Amuc-1100 polypeptide variants for influencing immune signaling and / or influencing gut barrier function and / or modulating metabolic status

Abstract

The present invention provides polypeptide variants of an extracellular polypeptide of Akkermansia muciniphila capable of modulating and / or promoting the function of the intestinal mucosal immune system and / or maintaining and / or restoring the metabolic status and / or increasing the physical integrity of the intestinal mucosal barrier in a mammal. The polypeptide variants or host cells comprising the polypeptide variants can be used for the prevention and / or treatment of various disorders that benefit from an increased physical integrity of the intestinal mucosal barrier and / or an improved function of the intestinal mucosal immune system and metabolic status.

Description

Technical Field

[0001] This invention relates to the fields of intestinal mucosal immune system, intestinal mucosal barrier, pharmaceutical, food, or feed compositions comprising peptides and / or host cells, wherein the peptides and / or host cells are capable of modulating and / or promoting the function of the intestinal mucosal immune system and / or maintaining and / or restoring and / or increasing the physical integrity of the intestinal mucosal barrier, and / or maintaining, restoring, or improving glucose and / or cholesterol and / or triglyceride homeostasis in mammals (e.g., humans). Background Technology

[0002] Increased permeability or hyperpermeability of the intestinal mucosal barrier is believed to play a role in a variety of diseases and conditions, such as bowel-related diseases, autoimmune diseases, allergic reactions, cancer, type 2 diabetes, obesity, depression, anxiety, and many others. Therefore, there is growing interest in the role of intestinal mucosal barrier dysfunction in the pathogenesis of many gastrointestinal (GI) diseases in mammals.

[0003] Under normal conditions, the intestinal mucosal barrier acts as a selective barrier, allowing the absorption of nutrients, electrolytes, and water while preventing exposure to harmful macromolecules, microorganisms, diet, and microbial antigens (such as food allergens). The intestinal mucosal barrier is essentially composed of a layer of mucus and a lower layer of epithelial cells (referred to herein as "intestinal epithelial cells"). These intestinal epithelial cells are tightly connected to each other through so-called "tight junctions," which are essentially the physical connections between the membranes of two intestinal epithelial cells. Maintaining the intestinal mucosal barrier, particularly the physical integrity of the intestinal epithelial cell layer (i.e., maintaining tight junctions between cells), is crucial for protecting the host from the migration of pathogenic microorganisms, antigens, and other undesirable agents from the intestine into the bloodstream.

[0004] The intestinal mucosal barrier is also about 10 12 -10 14A large number of symbiotic microorganisms colonize the intestinal mucosal barrier, primarily anaerobic or microaerophilic bacteria, most of which live in symbiosis with their host. These bacteria are beneficial to their host in many ways. They provide protection against pathogens within the host and play a nutritional role by synthesizing vitamin K and some components of the B vitamins. Furthermore, the intestinal mucosal barrier has evolved a complex "intestinal mucosal immune system" to distinguish symbiotic bacteria (i.e., beneficial bacteria) from pathogens and other harmful agents. The intestinal mucosal immune system is a component of the intestinal mucosal barrier, comprising lymphoid tissue and specific immune cells (i.e., lymphocytes and plasma cells), which are widely distributed throughout the intestinal mucosal barrier. One of the microorganisms that naturally colonizes the mucosa of healthy individuals is *Akkermansia muciniphila*, which has been shown to enhance intestinal barrier function (Everard et al., PNAS 110 (2013) 9066-71; Reunanen et al., Appl Enⅵron Microbiol March 2020 15), thereby influencing diseases associated with impaired intestinal barrier function.

[0005] In some cases, the intestinal mucosal barrier may be vulnerable to attack by various infectious organisms or agents that are normally unable to cross it, but may still manage to do so (e.g., through gaps created by loose tight junctions between intestinal epithelial cells). Organisms or other agents that cross the intestinal mucosal barrier may cause disease or other undesirable conditions in the host (e.g., allergies). Examples of such diseases include obesity, metabolic syndrome, diseases related to insulin deficiency or insulin resistance, type 2 diabetes, type 1 diabetes, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, heart disease, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, 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, alcohol dependence, cardiovascular disease, high cholesterol, elevated triglycerides, atherosclerosis, sleep apnea, osteoarthritis, gallbladder disease, and cancer.

[0006] Conversely, conditions such as those described above, as well as other conditions such as food allergies, intestinal immaturity due to premature birth, exposure to radiation, chemotherapy and / or toxins, autoimmune diseases, malnutrition, sepsis, etc., may alter the physical integrity of the intestinal mucosal barrier (i.e., cause the loosening of the tight junctions between intestinal epithelial cells), which in turn may allow unwanted microorganisms or other factors to cross the host intestinal mucosal barrier.

[0007] Over the years, several vaccines and / or antibodies have been developed targeting such microorganisms or agents. However, the success rate of such approaches has been reduced because vaccines or antibodies are not effective at targeting or eradicating certain microorganisms or pathogens.

[0008] Other approaches aimed at preventing harmful microorganisms and other factors from crossing the host intestinal mucosal barrier and / or at preventing excessive permeability of the intestinal mucosal barrier have also been explored. For example, compositions containing glutamate have been developed to prevent and / or treat conditions associated with excessive intestinal mucosal barrier permeability (WO 01 / 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, pages 639-654). Formulations containing arabinoxylan have also been developed to enhance beneficial effects on GI bacteria living near the intestinal mucosal barrier to modulate the intestinal mucosal barrier (US2012 / 0230955).

[0009] WO2016177797 discloses a polypeptide derived from Akkermansia myxophilus, namely polypeptide Amuc-1100, which can maintain, restore or increase the physical integrity of the intestinal mucosal barrier in mammals and / or maintain, restore or improve glucose and / or cholesterol and / or triglyceride homeostasis, and / or improve the metabolic or immune status of mammals, particularly by interacting with toll-like receptor 2 (TLR2) and / or regulating TLR2 and / or NFk-B-dependent signaling pathways, and / or promoting the release of cytokines (e.g., IL-6, IL-8 and IL-10) from immune cells located near the mucosal intestinal barrier in mammals (e.g., humans).

[0010] One object of the present invention is to provide new or improved factors and / or compositions containing such factors, suitable for maintaining and / or restoring and / or increasing the physical integrity of the intestinal mucosal barrier and / or preventing excessive permeability of the intestinal mucosal barrier in mammals (e.g., humans), and / or maintaining and / or restoring and / or improving homeostasis of glucose and / or cholesterol and / or triglycerides in mammals, and preferably thereby preventing or treating diseases or conditions in said mammals associated with poor permeability of the intestinal mucosal barrier and / or imbalance of glucose and / or cholesterol and / or triglyceride homeostasis. Optionally or additionally, an object of the present invention is to provide further or improved factors and / or compositions containing such factors, suitable for modulating and / or promoting the function of the intestinal mucosal immune system in mammals. Summary of the Invention

[0011] The inventors have identified a distal variant of the polypeptide Amuc-1100 from Akkermansia glycaniphila that modulates and / or promotes the function of the intestinal immune system in mammals (e.g., humans) and / or maintains and / or restores and / or increases the physical integrity of the intestinal mucosal barrier, and / or maintains and / or restores and / or improves glucose and / or cholesterol and / or triglyceride homeostasis. This is surprising because previous studies have reported that Akkermansia glycaniphila has no homolog of Amuc-1100 (see Xing et al. (2019; Genes & Genomics 41:1253–1264)).

[0012] Without wishing to be bound by any theory, it is believed that the beneficial effects of the peptides of the present invention derive from their ability to interact with the TLR2 signaling pathway present on the surface of immune cells located near the intestinal mucosal barrier in mammals. More specifically, the inventors have discovered that the peptides taught herein are capable of interacting with TLR2 present on the surface of immune cells and / or regulating and / or stimulating the TLR2 signaling pathway in immune cells located near the intestinal mucosal barrier, thereby stimulating said immune cells to secrete cytokines (e.g., IL-6, IL-8, and IL-10).

[0013] Furthermore, the inventors have discovered that the peptides taught herein can modulate and / or increase transepithelial resistance of the intestinal mucosal barrier in mammals. Since increased transepithelial resistance is a measure of decreased intestinal mucosal barrier permeability, it is believed that the peptides taught herein, including their variants, can modulate the physical integrity of the intestinal mucosal barrier, particularly at the level of tight junctions between epithelial cells.

[0014] These effects are believed to combine to lead to improvements or increases in the function of the intestinal mucosal immune system (e.g., the release of more cytokines at the intestinal mucosal barrier) and in the physical integrity of the intestinal mucosal barrier, particularly the level of connectivity between intestinal epithelial cells (i.e., through tighter intercellular junctions).

[0015] Furthermore, treatment of HFD-fed mice with the peptide according to the invention resulted in a significant reduction in the increase in body weight and fat mass without affecting food intake. Treatment with this peptide also corrected HFD-induced hypercholesterolemia, with a significant reduction in serum high-density lipoprotein cholesterol and a similar trend in low-density lipoprotein cholesterol. In addition, administration of this peptide reduced glucose intolerance with the same or better efficacy as the Amuc-1100 peptide from Akkermansia myxophilus.

[0016] Finally, it is known that metformin stimulates the growth of Akkermansia (Lee HandKo G, Appl Englion Microbiol. 2014 Oct; 80(19): 5935-43), therefore Akkermansia and its extracellular peptides with similar functions to this polypeptide may have similar effects on gestational diabetes and preeclampsia as metformin (Syngelaki et al. N Engl J Med. 2016 Feb4; 374(5): 434-43).

[0017] polypeptide

[0018] This invention teaches the isolation of polypeptides, characterized in that the isolated polypeptides

[0019] a) Has 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 (over the entire length) with SEQ ID NO:9;

[0020] b) Contains at least 1, 2, 3, 4, 5, 6, or 7 of the following amino acid residue groups.

[0021] i. R, S, I, S, A and / or P (or their conservative substitutes), which are located at positions 1, 2, 8, 20, 23 and / or 27 in SEQ ID NO:9, respectively;

[0022] ii. C, K, K, I and / or T (or their conservative substitutes), whose positions correspond to positions 92, 93, 95, 97 and / or 100 in SEQ ID NO:9, respectively;

[0023] iii. W, L, G and / or F (or their conservative substitutes), which are located at positions 105, 106, 107 and / or 108 in SEQ ID NO:9, respectively;

[0024] iv. F and / or E (or their conservative substitutes), which are located at positions 126 and / or 127 in SEQ ID NO:9, respectively;

[0025] v. V, Y and / or R (or their conservative substitutes), which are located at positions 149, 150 and / or 151 in SEQ ID NO:9, respectively;

[0026] ⅵ. P, E, I, F, Q, R, S and / or V (or their conservative substitutes), whose positions correspond to positions 179, 181, 182, 184, 185, 188, 190 and / or 191 in SEQ ID NO:9, respectively;

[0027] ⅶ. P, P, P, A, A, P, G, T, A, E, A, P, Q, K, G and / or E (or their conservative substitutes), which are located at positions 220, 222, 229, 230, 231, 234, 248, 258, 260, 262, 264, 172, 175, 279, 283 and / or 285 in SEQ ID NO:9, respectively.

[0028] The peptides defined above can affect immune signaling and / or intestinal barrier function and / or glucose and / or cholesterol and / or triglyceride homeostasis. Preferably, the isolated peptides do 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 peptides taught herein are capable of binding to Toll-like receptor 2 (TLR2).

[0029] In one embodiment, the polypeptide as defined above is contained in the composition, preferably further comprising a carrier, such as a physiologically acceptable, pharmaceutically acceptable, food-acceptable, or nutritionally acceptable carrier. The carrier can be any inert carrier. For example, non-limiting examples of suitable physiologically or pharmaceutically acceptable carriers include any known physiological or pharmaceutical carriers, buffers, diluents, and excipients.

[0030] In one embodiment, the peptides and variants taught herein are capable of stimulating the TLR2 signaling pathway in cells, stimulating the release of cytokines (e.g., IL-6, IL-8, IL-10, etc.) from cells and / or increasing transepithelial resistance (TER) of mammalian (e.g., human) cells, and / or improving the metabolic or immune status of mammals (e.g., mice or humans).

[0031] As described under a), the polypeptides taught herein may also include variants of the amino acid sequence of SEQ ID NO:9, said 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 substitution, deletion, or insertion of one or more amino acids. Preferably, such polypeptides contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, up to 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions, or insertions, compared to the polypeptide having the amino acid sequence of SEQ ID NO:9. As described above, the polypeptide may have 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, for example, at least 50% sequence identity with SEQ ID NO:9. The polypeptide according to the invention may or may not contain a leader sequence.

[0032] In one embodiment, the polypeptide according to the invention comprises:

[0033] - At least 5 amino acid residues (or their conserved substitutes) as defined in i);

[0034] - At least four amino acid residues (or their conserved substitutes) as defined in ii);

[0035] - At least 3 amino acid residues (or their conserved substitutes) as defined in iii);

[0036] - At least one amino acid residue (or its conserved substitute) as defined in iv);

[0037] - At least two amino acid residues (or their conserved substitutes) as defined by v);

[0038] - At least 7 amino acid residues (or their conserved substitutes) as defined in (vi); and / or

[0039] At least 15 (or at least 12) amino acid residues (or their conserved substitutes) as defined in (vii). Additionally, or concurrently, the polypeptides taught herein may specifically comprise the following groups of amino acid residues as defined above.

[0040] -i);

[0041] -ⅰ) and ⅶ);

[0042] -i), ii), vi) and vi);

[0043] -i), iii), iv) and vi);

[0044] -ⅰ), ⅱ), ⅲ), ⅳ), v), ⅵ), ⅶ).

[0045] Additionally, or simultaneously, the polypeptides taught herein may have at least 75% sequence identity with SEQ ID NO:9, for example, over the entire length.

[0046] In a preferred embodiment, the isolated polypeptide according to the invention further comprises amino acid residues S, N, E, N, (A), P, Q, L and / or L (or their conserved substitutions) at positions corresponding to 28, 29, 35, 37, (40), 71, 78, 81 and / or 88 in SEQ ID NO:9, respectively. Preferably, it comprises at least eight of the amino acid residues.

[0047] In another preferred embodiment, the isolated polypeptide according to the invention 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 116, 124, 136, 142, 148, 175, 198, 204, 212, 213, 289, 295, 298 and / or 301 in SEQ ID NO:9. Preferably, it comprises at least 13 (or at least 11) of the amino acid residues.

[0048] The isolated polypeptide according to the present invention can be a natural variant of the polypeptide according to SEQ ID NO:9, such as a naturally occurring polypeptide with the same function or a synthetic polypeptide with the same function, i.e., it can affect immune signaling and / or intestinal barrier function and / or glucose and / or cholesterol and / or triglyceride homeostasis. The polypeptide is capable of binding to Toll-like receptor 2 (TLR2).

