MICROORGANISMS SYNTHESIZING ANTI-INFLAMMATORY MOLECULES

Abstract

The present invention relates to the field of prevention and/or treatment of Inflammatory Bowel Diseases such as Crohn's Disease and Ulcerative Colitis.

Claims

1. A microorganism comprising a microbial anti-inflammatory molecule (MAM) gene and one or more gene(s) encoding polysaccharide A biosynthesis pathway for use in the prevention and/or treatment of Inflammatory Bowel Diseases, wherein one or more gene(s) encoding a metabolic pathway is selected from the group consisting of one or more of the genes encoding UpaY, UpaZ, flippase, galactopyranose mutase, glycosyltransferase, amino sugar synthetase, undecaprenyl-phosphate galactose phosphotransferase, protein disulfide isomerase and methyltransferase protein.

2. The microorganism according to claim 1, wherein the microorganism is bacteria, yeast or fungi.

3. The microorganism of claim 2, wherein the bacteria, yeast or fungi is produced synthetically by genetic engineering.

4. The microorganism of claim 2, wherein the MAM gene or the one or more gene(s) encoding polysaccharide A biosynthesis pathway is transferred in vitro into the bacteria, yeast or fungi.

5. The microorganism of claim 1, wherein the Inflammatory Bowel Disease is Crohn's Disease or Ulcerative Colitis.

6. The microorganism of claim 1, wherein the gene encoding MAM gene has at least 70% sequence identity with the Seg ID. No. 1.

7. The microorganism of claim 1, wherein one or more of the genes encoding the polysaccharide A biosynthesis pathway has at least 70% sequence identity with one or more sequence(s) selected from the group consisting of Seg ID. No. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25.

8. The microorganism of claim 1, wherein the microorganism is in a physiologically acceptable carrier composition.

9. The microorganism of claim 8, wherein the composition is a food composition.

10. The microorganism of claim 8, wherein the composition is a pharmaceutical composition.

11. The microorganism of claim 8, wherein the microorganism is present in the composition in lyophilized or microencapsulated form.

12. The microorganism of claim 8, wherein the composition is a probiotic to be administered.

13. The microorganism of claim 9, wherein the food composition comprises a dairy product or a fermented dairy product.

14. The microorganism of claim 13, wherein the food composition comprises a yogurt or a yogurt drink.

15. The microorganism of claim 10, wherein the pharmaceutical composition is in a solid dosage form.

16. The microorganism of claim 15, wherein the pharmaceutical composition is in the form of a capsule, a tablet or a powder.

Description

BRIEF DESCRIPTION OF THE FIGURE

[0030] FIG. 1: Plasma IL-10 levels (pg/ml) of the observed subgroups. People having the Microbial Anti-Inflammatory Molecule (MAM) gene and polysaccharide A biosynthetic gene cluster in their gut microbiome experience statistically significantly higher IL-10 secretion compared to the people lacking said pathway.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The preferred alternatives of the embodiments of invention, which are mentioned in this detailed description, are only intended for providing a better understanding of the subject-matter, and should not be construed in any restrictive sense.

Microorganism

[0032] Primarily, the invention is directed to a microorganism comprising a gene encoding MAM and a gene cluster encoding the polysaccharide A biosynthesis pathway, which enables the microorganism to produce MAM and polysaccharide A molecules, preferablyunder anaerobic conditions. The microorganism may be an isolate belonging to the bacterial, archaeal, or fungal kingdoms.

[0033] The gene encoding the microbial anti-inflammatory molecule protein is the mam gene disclosed herein. The gene cluster belonging to the pathway synthesizing the polysaccharide A molecule must contain at least one of the genes encoding the following proteins: UpaY, UpaZ, flippase, galactopyranose mutase, glycosyltransferase, amino sugar synthetase, undecaprenyl-phosphate galactose phosphotransferase, protein disulfide isomerase, methyltransferase.

[0034] Said microorganism may also be a genetically modified bacterium, archaea or fungus, to which the above-mentioned genes have been transferred in vitro.

Genes:

[0035] The gene encoding the microbial anti-inflammatory molecule has at least 70, 80, 90, 95, 99, 100% identity with Sequence ID No. 1. The respective amino acid sequence is shown in Sequence ID. No. 2.

[0036] The gene encoding the UpaY has at least 70, 80, 90, 95, 99, 100% identity with Sequence ID No. 3. The respective amino acid sequence is shown in Sequence ID. No. 4.

[0037] The gene encoding the UpaZ have at least 70, 80, 90, 95, 99, 100% identity with Sequence ID No. 5. The respective amino acid sequence is shown in Sequence ID. No. 6.

[0038] The gene encoding the flippase has at least 70, 80, 90, 95, 99, 100% identity with Sequence ID No. 7. The respective amino acid sequence is shown in Sequence ID. No. 8.

