Korean J Physiol Pharmacol.  2024 Mar;28(2):153-164. 10.4196/kjpp.2024.28.2.153.

Profiling of endogenous metabolites and changes in intestinal microbiota distribution after GEN-001 (Lactococcus lactis) administration

• Kim MG
• Kim S
• Jeon JY
• Moon SJ
• Kwak YG
• Na JY
• Lee S
• Park KM
• Kim HJ
• Lee SM
• Choi SY
• Shin KH

• KMID: 2553527
• DOI: http://doi.org/10.4196/kjpp.2024.28.2.153

Abstract

This study aimed to identify metabolic biomarkers and investigate changes in intestinal microbiota in the feces of healthy participants following administration of Lactococcus lactis GEN-001. GEN-001 is a single-strain L. lactis strain isolated from the gut of a healthy human volunteer. The study was conducted as a parallel, randomized, phase 1, open design trial. Twenty healthy Korean males were divided into five groups according to the GEN-001 dosage and dietary control. Groups A, B, C, and D1 received 1, 3, 6, and 9 GEN-001 capsules (1 × 10¹¹ colony forming units), respectively, without dietary adjustment, whereas group D2 received 9 GEN-001 capsules with dietary adjustment. All groups received a single dose. Fecal samples were collected 2 days before GEN-001 administration to 7 days after for untargeted metabolomics and gut microbial metagenomic analyses; blood samples were collected simultaneously for immunogenicity analysis. Levels of phenylalanine, tyrosine, cholic acid, deoxycholic acid, and tryptophan were significantly increased at 5–6 days after GEN-001 administration when compared with predose levels. Compared with predose, the relative abundance (%) of Parabacteroides and Alistipes significantly decreased, whereas that of Lactobacillus and Lactococcus increased; Lactobacillus and tryptophan levels were negatively correlated. A single administration of GEN-001 shifted the gut microbiota in healthy volunteers to a more balanced state as evidenced by an increased abundance of beneficial bacteria, including Lactobacillus, and higher levels of the metabolites that have immunogenic properties.

Keyword

Biological products; Gastrointestinal microbiome; Lactococcus lactis; Metabolomics; Microbiota

Figure

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  Fig. 1 Study design and sample collection time points. LBP, live biotherapeutic product.

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  Fig. 2 Pathway and network analyses. (A) Pathway analysis results were significant at p < 0.05 and a pathway impact of 0.1. (B) Network analysis results were significant at a degree of ≥ 2, betweenness ≥ 1.0, and p < 0.05. Color boxes and numbers are indicated in the function table.

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  Fig. 3 Alterations in the relative abundance of Lactococcus lactis and Spearman’s correlation analysis between L. lactis and metabolites. Alterations in the relative abundance of L. lactis and Spearman’s correlation analysis between L. lactis and metabolites, (A) before and after GEN-001 administration, (B) with different GEN-001 doses, and (C) with or without dietary control. (D) Correlation analysis between L. lactis composition and metabolites in fecal samples 1–2 days after GEN-001 administration vs. predose. Values are presented as mean with standard deviation. Group A, four healthy adult men who take 1 × 1011 CFU; Group B, four healthy adult men who take 3 × 1011 CFU; Group C, four healthy adult men who take 6 × 1011 CFU; Group D1, four healthy adult men who take 9 × 1011 CFU without dietary control; Group D2, four healthy adult men who take 9 × 1011 CFU with dietary control; CFU, colony forming unit. *p < 0.05 and **p < 0.01.

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  Fig. 4 Genus level changes in microbial distribution. (A) Gut microbial composition at genus levels before and after GEN-001 administration over time (groups A–D1). (B) Changes in the relative abundance (%) of Alistipes and Parabacteroides in the top 10 genera. (C) Changes in the relative abundance of genera used in probiotics (Lactobacillus, Lactococcus, and Bifidobacterium) after GEN-001 administration. (D) Spearman’s correlation analysis between the composition of the three probiotic genera and metabolites in fecal samples. Values are presented as mean with standard deviation. *p < 0.05.