[0049] The polypeptides taught herein may be preceded by an N-terminal signal sequence that stimulates the polypeptide to be secreted from cells. 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 naturally occurring N-terminal signal sequence of the predicted Amuc-1100 polypeptide. However, other N-terminal signal sequences capable of causing Amuc-1100 to be secreted from cells may also be used. For example, a truncated or extended version of the naturally occurring N-terminal signal sequence of the predicted Amuc-1100 polypeptide may be used, as long as such N-terminal signal sequence allows Amuc-1100 to be secreted from cells. Alternatively, a non-naturally occurring N-terminal signal sequence may be employed. Those skilled in the art can identify N-terminal signal sequences suitable for the present invention. Therefore, the polypeptides of the present invention may comprise the N-terminal amino acid sequence of SEQ ID NO:3 from their amino acid sequence.

[0050] Amino acid sequence identity can be determined by any suitable method available in the art. For example, amino acid sequence identity as defined above can be determined by pairwise alignment using the Needleman and Wunsch algorithm with the GAP default parameters. It should also be understood that many methods can be used to identify, synthesize, or isolate variants of the peptides taught herein, such as Western blotting, immunohistochemistry, ELISA, amino acid synthesis, etc.

[0051] It should also be understood that any variant of the polypeptides taught herein performs the same function and / or has the same activity as the polypeptides taught herein. The function or activity of any variant can be determined by any method known in the art, which will be considered suitable for these purposes by those skilled in the art.

[0052] Polynucleotides

[0053] The present invention also teaches a nucleic acid molecule, such as an isolated, synthetic, or recombinant nucleic acid molecule, comprising a nucleic acid sequence encoding a polypeptide taught herein, such as the nucleic acid sequence shown in SEQ ID NO:29 or SEQ ID NO:33, or a nucleic acid sequence 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.

[0054] The term "isolated nucleic acid molecule" (e.g., cDNA, genomic DNA, or RNA) includes naturally occurring, artificial, or synthetic nucleic acid molecules. Nucleic acid molecules can encode any polypeptide taught herein. These nucleic acid molecules can be used to produce the polypeptides taught herein. Due to the degeneracy of the genetic code, different nucleic acid molecules may encode the same polypeptide (e.g., a polypeptide containing the amino acid sequence of SEQ ID NO:9).

[0055] It should also be understood that many methods can be used to identify, synthesize, or isolate variants of the polynucleotides taught in this article, such as nucleic acid hybridization, PCR techniques, in silico analysis, and nucleic acid synthesis.

[0056] The nucleic acid molecules taught herein may include nucleic acid molecules encoding an N-terminal signal sequence adapted to stimulate the secretion of the polypeptide taught herein from its host cell. The nucleic acid molecule encoding the N-terminal signal sequence may comprise a nucleic acid sequence as described in SEQ ID NO:4.

[0057] In one embodiment, the nucleic acid molecule taught herein can be included in a chimeric gene, wherein the nucleic acid molecule is operatively linked to a promoter. Therefore, the present invention also relates to chimeric genes comprising the nucleic acid molecule taught herein.

[0058] Any promoter known in the art and suitable for linking with the nucleic acid molecules taught herein can be used. Non-limiting examples of suitable promoters include promoters that allow constitutive or regulatory expression, weak expression, and strong expression, etc. Any method known in the art can be used to include the nucleic acid molecules taught herein in chimeric genes.

[0059] It may be advantageous to operatively link the nucleic acid molecules taught in this article to so-called “constitutive promoters”.

[0060] Furthermore, it may be advantageous to operatively link the polynucleotides and their variants taught in this paper to so-called “inducible promoters.” Inducible promoters can be physiologically regulated promoters (e.g., through external application of certain compounds).

[0061] The chimeric genes taught herein can be contained in a “vector” or “nucleic acid construct”. Therefore, the present invention also relates to vectors containing chimeric genes or nucleic acid molecules taught herein.

[0062] In one aspect, the present invention relates to host cells that have been genetically modified to include, for example, nucleic acid molecules, chimeric genes, or vectors taught herein in their genome.

[0063] The genetically modified host cells taught herein can be used for semi-in vivo (ex-vivo) and / or in vitro (in-vivtro) production of the polypeptides and their variants taught herein, either intracellularly or by any means released from the cell. In particular, the polypeptides taught herein can be expressed as soluble or secreted molecules. The genetically modified host cells taught herein can be any host cell suitable for transformation or genetic engineering procedures. Non-limiting examples of suitable host cells include culturable cells, such as any prokaryotic or eukaryotic cell. In one embodiment, the polypeptides according to the invention are expressed in bacteria, such as *Escherichia coli*.

[0064] In one implementation, the host cell taught herein can be any cell that naturally expresses the peptide taught herein or a variant thereof. In this case, the host cell may overexpress the peptide taught herein or a variant thereof.

[0065] In yet another implementation, the host cell taught herein can be any cell that does not naturally express the polypeptide or a variant thereof taught herein.

[0066] In one implementation, the host cells taught herein do not belong to the Akkermansia myxophilia or Akkermansia polysaccharide species.

[0067] In another embodiment, the host cell may belong to the species *Ackermania myxophila* or *Ackermania polymorpha* and be genetically modified to contain an additional copy of the nucleic acid molecule taught herein, or to contain the chimeric gene or vector taught herein. Such *Ackermania myxophila* or *Ackermania polymorpha* cells may overexpress the polypeptides taught herein or variants thereof.

[0068] The host cells taught herein can be genetically modified using any method known in the art. For example, the host cells or organisms taught herein can be genetically modified using a method including the following steps.

[0069] a) Transform host cells with the nucleic acid molecules taught in this paper, such as nucleic acid sequences that can encode the polypeptides and their variants taught in this paper;

[0070] b) Culture the host cells under conditions suitable for allowing the expression of nucleic acid molecules taught herein and / or the production of polypeptides or variants thereof taught herein;

[0071] c) Optionally, host cells capable of expressing the nucleic acid molecules taught herein and / or producing the polypeptides or variants thereof taught herein are selected.

[0072] In one embodiment, the genetically modified host cells taught herein may belong to bacterial species that are naturally occurring or live near or within the intestinal mucosal barrier of mammals. Such bacterial species are generally referred to as “intestinal mucosa-associated bacterial species.” Non-limiting examples of “intestinal mucosa-associated bacterial species” include *Ackermania myxophilus* (ATTC BAA-835), *Faecalibacterium prausnitzⅱ* (A2-165), *Lactobacillus rhamnosus* (ATCC 53103), and *Bifidobacterium breve* (DSM-20213).

[0073] In some embodiments, it is advantageous to genetically modify intestinal mucosa-associated bacteria with any polynucleotides taught herein and their variants, such as by expressing or overexpressing the polynucleotides taught herein or by producing or overproducing the polypeptides taught herein, directly into or within the intestinal mucosal barrier of mammals (e.g., humans). In a preferred embodiment, the intestinal mucosa-associated bacteria can be any bacteria from *Ackermania myxophila* or *Ackermania polyglucosi* species. This overproduction can be achieved by genetic modification tools involving recombinant DNA technology, genome editing (e.g., by using tools based on CRISPR / Cas-like systems), or by classical mutational selection systems.

[0074] In one embodiment, the genetically modified host cell can be any bacteria, particularly those not belonging to species that naturally exist or live near or within the intestinal mucosal barrier of mammals. Non-limiting examples of such bacteria include any beneficial isolated strains of intestinal bacteria, such as probiotics, particularly strains selected from the genera *Lactococcus*, *Lactobacillus*, or *Bifidobacterium*. Additionally, strictly anaerobic intestinal bacteria, such as those genera known to exist in the human gut, can also be used (Rajilic-Stojanoⅵc&de Vos, The first 1000 cultured species of the human gastrointestinal microbiota. FEMS Microbiol Rev. 38:996-1047).

[0075] Methods for producing peptides

[0076] In another aspect, the present invention relates to a method for generating polypeptides (including variants) taught herein, comprising the following steps:

[0077] (a) Culturing host cells as taught herein under conditions that allow the production of the polypeptides or variants thereof; and

[0078] (b) Optionally, the polypeptide produced in step (a) is separated.

[0079] In step (a), the host cells taught herein can be cultured on any known culture medium according to any known culture method. Those skilled in the art will be able to select suitable host cells and will be able to establish suitable conditions that allow for peptide production.

[0080] Alternatively, peptides can be produced by a method including the following steps:

[0081] (a) Culture Akkermansia muciniphila or Akkermansia spp. in a suitable culture medium; and

[0082] (b) Optionally, the polypeptide produced in step (a) is separated.

[0083] The polypeptide produced in step (a) of the above method can be isolated by any method known in the art. Those skilled in the art will be able to isolate the polypeptide produced from such a culture medium.

[0084] Suitable culture media were taught, for example, by Derrien et al. (2004, Int. J. Syst. Evol. Microbiol. 54:1469-76). Derrien et al. taught the isolation of *Ackermania muc*. T The strain was trained to grow on a basal anaerobic medium containing hog gastric mucin as the sole carbon and nitrogen source. The authors also taught that *Ackermannii myxotroph* can grow on nutrient-rich media, such as Columbia broth (CB) and Brain Heart Infusion (BHI) broth, or on a basal medium containing glucose and high concentrations of casitone and yeast extract. Similarly, Lukovac et al. (mBio) taught the growth of *Ackermannii myxotroph* on a basal medium containing glucose and fucose, along with a high amount of casitone (2014, mBio 01438-14). Similar methods can be used for *Ackermannii polysaccharide*.

[0085] Composition

[0086] In another respect, the present invention relates to compositions comprising any polypeptides taught herein.

[0087] In another aspect, the present invention relates to compositions comprising host cells as taught herein. The host cells can be in quantities of about 10... 4 To about 1015 The amount present is approximately 10 colony-forming units (CFU). For example, the effective amount in host cells could be approximately 10 5 CFU to approximately 10 14 One CFU, preferably about 10 6 CFU to approximately 10 13 One CFU, preferably about 10 7 CFU to approximately 10 12 One CFU, more preferably about 10 8 CFU to approximately 10 12 The amount is one CFU. The host cell can be alive or dead. The effectiveness of the host cell is related to the presence of the peptide taught in this article.

[0088] In one embodiment, the compositions taught herein further comprise a carrier, such as a physiologically acceptable, pharmaceutically acceptable, food-acceptable, or nutritionally acceptable carrier. The carrier can be any inert carrier. For example, non-limiting examples of suitable physiologically or pharmaceutically acceptable carriers include any well-known physiological or pharmaceutical carriers, buffers, diluents, and excipients. It should be understood that the selection of a suitable physiological or pharmaceutical carrier, food carrier, or nutritional carrier will depend on the intended mode of administration (e.g., oral) and the intended form of the composition (e.g., beverage, yogurt, powder, capsule, etc.). Those skilled in the art know how to select a suitable carrier, such as a physiologically acceptable, nutritionally acceptable, or pharmaceutically acceptable carrier, that is suitable for or compatible with the compositions taught herein.

[0089] In one embodiment, the compositions taught herein can be nutritional or food compositions. For example, the compositions taught herein can be foods, food supplements, feeds, or feed additives, such as dairy products, such as fermented dairy products, such as yogurt or yogurt beverages. In this case, the composition can contain a nutritionally acceptable or food-acceptable carrier, which can be a suitable food base.

[0090] In one embodiment, the composition taught herein may be a pharmaceutical composition. The pharmaceutical composition may also be used as a supplement (e.g., a food supplement). In addition to the peptides and / or host cells taught herein, the pharmaceutical compositions taught herein may contain a pharmaceutically, nutritionally, or dietaryly or physiologically acceptable carrier. Preferred forms depend on the intended administration route and (therapeutic) application. The carrier may be any compatible, physiologically acceptable, non-toxic substance suitable for delivering the peptides and / or host cells taught herein to the gastrointestinal tract of a mammal (e.g., a human), preferably near or within the intestinal mucosal barrier (more preferably the colonic mucosal barrier). For example, sterile water or inert solids may be used as carriers, typically supplemented with pharmaceutically acceptable adjuvants, buffers, dispersants, etc.

[0091] The compositions taught herein may be in liquid form, such as a stable suspension of the peptides or host cells taught herein, or in solid form, such as a powder of lyophilized host cells taught herein. In the case that the host cells taught herein are lyophilized, cryoprotectants such as lactose, trehalose, or glycogen may be used. For oral administration, the peptides or lyophilized host cells 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 peptides or host cells taught herein may be encapsulated together with inactive ingredients and powder carriers (e.g., glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate, etc.) in capsules (e.g., gelatin capsules).

[0092] In one embodiment, the compositions taught herein may comprise one or more components adapted to promote the survival and / or viability and / or maintain the integrity of the peptides and / or host cells taught herein during storage and / or exposure to bile and / or passage through the gastrointestinal tract of mammals (e.g., humans). Non-limiting examples of such components include enteric coatings and controlled-release agents that allow passage through the stomach. Those skilled in the art know how to select suitable components to ensure that the active ingredient (whether a peptide or a host cell) reaches its intended destination and functions there.

[0093] In one embodiment, the compositions taught herein may further comprise a mucosal binder or a mucosal binding peptide. As used herein, the terms "mucosal binder" or "mucosal binding peptide" refer to agents or peptides capable of attaching to the intestinal mucosal barrier of mammals, such as humans.

[0094] Additionally, specific docking systems can be used to connect the peptides taught herein, the cells that produce said peptides, or even living or dead non-producing cells. Binding can occur at the C-terminus or N-terminus, whichever appears most efficient, and the use of spacer peptides is also described. Examples include the use of a LysM-based peptidoglycan binding system (Visweswaran GR et al. 2014, Appl Microbiol Biotechnol. 98:4331-45.). Furthermore, various mucosa-binding peptides have been disclosed in the art. Non-limiting examples of mucosa-binding peptides include bacterial toxin membrane-binding subunits, including, for example, the B subunit of cholera toxin, the B subunit of heat-labile enterotoxin from Escherichia coli, the S2, S3, S4 and / or S5 subunits of Bordetella pertussis toxin, the B fragment of diphtheria toxin, and membrane-binding subunits of Shiga toxin or Shiga-like toxins. Other suitable mucosa-binding peptides include bacterial fimbriae, such as *Escherichia coli* fimbriae K88, K99, 987P, F41, FAIL, CFAⅲICES1, CS2 and / or CS3, CFAⅱVICS4, CS5 and / or CS6, and P fimbriae. Other non-limiting examples of fimbriae include *Bordetella pertussis* filamentous hemagglutinin, *Vibrio cholerae* toxin-coregulate pilus (TCP), mannose-sensitive hemagglutinin (MSHA), and fucose-sensitive hemagglutinin (PSHA). Other mucosal binding agents include viral attachment proteins, including influenza virus and Sendai virus hemagglutinins, and animal agglutinins or agglutinin-like molecules, including immunoglobulin molecules or fragments thereof, calcium-dependent (type C) agglutinins, selectins, collagen agglutinins or helixpomatishemagglutinin, and phytohemagglutinins with mucosal binding subunits, including concanavalin A, wheat germ agglutinin, phytohemagglutinin, abrin, ricin, etc. The advantage of this delivery method is that it avoids the use of live recombinant organisms.