[0039] The gene encoding the galactopyranose mutase has at least 70, 80, 90, 95, 99, 100% identity with Sequence ID No. 9. The respective amino acid sequence is shown in Sequence ID. No. 10.

[0040] The gene encoding the glycosyltransferase must be a gene with at least 70, 80, 90, 99, 100% identity with Sequence ID No. 11 or Sequence ID No. 13 or Sequence ID No. 15 or Sequence ID No. 17. The respective amino acid sequence are shown in Sequence ID. No. 12, 14, 16 and 18, respectively.

[0041] The gene encoding the amino sugar synthetase has at least 70, 80, 90, 95, 99, 100% identity with Sequence ID No. 19. The respective amino acid sequence is shown in Sequence ID. No. 20.

[0042] The gene encoding the undecaprenyl-phosphate galactose phosphotransferase has at least 70, 80, 90, 95, 99, 100% identity with Sequence ID No. 21. The respective amino acid sequence is shown in Sequence ID. No. 22.

[0043] The gene encoding the protein disulfide isomerase has at least 70, 80, 90, 95, 99, 100% identity with Sequence ID No. 23. The respective amino acid sequence is shown in Sequence ID. No. 24.

[0044] The gene encoding the methyltransferase has at least 70, 80, 90, 95, 99, 100% identity with Sequence ID No. 25. The respective amino acid sequence is shown in Sequence ID. No. 26.

Composition:

[0045] The present invention also includes a composition comprising a bacterium according to the present disclosure and optionally a physiologically acceptable carrier. The physiologically acceptable carrier may be any suitable carrier for keeping the microorganism with said properties alive until it is consumed by a target organism (e.g., human or animal). For example, examples of acceptable carriers suitable for this purpose include any of the well known physiological or pharmaceutical carriers, buffers, and intermediates. The selection of an appropriate physiological or pharmaceutical carrier may vary depending on the intended mode of administration of the composition (e.g., oral) and the intended form of the composition (e.g., beverage, yogurt, powder, capsules, etc.) as discussed herein.

[0046] The composition containing said microorganisms may be in the form of food (food), feed, food formulation, food supplement composition or pharmaceutical composition.

[0047] Preferably said composition is a food or food supplement composition. The food or food supplement composition may be selected from the group consisting of a liquid, liquid beverage (including milk beverage and fermented beverage), yogurt, cheese, gel, gelatin, gelatin capsule, powder, paste, pressed tablet, and gel cap. The food or food supplement composition may be a dairy product, preferably a fermented dairy product, preferably a yogurt or a yogurt drink.

[0048] Preferably, said composition may be a probiotic composition. Such probiotic composition may include (isolated) bacteria, fungi, or a strain synthetically derived therefrom, as discussed herein. Preferably, the composition may further comprise one or more additional beneficial isolated strains of intestinal microorganism.

[0049] Preferably said composition may contain one or more prebiotic ingredients, any prebiotic ingredient suitable for increasing the activity of the microorganism and/or stimulating its growth. Examples of suitable prebiotic ingredients include fibers such as inulin, pectin, and resistant starch, as well as inositol, cellobiose, maltose, mannose, salicin, trehalose, amygdalin, arabinose, melibiose, sorbitol, rhamnose and/or xylose.

[0050] Said microorganism may be contained in lyophilized form, microencapsulated form (see for example Solanki et al., BioMed Res. Int. 2013, Article ID 620719), or any other form that protects the viability and activity of the microorganism species.

[0051] Preferably, said composition may be a pharmaceutical composition. The pharmaceutical composition may be produced for use as a supplement. A pharmaceutical composition will generally contain a pharmaceutical carrier in addition to the microorganism as mentioned herein. The carrier is preferably an inactive carrier. The preferred form depends on the intended mode of administration and (therapeutic) application. A pharmaceutical carrier may be any compatible, non-toxic substance suitable for delivering the organism mentioned herein into the body of a subject. For example, sterile water or inactive solids can be used as a carrier, often complemented by a pharmaceutically acceptable adjuvant, buffering agent, dispersing agent, and the like. The pharmaceutical composition may be in liquid form (e.g., a stabilized bacterial suspension of said microorganism) or in solid form (e.g., powder of said lyophilized microorganism). When said microorganism is lyophilized, a cryoprotectant such as lactose, trehalose or glycogen can be used. For example, for oral administration, said microorganism may be administered in solid dosage forms such as capsules, tablets and powders containing lyophilized microorganism, or in liquid dosage forms such as syrups and suspensions. Said microorganism may be carried, for example, in lyophilized form, in capsules such as gelatin capsules, in the form of inactive ingredients and, for example, glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate and the like.