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  Fig. 5 Changes in concentrations of metabolites after GEN-001 administration. (A) p-cresol sulfate (tyrosine metabolite), (B) Indole-3-acetic acid (tryptophan metabolite). Values are presented as mean with standard deviation. *p < 0.05.

Reference

1. Candelli M, Franza L, Pignataro G, Ojetti V, Covino M, Piccioni A, Gasbarrini A, Franceschi F. 2021; Interaction between lipopolysaccharide and gut microbiota in inflammatory bowel diseases. Int J Mol Sci. 22:6242. DOI: 10.3390/ijms22126242. PMID: 34200555. PMCID: PMC8226948.

2. Vatanen T, Kostic AD, d'Hennezel E, Siljander H, Franzosa EA, Yassour M, Kolde R, Vlamakis H, Arthur TD, Hämäläinen AM, Peet A, Tillmann V, Uibo R, Mokurov S, Dorshakova N, Ilonen J, Virtanen SM, Szabo SJ, Porter JA, Lähdesmäki H, et al. DIABIMMUNE Study Group. Xavier RJ. 2016; Variation in microbiome LPS immunogenicity contributes to autoimmunity in humans. Cell. 165:842–853. Erratum in: Cell. 2016;165:1551. DOI: 10.1016/j.cell.2016.04.007. PMID: 27133167. PMCID: PMC4950857.

3. Orivuori L, Mustonen K, de Goffau MC, Hakala S, Paasela M, Roduit C, Dalphin JC, Genuneit J, Lauener R, Riedler J, Weber J, von Mutius E, Pekkanen J, Harmsen HJM, Vaarala O. PASTURE Study Group. 2015; High level of fecal calprotectin at age 2 months as a marker of intestinal inflammation predicts atopic dermatitis and asthma by age 6. Clin Exp Allergy. 45:928–939. DOI: 10.1111/cea.12522. PMID: 25758537.

4. Yang W, Cong Y. 2021; Gut microbiota-derived metabolites in the regulation of host immune responses and immune-related inflammatory diseases. Cell Mol Immunol. 18:866–877. DOI: 10.1038/s41423-021-00661-4. PMID: 33707689. PMCID: PMC8115644.

5. Tremaroli V, Bäckhed F. 2012; Functional interactions between the gut microbiota and host metabolism. Nature. 489:242–249. DOI: 10.1038/nature11552. PMID: 22972297.

6. Postler TS, Ghosh S. 2017; Understanding the holobiont: how microbial metabolites affect human health and shape the immune system. Cell Metab. 26:110–130. DOI: 10.1016/j.cmet.2017.05.008. PMID: 28625867. PMCID: PMC5535818.

7. Carding S, Verbeke K, Vipond DT, Corfe BM, Owen LJ. 2015; Dysbiosis of the gut microbiota in disease. Microb Ecol Health Dis. 26:26191. DOI: 10.3402/mehd.v26.26191. PMID: 25651997. PMCID: PMC4315779.

8. Li SX, Armstrong A, Neff CP, Shaffer M, Lozupone CA, Palmer BE. 2016; Complexities of gut microbiome dysbiosis in the context of HIV infection and antiretroviral therapy. Clin Pharmacol Ther. 99:600–611. DOI: 10.1002/cpt.363. PMID: 26940481. PMCID: PMC4927263.

9. Manichanh C, Borruel N, Casellas F, Guarner F. 2012; The gut microbiota in IBD. Nat Rev Gastroenterol Hepatol. 9:599–608. DOI: 10.1038/nrgastro.2012.152. PMID: 22907164.

10. Federico A, Dallio M, DI Sarno R, Giorgio V, Miele L. 2017; Gut microbiota, obesity and metabolic disorders. Minerva Gastroenterol Dietol. 63:337–344. DOI: 10.23736/S1121-421X.17.02376-5. PMID: 28927249.

11. Wang Z, Wang Q, Zhao J, Gong L, Zhang Y, Wang X, Yuan Z. 2019; Altered diversity and composition of the gut microbiome in patients with cervical cancer. AMB Express. 9:40. DOI: 10.1186/s13568-019-0763-z. PMID: 30904962. PMCID: PMC6431307.