[0095] Although not required, it is advantageous to add one or more mucosal binding agents or mucosal binding peptides to the compositions taught herein in order to target the peptides or host cells taught herein to the intestinal mucosal barrier.

[0096] The compositions taught herein may further comprise ingredients selected from prebiotics, probiotics, carbohydrates, peptides, lipids, vitamins, minerals, pharmaceuticals, preservatives, antibiotics, or any combination thereof.

[0097] In one embodiment, the compositions taught herein may further comprise one or more ingredients that further enhance the nutritional and / or therapeutic value of the compositions taught herein. For example, the addition of one or more ingredients selected from proteins, amino acids, enzymes, mineral salts, vitamins (e.g., thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, niacin, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, vitamin B12, para-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, trace element components (e.g., compounds of cobalt, copper, manganese, iron, zinc, tin, nickel, chromium, molybdenum, iodine, chlorine, silicon, vanadium, selenium, calcium, magnesium, sodium, and potassium, etc.) (e.g., nutrients, veterinary drugs, or pharmaceuticals, etc.) may be advantageous. Those skilled in the art are familiar with methods and ingredients suitable for enhancing the nutritional and / or therapeutic / pharmaceutical value of the compositions described herein.

[0098] In one implementation, the host cell may be incorporated in lyophilized or microencapsulated form, or in any other form that preserves the activity and / or viability of the host cell (such as a bacterial strain) (as reviewed, for example, by Solanki et al. BioMedRes.Int.2013, Article ID 620719).

[0099] Treatment

[0100] On the other hand, the present invention relates to methods for treating and / or preventing diseases or conditions selected from: obesity, metabolic syndrome, insulin deficiency or insulin resistance-related conditions, type 2 diabetes, type 1 diabetes, gestational diabetes, preeclampsia, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, heart disease, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, immune system dysfunction related to obesity (weight gain), allergic reactions, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other diseases related to impaired barrier function, wound healing, behavioral disorders. Methods for promoting weight loss in mammals; methods for promoting anti-inflammatory activity in the mammalian gut; methods for promoting the function of the intestinal mucosal immune system in mammals; methods for maintaining, restoring and / or improving glucose and / or cholesterol and / or triglyceride homeostasis; and methods for maintaining, restoring and / or increasing the physical integrity of the intestinal mucosal barrier in mammals. The methods include the step of administering an effective amount of the peptides, host cells, or compositions taught herein to a mammal in need.

[0101] 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, enterically, or parenterally. It should be understood that the manner or route of administration will depend on the current circumstances (e.g., individual age, desired site of action, disease condition, etc.) and the intended form of the composition (e.g., pills, liquids, powders, etc.).

[0102] In a preferred embodiment, the polypeptide, host cell, or composition taught herein are administered orally.

[0103] use

[0104] In another aspect, the present invention relates to the use of the nucleic acid molecules, chimeric genes, and / or vectors taught herein for the production of peptides and / or for the production of host cells taught herein. The peptides and / or host cells taught herein, compared to host cells (e.g., bacteria) not genetically modified with the polynucleotides, chimeric genes, or vectors taught herein, may have an enhanced ability to interact with TLR2 receptors on cells and / or an enhanced ability to stimulate TLR2 signaling pathways in cells, and / or an enhanced ability to stimulate the production of cytokines (particularly IL-1β, IL-6, IL-8, IL-10, and TNF-α) from cells, and / or an enhanced ability to increase TER in mammalian (e.g., human) cells.

[0105] On the other hand, the present invention relates to the use of the polypeptides, host cells, or compositions taught herein as medicines; particularly for promoting the function of the intestinal mucosal immune system in mammals or for maintaining, restoring, and / or increasing the physical integrity of the intestinal mucosal barrier; for maintaining, restoring, and / or improving glucose and / or cholesterol and / or triglyceride homeostasis in mammals; for preventing and / or treating diseases or conditions selected from: obesity in mammals, such as diet-induced obesity, metabolic syndrome, insulin deficiency or insulin resistance-related conditions, type 2 diabetes, type 1 diabetes, gestational diabetes, preeclampsia, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, heart disease, stroke, non-alcoholic lipoproteinemia. Fatty liver disease, alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, immune system dysfunction associated with obesity (weight gain), allergic reactions, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other diseases associated with impaired barrier function, wound healing, behavioral disorders, alcohol dependence, 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, intestinal immaturity (e.g., due to premature birth), exposure to radiation, chemotherapy and / or toxins, autoimmune diseases, malnutrition, sepsis, etc.; use for promoting anti-inflammatory activity in the mammalian gut; or use for promoting weight loss in mammals.

[0106] In one implementation, the mammal, such as a human, can be of any age group (e.g., infant, adult, elderly) and any sex (male and female). In another implementation, the mammal can be an infant (e.g., newborn, baby, toddler, etc.), especially a premature infant.

[0107] The mammal can be any mammal, such as humans, non-human primates, rodents, cats, dogs, cows, horses, etc. In a preferred embodiment, the mammal is a human.

[0108] The isolated polypeptide of the present invention is further characterized in that the polypeptide may alternatively have the following features

[0109] a) Has 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 (over the entire length) with SEQ ID NO:5;

[0110] b) Contains at least 1, 2, 3, 4, 5, 6, or 7 of the following amino acid residue groups.

[0111] i. R, S, I, S, A and / or P (or their conservative substitutes), which are located at positions 6, 7, 13, 22, 25 and / or 30 in SEQ ID NO:5, respectively;

[0112] ii. C, K, K, I and / or T (or their conservative substitutes), which are located at positions 88, 89, 91, 93 and / or 96 in SEQ ID NO:5, respectively;

[0113] iii. W, L, G and / or F (or their conservative substitutes), which are located at positions 101, 102, 103 and / or 104 in SEQ ID NO:5, respectively;

[0114] iv. F and / or E (or their conservative substitutes), which are located at positions 122 and / or 123 in SEQ ID NO:5, respectively;

[0115] v. V, Y and / or R (or their conservative substitutes), which are located at positions 145, 146 and / or 147 in SEQ ID NO:5, respectively;

[0116] ⅵ. P, E, I, F, Q, R, S and / or V (or their conservative substitutes), whose positions correspond to positions 174, 176, 177, 179, 180, 183, 185 and / or 186 in SEQ ID NO:5, respectively;

[0117] ⅶ. P, P, P, A, A, P, G, T, A, E, A, P, Q, K, G and / or E (or their conservative substitutes), which are located at positions 215, 217, 221, 222, 223, 226, 234, 239, 241, 243, 245, 150, 153, 257, 261 and / or 263 in SEQ ID NO:5, respectively.

[0118] The aforementioned polypeptide can affect immune signaling and / or intestinal barrier function and / or 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. This polypeptide is capable of binding to Toll-like receptor 2 (TLR2).

[0119] In one embodiment, the polypeptide as defined above is contained in the composition, preferably further comprising a carrier, such as a physiologically acceptable, pharmaceutically acceptable, food-acceptable, or nutritionally acceptable carrier. The carrier can be any inert carrier. For example, non-limiting examples of suitable physiologically or pharmaceutically acceptable carriers include any well-known physiological or pharmaceutical carriers, buffers, diluents, and excipients.

[0120] As described under a), the polypeptide may further comprise a variant of the amino acid sequence of SEQ ID NO:5, the amino acid sequence of which has more than 25% sequence identity with the amino acid sequence of SEQ ID NO:5. The variant of the polypeptide also includes polypeptides derived from the polypeptide having the amino acid sequence of SEQ ID NO:5 by substitution, deletion, or insertion of one or more amino acids. Preferably, such polypeptides contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 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 previously stated, the polypeptide may have 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 over its entire length, for example, at least 50% sequence identity with SEQ ID NO:5. The polypeptide according to the invention may or may not contain a leader sequence.

[0121] In one embodiment, the polypeptide according to the invention comprises:

[0122] - At least 5 amino acid residues (or their conserved substitutes) as defined in i);

[0123] - At least four amino acid residues (or their conserved substitutes) as defined in ⅱ);

[0124] - At least three amino acid residues (or their conserved substitutes) as defined in iii);

[0125] - At least one amino acid residue (or its conserved substitute) as defined in iv);

[0126] - At least two amino acid residues (or their conserved substitutes) as defined in (v);

[0127] - At least 7 amino acid residues (or their conserved substitutions) as defined in (vi); and / or

[0128] - At least 15 (or at least 12) amino acid residues (or their conserved substitutes) as defined in (vii).

[0129] Additionally, or concurrently, the polypeptides taught herein may specifically comprise the following groups of amino acid residues as defined above:

[0130] -i);

[0131] -i) and ⅶ);

[0132] -i), ⅱ), ⅵ) and ⅶ);

[0133] -i), ⅲ), ⅳ) and ⅶ);

[0134] -i), ⅱ), ⅲ), ⅳ), v), ⅵ), ⅶ).

[0135] Additionally, or simultaneously, the polypeptides taught herein may have at least 75% sequence identity with SEQ ID NO:5, for example, over the entire length.

[0136] In a preferred embodiment, the isolated polypeptide according to the invention 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 in SEQ ID NO:5, respectively. Preferably, it comprises at least eight of the amino acid residues.

[0137] In another preferred embodiment, the isolated polypeptide according to the invention further comprises 112, 120, 132, 138, 144, 170, 193, respectively corresponding to SEQ ID NO:5. Amino acid residues P, L, N, G, K, W, I, Y, R, I, V, L, F and / or P (or their conserved substitutes) at positions 199, 207, 208, 297, 273, 276 and / or 279. Preferably, it contains at least 13 (or at least 11) of the amino acid residues.

[0138] The isolated polypeptide according to the present invention can be a natural variant of the polypeptide according to SEQ ID NO:5, such as a naturally occurring polypeptide with the same function or a synthetic polypeptide with the same function, i.e., it can affect immune signaling and / or intestinal barrier function and / or glucose and / or cholesterol and / or triglyceride homeostasis. The polypeptide is capable of binding to Toll-like receptor 2 (TLR2).

[0139] The polypeptide isolated according to the present invention can be selected from:

[0140] - A separate polypeptide (over the entire length) 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:5;

[0141] - A separate polypeptide (over the entire length) 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;

[0142] - A separate polypeptide (over the entire length) 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:7;

[0143] - A separated polypeptide (overall length) 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:8; and

[0144] - A separate polypeptide (overall length) 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:9.

[0145] Preferably, it contains (groups of) (conserved) amino acid residues taught herein.

[0146] General definition

[0147] In the context of this invention, the term "peptide" is equivalent to the term "protein." A polypeptide has a specific amino acid sequence. "Variants" of the polypeptides of this invention preferably have an amino acid sequence with at least 25% sequence identity to a reference polypeptide. The polypeptide of this invention is isolated when it is no longer in its native environment, i.e., when it is no longer in a fimbriae environment, and / or when it is no longer in a cellular environment, such as Akkermansia muciniphila cells or Akkermansia glycanophila cells. A leader sequence is a region (encoded) between the promoter and the coding region that participates in the regulation of expression. A leader sequence (or a portion thereof) can be translated into a leader peptide, but unlike a signal peptide, a leader peptide is never part of a structural protein.

[0148] As used in this article, "conserved substitutions" can refer to the substitution of one or more amino acids in a polypeptide without substantially losing its functionality. It is well known that one amino acid can be substituted for another without sacrificing the polypeptide's activity. For example, the following amino acids can typically be interchanged:

[0149] Ala, Ser, Thr, Gly (small aliphatic, nonpolar, or slightly polar residues)

[0150] Asp, Asn, Glu, Gln (polar, negatively charged residues and their amides)

[0151] His, Arg, Lys (polar, positively charged residues)

[0152] Met, Leu, Ile, Val (Cys) (large aliphatic, nonpolar residues)

[0153] Phe, Ty, Trp (large aromatic residues)

[0154] (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)

[0155] Preferred "substitutions" are those that are conservative, i.e., in which residues are substituted by another of the same general types. When making changes, the hydrophobicity index of the amino acid can be taken into account (see, for example, Kyte et al., J. Mol. Biol. 157, 105-132 (1982)). It is known in the art that certain amino acids can be substituted by other amino acids having similar hydrophobicity indices or scores and still produce polypeptides with similar biological activities. When making such changes, substitution of amino acids with a hydrophobicity index within ±2 is preferred, within ±1 is more preferred, and within ±0.5 is even more preferred. Similarly, the selected amino acid can be substituted by other amino acids having similar hydrophilicity, as described in U.S. Patent No. 4,554,101. When making such changes, as with the hydrophobicity index, substitution of amino acids with a hydrophobicity index within ±2 is preferred, within ±1 is more preferred, and within ±0.5 is even more preferred.

[0156] As used herein, the terms "sequence identity" or "sequence similarity" refer to the situation where an amino acid or nucleic acid sequence shares sequence identity or sequence similarity with another reference amino acid or nucleic acid sequence. "Sequence identity" or "sequence similarity" can be determined by aligning two polypeptide or two nucleotide sequences using a global or local alignment algorithm. Sequences are described as "substantially identical" or "substantially similar" when they share at least a minimum percentage of sequence identity (as defined below) using programs such as GAP or BESTFIT with default parameters. GAP employs the Needleman and Wunsch global alignment algorithm, aligning the two sequences across their entire length to maximize the number of matches and minimize the number of gaps. Typically, GAP default parameters are used, where the gap creation penalty is 50 (nucleotides) / 8 (protein) and the gap extension penalty is 3 (nucleotides) / 2 (protein). For nucleotides, the default scoring matrix used is nwsgapdna; for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignment and percentage sequence identity scores can be determined using computer programs such as the GGWisconsin software package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-375 USA, or EmbossWin version 2.10.0 (using the program “needle”). Alternatively, the percentage of similarity or identity can be determined by searching a database using algorithms such as FASTA, BLAST, etc. Preferably, sequence identity refers to sequence identity across the entire length of the sequence.

[0157] Transepithelial resistance (TER) is a measure of the permeability of the epithelial cell layer in vitro. Increased epithelial cell permeability is associated with weakened tight junctions and decreased TER.

[0158] As used herein, the term "chimeric gene" refers to any gene that is not naturally occurring, i.e., a gene that is not typically found in species in nature, particularly a gene in which one or more portions of its nucleic acid sequence are not associated with each other in nature. For example, a promoter that is inherently unrelated to part or all of the transcribed region or to another regulatory region. The term "chimeric gene" should be understood to include expression constructs in which a heterologous promoter or transcriptional regulatory sequence is operatively linked to one or more coding sequences and an optional 3'-untranslated region (3'-UTR). Additionally, chimeric genes may contain promoters, coding sequences, and optional 3'-UTRs derived from the same species, but they do not occur naturally in this combination.

