Skip to main content
SpringerLink
Log in

Genome analysis and 2’-fucosyllactose utilization characteristics of a new Akkermansia muciniphila strain isolated from mice feces

  • Original Article
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

Akkermansia muciniphila is considered to be a next-generation probiotic, and closely related to host metabolism and immune response. Compared with other probiotics, little is known about its genomic analysis. Therefore, further researches about isolating more A. muciniphila strains and exploring functional genes are needed. In the present study, a new strain isolated from mice feces was identified as A. muciniphila (MucX). Whole-genome sequencing and annotation revealed that MucX possesses key genes necessary for human milk oligosaccharides (HMO) utilization, including α-l-fucosidases, β-galactosidases, exo-α-sialidases, and β-acetylhexosaminidases. The complete metabolic pathways for γ-aminobutyric acid and squalene and genes encoding functional proteins, such as the outer membrane protein Amuc_1100, were annotated in the MucX genome. Comparative genome analysis was used to identify functional genes unique to MucX compared to six other A. muciniphila strains. Results showed MucX genome possesses unique genes, including sugar transporters and transferases. Single-strain incubation revealed faster utilization of 2′-fucosyllactose (2′-FL), galacto-oligosaccharides, and lactose by MucX than by A. muciniphila DSM 22959. This study isolated and identified an A. muciniphila strain that can utilize 2′-FL, and expolored the genes related to HMO utilization and special metabolites, which provided a theoretical basis for the further excavation of A. muciniphila function and the compound application with fucosylated oligosaccharides.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Aakko J, Kumar H, Rautava S, Wise A, Autran C, Bode L, Isolauri E, Salminen S (2017) Human milk oligosaccharide categories define the microbiota composition in human colostrum. Benef Microbes 8:563–567

    Article  CAS  PubMed  Google Scholar 

  • Arnold JW, Roach J, Fabela S, Moorfield E, Ding S, Blue E, Dagher S, Magness S, Tamayo R, Bruno-Barcena JM, Azcarate-Peril MA (2021) The pleiotropic effects of prebiotic galacto-oligosaccharides on the aging gut. Microbiome 9:31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bertelli C, Tilley KE, Brinkman FSL (2019) Microbial genomic island discovery, visualization and analysis. Brief Bioinform 20(5):1685–1698

    Article  CAS  PubMed  Google Scholar 

  • Bian X, Wu W, Yang L, Lv L, Wang Q, Li Y, Ye J, Fang D, Wu J, Jiang X, Shi D, Li L (2019) Administration of Akkermansia muciniphila ameliorates dextran sulfate sodium-induced ulcerative colitis in mice. Front Microbiol 10:2259

    Article  PubMed  PubMed Central  Google Scholar 

  • Bisson LF, Fan Q, Walker GA (2016) Sugar and glycerol transport in saccharomyces cerevisiae. Adv Exp Med Biol 892:125–168

    Article  CAS  PubMed  Google Scholar 

  • Buchfink B, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12:59–60

    Article  CAS  PubMed  Google Scholar 

  • Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The carbohydrate-active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37:D233-238

    Article  CAS  PubMed  Google Scholar 

  • Chapman GB, Hillier J (1953) Electron microscopy of ultra-thin sections of bacteria I. Cellular division in Bacillus cereus. J Bacteriol 66:362–373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen LQ, Cheung LS, Feng L, Tanner W, Frommer WB (2015) Transport of sugars. Annu Rev Biochem 84:865–894

    Article  CAS  PubMed  Google Scholar 

  • Curiel J, Peirotén Á, Landete J, Bastida A, Langa S, Arqués J (2021) Architecture insight of bifidobacterial α-l-fucosidases. Int J Mol Sci 22(16):8462

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Delcher AL, Bratke KA, Powers EC, Salzberg SL (2007) Identifying bacterial genes and endosymbiont DNA with glimmer. Bioinformatic 23(6):673–679

    Article  CAS  Google Scholar 

  • Derrien M, Vaughan EE, Plugge CM, de Vos WM (2004) Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium. Int J Syst Evol Microbiol 54:1469–1476

    Article  CAS  PubMed  Google Scholar 

  • Duranti S, Ruiz L, Lugli GA, Tames H, Milani C, Mancabelli L, Mancino W, Longhi G, Carnevali L, Sgoifo A, Margolles A, Ventura M, Ruas-Madiedo P, Turroni F (2020) Bifidobacterium adolescentis as a key member of the human gut microbiota in the production of GABA. Sci Rep 10(1):14112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Flemming HC (2016) EPS-then and now. Microorganisms 4:41

    Article  PubMed Central  Google Scholar 

  • Ghimire GP, Thuan NH, Koirala N, Sohng JK (2016) Advances in biochemistry and microbial production of squalene and its derivatives. J Microbiol Biotechnol 26(3):441–451

