally administered daily 2 × 10⁶ of the suspended solution in sterile anaerobic PBS. 8 Further administration of A. muciniphila at cfu / 0.15 ml (HFD Akk), which contains a 10-fold dilution of A. muciniphila, yielded a final concentration of 2.5% glycerol. As previously demonstrated by Everard et al., the ND and HFD groups were treated daily with oral forced administration of sterile anaerobic PBS containing equivalent volumes of 2.5% glycerol. Another subpopulation of HFD-treated mice was further administered Amuc-1100 peptide, delivered by daily oral forced administration of 3.1 μg of protein Amuc_1100 in equivalent volumes of sterile PBS containing 2.5% glycerol. Treatment of HFD-treated mice with Amuc-1100 resulted in a similar or even greater reduction in body weight and fat mass compared to live A. muciniphila (Figures 1A and 1B), without affecting food intake (Figure 1C). Treatment with A. muciniphila or Amuc-1100 also cured HFD-induced hypercholesterolemia, accompanied by a significant decrease in serum HDL cholesterol and a similar trend with respect to LDL cholesterol (Figure 1D).

[0154] Notably, treatment with Amuc-1100 resulted in a significant decrease in serum triglycerides compared to untreated mice given HFD. Furthermore, Amuc-1100 treatment also reduced the average diameter of adipocytes in HFD-treated mice from 38 micrometers to 29 micrometers, which is similar to the diameter (27 micrometers) seen in untreated mice.

[0155] Interestingly, administration of Amuc-1100 reduced impaired glucose tolerance with the same potency as live bacteria (Figures 1E to 1F).

[0156] To further investigate glucose metabolism, the inventors investigated insulin sensitivity by injecting insulin into the portal vein. The inventors investigated the threonine (Akt) of insulin receptors (IRs) and their downstream mediator Akt in the liver. thr ) Site and serine (Akt ser Insulin-induced phosphorylation at the ) site was analyzed (Figure 1G). HFD administration reduced the phosphorylation of all proteins compared to mice fed a control diet, and Akt thr In this case, a significant improvement was achieved (Figure 1H). Treatment with live A. muciniphila or Amuc-1100 counteracted these effects, and compared to untreated mice given HFD, mice treated with Amuc-1100 showed improved p-IR and p-Akt levels. thr The level was remarkably high (Figures 1G-1H), and in mice treated with live bacteria, p-Akt ser The level was remarkably high (Figure 1I). Similar results were obtained with polypeptides of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0157] Example 6: Comparative analysis of Amuc-1100 natural variants and mutants Objectives and Approach This study aims to understand the signaling ability of Amuc-1100 to Toll-like receptor 2 (TLR2) from a structural activity perspective (Derrien et al., 2004; Plovier et al., 2007). This is related to Ackermansia muciniphila Amuc T This study addresses the issue by measuring the TLR2 signaling ability of native variants exhibiting diverse sequence identity compared to the reference strain Amuc-1100 protein, as well as Amuc-1100 knockout mutants. All proteins, including Amuc-1100 and both native and structural variants, were expressed without an N-terminal membrane anchor containing a signal peptide (ΔSP) sequence to ensure solubility in the cytosol of the expression host, E. coli.

[0158] Natural variant In closely related A. muciniphila strains, Amuc T We identified four proteins that share more than 80% amino acid identity with the Amuc_1100 protein (pTH008, SEQ ID NO: 5, pTH009, SEQ ID NO: 6, pTH010, SEQ ID NO: 7, pTH011, SEQ ID NO: 8). Furthermore, we identified an even more distant variant of Ackermansia glycanipira with only 28% sequence identity (pTH012, SEQ ID NO: 9).

[0159] The inventors refer to these five proteins as natural variants of Amuc_1100. Table 2 and Figure 2 show the conserved residues of the natural variants investigated.

[0160] [Table 2A]

[0161] [Table 2B]

[0162] Gene synthesis and cloning Next, the inventors designed the DNA coding sequence for the protein sequence of the native variant into pTN0003 by excluding the predicted signal peptide detected using SignalP 5.0 (Almagro Armenteros et al., 2019), ultimately obtaining pTN0005. This plasmid (pTN0005) contains Amuc T The exact coding sequence of Amuc-1100 was used because it has been shown to result in significant overexpression in E. coli, as previously demonstrated (Plovier et al., 2017).