[0159] As used herein, the term "genetically modified host cell" refers to a cell that has been genetically modified, for example, by introducing a foreign nucleic acid sequence or by specifically altering an endogenous gene sequence. Such cells can be genetically modified by introducing, for example, one or more mutations, insertions and / or deletions in the endogenous gene and / or by inserting genetic constructs (e.g., vectors or chimeric genes) into the genome. Genetically modified host cells can refer to isolated or cultured cells. Genetically modified cells can be "transduced cells," wherein the cells have been infected with, for example, a modified virus; retroviruses can be used, but other suitable viruses, such as lentiviruses, may also be considered. Non-viral methods, such as transfection, can also be used. Therefore, genetically modified host cells can also be "stable transfected cells" or "transiently transfected cells." Transfection refers to the transfer of DNA (or RNA) into a cell using non-viral methods to enable gene expression. Transfection methods are well known in the art, such as calcium phosphate transfection, PEG transfection, and liposome or liposome transfection of nucleic acids. This transfection can be transient or stable, in which cells that have already integrated the gene construct into their genome can be selected.

[0160] As used herein, the term "effective amount" refers to the amount required to achieve the effects taught herein. For example, the effective amount of the peptide or genetically engineered host cell taught herein is the amount effective for modulating and / or promoting the function of the intestinal mucosal immune system and / or maintaining and / or restoring and / or increasing the physical integrity of the intestinal mucosal barrier (e.g., promoting tighter junctions between intestinal epithelial cells), and / or for modulating and / or stimulating the Toll-like receptor signaling pathway (i.e., the TLR2 pathway) in immune cells and / or for increasing the production of cytokines (e.g., IL-6, IL-8, and IL-10) in immune cells, and / or for preventing and / or treating diseases or conditions such as obesity, metabolic syndrome, insulin deficiency or insulin resistance-related conditions, type 2 diabetes, type 1 diabetes, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), glucose intolerance, and abnormal lipid metabolism. Acne, atherosclerosis, hypertension, heart disease, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, immune system dysfunction related to obesity (weight gain), allergic reactions, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other diseases related to impaired barrier function, wound healing, behavioral disorders, alcohol dependence, 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, intestinal immaturity (e.g., due to premature birth), exposure to radiation, chemotherapy and / or toxins, autoimmune diseases, malnutrition, sepsis, etc.

[0161] As used herein, the terms "physiologically acceptable carrier" or "food-acceptable carrier," "nutritionally acceptable carrier," or "pharmaceutically acceptable carrier" refer to physiologically acceptable or food-acceptable carriers or nutritionally acceptable or pharmaceutically acceptable carrier materials, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, relating to providing a drug delivery form of the polypeptides or host cells of the present invention. Each carrier must be... "Acceptable" means compatible with the other components of the composition and does not cause harm to an individual, i.e., suitable for consumption or nutritionally acceptable. The terms "suitable for consumption" or "nutritionalally acceptable" refer to ingredients or substances that are generally considered safe for human (and other mammals) consumption. Non-limiting examples of materials that can be used as physiologically acceptable or nutritionally acceptable or pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered astragalus gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, and safflower oil. Oils, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; (21) other non-toxic and compatible substances used in pharmaceutical preparations, etc. Furthermore, the terms “nutritionally acceptable” and “pharmaceuticalally acceptable” as used herein refer to compositions or pharmaceutical preparations, combinations of materials or compositions, and / or their dosage forms that, within reasonable medical judgment, are suitable for contact with tissues in humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0162] The term "homeostasis" refers to the characteristic of a system in which variables are regulated to maintain stable and relatively constant internal conditions. All animals regulate their blood glucose concentration. The body's regulation of glucose is the process of maintaining "glucose homeostasis." Mammals use different hormones (such as insulin, glucagon, glucagon-like peptide-1, catecholamines, etc.) and different neural pathways (such as neural relays and the gut-brain-peripheral organ axis). The human body maintains a constant glucose level for most of the day, even after a 24-hour fast. Even during prolonged fasting, blood glucose levels decrease only slightly. Insulin, secreted by the beta cells of the pancreas, efficiently transports glucose to these cells by instructing them to conserve more glucose for their own use. If intracellular glucose levels are high, cells convert it into insoluble glycogen to prevent soluble glucose from interfering with cellular metabolism. Ultimately, this lowers blood glucose levels, and insulin helps prevent hyperglycemia. Diabetes occurs when insulin is deficient or cells develop resistance to it. Glucagon, secreted by the α cells of the pancreas, prompts cells to break down stored glycogen or convert non-carbohydrate carbon sources into glucose via gluconeogenesis, thereby preventing hypoglycemia. The control of glucose metabolism also involves many other factors and hormones (such as glucagon-like peptide-1 and catecholamines). Different mechanisms involving neural pathways also contribute to this complex regulation.

[0163] Cholesterol homeostasis is a mechanism that helps maintain a balanced cholesterol level in living organisms. Cholesterol is an essential biomolecule in the human body, performing various physiological functions, such as serving as a precursor for the production of bile acids, vitamin D, and steroid hormones. It is also a key structural element on the cell membrane of every cell in the body. Despite its beneficial and necessary functions, dysregulation 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 controlling cholesterol homeostasis because it not only biosynthesizes cholesterol released into the circulatory system but also breaks down potentially harmful free-floating cholesterol in the blood. High-density lipoproteins (HDLs) are beneficial for maintaining cholesterol homeostasis because they absorb and directly return potentially dangerous cholesterol to the liver, where it is synthesized into harmless bile acids for use by the digestive system. Low-density lipoproteins (LDLs) have fewer beneficial effects because they tend to deposit cholesterol on body cells and arterial walls. Studies show that it is excessively high LDL levels that increase the risk of cardiovascular disease. In healthy individuals, cholesterol homeostasis is tightly regulated by complex feedback loops. In this scenario, if a healthy individual ingests a large amount of dietary cholesterol, the liver's biosynthesis is significantly reduced to maintain balance. In healthy individuals with high baseline LDL cholesterol levels, whether due to years of poor dietary habits or other genetic or medical conditions, the feedback loops and systemic coping mechanisms may not be able to withstand the same high intake, leading to a dangerous disruption of homeostasis.

[0164] "Triglyceride homeostasis" is a mechanism that helps maintain the internal balance of triglycerides in a living organism. Triglyceride metabolism has important clinical significance. Hypertriglyceridemia indicates high (excessively high) blood or serum levels of triglycerides (the most abundant fat molecule). Even in the absence of hypercholesterolemia (high cholesterol levels), elevated triglyceride levels are associated with atherosclerosis and increase the risk of cardiovascular disease. High triglyceride levels also increase the risk of acute pancreatitis. Furthermore, over time, increases in triglyceride (TG) levels increase the risk of diabetes. Studies have shown that insulin resistance is associated with high triglyceride (TG) levels.

[0165] As used herein, the term “about” refers to a normal tolerance range in the art, such as within two standard deviations of the mean. The term “about” can be understood to include values ​​that deviate from the specified value by up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%.

[0166] As used herein, the terms “including” or “contains”, and their variations, refer to a situation where the term is used in its non-restrictive sense to indicate that the items following the word are included, but not excluded unless specifically mentioned. It also includes the more restrictive verbs “consistently of” and “comprises of”.

[0167] Referring to an element with the indefinite article "a" or "an" does not preclude the possibility of multiple elements, unless the context explicitly requires the presence of one and only one element. Therefore, the indefinite article "a" or "an" usually means "at least one". Attached Figure Description

[0168] Figure 1 The following data are shown: A) Total weight gain (g) (n = 8-10). B) Total fat mass gain (g) measured by time-domain nuclear magnetic resonance (NMR) (n = 8-10). C) Daily food intake. D) Plasma VLDL, LDL, and HDL cholesterol levels (n = 8-10). E) Plasma glucose (mg / dL) measured between -30 minutes and 120 minutes after glucose loading. -1 The curve and the mean area under the curve (AUC) (mg dl) -1 .minute -1 G) The ratio of the control to insulin-stimulated p-IRβ on the loading control, determined by densitometric assay (n = 3-5). H and I) The ratio of the control to insulin-stimulated p-Akt on the loading control, determined by densitometric assay. thr308 and p-Akt ser473 The ratio (n = 3-5).

[0169] Figure 2 Conserved residues in the natural variants of Amuc-1100 (SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9) are shown. Viewed from top to bottom and left to right, the first box indicates outward-pointing conserved residues, representing their potential role in interactions. The second, third, and fourth boxes indicate hydrophobic residues, possibly related to structural integrity. The fifth box indicates outward-pointing conserved residues, representing their potential role in interactions. The sixth box indicates a loop.

[0170] Figure 3 The sequence of Amuc-1100 (SEQ ID NO:1) is shown. Conserved residues are marked with circles, and missing portions are marked with gray.

[0171] Figure 4 – Bicistronic design used in expression plasmids. Short peptide translation driven by RBS1 ensures the accessibility of RBS2, which in turn drives the translation of the target protein. Linearization of RBS2 ensures the removal of potentially repressive secondary structures in the 5'UTR, improving translation efficiency (Mutalik et al., 2013; Nieuwkoop et al., 2019).

[0172] Figure 5 SEAP activity (in AU) of positive control (Pam3CSK4, 1 μg / mL), negative control (PBS and DMEM), and various Amuc_1100 variants purified after TEV lysis (all 50 μg / mL). For native variants (above), pTH00x ID refers to the plasmid name used to purify the corresponding protein.

[0173] sequence list

[0174] SEQ ID NO:1: Amino acid sequence of the Amuc-1100 peptide (underlined residues)

[0175] IVNSK RS ELDKK I SIAAKEIK S AN A AEIT P SRS SN EELEK E L N RY A KAVGSLETAYKPFLASSALV P TTPTAF Q NE L KTFRDS L ISS CK K K N I LI T DTSS WLGF QVYSTQA P SVQAAST L G FE LKAINSLV N KLAEC G LSKFI KVY R PQLPIETPANNPEESDEADQAP W TPM P L EI A FQ GD R E SV LKAMNAI TGMQD Y LFTVNSI RI RNERMM P P P IAN PAA AK P AAAQPAT G AASL T P A D E A A APAA P AI Q QVI K PYM G K E QVF V QVSLN L VH F NQ P KAQEPSED

[0176] SEQ ID NO:2: Nucleotide sequence encoding the Amuc-1100 polypeptide

[0177] atcgtcaattccaaacgcagtgaactggacaaaaaaatcagcatcgccgccaaggaaatcaagtccgccaatgctgcggaaatcactccgagccgatcatccaacgaagagctggaaaaagaactgaaccgctatgccaaggccgtgggcagcctggaaacggcctacaagcccttccttgcctcctccgcgctggtccccaccacgcccacggcattccagaatgaactgaaaacattcagggattccctgatctcctcctgcaagaaaaagaacattctcataacggacacatcctcctggctcggtttccaggtttacagcacccaggctccctctgttcaggcggcctccacgctgggttttgaattgaaagccatcaacagcctggtcaacaaactggcggaatgcggcctgtccaaattcatcaaggtgtaccgcccccagctccccattgaaaccccggcgaacaatccggaagaatcggacgaagccgaccaggccccatggactcccatgcctctggaaatagccttccagggcgaccgggaaagtgtattgaaagccatgaacgccataaccggcatgcaggactatctgttcacggtcaactccatccgtatccgcaacgaacggatgatgccccctcccatcgccaatccggcagccgccaaacctgccgcggcccaacccgccacgggtgcggcttccctgactccggcggatgaggcggctgcacctgcagccccggccatccagcaagtcatcaagccttacatgggcaaggagcaggtctttgtccaggtctccctgaatctggtccacttcaaccagcccaaggctcaggaaccgtctgaagattaa

[0178] SEQ ID NO: 3: Amino acid sequence of the predicted N-terminal signal sequence of Amuc-1100 polypeptide

[0179] MSNWITDNKPAAMVAGVGLLLFLGLSATGY

[0180] SEQ ID NO:4: Nucleotide sequence for predicting the N-terminal signal sequence of the Amuc-1100 peptide

[0181] atgagcaattggattacagacaacaagcccgccgccatggtcgcgggcgtgggacttctcttattcctggggttatccgcgacagggtac

[0182] SEQ ID NO:5: Amino acid sequence of the protein WP_094137363.1 of Akkermansia myxophilus (pTH008, underlined residues)

[0183] IVNSK RS ELDKK I SIAAKEIK S AN A AEIT P CRS SN EDLEK E L N RY A KAVNSLETAYKPFLASSALV P TTPTAF Q NE L KTFRDS L ISS CK K K N I LI T DTSN WLGF QVYSTQA P SVQAAST L G FE LKAINSLV N KLTEC G LSKFI KVY R PQLPIETPANNPEESDEADQSP W TPM P L EI A FQ GD R E SV LNAINA I TGMQD Y LFTINSI RI RNERMM P P P IAN PAA AK P AADQPAT G AASL T P A DE A A APAA P AI Q QVI K PYM G K E QIF V QVSLN L IH F NQ P KAQEPSED

[0184] SEQ ID NO:6: Amino acid sequence of Akkermansia myxophilus protein WP_022398192.1 (pTH009, underlined residues)

[0185] IVNSK RS ELDKK I SVASKEIK S AN A AEIT P SRA SN EELEK E L N RY A KAVTSLETAYKPFLASSALV P TTPTAF Q NE L KTFRDA L IAS CK K K N I LI T DTSS WLGF QVYSTQA P SVQAAST L G FE LKAVNSLV N KLTDC G LSKFI KVY R PQLPIENPANNPEEDADEPNQAP W TPM P L EI A FQ GN R E SV LKAMNAITDSQD Y LFTVNSI R IRNERMM P P P IAN PA A AK P AAAQPAA G AASL T PA D E A A APAA P AI Q QLI K PYM G K E QIF V QVSLN L VH F NQ P KAQEPSED

[0186] SEQ ID NO:7: Amino acid sequence of Akkermansia mycotoxin protein WP_102725837.1 (pTH010, underlined residues)

[0187] MVNSK RS ELDK K ISVASKEIK S AN A AEIT P SRT SN NELEK E L N RY A KAVTNLETAYKPFLASSALV P TTPTAF Q NE L KTFRDA L IAA CK K K N I QI T DTSS WLGF QVYSTQA P SVQAAST L G FE LKAVNSLA N KLTDC G LTKFI KVY R PQLPIENPANNPEEEAEEPNQAP W SPM P L E IA FQ GD R E SV LKAMNA I TDSQD Y LFTVNSI R IRNERMM P P P IAG PA A PK P AAAQSAAG AADLRP A D E A A AQSAA P AI Q QVI K PYM G K E QIF V QVSLN L VH F NQ P KAQEPSED

[0188] SEQ ID NO:8: Amino acid sequence of Akkermansia spp. KLE1797 protein WP_067981703.1 (pTH011, underlined residues)