12. Sims TT, Colbert LE, Zheng J, Delgado Medrano AY, Hoffman KL, Ramondetta L, Jazaeri A, Jhingran A, Schmeler KM, Daniel CR, Klopp A. 2019; Gut microbial diversity and genus-level differences identified in cervical cancer patients versus healthy controls. Gynecol Oncol. 155:237–244. DOI: 10.1016/j.ygyno.2019.09.002. PMID: 31500892. PMCID: PMC6825899.

13. Kang GU, Jung DR, Lee YH, Jeon SY, Han HS, Chong GO, Shin JH. 2020; Dynamics of fecal microbiota with and without invasive cervical cancer and its application in early diagnosis. Cancers (Basel). 12:3800. DOI: 10.3390/cancers12123800. PMID: 33339445. PMCID: PMC7766064.

14. Cordaillat-Simmons M, Rouanet A, Pot B. 2020; Live biotherapeutic products: the importance of a defined regulatory framework. Exp Mol Med. 52:1397–1406. DOI: 10.1038/s12276-020-0437-6. PMID: 32908212. PMCID: PMC8080583.

15. Paquet JC, Claus SP, Cordaillat-Simmons M, Mazier W, Rawadi G, Rinaldi L, Elustondo F, Rouanet A. 2021; Entering first-in-human clinical study with a single-strain live biotherapeutic product: input and feedback gained from the EMA and the FDA. Front Med (Lausanne). 8:716266. DOI: 10.3389/fmed.2021.716266. PMID: 34458291. PMCID: PMC8385711.

16. Dreher-Lesnick SM, Stibitz S, Carlson PE Jr. 2017; U.S. regulatory considerations for development of live biotherapeutic products as drugs. Microbiol Spectr. 5:BAD–0017. DOI: 10.1128/microbiolspec.BAD-0017-2017. PMID: 28975881.

17. U.S. Food and Drug Administration. 2016. Early Clinical Trials with Live Biotherapeutic Products: Chemistry, Manufacturing, and Control Information [Internet]. U.S. Food and Drug Administration. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/early-clinical-trials-live-biotherapeutic-products-chemistry-manufacturing-and-control-information. cited 2023 Dec 4.

18. Yu JS, Youn GS, Choi J, Kim CH, Kim BY, Yang SJ, Lee JH, Park TS, Kim BK, Kim YB, Roh SW, Min BH, Park HJ, Yoon SJ, Lee NY, Choi YR, Kim HS, Gupta H, Sung H, Han SH, et al. 2021; Lactobacillus lactis and Pediococcus pentosaceus-driven reprogramming of gut microbiome and metabolome ameliorates the progression of non-alcoholic fatty liver disease. Clin Transl Med. 11:e634. DOI: 10.1002/ctm2.634. PMID: 34965016. PMCID: PMC8715831.

19. Ye J, Erland LAE, Gill SK, Bishop SL, Verdugo-Meza A, Murch SJ, Gibson DL. 2021; Metabolomics-guided hypothesis generation for mechanisms of intestinal protection by live biotherapeutic products. Biomolecules. 11:738. DOI: 10.3390/biom11050738. PMID: 34063522. PMCID: PMC8156236.

20. Luerce TD, Gomes-Santos AC, Rocha CS, Moreira TG, Cruz DN, Lemos L, Sousa AL, Pereira VB, de Azevedo M, Moraes K, Cara DC, LeBlanc JG, Azevedo V, Faria AMC, Miyoshi A. 2014; Anti-inflammatory effects of Lactococcus lactis NCDO 2118 during the remission period of chemically induced colitis. Gut Pathog. 6:33. DOI: 10.1186/1757-4749-6-33. PMID: 25110521. PMCID: PMC4126083.