    Article  CAS  PubMed  Google Scholar 

  • Grześkowiak Ł, Collado MC, Mangani C, Maleta K, Laitinen K, Ashorn P, Isolauri E, Salminen S (2012) Distinct gut microbiota in southeastern African and northern European infants. J Pediatr Gastroenterol Nutr 54:812–816

    Article  PubMed  Google Scholar 

  • Guo X, Li S, Zhang J, Wu F, Li X, Wu D, Zhang M, Ou Z, Jie Z, Yan Q, Li P, Yi J, Peng Y (2017) Genome sequencing of 39 Akkermansia muciniphila isolates reveals its population structure, genomic and functional diversity, and global distribution in mammalian gut microbiotas. BMC Genomics 18(1):800

    Article  PubMed  PubMed Central  Google Scholar 

  • Hagi T, Geerlings SY, Nijsse B, Belzer C (2020) The effect of bile acids on the growth and global gene expression profiles in Akkermansia muciniphila. Appl Microbiol Biotechnol 104:10641–10653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hasan MM, Khan SN, Karim MM, Begum A, Hoq MM (2019) Complete genome and plasmid sequence of a novel Bacillus sp. BD59S, a parasporal protein synthesizing bacterium. 3 Biotech 9:318

    Article  PubMed  PubMed Central  Google Scholar 

  • Henrick BM, Rodriguez L, Lakshmikanth T, Pou C, Henckel E, Arzoomand A, Olin A, Wang J, Mikes J, Tan Z, Chen Y, Ehrlich AM, Bernhardsson AK, Mugabo CH, Ambrosiani Y, Gustafsson A, Chew S, Brown HK, Prambs J, Bohlin K, Mitchell RD, Underwood MA, Smilowitz JT, German JB, Frese SA, Brodin P (2021) Bifidobacteria-mediated immune system imprinting early in life. Cell 184:3884–3898

    Article  CAS  PubMed  Google Scholar 

  • Higarza SG, Arboleya S, Arias JL, Gueimonde M, Arias N (2021) Akkermansia muciniphila and environmental enrichment reverse cognitive impairment associated with high-fat high-cholesterol consumption in rats. Gut Microbes 13:1880240

    Article  PubMed Central  Google Scholar 

  • Karcher N, Nigro E, Punčochář M, Blanco-Míguez A, Ciciani M, Manghi P, Zolfo M, Cumbo F, Manara S, Golzato D, Cereseto A, Arumugam M, Bui TPN, Tytgat HLP, Valles-Colomer M, de Vos WM, Segata N (2021) Genomic diversity and ecology of human-associated Akkermansia species in the gut microbiome revealed by extensive metagenomic assembly. Genome Biol 22:209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Korpela K, Salonen A, Hickman B, Kunz C, Sprenger N, Kukkonen K, Savilahti E, Kuitunen M, de Vos W (2018) Fucosylated oligosaccharides in mother’s milk alleviate the effects of caesarean birth on infant gut microbiota. Sci Rep 8:13757

    Article  PubMed  PubMed Central  Google Scholar 

  • Koskiniemi S, Lamoureux JG, Nikolakakis KC, de Roodenbeke C, Kaplan MD, Low DA, Hayes CS (2013) Rhs proteins from diverse bacteria mediate intercellular competition. Proc Natl Acad Sci USA 110:7032–7037

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kostopoulos I, Elzinga J, Ottman N, Klievink JT, Blijenberg B, Aalvink S, Boeren S, Mank M, Knol J, de Vos WM, Belzer C (2020) Akkermansia muciniphila uses human milk oligosaccharides to thrive in the early life conditions in vitro. Sci Rep 10:14330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 70 for bigger datasets. Mol Biol Evol 33(7):1870–1874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lawson MAE, O’Neill IJ, Kujawska M, Gowrinadh Javvadi S, Wijeyesekera A, Flegg Z, Chalklen L, Hall LJ (2020) Breast milk-derived human milk oligosaccharides promote Bifidobacterium interactions within a single ecosystem. ISME J 14:635–648

    Article  CAS  PubMed  Google Scholar 

  • Li N, Fu X, Xiao M, Wei X, Yang M, Liu Z, Mou H (2020) Enzymatic preparation of a low-molecular-weight polysaccharide rich in uronic acid from the seaweed Laminaria japonica and evaluation of its hypolipidemic effect in mice. Food Funct 11:2395–2405

    Article  CAS  PubMed  Google Scholar 

  • Li N, Liu B, Diao M, Lu J, Liu W, Tong Y (2021) Construction of squalene producing cell factories and screening, cloning and expression of key genes. Chin J Biotechnol 37:2813–2824