[0052] With the invention, it is intended to be used for the protection, restoration and/or improvement of gastrointestinal system health in general and/or for the prevention and/or treatment of Inflammatory Bowel Diseases such as Crohn's Disease and Ulcerative Colitis. It is intended herein to observe the expected efficacy by administering an effective amount of said microorganism to said subject by said means.

[0053] The following example that follows further illustrates the invention but should not be construed to limit the scope of the invention in any way.

EXAMPLE

[0054] The discovery of phenotypic effects of the putative gene cluster was conducted via an observational study. The observation cohort consisted of 314 individuals with inflammatory bowel disease (171 ulcerative colitis and 143 Crohn's Disease patients). Stool samples of each participant were collected using appropriate fecal sampling techniques at the baseline. At the same time window, biochemical and immunological monitoring of each individual from peripheral blood was conducted. The fecal samples were subject to total DNA isolation. In order to evaluate the gut microbiome, shotgun metagenome sequencing was performed. Raw DNA sequencing data were analyzed using bioinformatic techniques. Each metagenome sample were mapped to reference genomes of known gut microbes and the relative coverage of each genomic regions were estimated. Characterization of the metabolic pathways were achieved by using KEGG patways database. It was observed that a individuals harboring both Microbial Anti-Inflammatory Molecule (MAM) gene and polysaccharide A biosynthetic gene cluster have significantly more pronounced anti-inflammatory activity monitored based on plasma Inteleukin-10 levels (FIG. 1). The plasma IL-10 level of each individual was compared based on harboring the defined gene cluster. The observation cohort was divided into two subgroups which are the gene cluster deleted group (the genomic region containing the gene/pathway has less than 5% coverage of the rest of the genome) and the gene cluster harboring group. It was observed that the gene cluster deletion group experiences Hower level of IL-10 compared to the people having a gut microbiome that contains the genes (p<10.sup.5, Mann-Whitney U-test).

[0055] Based on the experiments conducted in this cohort, it is hypothesized that people harboring microorganisms that are able to the corresponding inti-inflammatory molecules in their gut microbiome are in a trend of having more pronounced anti-inflammatory indicators. In case that organisms having that function are administired in a appropriate way, they can provide health benefits to the recepient experiencing pro-inflammation in inflammatory bowel disease. Furthermore, organisms with that property can be positioned as probiotics/biotherapeutics for prevention and treatment of inflammatory bowel diseases.

REFERENCES

[0056] Wen Z, Fiocchi C. Inflammatory bowel disease: autoimmune or immune-mediated pathogenesis?. Clinical and Developmental Immunology. 2004 Sep 1; 11(3-4):195-204.

[0057] Khan I, Ullah N, Zha L, Bai Y, Khan A, Zhao T, Che T, Zhang C. Alteration of gut microbiota in inflammatory bowel disease (IBD): cause or consequence? IBD treatment targeting the gut microbiome. Pathogens. 2019 Sep; 8(3):126.

[0058] Pratt M, Forbes J D, Knox N C, Bernstein C N, Van Domselaar G. Microbiome-Mediated Immune Signaling in Inflammatory Bowel Disease and Colorectal Cancer: Support From Meta-omics Data. Frontiers in Cell and Developmental Biology. 2021; 9.

[0059] Breyner N M, Michon C, de Sousa C S, Vilas Boas P B, Chain F, Azevedo V A, Langella P, Chatel J M. Microbial anti-inflammatory molecule (MAM) from Faecalibacterium prausnitzii shows a protective effect on DNBS and DSS-induced colitis model in mice through inhibition of NF-B pathway. Frontiers in microbiology. 2017 Feb. 1; 8:114.

[0060] Ramakrishna C, Kujawski M, Chu H, Li L, Mazmanian S K, Cantin E M. Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis. Nature communications. 2019 May 14; 10(1):1-3.

[0061] Li M C, He S H. IL-10 and its related cytokines for treatment of inflammatory bowel disease. World Journal of Gastroenterology. 2004 Mar 1; 10(5):620.

[0062] Herfarth H, Schlmerich J. IL-10 therapy in Crohn's disease: at the crossroads. Gut. 2002 Feb 1; 50(2):146-7.

[0063] Solanki H K, Pawar D D, Shah D A, Prajapati V D, Jani G K, Mulla A M, Thakar P M. Development of microencapsulation delivery system for long-term preservation of probiotics as biotherapeutics agent. BioMed research international. Jan 2013 1; 2013.

Sequence Listing XML Reference

[0064] The disclosure herein incorporates by reference, in its entirety, the XML file containing a Sequence Listing: [0065] Name of XML file: MICROORGANISMS SYNTHESIZING ANTI-INFLAMMATORY MOLECULES seq 220719 applicant name.xml [0066] Date of creation: Jul. 19, 2022 [0067] Size of the XLM file: 38 KB