[0163] The pTN0003 vector, used as the backbone for all expression constructs, contains the p15A origin, kanamycin resistance gene, T7 promoter, and bicistronic design, followed by a transcription termination sequence (Mutalik et al., 2013; Nieuwkoop et al., 2019) (see Figure 4 for an overview). A summary of the elements in the pTN0003 expression plasmid backbone is shown in Table 3.

[0164] [Table 3]

[0165] The inventors back-translated the protein sequences for five native variants (pTH008, pTH009, pTH010, pTH011, and pTH012) and optimized the DNA coding sequences for expression in E. coli using Benchling's codon optimization tool (based on DNAChisel) (Benchling, 2018). DNA for these native variants of pTN0005 and the Amuc-1100 sequence was ordered as gBlocks (Integrated DNA Technologies; https: / / eu.idtdna.com / DT). Subsequently, the DNA fragments were cloned into a PCR-amplified linear pTN0003 vector by Gibson Assembly (primers in Table 4). The protein coding sequences ultimately incorporated into the expression plasmid for each variant are shown in Table 5.

[0166] [Table 4]

[0167] [Table 5A]

[0168] [Table 5B]

[0169] [Table 5C]

[0170] All expression plasmids were transformed into BL21(DE3) competent E. coli cells (New England Biolabs). After cloning, the expression construct sequences were validated.

[0171] Protein expression and purification Protein expression The bacterial strain containing the expression plasmid was pre-cultured in LB medium supplemented with kanamycin (50 ug / mL). 10 mL of the pre-culture was inoculated into a 5 L Erlenmeyer flask containing 1.5 L of LB medium supplemented with kanamycin (50 ug / mL), and incubated at 37°C and 120 rpm until the culture reached an OD600 of 0.6–0.8. Prior to induction, the flask was placed on ice for 30 minutes. After induction with 0.4 mM IPTG (final concentration), the culture was further incubated at 20°C and 120 rpm for 18 hours. Cells were collected by centrifugation, and the pellet was washed in 25 mL of a washing buffer (50 mM NaH2PO4). 4、 The cells were washed with 300 mM NaCl, 20 mM imidazole (pH 8.0). The cell pellet was stored at -80°C.

[0172] Protein purification The cell pellet was thawed in 25 mL of washing buffer containing a protease inhibitor tablet (Roche cOmplete®). The resuspended cells were sonicated (Bandelin Sonopuls, VS 70 / T probe, 25% intensity, 1 second on, 2 seconds off for a total of 10 minutes, on ice). Lysized cells were centrifuged (15 minutes, 30000 × g, 4°C) and filtered (0.45 μm) to remove cell fragments.

[0173] The protein was further purified using a 5 mL HisTrap HP column (GE Healthcare) with an Akta FPLC system, utilizing the N-terminal His tag. The protein was then purified in 50 mM NaH2PO4. 4、 The protein was eluted in 300 mM NaCl, 500 mM imidazole, and pH 8.0. The His tag was cleaved using 0.7 mg of His-tagged TEV protease during overnight dialysis (14k MWCO) at 4°C against a 1:500 ratio washing buffer. To remove the TEV protease from the Amuc_1100 protein, these were passed through a HisTrap column a second time. At this point, the pass-through fraction containing the target protein was recovered, but the His-tagged TEV protease remained bound to the HisTrap column.

[0174] In vitro culture and stimulation of human HEK-Blue hTLR2 cell line We screened for TLR2 activation using HEK-Blue hTLR2 cells (Invivogen, CA, USA). In this cell line, TLR2 stimulation, followed by activation of NF-κB and AP-1, induces the production of spectrophotometrically quantifiable secreted embryonic alkaline phosphatase (SEAP).