    CAS  Google Scholar 

  • Liu N, Zhu L, Zhang Z, Huang H, Jiang L (2019) Draft genome sequence of a multidrug-resistant blaOXA-69-producing Acinetobacter baumannii L13 isolated from Tarim River sample in China. J Global Antimicrob Resist 18:145–147

    Article  Google Scholar 

  • Lowe TM, Chan PP (2016) tRNAscan-SE on-line: Search and contextual analysis of transfer RNA genes. Nucleic Acids Res 44:W54-57

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar BA, Lai T, Steppi S, Jobb G, Forster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, Konig A, Liss T, Lussmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer KH (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Luna E, Parkar SG, Kirmiz N, Hartel S, Hearn E, Hossine M, Kurdian A, Mendoza C, Orr K, Padilla L, Ramirez K, Salcedo P, Serrano E, Choudhury B, Paulchakrabarti M, Parker CT, Huynh S, Cooper K, Flores GE (2022) Utilization efficiency of human milk oligosaccharides by human-associated Akkermansia is strain dependent. Appl Environ Microbiol 88(1):e01487-e1521

    Article  CAS  PubMed Central  Google Scholar 

  • Meng X, Zhang J, Wu H, Yu D, Fang X (2020) Akkermansia muciniphila aspartic protease Amuc_1434* inhibits human colorectal cancer LS174T cell viability via TRAIL-mediated apoptosis pathway. Int J Mol Sci 21:3385

    Article  CAS  PubMed Central  Google Scholar 

  • Olson CA, Vuong HE, Yano JM, Liang QY, Nusbaum DJ, Hsiao EY (2018) The gut microbiota mediates the anti-seizure effects of the ketogenic diet. Cell 173:1728–1741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ottman N (2015) Host immunostimulation and substrate utilization of the gut symbiont Akkermansia muciniphila. PhD thesis. Wageningen University, Wageningen, the Netherlands

  • Ottman N, Davids M, Suarez-Diez M, Boeren S, Schaap PJ, Martins Dos Santos VAP, Smidt H, Belzer C, de Vos WM (2017) Genome-scale model and omics analysis of metabolic capacities of Akkermansia muciniphila reveal a preferential mucin-degrading lifestyle. Appl Environ Microbiol 83:e01014-e1017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ouwerkerk JP, Aalvink S, Belzer C, de Vos WM (2016) Akkermansia glycaniphila sp. nov., an anaerobic mucin-degrading bacterium isolated from reticulated python faeces. Int J Syst Evol Microbiol 66(11):4614–4620

    Article  CAS  PubMed  Google Scholar 

  • Plovier H, Everard A, Druart C, Depommier C, Van Hul M, Geurts L, Chilloux J, Ottman N, Duparc T, Lichtenstein L, Myridakis A, Delzenne NM, Klievink J, Bhattacharjee A, van der Ark KC, Aalvink S, Martinez LO, Dumas ME, Maiter D, Loumaye A, Hermans MP, Thissen JP, Belzer C, de Vos WM, Cani PD (2017) A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice. Nat Med 23:107–113

    Article  CAS  PubMed  Google Scholar 

  • Régnier M, Rastelli M, Morissette A, Suriano F, Le Roy T, Pilon G, Delzenne NM, Marette A, Van Hul M, Cani PD (2020) Rhubarb supplementation prevents diet-induced obesity and diabetes in association with increased Akkermansia muciniphila in mice. Nutrients 12:2932

    Article  PubMed Central  Google Scholar 

  • Ribo S, Sánchez-Infantes D, Martinez-Guino L, García-Mantrana I, Ramon-Krauel M, Tondo M, Arning E, Nofrarías M, Osorio-Conles Ó, Fernández-Pérez A, González-Torres P, Cebrià J, Gavaldà-Navarro A, Chenoll E, Isganaitis E, Villarroya F, Vallejo M, Segalés J, Jiménez-Chillarón JC, Bottiglieri T, Demerath EW, Fields DA, Collado MC, Lerin C (2021) Increasing breast milk betaine modulates Akkermansia abundance in mammalian neonates and improves long-term metabolic health. Sci Transl Med 13:eabb0322

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Richter M, Rosselló-Móra R, Glöckner FO, Peplies J (2015) JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 32(6):929–931

    Article  PubMed  PubMed Central  Google Scholar 

  • Sánchez-Fidalgo S, Villegas I, Rosillo MÁ, Aparicio-Soto M, de la Lastra CA (2015) Dietary squalene supplementation improves DSS-induced acute colitis by downregulating p38 MAPK and NFkB signaling pathways. Mol Nutr Food Res 59:284–292

    Article  PubMed  Google Scholar 

  • Sela DA, Chapman J, Adeuya A, Kim JH, Chen F, Whitehead TR, Lapidus A, Rokhsar DS, Lebrilla CB, German JB, Price NP, Richardson PM, Mills DA (2008) The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome. Proc Natl Acad Sci USA 105:18964–18969