21. Jin SW, Lee GH, Jang MJ, Hong GE, Kim JY, Park GD, Jin H, Kim HS, Choi JH, Choi CY, Lee SG, Jeong HG, Hwang YP. 2020; Immunomodulatory activity of Lactococcus lactis GCWB1176 in cyclophosphamide-induced immunosuppression model. Microorganisms. 8:1175. DOI: 10.3390/microorganisms8081175. PMID: 32748895. PMCID: PMC7464527.

22. Peng X, Zhang R, Duan G, Wang C, Sun N, Zhang L, Chen S, Fan Q, Xi Y. 2018; Production and delivery of Helicobacter pylori NapA in Lactococcus lactis and its protective efficacy and immune modulatory activity. Sci Rep. 8:6435. DOI: 10.1038/s41598-018-24879-x. PMID: 29691472. PMCID: PMC5915382.

23. Kim S, Kim Y, Lee S, Kim Y, Jeon B, Kim H, Park H. 2022; Live biotherapeutic Lactococcus lactis GEN3013 enhances antitumor efficacy of cancer treatment via modulation of cancer progression and immune system. Cancers (Basel). 14:4083. DOI: 10.3390/cancers14174083. PMID: 36077619. PMCID: PMC9455052.

24. Kim SY, Kim JE, Lee KW, Lee HJ. 2009; Lactococcus lactis ssp. lactis inhibits the proliferation of SNU-1 human stomach cancer cells through induction of G0/G1 cell cycle arrest and apoptosis via p53 and p21 expression. Ann N Y Acad Sci. 1171:270–275. DOI: 10.1111/j.1749-6632.2009.04721.x. PMID: 19723065.

25. Kim JY, Woo HJ, Kim YS, Kim KH, Lee HJ. 2003; Cell cycle dysregulation induced by cytoplasm of Lactococcus lactis ssp lactis in SNUC2A, a colon cancer cell line. Nutr Cancer. 46:197–201. DOI: 10.1207/S15327914NC4602_13. PMID: 14690796.

26. Bohlul E, Hasanlou F, Taromchi AH, Nadri S. 2019; TRAIL-expressing recombinant Lactococcus lactis induces apoptosis in human colon adenocarcinoma SW480 and HCT116 cells. J Appl Microbiol. 126:1558–1567. DOI: 10.1111/jam.14237. PMID: 30815963.

27. Abooshahab R, Hooshmand K, Razavi SA, Gholami M, Sanoie M, Hedayati M. 2020; Plasma metabolic profiling of human thyroid nodules by gas chromatography-mass spectrometry (GC-MS)-based untargeted metabolomics. Front Cell Dev Biol. 8:385. DOI: 10.3389/fcell.2020.00385. PMID: 32612989. PMCID: PMC7308550.

28. Land MH, Rouster-Stevens K, Woods CR, Cannon ML, Cnota J, Shetty AK. 2005; Lactobacillus sepsis associated with probiotic therapy. Pediatrics. 115:178–181. DOI: 10.1542/peds.2004-2137. PMID: 15629999.

29. Kalia VC, Patel SKS, Cho BK, Wood TK, Lee JK. 2022; Emerging applications of bacteria as antitumor agents. Semin Cancer Biol. 86:1014–1025. DOI: 10.1016/j.semcancer.2021.05.012. PMID: 33989734.

30. Blachier F, Andriamihaja M. 2022; Effects of the L-tyrosine-derived bacterial metabolite p-cresol on colonic and peripheral cells. Amino Acids. 54:325–338. DOI: 10.1007/s00726-021-03064-x. PMID: 34468872.

31. Wypych TP, Pattaroni C, Perdijk O, Yap C, Trompette A, Anderson D, Creek DJ, Harris NL, Marsland BJ. 2021; Microbial metabolism of L-tyrosine protects against allergic airway inflammation. Nat Immunol. 22:279–286. DOI: 10.1038/s41590-020-00856-3. PMID: 33495652.

32. Musso NR, Brenci S, Indiveri F, Lotti G. 1997; L-tyrosine and nicotine induce synthesis of L-Dopa and norepinephrine in human lymphocytes. J Neuroimmunol. 74:117–120. DOI: 10.1016/S0165-5728(96)00212-3. PMID: 9119963.