[0175] Cell lines were grown in maintenance medium of Dulbecco's modified Eagle medium (DMEM) supplemented with GlutaMAX®, 4.5 g / L D-glucose, 100 U / mL penicillin, 100 μg / mL streptomycin, 100 μg / mL normocin, 10% (v / v) heat-inactivated FBS, and HEK-Blue® Selection (Invivogen), and subcultured to a maximum concentration of 70-80%. Cells were maintained to the maximum extent of 25 subcultures. HEK-Blue cells in flat-bottom 96-well plates were inoculated into maintenance medium without HEK-Blue® Selection, and TLR2 activation was tested three times by stimulating the cells with the addition of 20 μL of the target protein (concentration of 50 ug / mL) after 24 hours. The 96-well plates were incubated in a 5% CO2 incubator at 37°C for 20-24 hours. Receptor ligand Pam3CSK4 was used as a positive control, and PBS (dilution reagent for the target protein) was used as a negative control. Secretory embryonic alkaline phosphatase (SEAP) activity was detected by measuring the absorbance at 600 nm 1 hour after adding 20 μL of induced HEK-Blue hTLR2 supernatant to 180 μL of QUANTI-Blue (Invivogen) (Synergy® Mx, BioTek Instruments, Inc., VT, USA), and expressed in arbitrary units (AU).

[0176] Results and Conclusions Amuc T The ability of purified Amuc-1100 protein and its native variant to activate TLR2 was measured as described above. The results are shown in Figure 5.

[0177] The activity of 1 ug / ml Pam3CSK4 against positive control TLR2 cells reached approximately 4.0 AU, while that of negative controls PBS and DMEM was lower than 1.0 AU. T The Amuc1100 protein (1100) showed significant activity against TLR2 cells at background levels exceeding approximately 2.0 AU.

[0178] As can be seen in Figure 5, all of the natural variants tested here were able to activate the TLR2 receptor. Surprisingly, the TLR2 activation ability of the Amuc-1100 natural variant (Figure 5) was higher than that of the positive control, reaching approximately 4.5 AU.

[0179] 3D modeling results (data not shown) indicated that deletions in each region may reduce the ability of proteins to interact with the TLR2 receptor. These results point to the importance of dimerization and the presence of long, irregular loops in Amuc-1100 as structural features that improve TLR signaling activity.

[0180] Therefore, the relationship between the deletion of a specific conserved region and its effect on the ability to activate TLR2 is evaluated in Table 6.

[0181] [Table 6]

[0182] In particular, the presence of a beta strand at the N-terminus and a long irregular loop for dimerization is important for (improved) TLR signaling activity.

[0183] (References) TIFF0007914014000029.tif150170

Claims

1. A composition comprising an isolated polypeptide and a pharmaceutically or digestibly acceptable carrier, wherein the isolated polypeptide has an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, A composition in which the polypeptide induces immune signaling and / or affects intestinal barrier function and / or affects glucose and / or cholesterol and / or triglyceride homeostasis.

2. The composition according to claim 1, which is a nutritional composition or a pharmaceutical composition.

3. The composition according to claim 1 or 2, for use in promoting intestinal mucosal immune system function, for maintaining, restoring or improving glucose and / or cholesterol and / or triglyceride homeostasis, or for maintaining, restoring and / or enhancing the physical integrity of the intestinal mucosal barrier in mammals.

4. In mammals, obesity, metabolic syndrome, insulin deficiency or insulin resistance-related disorders, type 2 diabetes, type 1 diabetes, gestational diabetes, pre-eclampsia, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), impaired glucose tolerance, abnormal lipid metabolism, atherosclerosis, hypertension, heart conditions, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, fatty liver, lipid metabolism disorders, immune system dysfunction associated with obesity (weight gain), allergies, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, impaired brain function The composition according to claim 1 or 2 for use in preventing and / or treating other diseases associated with renal function, wound healing, behavioral disorders, alcoholism, cardiovascular disease, high cholesterol, elevated triglycerides, atherosclerosis, sleep apnea, osteoarthritis, gallbladder disease, cancer, and conditions that alter the physical integrity of the intestinal mucosal barrier, such as food allergies, for example, underdeveloped intestines due to premature birth of an infant, exposure to radiation, chemotherapy and / or toxins, autoimmune disorders, malnutrition, sepsis, etc.

5. A composition according to claim 1 or 2, for use in promoting anti-inflammatory activity in the intestines of mammals.

6. A composition according to claim 1 or 2 for use in promoting weight loss in mammals.

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