[0189] MANSE RS DLDKK I KSASQEIK S AN A AAIT P SHT SN KELEK E L N RY A KAIGNLETAYKPFMASSVLA P TTPTAF Q NE L KAFRES L IAS CK E K N I QI T DTSS WLGF QLYSTQA P SVQATPT L T FE MKAINSLV N KLTDC G LTKFI KVY R SQLPIENPARNTEDEEDSDQKAP W TGM P L E IA FQ GD R G SV LKAMNA I TDSQE Y LFTVNSI R IRNERMM P P P ITN PA A AQP ASAQPQT G AASL T P A G E A A APAE P PI Q QII K PYM G K E QVM V QVSLN L VH F AQ P KAQEPSED

[0190] SEQ ID NO:9: Amino acid sequence of Akkermansia WP_067777749.1 (pTH012, underlined residues)

[0191] RS ASQDN I ASIEEGQSTLD S DR A KRF PSN EQSLP E V N AA A TRAAAIKEQILASTASFGQTVETATTVDGR P INGKEL Q DK L NTLHNK L EQL CK E K D I KL T PEAS WLGF SAFRSVT P NESDAPD L S FE LSGIDHFV N TVAAN G AVSIT KVYR PTVSEPADKTGKPKPAAKKNTGD W NTL P F EI S FQ AK R G SV GSILES I AQDKE Y CYYITGM RI ASDLTT P V P LDPFKK PAA PQ PEETATAVSDIIDD G LGGGDPLGG T P A A E P A PAPER P AA Q TVA K QIL G N E TIR V YIACE L VR F NT P

[0192] SEQ ID NO:13

[0193] TAATACGACTCACTATAGGGGCCCAAGTTCACTTAAAGGAGATCAACAATGAAAGCAATTTTCGTACTGAAACATCTTAATCAGGGGGAGGTTTCTA

[0194] SEQ ID NO:14

[0195] ATGCATCATCATCATCATCATCATGAAAACCTGTACTTCCAATCC

[0196] SEQ ID NO:15

[0197] TGCCGACTCAGTTGCTGCTTCTACTGGGCGCCCCGCTCGGCGGGTTTTTTT

[0198] SEQ ID NO:16

[0199] gccgactcagttgctgc

[0200] SEQ ID NO:17

[0201] ggattgaagtacaggttttcatg

[0202] SEQ ID NO:29(pTH008)

[0203] ATCGTCAATAGTAAACGCTCTGAGCTGGATAAGAAGATCTCTATCGCCGCTAAGGAGATCAAGTCGGCTAATGCCGCCGAGATCACCCCCTGTCGCTCTAGTAATGAGGATCTTGAGAAAGAGCTGAATCGCTATGCCAAGGCGGTCAATAGTCTGGAGACCGCCTATAAACCTTTTCTGGCCTCGAGTGCCCTTGTTCCCACCACCCCTACCGCCTTTCAGAATGAACTGAAAACATTCAGAGACAGCCTAATAAGCAGCTGCAAAAAAAAAAACATACTAATAACAGACACAAGCAACTGGCTAGGATTCCAAGTATACAGCACACAAGCACCAAGCGTACAAGCAGCAAGCACACTAGGATTCGAACTAAAAGCAATAAACAGCCTAGTAAACAAACTAACAGAATGCGGACTAAGCAAATTCATAAAAGTATACAGACCACAACTACCAATAGAAACACCAGCAAACAACCCAGAAGAAAGCGACGAAGCAGACCAAAGCCCATGGACACCAATGCCACTAGAAATAGCATTCCAAGGAGACAGAGAAAGCGTACTAAACGCAATAAACGCAATAACAGGAATGCAAGACTACCTATTCACAATAAACAGCATAAGAATAAGAAACGAAAGAATGATGCCACCACCAATAGCAAACCCAGCAGCAGCAAAACCAGCAGCAGACCAACCAGCAACAGGAGCAGCAAGCCTAACACCAGCAGACGAAGCAGCAGCACCAGCAGCACCAGCAATACAACAAGTAATAAAACCATACATGGGAAAAGAACAAATATTCGTACAAGTAAGCCTAAACCTAATACACTTCAACCAACCAAAAGCACAAGAACCAAGCGAAGACTAA

[0204] SEQ ID NO:30(pTH009)

[0205] ATCGTCAATTCGAAGCGCTCAGAGCTGGATAAGAAGATCTCCGTTGCCAGTAAAGAGATCAAGAGTGCCAATGCCGCTGAGATCACCCCCTCGCGCGCCTCTAATGAGGAACTGGAGAAAGAACTTAATCGCTATGCCAAAGCCGTTACCAGTCTGGAGACCGCCTATAAGCCCTTTCTGGCCTCTTCTGCCCTGGTCCCCACCACTCCTACCGCCTTTCAGAATGAGCTGAAAACATTCAGAGACGCACTAATAGCAAGCTGCAAAAAAAAAAACATACTAATAACAGACACAAGCAGCTGGCTAGGATTCCAAGTATACAGCACACAAGCACCAAGCGTACAAGCAGCAAGCACACTAGGATTCGAACTAAAAGCAGTAAACAGCCTAGTAAACAAACTAACAGACTGCGGACTAAGCAAATTCATAAAAGTATACAGACCACAACTACCAATAGAAAACCCAGCAAACAACCCAGAAGAAGACGCAGACGAACCAAACCAAGCACCATGGACACCAATGCCACTAGAAATAGCATTCCAAGGAAACAGAGAAAGCGTACTAAAAGCAATGAACGCAATAACAGACAGCCAAGACTACCTATTCACAGTAAACAGCATAAGAATAAGAAACGAAAGAATGATGCCACCACCAATAGCAAACCCAGCAGCAGCAAAACCAGCAGCAGCACAACCAGCAGCAGGAGCAGCAAGCCTAACACCAGCAGACGAAGCAGCAGCACCAGCAGCACCAGCAATACAACAACTAATAAAACCATACATGGGAAAAGAACAAATATTCGTACAAGTAAGCCTAAACCTAGTACACTTCAACCAACCAAAAGCACAAGAACCAAGCGAAGACTAA

[0206] SEQ ID NO:31(pTH010)

[0207] ATGGTCAATTCGAAACGCAGTGAGCTGGATAAAAAGATCTCCGTCGCCTCTAAAGAGATCAAGAGTGCCAATGCCGCCGAGATCACCCCTAGTCGCACCTCTAATAATGAGCTGGAGAAAGAGCTGAATCGCTATGCCAAGGCCGTGACCAATCTGGAAACCGCCTATAAGCCCTTTCTGGCCTCTTCGGCCCTGGTTCCTACCACCCCCACCGCCTTTCAGAATGAGCTGAAAACATTCAGAGACGCACTAATAGCAGCATGCAAAAAAAAAAACATACAAATAACAGACACAAGCAGCTGGCTAGGATTCCAAGTATACAGCACACAAGCACCAAGCGTACAAGCAGCAAGCACACTAGGATTCGAACTAAAAGCAGTAAACAGCCTAGCAAACAAACTAACAGACTGCGGACTAACAAAATTCATAAAAGTATACAGACCACAACTACCAATAGAAAACCCAGCAAACAACCCAGAAGAAGAAGCAGAAGAACCAAACCAAGCACCATGGAGCCCAATGCCACTAGAAATAGCATTCCAAGGAGACAGAGAAAGCGTACTAAAAGCAATGAACGCAATAACAGACAGCCAAGACTACCTATTCACAGTAAACAGCATAAGAATAAGAAACGAAAGAATGATGCCACCACCAATAGCAGGACCAGCAGCACCAAAACCAGCAGCAGCACAAAGCGCAGCAGGAGCAGCAGACCTAAGACCAGCAGACGAAGCAGCAGCACAAAGCGCAGCACCAGCAATACAACAAGTAATAAAACCATACATGGGAAAAGAACAAATATTCGTACAAGTAAGCCTAAACCTAGTACACTTCAACCAACCAAAAGCACAAGAACCAAGCGAAGACTAA

[0208] SEQ ID NO:32(pTH011)

[0209] ATGGCTAATTCGGAGCGCAGTGATCTGGATAAGAAAATCAAGTCTGCTAGTCAGGAGATCAAGAGTGCCAATGCCGCCGCCATCACCCCCTCGCATACCTCGAATAAAGAGCTGGAGAAAGAGCTGAATCGCTATGCTAAGGCTATCGGCAATCTTGAGACCGCTTATAAGCCCTTTATGGCCTCTTCGGTTCTGGCTCCCACCACCCCTACCGCCTTTCAGAATGAGCTGAAAGCATTCAGAGAAAGCCTAATAGCAAGCTGCAAAGAAAAAAACATACAAATAACAGACACAAGCAGCTGGCTAGGATTCCAACTATACAGCACACAAGCACCAAGCGTACAAGCAACACCAACACTAACATTCGAAATGAAAGCAATAAACAGCCTAGTAAACAAACTAACAGACTGCGGACTAACAAAATTCATAAAAGTATACAGAAGCCAACTACCAATAGAAAACCCAGCAAGAAACACAGAAGACGAAGAAGACAGCGACCAAAAAGCACCATGGACAGGAATGCCACTAGAAATAGCATTCCAAGGAGACAGAGGAAGCGTACTAAAAGCAATGAACGCAATAACAGACAGCCAAGAATACCTATTCACAGTAAACAGCATAAGAATAAGAAACGAAAGAATGATGCCACCACCAATAACAAACCCAGCAGCAGCACAACCAGCAAGCGCACAACCACAAACAGGAGCAGCAAGCCTAACACCAGCAGGAGAAGCAGCAGCACCAGCAGAACCACCAATACAACAAATAATAAAACCATACATGGGAAAAGAACAAGTAATGGTACAAGTAAGCCTAAACCTAGTACACTTCGCACAACCAAAAGCACAAGAACCAAGCGAAGACTAA

[0210] SEQ ID NO:33(pTH012)

[0211] ATGGCTAATTCGGAGCGCAGTGATCTGGATAAGAAAATCAAGTCTGCTAGTCAGGAGATCAAGAGTGCCAATGCCGCCGCCATCACCCCCTCGCATACCTCGAATAAAGAGCTGGAGAAAGAGCTGAATCGCTATGCTAAGGCTATCGGCAATCTTGAGACCGCTTATAAGCCCTTTATGGCCTCTTCGGTTCTGGCTCCCACCACCCCTACCGCC TTTCAGAATGAGCTGAAAGCATTCAGAGAAAGCCTAATAGCAAGCTGCAAAGAAAAAAACATACAAATAACAGACACAAGCAGCTGGCTAGGATTCCAACTATACAGCACACAAGCACCAAGCGTACAAGCAACACCAACACTAACATTCGAAATGAAAGCAATAAACAGCCTAGTAAACAAACTAACAGACTGCGGACTAACAAAATTCATAAAAG TATACAGAAGCCAACTACCAATAGAAAACCCAGCAAGAAACACAGAAGACGAAGAAGACAGCGACCAAAAAGCACCATGGACAGGAATGCCACTAGAAATAGCATTCCAAGGAGACAGAGGAAGCGTACTAAAAGCAATGAACGCAATAACAGACAGCCAAGAATAACCTATTCACAGTAAACAGCATAAGAATAAGAAACGAAAGAATGATGCCACC ACCAATAACAAACCCAGCAGCAGCACAACCAGCAAGCGCACAACCACAAACAGGAGCAGCAAGCCTAACACCAGCAGGAGAAGCAGCAGCACCAGCAGAACCACCAATACAACAAATAATAAAACCATACATGGGAAAAGAACAAGTAATGGTACAAGTAAGCCTAAACCTAGTACACTTCGCACAACCAAAAGCACAAGAACCAAGCGAAGACTAA Detailed Implementation

[0212] Example

[0213] Example 1: Genetically modified bacteria to produce Amuc-1100 protein.

[0214] method:

[0215] Under the control of the inducible T7 promoter of the pET28-derived compound, a polynucleotide encoding mature Amuc-1100 (nucleotide sequence of SEQ ID NO:2) with a C-terminal His-Tag was cloned into *E. coli* TOP10 and introduced into *E. coli* BL21(DE3) for overproduction. For this purpose, the ATG start codon was added to SEQ ID NO:2, so that the resulting peptide begins with the amino acid sequence MIVNS. All constructs were confirmed by Sanger sequence analysis. Constructs carrying overexpressed Amuc-1100 resulted in the overproduction of soluble Amuc-1100 protein, which was purified to apparent homogeneity by Ni column affinity chromatography and used at concentrations of 100–300 μg / mL. Antibodies were generated in rabbits using the purified Amuc-1100, essentially as described previously (Reunanen J et al. 2012, *Appl Environ Microbiol* 78:2337-44).

[0216] result:

[0217] The results showed that *E. coli* transformed with the polynucleotide (SEQ ID NO:2) produced a soluble form of the Amuc-1100 protein, which could be easily separated using the Ni column chromatography method described above (Tailford LE et al. 2015, NatCommun. 6:7624). Similar results were obtained using the polynucleotides of SEQ ID NO:29 or SEQ ID NO:33.

[0218] Example 2: Interactions and Stimulations of the TLR2 Signaling Pathway

[0219] method:

[0220] To test the ability of Amuc-1100 to bind to TLR2 and other TLR receptors and subsequently stimulate TLR2 and other TLR signaling pathways, reporter cell lines expressing TLR2 and TLR4 receptors were prepared. The ability of Amuc-1100 to bind to cell lines expressing TLR2 or TLR4 and subsequently stimulate TLR2 and / or TLR4 signaling pathways in vitro was tested by measuring NK-kB produced from the reporter cells.

[0221] In short, hTLR2 and hTLR4 cell lines (Invivogen, California, USA) were used. Stimulation of the receptors with the corresponding ligands activated NF-κB and AP-1, thereby inducing the production of secretory embryonic alkaline phosphatase (SEAP), the levels of which were measured using a spectrophotometer (Spectramax). All cell lines were grown and passaged to 70–80% confluence using 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). Immunoimmunoassays were performed for each cell line by adding 20 μl of Amuc-1100 suspension. Reporter cells were incubated with Amuc-1100 at 37°C, 5% CO2 for 20–24 hours. Receptor ligands Pam3CSK4 (10 ng / ml for hTLR2) and LPS-EB (50 ng / ml for hTLR2) were used as positive controls, while maintenance medium without any selective antibiotics was used as a negative control. SEAP secretion was detected by measuring OD600 at 15 min, 1 h, 2 h, and 3 h after adding 180 μL of QUANTI-Blue (Invivogen, California, USA) to 20 μL of induced hTLR2 and hTLR4 supernatant. Experiments were performed in triplicate.

[0222] result:

[0223] The results showed that Amuc-1100 can interact with TLR2. Furthermore, the results indicated that Amuc-1100 exerts an immunostimulatory effect on reporter cells expressing TLR2, specifically stimulating the release of NF-κB from reporter cells. Similar results were obtained using peptides of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

[0224] Example 3. Stimulating peripheral blood mononuclear cells to release cytokines.