33. Duboc H, Rajca S, Rainteau D, Benarous D, Maubert MA, Quervain E, Thomas G, Barbu V, Humbert L, Despras G, Bridonneau C, Dumetz F, Grill JP, Masliah J, Beaugerie L, Cosnes J, Chazouillères O, Poupon R, Wolf C, Mallet JM, et al. 2013; Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases. Gut. 62:531–539. DOI: 10.1136/gutjnl-2012-302578. PMID: 22993202.

34. Brestoff JR, Artis D. 2013; Commensal bacteria at the interface of host metabolism and the immune system. Nat Immunol. 14:676–684. DOI: 10.1038/ni.2640. PMID: 23778795. PMCID: PMC4013146.

35. Dsouza M, Menon R, Crossette E, Bhattarai SK, Schneider J, Kim YG, Reddy S, Caballero S, Felix C, Cornacchione L, Hendrickson J, Watson AR, Minot SS, Greenfield N, Schopf L, Szabady R, Patarroyo J, Smith W, Harrison P, Kuijper EJ, et al. 2022; Colonization of the live biotherapeutic product VE303 and modulation of the microbiota and metabolites in healthy volunteers. Cell Host Microbe. 30:583–598.e8. DOI: 10.1016/j.chom.2022.03.016. PMID: 35421353.

36. Agus A, Planchais J, Sokol H. 2018; Gut microbiota regulation of tryptophan metabolism in health and disease. Cell Host Microbe. 23:716–724. DOI: 10.1016/j.chom.2018.05.003. PMID: 29902437.

37. Ghiboub M, Verburgt CM, Sovran B, Benninga MA, de Jonge WJ, Van Limbergen JE. 2020; Nutritional therapy to modulate tryptophan metabolism and aryl hydrocarbon-receptor signaling activation in human diseases. Nutrients. 12:2846. DOI: 10.3390/nu12092846. PMID: 32957545. PMCID: PMC7551725.

38. Lamas B, Richard ML, Leducq V, Pham HP, Michel ML, Da Costa G, Bridonneau C, Jegou S, Hoffmann TW, Natividad JM, Brot L, Taleb S, Couturier-Maillard A, Nion-Larmurier I, Merabtene F, Seksik P, Bourrier A, Cosnes J, Ryffel B, Beaugerie L, et al. 2016; CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands. Nat Med. 22:598–605. DOI: 10.1038/nm.4102. PMID: 27158904. PMCID: PMC5087285.

39. Zelante T, Iannitti RG, Cunha C, De Luca A, Giovannini G, Pieraccini G, Zecchi R, D'Angelo C, Massi-Benedetti C, Fallarino F, Carvalho A, Puccetti P, Romani L. 2013; Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity. 39:372–385. DOI: 10.1016/j.immuni.2013.08.003. PMID: 23973224.

40. Li Y, Innocentin S, Withers DR, Roberts NA, Gallagher AR, Grigorieva EF, Wilhelm C, Veldhoen M. 2011; Exogenous stimuli maintain intraepithelial lymphocytes via aryl hydrocarbon receptor activation. Cell. 147:629–640. DOI: 10.1016/j.cell.2011.09.025. PMID: 21999944.

41. Wang Q, Yang K, Han B, Sheng B, Yin J, Pu A, Li L, Sun L, Yu M, Qiu Y, Xiao W, Yang H. 2018; Aryl hydrocarbon receptor inhibits inflammation in DSSinduced colitis via the MK2/pMK2/TTP pathway. Int J Mol Med. 41:868–876. DOI: 10.3892/ijmm.2017.3262.

42. Nikolaus S, Schulte B, Al-Massad N, Thieme F, Schulte DM, Bethge J, Rehman A, Tran F, Aden K, Häsler R, Moll N, Schütze G, Schwarz MJ, Waetzig GH, Rosenstiel P, Krawczak M, Szymczak S, Schreiber S. 2017; Increased tryptophan metabolism is associated with activity of inflammatory bowel diseases. Gastroenterology. 153:1504–1516.e2. DOI: 10.1053/j.gastro.2017.08.028. PMID: 28827067.

43. Palego L, Betti L, Rossi A, Giannaccini G. 2016; Tryptophan biochemistry: structural, nutritional, metabolic, and medical aspects in humans. J Amino Acids. 2016:8952520. DOI: 10.1155/2016/8952520. PMID: 26881063. PMCID: PMC4737446.

44. Park J, Kim NE, Yoon H, Shin CM, Kim N, Lee DH, Park JY, Choi CH, Kim JG, Kim YK, Shin TS, Yang J, Park YS. 2021; Fecal microbiota and gut microbe-derived extracellular vesicles in colorectal cancer. Front Oncol. 11:650026. DOI: 10.3389/fonc.2021.650026. PMID: 34595105. PMCID: PMC8477046.

45. Moschen AR, Gerner RR, Wang J, Klepsch V, Adolph TE, Reider SJ, Hackl H, Pfister A, Schilling J, Moser PL, Kempster SL, Swidsinski A, Orth Höller D, Weiss G, Baines JF, Kaser A, Tilg H. 2016; Lipocalin 2 protects from inflammation and tumorigenesis associated with gut microbiota alterations. Cell Host Microbe. 19:455–469. DOI: 10.1016/j.chom.2016.03.007. PMID: 27078067.

46. Tilg H, Adolph TE, Gerner RR, Moschen AR. 2018; The intestinal microbiota in colorectal cancer. Cancer Cell. 33:954–964. DOI: 10.1016/j.ccell.2018.03.004. PMID: 29657127.

47. Park BH, Kim IS, Park JK, Zhi Z, Lee HM, Kwon OW, Lee BC. 2021; Probiotic effect of Lactococcus lactis subsp. cremoris RPG-HL-0136 on intestinal mucosal immunity in mice. Appl Biol Chem. 64:93. DOI: 10.1186/s13765-021-00667-6.

48. Nishiyama K, Kobayashi T, Sato Y, Watanabe Y, Kikuchi R, Kanno R, Koshizuka T, Miyazaki N, Ishioka K, Suzutani T. 2018; A double-blind controlled study to evaluate the effects of yogurt enriched with Lactococcus lactis 11/19-B1 and Bifidobacterium lactis on serum low-density lipoprotein level and antigen-specific interferon-γ releasing ability. Nutrients. 10:1778. DOI: 10.3390/nu10111778. PMID: 30453487. PMCID: PMC6266548.

49. Pan H, Sun T, Cui M, Ma N, Yang C, Liu J, Pang G, Liu B, Li L, Zhang X, Zhang W, Chang J, Wang H. 2022; Light-sensitive Lactococcus lactis for microbe-gut-brain axis regulating via upconversion optogenetic micro-nano system. ACS Nano. 16:6049–6063. DOI: 10.1021/acsnano.1c11536. PMID: 35362965.

50. Wang S. 2023; Multiscale adaptive differential abundance analysis in microbial compositional data. Bioinformatics. 39:btad178. DOI: 10.1093/bioinformatics/btad178. PMID: 37018137. PMCID: PMC10112958.

51. Bruijning M, Ayroles JF, Henry LP, Koskella B, Meyer KM, Metcalf CJE. 2023; Relative abundance data can misrepresent heritability of the microbiome. Microbiome. 11:222. DOI: 10.1186/s40168-023-01669-w. PMID: 37814275. PMCID: PMC10561453.

52. Taguer M, Maurice CF. 2016; The complex interplay of diet, xenobiotics, and microbial metabolism in the gut: Implications for clinical outcomes. Clin Pharmacol Ther. 99:588–599. DOI: 10.1002/cpt.366. PMID: 26950037.

53. Zeng Z, Liu W, Luo S, Hu C, Xu N, Huang A, Xi T, Xing Y. 2019; Shape of gastrointestinal immunity with non-genetically modified Lactococcus lactis particles requires commensal bacteria and myeloid cells-derived TGF-β1. Appl Microbiol Biotechnol. 103:3847–3861. DOI: 10.1007/s00253-019-09716-z. PMID: 30852661.