[0225] method:

[0226] The ability of Amuc-1100 to stimulate peripheral blood mononuclear cells (PBMCs) to produce or release cytokines was tested in vitro. In short, peripheral blood from three healthy donors was obtained from the Sanquin Blood Bank in Radboud, Netherlands. PBMCs were isolated from the blood of healthy donors using Ficoll-Paque Plus gradient centrifugation according to the manufacturer's protocol (Amersham Biosciences, Uppsala, Sweden). After centrifugation, the mononuclear cells were collected, washed in Iscove's modified Dulbecco medium (IMDM) + glutamine (Glutamax) (Invitrogen, Bleda, Netherlands), and adjusted to 0.5 × 10⁻⁶ mol / L in IMDM + glutamine (Glutamax) supplemented with penicillin (100 U / ml) (Invitrogen), streptomycin (100 μg / ml) (Invitrogen), and 10% heat-inactivated FBS (Lonza, Basel, Switzerland). 6 Cells / ml. PBMCs (0.5) ×10 6 Cells per well were seeded into 48-well tissue culture plates. A negative control (culture medium only) was used for each donor.

[0227] PBMCs were stimulated for 1 day with live Akkermansia muciniphilis cells (1:10 ratio with PBMCs) or Amuc-1100, or heated at 99°C for 10 min. The production of cytokines IL-6, IL-8, IL-10, TNF-α, IL-1β, and IL-12p70 in the culture supernatant was then determined using multiplex analysis (Human Inflammation CBA Kit, Becton and Dickinson) according to the manufacturer's FACS CantoII (Becton and Dickinson) protocol, and analyzed using BD FCAP software (Becton and Dickinson). The detection limits, according to the manufacturer, were as follows: IL-8 3.6 pg / ml, IL-1β 7.2 pg / ml, IL-6 2.5 pg / ml, IL-10 3.3 pg / ml, TNF-α 3.7 pg / ml, and IL-12p70 1.9 pg / ml.

[0228] result

[0229] The results showed that, compared with the control (culture medium only), Amuc-1100 could stimulate the production of cytokines, specifically, increased levels of IL-1β, IL-6, IL-8, IL-10, and TNF-α were observed. The cytokine levels induced by 4.5 μg / ml Amuc-1100 were significantly higher than those induced by 5 × 10⁻⁶ μg / ml Amuc-1100.6 The levels of live or heat-inactivated Akkermansia myxophilus cells were similar (see Table 1 below).

[0230] Table 1. Levels of cytokines induced by Amuc-1100 and live or heat-inactivated forms of Akkermansia muciniphila.

[0231] Similar results can be obtained using peptides of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

[0232] Example 4: Regulation of transepithelial resistance (TER)

[0233] method:

[0234] The ability of Amuc-1100 to promote the integrity of the intestinal epithelial cell layer was evaluated by measuring its ability to stimulate or increase the TER (thermal activity) of Caco-2 cells in vitro. In short, Caco-2 cells (5 × 10⁻⁶) were used to... 4 10 cells / inserts were seeded into Millicell cell culture inserts (3μm pore size; Millipore) and cultured for 8 days. The bacterial cells were washed once with RPMI 1640 and incubated at 0.25 (approximately 10) g / mL in RPMI 1640. 8 The OD600 nm value of each cell was applied to the plug. Purified Amuc-1100 was applied to the plug at concentrations of 0.05, 0.5, and 5 μg / ml. Transepithelial resistance of cell cultures at 0 and 24 hours after Amuc-1100 addition was determined using a Millicell ERS-2TER instrument (Millipore).

[0235] result:

[0236] The results showed that after co-culturing with Caco-2 cells for 24 hours, 0.05 μg / ml Amuc-1100 significantly increased TER levels, with levels comparable to approximately 10. 8 Similar to *Ackermania* cells containing the same myxotropic protein. Similar results can also be obtained using peptides from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

[0237] Example 5: Regulation of diet-induced metabolic dysfunction

[0238] As previously described by Everard et al. (2013.PNAS.Vol.110(22):9066-9071), a group of 10-11 week old C57BL / 6J mice (n=10 per subgroup) were fed either the control diet (ND) or the HF diet (HFD; 60% fat and 20% carbohydrate (kcal / 100g) D12492i, Research Diet, New Brunswick, NJ, USA). As described by Lukovac et al. (2014, mBio01438-14), *Akermansia muciniphila* Muciniphila was cultured on a synthetic medium (containing per liter of deionized water: 0.4 g KH₂PO₄, 0.669 g Na₂HPO₄·2H₂O, 0.3 g NH₄Cl, 0.3 g NaCl, 0.1 g MgCl₂·6H₂O, 10 g tyrosine, 1 mM L-threonine, 1 ml trace mineral solution, 5 mM L-fucose, and 5 mM D-glucose) T The concentrate was then prepared in PBS containing 25% glycerol and stored at -80°C as described by Everard et al. above. A group of mice receiving HFD also received an additional 2 × 10⁻⁶ mmol / L orally daily. 8 CFU / 0.15 ml of Akkermansia myxophilus (HFDAkk) was suspended in sterile, anaerobic PBS—this resulted in a final concentration of 2.5% glycerol because it included a 10-fold dilution of Akkermansia myxophilus. The ND and HFD groups were administered an equal volume of sterile, anaerobic PBS containing 2.5% glycerol orally once daily, as previously described by Everard et al. above. Another group of mice receiving HFD additionally received the Amuc-1100 peptide, delivered daily by oral administration of 3.1 μg of protein Amuc_1100 (in an equal volume of sterile PBS containing 2.5% glycerol). This was in contrast to live Akkermansia myxophilus ( Figure 1 Compared to A and B), treatment with Amuc-1100 in HFD-fed mice resulted in similar or even more significant decreases in body weight and fat mass gain, without affecting food intake. Figure 1 Treatment with Akkermansia myxophilus or Amuc-1100 also corrected HFD-induced hypercholesterolemia, with a significant decrease in serum HDL-cholesterol and a similar trend in LDL-cholesterol. Figure 1 (D).

[0239] Notably, Amuc-1100 treatment resulted in a significant reduction in serum triglycerides compared to untreated HFD-fed mice. Furthermore, Amuc-1100 treatment also reduced the average diameter of adipocytes in HFD-fed mice from 38 μm to 29 μm, similar to the diameter found in untreated mice (27 μm).

[0240] Interestingly, administration of Amuc-1100 reduced glucose intolerance with the same potency as live bacteria. Figure 1 EF).

[0241] To further investigate glucose metabolism, the inventors studied insulin sensitivity by injecting insulin into the portal vein. The inventors analyzed the insulin-induced insulin receptor (IR) and its downstream mediator Akt in the liver at threonine (Akt) levels. thr ) and serine (Akt) ser Phosphorylation at site ( Figure 1 (G). Compared with mice fed a control diet, administration of HFD resulted in decreased phosphorylation of all proteins, particularly Akt. thr Significant results were achieved in the case of ( Figure 1 These effects were counteracted by treatment with live Akkermansia myxophilus or Amuc-1100, and mice treated with Amuc-1100 showed significantly higher p-IR and p-Akt levels compared to untreated HFD-fed mice. thr The level increased significantly ( Figure 1 GH), p-Akt in mice treated with live bacteria ser The level increased significantly ( Figure 1 Similar results can be obtained using peptides of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

[0242] Example 6: Comparative Analysis of Natural Variant and Mutant of Amuc-1100

[0243] Objectives and Methods

[0244] This study aimed to understand the signal transduction ability of Amuc-1100 to Toll-like receptor 2 (TLR2) from a structure-activity perspective (Derrien et al., 2004; Plovier et al., 2007). This was achieved by measuring the signal transduction ability of Amuc-1100 to Toll-like receptor 2 (TLR2) from the perspective of structure-activity. T The TLR2 signaling ability was achieved by comparing the Amuc-1100 protein of the strain with natural variants having different sequence identity and Amuc-1100 deletion mutants. All proteins (including Amuc-1100 and natural and structural variants) were expressed without the N-terminal membrane anchor signal peptide (ΔSP) sequence to ensure their solubility in the cytoplasm of the expression host *E. coli*.

[0245] Natural variants

[0246] Four proteins were identified in Akkermansia myxophilus-associated strains, which are related to Amuc... T The amino acid identity of the Amuc_1100 protein was greater than 80% (pTH008, SEQ ID NO:5, pTH009, SEQ ID NO:6, pTH010, SEQ ID NO:7, pTH011, SEQ ID NO:8). Furthermore, a more distant variant from Akkermansia globulus (pTH012, SEQ ID NO:9) was identified, with only 28% sequence identity.

[0247] The inventors refer to these five proteins as natural variants of Amuc_1100. (Table 2 and...) Figure 2 Conserved residues in the natural variants studied are shown.

[0248] Table 2. Conserved residues in the natural variants studied (numbered according to Amuc-1100, SEQ ID NO:1).

[0249] Gene Synthesis and Cloning

[0250] Next, by excluding the predicted signal peptide detected using SignalP 5.0 (AlmagroArmenteros et al., 2019), the inventors designed the DNA coding sequence of the protein sequence of the natural variant and converted it into pTN0003, ultimately obtaining pTN0005. In this plasmid (pTN0005), Amuc was used... T The exact coding sequence of Amuc-1100, as previously described (Plovier et al., 2017), has been shown to lead to significant overexpression in Escherichia coli.

[0251] The pTN0003 vector, used as the backbone for all expression constructs, contains a p15A starter, a kanamycin resistance gene, a T7 promoter, and a bicistronic design, followed by a terminator sequence (Mutalik et al., 2013; Nieuwkoop et al., 2019) (see overview). Figure 4 ).

[0252] For five natural variants (pTH008, pTH009, pTH010, pTH011, and pTH012), the inventors reverse-translated the protein sequences using Benchling's codon optimization tool (based on DNAChisel) (Benchling, 2018) and optimized the DNA coding sequences for expression in *E. coli* using Benchling's codon optimization tool (based on DNAChisel). For these natural variants and the Amuc-1100 sequence of pTN0005, the DNA was arranged in gBlocks order (IntegratedDNAtechnologies; https: / / eu.idtdna.com / DT). The DNA fragments were then cloned into the PCR-amplified linear pTN0003 vector via Gibson assembly.

[0253] All expression plasmids were transformed into BL21(DE3) competent E. coli cells (New England Biolabs). After cloning, the expression constructs were sequence validated.

[0254] Protein expression and purification

[0255] protein expression

[0256] The strain carrying the expression plasmid was pre-cultured in LB medium supplemented with kanamycin (50 μg / mL). 10 mL of the pre-culture was inoculated into 5 L Erlenmeyers flasks containing 1.5 L of LB medium supplemented with kanamycin (50 μg / mL) and incubated at 37 °C and 120 rpm until the culture reached an OD600 of 0.6–0.8. Before induction, the flasks were placed on ice for 30 min. After induction with 0.4 mM IPTG (final concentration), the cultures were further incubated at 20 °C and 120 rpm for 18 h. Cells were collected by centrifugation, and the pellet was washed with 25 mL of washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0). The cell pellet was stored at -80 °C.

[0257] Protein purification

[0258] The cell precipitate was supplemented with protease inhibitor tablets (Roche cOmplete). TMThaw the cells in 25 mL of wash buffer. Sonicate the resuspended cells (Bandelin Sonopuls, VS 70 / T probe, 25% intensity, 1 second on, 2 seconds off, total 10 minutes, on ice). Centrifuge the lysed cells (15 minutes, 30000×g, 4°C) and filter (0.45 μm) to remove cell debris.

[0259] Proteins were further purified using an AktaFPLC system with an N-terminal His-tag on a 5 mL HisTrap HP column (GE Healthcare). Proteins were eluted in 50 mM NaH₂PO₄, 300 mM NaCl, 500 mM imidazole, pH 8.0. During overnight dialysis (14 kWh CO₂) at 4°C with the wash buffer, the His-tag was cleaved at a ratio of 1:500 using 0.7 mg of His-labeled TEV protease. To remove the TEV protease from Amuc_1100 proteins, the proteins were run a second time on the HisTrap column. This time, the flowstream containing the target protein was collected while the His-labeled TEV protease remained bound to the HisTrap column.

[0260] In vitro culture and stimulation of human HEK-BluehTLR2 cell line

[0261] TLR2 activation was screened using HEK-Blue hTLR2 cells (Invincible, California, USA). In this cell line, TLR2 stimulation and subsequent activation of NF-κB and AP-1 induced the production of secretory embryonic alkaline phosphatase (SEAP), which could be quantified spectrophotometrically.

[0262] Cell lines supplemented with GlutaMAX TM 4.5 g / mL glucose, 100 U / mL penicillin, 100 μg / mL streptomycin, 100 μg / mL normocin, 10% (v / v) heat-inactivated FBS and HEK-Blue TM Selection (Invincible) Dulbecco's modified Eagle Medium (DMEM) maintenance medium was used for growth and passage to 70–80% confluency. Cells were maintained to a maximum extent until passage 25. HEK-blue cells were seeded in HEK-blue-free medium. TMTLR2 activation was detected by stimulating the wells with 20 μL of the target protein (50 μg / mL) in Triplelo after 24 hours in maintenance medium of Selection. The 96-well plates were incubated at 37°C for 20–24 hours in a 5% CO2 incubator. The receptor ligand Pam3CSK4 was used as a positive control, and PBS (a dilution reagent for the target protein) was used as a negative control. Secretory embryonic alkaline phosphatase (SEAP) activity was detected by measuring absorbance at 600 nm (Synergy™ Mx, BioTek Instruments, Inc., Vermont, USA) after 1 hour in 180 μL of QUANTI-Blue (Invivogen). The activity is expressed in arbitrary units (AU).

[0263] Results and Conclusions

[0264] As described above, measurements were taken from Amuc T The ability of the purified Amuc-1100 protein and its native variant to activate TLR2. Results are as follows: Figure 5 As shown.

[0265] The TLR2 cell viability of the 1ug / ml Pam3CSK4 positive control reached approximately 4.0 AU, while the viability of the negative controls PBS and DMEM was less than 1.0 AU. (Source: Amuc) T The Amuc1100 protein (1100) showed significant activity against TLR2 cells, exceeding the background value by approximately 2.0 AU.

[0266] from Figure 5 As can be seen, all natural variants tested here were able to activate the TLR2 receptor. The TLR2 activation capacity of the Amuc-1100 natural variant ( Figure 5 Surprisingly, it was higher than the positive control, reaching approximately 4.5 AU.

[0267] The results of 3D modeling (data not shown) indicate that deletion of the corresponding regions may reduce the protein's ability to interact with the TLR2 receptor. These results suggest that the dimerization and presence of long, disordered loops in Amuc-1100 are structural features that enhance TLR signaling activity. Therefore, the relationship between the deletion of specific conserved regions and their effect on the ability to activate TLR2 is evaluated in Table 3:

[0268] Table 3. Evaluation of the relationship between the absence of specific conserved regions and their negative impact on the ability to activate TLR2.

[0269] In particular, the presence of N-termini with β chains and long disordered rings for dimerization is important for (improving) TLR signaling activity.

[0270] References Almagro Armenteros,JJ,Tsirigos,KD, CK,Petersen,TN,Winther,O.,Brunak,S.,…Nielsen,H.(2019).SignalP 5.0 improves signal peptidepredictions using deep neural networks.Nature Biotechnology.https: / / doi.org / 10.1038 / s41587-019-0036-z Benchling(2018).Benchling for Academics·Benchling.RetrievedSeptember 13,2019,from www.benchling.com Derrien, MEEVaughan, CMPlugge&W.M.de Vos(2004)Akkermansiamuciniphila gen.nov.,sp.nov.,a human intestinal mucin-degrading bacterium.Int.J.Syst.Evol.Microbiol.54:1469-76. Mutalik, VK, Guimaraes, JC, Cambray, G., Lam, C., Christoffersen, MJ, Mai, QA,… Endy, D. (2013). Precise and reliable gene expressionⅵa standardtranscription and translation initiation elements. Nature Methods, 10(4), 354–360. https: / / doi.org / 10.1038 / nmeth.2404 Nieuwkoop, T., Claassens, NJ, & van der Oost, J. (2019). Improved protein production and codon optimization analyzes in Escherichia coli by bicistronicdesign. Microbial Biotechnology, 12(1), 173–179. https: / / doi.org / 10.1111 / 1751-7915.13332. Plovier,H.,Everard,A.,Druart,C.,Depommier,C.,Van Hul,M.,Geurts,L.,…Cani,PD(2017).A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabeticmice.Nature Medicine, 23(1). https: / / doi.org / 10.1038 / nm.4236. <110> Wageningen University <120> Amuc-1100 peptide variants for influencing immune signaling and / or intestinal barrier function and / or regulating metabolic state <130> KHP222111925.5 <160> 33 <170> PatentIn version 3.5 <210> 1 <211> 287 <212> PRT <213> Akkermansia muciniphila <400> 1 Ile Val Asn Ser Lys Arg Ser Glu Leu Asp Lys Lys Ile Ser Ile Ala 1 5 10 15 Ala Lys Glu Ile Lys Ser Ala Asn Ala Ala Glu Ile Thr Pro Ser Arg 20 25 30 Ser Ser Asn Glu Glu Leu Glu Lys Glu Leu Asn Arg Tyr Ala Lys Ala 35 40 45 Val Gly Ser Leu Glu Thr Ala Tyr Lys Pro Phe Leu Ala Ser Ser Ala 50 55 60 Leu Val Pro Thr Thr Pro Thr Ala Phe Gln Asn Glu Leu Lys Thr Phe 65 70 75 80 Arg Asp Ser Leu Ile Ser Ser Cys Lys Lys Lys Asn Ile Leu Ile Thr 85 90 95 Asp Thr Ser Ser Trp Leu Gly Phe Gln Val Tyr Ser Thr Gln Ala Pro 100 105 110 Ser Val Gln Ala Ala Ser Thr Leu Gly Phe Glu Leu Lys Ala Ile Asn 115 120 125 Ser Leu Val Asn Lys Leu Ala Glu Cys Gly Leu Ser Lys Phe Ile Lys 130 135 140 Val Tyr Arg Pro Gln Leu Pro Ile Glu Thr Pro Ala Asn Asn Pro Glu 145 150 155 160 Glu Ser Asp Glu Ala Asp Gln Ala Pro Trp Thr Pro Met Pro Leu Glu 165 170 175 Ile Ala Phe Gln Gly Asp Arg Glu Ser Val Leu Lys Ala Met Asn Ala 180 185 190 Ile Thr Gly Met Gln Asp Tyr Leu Phe Thr Val Asn Ser Ile Arg Ile 195 200 205 Arg Asn Glue Arg Met Met Pro Pro Ile Wing Asn Pro Ala Wing 210 215 220 Lys Pro AL Gln Pro AL Gln Pro AL Thr Gly AL Ser Leu Thr Pro 225 230 235 240 Wing Asp Glu Wing Wing Pro Wing Wing Pro Wing Ile Gln Gln Val Ile 245 250 255 Lys Pro Tyr Met Gly Lys Glu Gln Val Phe Val Gln Val Ser Leu Asn 260 265 270 Leu Val His Phe Asn Gln Pro Lys Ala Gln Glu Pro Ser Glu Asp 275 280 285 <210> 2 <211> 864 <212> DNA <213> Ambermansia muciniphila (Akkermansia muciniphila) <400> 2 atcgtcaatt ccaacgcag tgaactggac aaaaaaatca gcatcgccgc CAggaatc 60 aagtccgcca atgctgcgga aatcactccg agccgatcat ccaacgaaga gctggaaaaa 120 gaactgaacc gctatgccaa ggccgtgggc agcctggaa cggcctaca gccctcctt 180 gcctcctccg cgctggtccc caccacgccc acggcattcc agaatgaact gaaaacattc 240 agggattccc tgatctcctc ctgcaagaaa aagaacattc tcataacgga cacatcctcc 300 tggctcggtt tccaggttta cagcacccag gctccctctg ttcaggcggc ctccacgctg 360 ggttttgaat tgaaagccat caacagcctg gtcaacaaac tggcggaatg cggcctgtcc 420 aaattcatca aggtgtaccg cccccagctc cccattgaaa ccccggcgaa caatccggaa 480 gaatcggacg aagccgacca ggccccatgg actcccatgc ctctggaaat agccttccag 540 ggcgaccggg aaagtgtatt gaaagccatg aacgccataa ccggcatgca ggactatctg 600 ttcacggtca actccatccg tatccgcaac gaacggatga tgccccctcc catcgccaat 660 ccggcagccg ccaaacctgc cgcggcccaa cccgccacgg gtgcggcttc cctgactccg 720 gcggatgagg cggctgcacc tgcagccccg gccatccagc aagtcatcaa gccttacatg 780 ggcaaggagc aggtctttgt ccaggtctcc ctgaatctgg tccacttcaa ccagcccaag 840 gctcaggaac cgtctgaaga ttaa 864 <210> 3 <211> 30 <212> PRT <213> Akkermansia muciniphila <400> 3 Met Ser Asn Trp Ile Thr Asp Asn Lys Pro Ala Ala Met Val Ala Gly 1 5 10 15 Val Gly Leu Leu Leu Phe Leu Gly Leu Ser Ala Thr Gly Tyr 20 25 30 <210> 4 <211> 90 <212> DNA <213> Akkermansia muciniphila <400> 4 atgagcaatt ggattacaga caacaagccc gccgccatgg tcgcgggcgt gggacttctc 60 ttatcctgg ggttatccgc gacagggtac 90 <210> 5 <211> 287 <212> PRT <213> Akkermansia muciniphila <400> 5 Ile Val Asn Ser Lys Arg Ser Glu Leu Asp Lys Lys Ile Ser Ile Ala 1 5 10 15 Ala Lys Glu Ile Lys Ser Ala Asn Ala Ala Glu Ile Thr Pro Cys Arg 20 25 30 Ser Ser Asn Glu Asp Leu Glu Lys Glu Leu Asn Arg Tyr Ala Lys Ala 35 40 45 Val Asn Ser Leu Glu Thr Ala Tyr Lys Pro Phe Leu Ala Ser Ser Ala 50 55 60 Leu Val Pro Thr Thr Pro Thr Ala Phe Gln Asn Glu Leu Lys Thr Phe 65 70 75 80 Arg Asp Ser Leu Ile Ser Ser Cys Lys Lys Lys Asn Ile Leu Ile Thr 85 90 95 Asp Thr Ser Asn Trp Leu Gly Phe Gln Val Tyr Ser Thr Gln Ala Pro 100 105 110 Ser Val Gln Ala Ala Ser Thr Leu Gly Phe Glu Leu Lys Ala Ile Asn 115 120 125 Ser Leu Val Asn Lys Leu Thr Glu Cys Gly Leu Ser Lys Phe Ile Lys 130 135 140 Val Tyr Arg Pro Gln Leu Pro Ile Glu Thr Pro Ala Asn Asn Pro Glu 145 150 155 160 Glu Ser Asp Glu Ala Asp Gln Ser Pro Trp Thr Pro Met Pro Leu Glu 165 170 175 Ile Ala Phe Gln Gly Asp Arg Glu Ser Val Leu Asn Ala Ile Asn Ala 180 185 190 Ile Thr Gly Met Gln Asp Tyr Leu Phe Thr Ile Asn Ser Ile Arg Ile 195 200 205 Arg Asn Glu Arg Met Met Pro Pro Pro Ile Ala Asn Pro Ala Ala Ala 210 215 220 Lys Pro Ala Ala Asp Gln Pro Ala Thr Gly Ala Ala Ser Leu Thr Pro 225 230 235 240 Ala Asp Glu Ala Ala Ala Pro Ala Ala Pro Ala Ile Gln Gln Val Ile 245 250 255 Lys Pro Tyr Met Gly Lys Glu Gln Ile Phe Val Gln Val Ser Leu Asn 260 265 270 Leu Ile His Phe Asn Gln Pro Lys Ala Gln Glu Pro Ser Glu Asp 275 280 285 <210> 6 <211> 288 <212> PRT <213> Akkermansia muciniphila (Akkermansia muciniphila) <400> 6 Ile Val Asn Ser Lys Arg Ser Glu Leu Asp Lys Lys Ile Ser Val Ala 1 5 10 15 Ser Lys Glu Ile Lys Ser Ala Asn Ala Ala Glu Ile Thr Pro Ser Arg 20 25 30 Ala Ser Asn Glu Glu Leu Glu Lys Glu Leu Asn Arg Tyr Ala Lys Ala 35 40 45 Val Thr Ser Leu Glu Thr Ala Tyr Lys Pro Phe Leu Ala Ser Ser Ala 50 55 60 Leu Val Pro Thr Thr Pro Thr Ala Phe Gln Asn Glu Leu Lys Thr Phe 65 70 75 80 Arg Asp Ala Leu Ile Ala Ser Cys Lys Lys Lys Asn Ile Leu Ile Thr 85 90 95 Asp Thr Ser Ser Trp Leu Gly Phe Gln Val Tyr Ser Thr Gln Ala Pro 100 105 110 Ser Val Gln Ala Ala Ser Thr Leu Gly Phe Glu Leu Lys Ala Val Asn 115 120 125 Ser Leu Val Asn Lys Leu Thr Asp Cys Gly Leu Ser Lys Phe Ile Lys 130 135 140 Val Tyr Arg Pro Gln Leu Pro Ile Glu Asn Pro Ala Asn Asn Pro Glu 145 150 155 160 Glu Asp Ala Asp Glu Pro Asn Gln Ala Pro Trp Thr Pro Met Pro Leu 165 170 175 Glu Ile Ala Phe Gln Gly Asn Arg Glu Ser Val Leu Lys Ala Met Asn 180 185 190 Ala Ile Thr Asp Ser Gln Asp Tyr Leu Phe Thr Val Asn Ser Ile Arg 195 200 205 Ile Arg Asn Glu Arg Met Met Pro Pro Pro Ile Ala Asn Pro Ala Ala 210 215 220 Ala Lys Pro Ala Ala Ala Gln Pro Ala Ala Gly Ala Ala Ser Leu Thr 225 230 235 240 Pro Ala Asp Glu Ala Ala Ala Pro Ala Ala Pro Ala Ile Gln Gln Leu 245 250 255 Ile Lys Pro Tyr Met Gly Lys Glu Gln Ile Phe Val Gln Val Ser Leu 260 265 270 Asn Leu Val His Phe Asn Gln Pro Lys Ala Gln Glu Pro Ser Glu Asp 275 280 285 <210> 7 <211> 289 <212> PRT <213> Akkermansia muciniphila (Akkermansia muciniphila) <400> 7 Met Val Asn Ser Lys Arg Ser Glu Leu Asp Lys Lys Ile Ser Val Ala 1 5 10 15 Ser Lys Glu Ile Lys Ser Ala Asn Ala Ala Glu Ile Thr Pro Ser Arg 20 25 30 Thr Ser Asn Asn Glu Leu Glu Lys Glu Leu Asn Arg Tyr Ala Lys Ala 35 40 45 Val Thr Asn Leu Glu Thr Ala Tyr Lys Pro Phe Leu Ala Ser Ser Ala 50 55 60 Leu Val Pro Thr Thr Pro Thr Ala Phe Gln Asn Glu Leu Lys Thr Phe 65 70 75 80 Arg Asp Ala Leu Ile Ala Ala Cys Lys Lys Lys Asn Ile Gln Ile Thr 85 90 95 Asp Thr Ser Ser Trp Leu Gly Phe Gln Val Tyr Ser Thr Gln Ala Pro 100 105 110 Ser Val Gln Ala Ala Ser Thr Leu Gly Phe Glu Leu Lys Ala Val Asn 115 120 125 Ser Leu Ala Asn Lys Leu Thr Asp Cys Gly Leu Thr Lys Phe Ile Lys 130 135 140 Val Tyr Arg Pro Gln Leu Pro Ile Glu Asn Pro Ala Asn Asn Pro Glu 145 150 155 160 Glu Glu Ala Glu Glu Pro Asn Gln Ala Pro Trp Ser Pro Met Pro Leu 165 170 175 Glu Ile Ala Phe Gln Gly Asp Arg Glu Ser Val Leu Lys Ala Met Asn 180 185 190 Ala Ile Thr Asp Ser Gln Asp Tyr Leu Phe Thr Val Asn Ser Ile Arg 195 200 205 Ile Arg Asn Glu Arg Met Met Pro Pro Pro Ile Ala Gly Pro Ala Ala 210 215 220 Pro Lys Pro Ala Ala Ala Gln Ser Ala Ala Gly Ala Ala Asp Leu Arg 225 230 235 240 Pro Ala Asp Glu Ala Ala Ala Gln Ser Ala Ala Pro Ala Ile Gln Gln 245 250 255 Val Ile Lys Pro Tyr Met Gly Lys Glu Gln Ile Phe Val Gln Val Ser 260 265 270 Leu Asn Leu Val His Phe Asn Gln Pro Lys Ala Gln Glu Pro Ser Glu 275 280 285 Asp <210> 8 <211> 288 <212> PRT <213> Akkermansia sp. KLE1797 <400> 8 Met Ala Asn Ser Glu Arg Ser Asp Leu Asp Lys Lys Ile Lys Ser Ala 1 5 10 15 Ser Gln Glu Ile Lys Ser Ala Asn Ala Ala Ala Ile Thr Pro Ser His 20 25 30 Thr Ser Asn Lys Glu Leu Glu Lys Glu Leu Asn Arg Tyr Ala Lys Ala 35 40 45 Ile Gly Asn Leu Glu Thr Ala Tyr Lys Pro Phe Met Ala Ser Ser Val 50 55 60 Leu Ala Pro Thr Thr Pro Thr Ala Phe Gln Asn Glu Leu Lys Ala Phe 65 70 75 80 Arg Glu Ser Leu Ile Ala Ser Cys Lys Glu Lys Asn Ile Gln Ile Thr 85 90 95 Asp Thr Ser Ser Trp Leu Gly Phe Gln Leu Tyr Ser Thr Gln Ala Pro 100 105 110 Ser Val Gln Ala Thr Pro Thr Leu Thr Phe Glu Met Lys Ala Ile Asn 115 120 125 Ser Leu Val Asn Lys Leu Thr Asp Cys Gly Leu Thr Lys Phe Ile Lys 130 135 140 Val Tyr Arg Ser Gln Leu Pro Ile Glu Asn Pro Ala Arg Asn Thr Glu 145 150 155 160 Asp Glu Glu Asp Ser Asp Gln Lys Ala Pro Trp Thr Gly Met Pro Leu 165 170 175 Glu Ile Ala Phe Gln Gly Asp Arg Gly Ser Val Leu Lys Ala Met Asn 180 185 190 Ala Ile Thr Asp Ser Gln Glu Tyr Leu Phe Thr Val Asn Ser Ile Arg 195 200 205 Ile Arg Asn Glu Arg Met Met Pro Pro Pro Ile Thr Asn Pro Ala Ala 210 215 220 Ala Gln Pro Ala Ser Ala Gln Pro Gln Thr Gly Ala Ala Ser Leu Thr 225 230 235 240 Pro Ala Gly Glu Ala Ala Ala Pro Ala Glu Pro Pro Ile Gln Gln Ile 245 250 255 Ile Lys Pro Tyr Met Gly Lys Glu Gln Val Met Val Gln Val Ser Leu 260 265 270 Asn Leu Val His Phe Ala Gln Pro Lys Ala Gln Glu Pro Ser Glu Asp 275 280 285 <210> 9 <211> 301 <212> PRT <213> Akkermansia glycaniphila <400> 9 Arg Ser Ala Ser Gln Asp Asn Ile Ala Ser Ile Glu Glu Gly Gln Ser 1 5 10 15 Thr Leu Asp Ser Asp Arg Ala Lys Arg Phe Pro Ser Asn Glu Gln Ser 20 25 30 Leu Pro Glu Val Asn Ala Ala Ala Thr Arg Ala Ala Ala Ile Lys Glu 35 40 45 Gln Ile Leu Ala Ser Thr Ala Ser Phe Gly Gln Thr Val Glu Thr Ala 50 55 60 Thr Thr Val Asp Gly Arg Pro Ile Asn Gly Lys Glu Leu Gln Asp Lys 65 70 75 80 Leu Asn Thr Leu His Asn Lys Leu Glu Gln Leu Cys Lys Glu Lys Asp 85 90 95 Ile Lys Leu Thr Pro Glu Ala Ser Trp Leu Gly Phe Ser Ala Phe Arg 100 105 110 Ser Val Thr Pro Asn Glu Ser Asp Ala Pro Asp Leu Ser Phe Glu Leu 115 120 125 Ser Gly Ile Asp His Phe Val Asn Thr Val Ala Ala Asn Gly Ala Val 130 135 140 Ser Ile Thr Lys Val Tyr Arg Pro Thr Val Ser Glu Pro Ala Asp Lys 145 150 155 160 Thr Gly Lys Pro Lys Pro Ala Ala Lys Lys Asn Thr Gly Asp Trp Asn 165 170 175 Thr Leu Pro Phe Glu Ile Ser Phe Gln Ala Lys Arg Gly Ser Val Gly 180 185 190 Ser Ile Leu Glu Ser Ile Ala Gln Asp Lys Glu Tyr Cys Tyr Tyr Ile 195 200 205 Thr Gly Met Arg Ile Ala Ser Asp Leu Thr Thr Pro Val Pro Leu Asp 210 215 220 Pro Phe Lys Lys Pro Ala Ala Pro Gln Pro Glu Glu Thr Ala Thr Ala 225 230 235 240 Val Ser Asp Ile Ile Asp Asp Gly Leu Gly Gly Gly Asp Pro Leu Gly 245 250 255 Gly Thr Pro Ala Ala Glu Pro Ala Pro Ala Pro Glu Glu Val Arg Pro 260 265 270 Ala Ala Gln Thr Val Ala Lys Gln Ile Leu Gly Asn Glu Thr Ile Arg 275 280 285 Val Tyr Ile Ala Cys Glu Leu Val Arg Phe Asn Thr Pro 290 295 300 <210> 13 <211> 103 <212> DNA <213> Artificial Sequence <220> <223> - <400> 13 taatacgact cactataggg gcccaagttc acttaaaaag gagatcaaca atgaaagcaa 60 ttttcgtact gaaacatctt aatcatgcag gggagggttt cta 103 <210> 14 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> - <400> 14 atgcatcatc atcatcatca tcatgaaaac ctgtacttcc aatcc 45 <210> 15 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> - <400> 15 tgccgactca gttgctgctt ctactgggcg ccccgcttcg gcggggtttt ttt 53 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> - <400> 16 gccgactcag ttgctgc 17 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> - <400> 17 ggattggaag tacaggtttt catgatg 27 <210> 18 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> - <400> 18 agccgatcat ccaacgaaga gc 22 <210> 19 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> - <400> 19 ggattggaag tacaggtttt catg 24 <210> 29 <211> 864 <212> DNA <213> Akkermansia muciniphila (Akkermansia muciniphila) <400> 29 atcgtcaata gtaaacgctc tgagctggat aagaagatct ctatcgccgc taaggagatc 60 aagtcggcta atgccgccga gatcaccccc tgtcgccta gtaatgagga tcttgagaaa 120 gagctgaatc gctatgccaa ggcggtcaat agtctggaga ccgcctataa accttttctg 180 gcctcgagtg cccttgttcc caccacccct accgccttc agaatgaact gaaaacattc 240 agagacagcc tataagcag ctgcaaaaaa aaaacatac tataacaga cacaagcaac 300 tggctaggat tccaagtata cagcacacaa gcaccaagcg tacaagcagc aagcacacta 360 ggattcgaac taaaagcaat aaacagccta gtaaacaaac taacagaatg cggactaagc 420 aaattcataa aagtatacag accacaacta ccaatagaaa caccagcaaa caacccagaa 480 gaaagcgacg aagcagacca aagcccatgg acaccaatgc cactagaaat agcattccaa 540 ggacagag aaagcgtact aaacgcaata aacgcaataa caggaatgca agactaccta 600 ttcacaataa acagcataag aataagaaac gaaagaatga tgccaccacc aatagcaaac 660 ccagcagcag caaaaccagc agcagaccaa ccagcaacag gagcagcaag cctaacacca 720 gcagacgaag cagcagcacc agcagcacca gcaatacaac aagtaataaa accatacatg 780 ggaaaagaac aaatattcgt acaagtaagc ctaaacctaa tacacttcaa ccaaccaaaa 840 gcacaagaac caagcgaaga ctaa 864 <210> 30 <211> 867 <212> DNA <213> Akkermansia muciniphila (Akkermansia muciniphila) <400> 30 atcgtcaatt cgaagcgctc agagctggat aagaagatct ccgttgccag taaaagagatc 60 aagagtgcca atgccgctga gatcacccc tcgcgcgcct ctaatgagga actggagaaa 120 gaacttaatc gctatgccaa agccgttacc agtctggaga ccgcctataa gccctttctg 180 gcctcttctg ccctggtccc caccactcct accgcctttc agaatgagct gaaaacattc 240 agagacgcac taatagcaag ctgcaaaaaa aaaaacatac taataacaga cacaagcagc 300 tggctaggat tccaagtata cagcacacaa gcaccaagcg tacaagcagc aagcacacta 360 ggattcgaac taaaagcagt aacagccta gtaacaac taacagactg cggactaagc 420 aaattcataa aagtatacag accacaa ccaatagaa acccagcaaa 480 gaagacgcag acgaaccaaa ccaagcacca tggacaccaa tgccactaga atagcattc 540 shy gagaaagcgt actaaaagcaaaaaackaaaaaaaaaaaaaaaaaaaaaggc 600 ctattcacag tAAacaccat agaataga aacgaaagaa tgatgccacc accatagca 660 aacccagcag cagcaaacc agcagcagca caggagcag caggagcagc aagcctaca 720 ccagcagacg aagcagcagc ccagcagca ccagcaatac aacaactaat aaaaccatac 780 atgggaaaag aaaaatatt cgtacaagta agcctaaacc tagtacactt spaacca 840 aaaaaaaaaaaaaaaaaaackact 867 <210> 31 <211> 870 <212> DNA <213> Ambermansia muciniphila (Akkermansia muciniphila) <400> 31 atggtcaatt cgaaacgcag tgagctgt aaaagatct ccgtcgccctc tAAagagatc 60 aagagtgcca atgccgccga gatcacccct agtcgcacct ctaataatga gctggagaaa 120 gagctgaatc gctatgccaa ggccgtgacc aatctggaaa ccgcctataa gccctttctg 180 gcctcttcgg ccctggttcc taccaccccc accgcctttc agaatgagct gaaaacattc 240 agagacgcac taatagcagc atgcaaaaaa aaaaacatac aataacaga cacaagcagc 300 tggctaggat tccaagtata cagcacacaa gcaccaagcg tacaagcagc aagcacta 360 ggattcgaac taaaagcagt aaacagccta gcaaacaaac taacagactg cggatcaaca 420 aaattcataa aagtatacag accacaacta ccaatagaaa acccagcaaa caacccagaa 480 gaagaagcag aagaaccaaa ccaagcacca tggagcccaa tgccaactaga aatagcattc 540 caaggaca gagaaagcgt actaaaagca atgaacgcaa taacagacag ccaagactac 600 660 ggaccagcag caccaaaacc agcagcagca caaagcgcag caggagcagc agacctaaga 720 ccagcagacg aagcagcagc acaaagcgca gcaccagcaa tacaacaagt aataaacca 780 tacatgggaa aagaacaaat attcgtacaa gtaagcctaa acctagtaca cttcaaccaa 840 ccaaaagcac agaaaccaag cgaagactaa 870 <210> 32 <211> 867 <212> DNA <213> KLE1797 (Akkermansia sp. KLE1797) <400> 32 atggctaatt cggagcgcag tgatctgat aagaaaatca agtctgctag tcaggagatc 60 aagagtgcca atgccgccgc catcaccccc tcgcatacct cgataaaga gctggagaaa 120 gagctgaatc gctatgctaa ggctatcggc aatctgaga ccgcttataa gccctttatg 180 gcctctcgg ttctggctcc caccacccct accgccttc agaatgagct gaaagcattc 240 agagaaagcc tatagcaag ctgchaagaa aaaaacatac aaatacaga cacaagcagc 300 tggctaggat tccaactata cagcacacaa gcaccaagcg tacaaccac accacacta 360 acattcgaaa tgaagcaat aaacagccta gtaacaac taacagactg cggactaca 420 aaattcataa aagtatacag aagccaacta ccaatagaa acccagcaag aaacacagaa 480 gacgaagaag acagcgacca aaagcacca tggacagga tgccactga atagcattc 540 shuffle gaggaagcgt accaaaagcaaaaaggaaaaaaaacaaaaaaaaackaaaaaack 600 ctattcacag taaacaccat agaataga acgaaagaa tgatgccacc accaataca 660 aacccagcag cagcacaacc agcagcgca caccacacaa caggagcagc aagcctaca 720 ccagcaggag aagcagcagc ccagcaggaa ccaccatac aaaaaaccatac 780 atgggaaaag aacaagtaat ggtacaagta agcctaaacc tagtacactt cgcacaacca 840 aaaaaaaaaaaaaaaaaaackact 867 <210> 33 <211> 867 <212> DNA <213> The woodpecker (Akkermansia glycaniphila) <400> 33 atggctaatt cggagcgcag tgatctgat aagaaaatca agtctgctag tcaggagatc 60 aagagtgcca atgccgccgc catcaccccc tcgcatacct cgataaaga gctggagaaa 120 gagctgaatc gctatgctaa ggctatcggc aatctgaga ccgcttataa gccctttatg 180 gcctctcgg ttctggctcc caccacccct accgccttc agaatgagct gaaagcattc 240 agagaaagcc tatagcaag ctgchaagaa aaaaacatac aaatacaga cacaagcagc 300 tggctaggat tccaactata cagcacacaa gcaccaagcg tacaaccac accacacta 360 acattcgaaa tgaagcaat aaacagccta gtaacaac taacagactg cggactaca 420 aaattcataa aagtatacag aagccaacta ccaatagaa acccagcaag aaacacagaa 480 gacgaagaag acagcgacca aaagcacca tggacagga tgccactga atagcattc 540 shuffle gaggaagcgt accaaaagcaaaaaggaaaaaaaacaaaaaaaaackaaaaaack 600 ctattcacag taaacaccat agaataga acgaaagaa tgatgccacc accaataca 660 aacccagcag cagcacaacc agcagcgca caccacacaa caggagcagc aagcctaca 720 ccagcaggag aagcagcagc ccagcaggaa ccaccatac aaaaaaccatac 780 atgggaaaag aacaagtaat ggtacaagta agcctaaacc tagtacactt cgcacaacca 840 aaaaaaaaaaaaaaaaaaackact 867

Claims

1. A composition comprising an isolated polypeptide and a pharmaceutically or food-acceptable carrier, characterized in that... The isolated polypeptide has 100% sequence identity with SEQ ID NO:9, and the polypeptide affects immune signaling and / or intestinal barrier function and / or glucose and / or cholesterol and / or triglyceride homeostasis.

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

3. A genetically modified host cell comprising a nucleic acid molecule, said nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide as defined in claim 1.

4. The genetically modified host cell according to claim 3, wherein the host cell does not belong to the species Akkermansia myxophilus (…). Akkermansia muciniphila ) or species Akermansia pyrophila ( Akkermansia glycaniphila ).

5. The genetically modified host cell according to claim 3, wherein the host cell belongs to the species Akkermansia myxophila or Akkermansia polysaccharide.

6. A method for producing the polypeptide as defined in claim 1, comprising the following steps: (a) Culture the host cells according to any one of claims 3-5 under conditions that allow the production of the polypeptide; and (b) Separate the polypeptide produced in step (a).

7. Use of the composition according to any one of claims 1-2 or the host cell according to any one of claims 3-5 in the preparation of a medicament for the prevention and / or treatment of a disease selected from the group consisting of obesity, type 2 diabetes, and glucose intolerance in mammals.

8. A method for producing the polypeptide as defined in claim 1, comprising the following steps: (a) Culturing the species *Akermansia pyrenoidosa* in a suitable culture medium, said *Akermansia pyrenoidosa* comprising a nucleic acid sequence encoding a polypeptide as defined in claim 1; and (b) Separate the polypeptide produced in step (a).

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