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shi L, Muthukumar V, Stachon T, Latta L, Elhawy MI, Gunaratnam G, Orosz E, Seitz B, Kiderlen AF, Bischoff M, Szentmáry N (2020) The effect of anti-amoebic agents and Ce6-PDT on Acanthamoeba castellanii trophozoites and cysts, in vitro. Transl vis Sci Technol 9:29

    Article  PubMed  PubMed Central  Google Scholar 

  • Spanova M, Zweytick D, Lohner K, Klug L, Leitner E, Hermetter A, Daum G (2012) Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae. Biochem Biophys Acta 1821:647–653

    CAS  PubMed  Google Scholar 

  • Takada H, Katoh T, Katayama T (2020) Sialylated O -Glycans from hen egg white ovomucin are decomposed by mucin-degrading gut microbes. J Appl Glycosci 67:31–39

    Article  CAS  Google Scholar 

  • Thanapreedawat P, Kobayashi H, Inui N, Sakamoto K, Kim M, Yoto A, Yokogoshi H (2013) GABA affects novel object recognition memory and working memory in rats. J Nutr Sci Vitaminol 59:152–157

    Article  CAS  PubMed  Google Scholar 

  • Urrutia A, García-Angulo VA, Fuentes A, Caneo M, Legüe M, Urquiza S, Delgado SE, Ugalde J, Burdisso P, Calixto A (2020) Bacterially produced metabolites protect C. elegans neurons from degeneration. PLoS Biol 18:e3000638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Van Passel MW, Kant R, Zoetendal EG, Plugge CM, Derrien M, Malfatti SA, Chain PS, Woyke T, Palva A, de Vos WM, Smidt H (2011) The genome of Akkermansia muciniphila, a dedicated intestinal mucin degrader, and its use in exploring intestinal metagenomes. PLoS ONE 6(3):e16876

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang L, Tang L, Feng Y, Zhao S, Han M, Zhang C, Yuan G, Zhu J, Cao S, Wu Q, Li L, Zhang Z (2020) A purified membrane protein from Akkermansia muciniphila or the pasteurised bacterium blunts colitis associated tumourigenesis by modulation of CD8+ T cells in mice. Gut 69:1988–1997

    Article  CAS  PubMed  Google Scholar 

  • Wu R, Zhou M, Wu H (2021) Purification and characterization of an active N-acetylglucosaminyltransferase enzyme complex from Streptococci. Appl Environ Microbiol 76:7966–7971

    Article  Google Scholar 

  • Wu J, Xu R, Lu J, Liu W, Yu H, Liu M, Li J, Yin M, Peng H, Zha L (2022) Molecular cloning and functional characterization of two squalene synthase genes in Atractylodes lancea. Planta 255:8

    Article  CAS  Google Scholar 

  • Xiao M, Ren X, Cui J, Li R, Liu Z, Zhu L, Kong Q, Fu X, Mou H (2022) A novel glucofucobiose with potential prebiotic activity prepared from the exopolysaccharides of Clavibacter michiganensis M1. Food Chem 377:132001

    Article  CAS  PubMed  Google Scholar 

  • Xu Y, Wang N, Tan HY, Li S, Zhang C, Feng Y (2020) Function of Akkermansia muciniphila in obesity: Interactions with lipid metabolism, immune response and gut systems. Front Microbiol 11:219

    Article  PubMed  PubMed Central  Google Scholar 

  • Yoon HS, Cho CH, Yun MS, Jang SJ, You HJ, Kim JH, Han D, Cha KH, Moon SH, Lee K, Kim YJ, Lee SJ, Nam TW, Ko G (2021) Akkermansia muciniphila secretes a glucagon-like peptide-1-inducing protein that improves glucose homeostasis and ameliorates metabolic disease in mice. Nat Microbiol 6:563–573

    Article  CAS  PubMed  Google Scholar 

  • Zhai R, Xue X, Zhang L, Yang X, Zhao L, Zhang C (2019) Strain-specific anti-inflammatory properties of two Akkermansia muciniphila strains on chronic colitis in mice. Front Cell Infect Microbiol 9:239

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu L, Zhang D, Zhu H, Zhu J, Weng S, Dong L, Liu T, Hu Y, Shen X (2017) Berberine treatment increases Akkermansia in the gut and improves high-fat diet-induced atherosclerosis in Apoe-/- mice. Atherosclerosis 268:117–126

    Article  PubMed  Google Scholar 

  • Zou Y, Wang J, Wang Y, Peng B, Liu J, Zhang B, Lv H, Wang S (2020) Protection of galacto-oligosaccharide against E coli O157 colonization through enhancing gut barrier function and modulating gut microbiota. Foods 9:1710

    Article  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31872893) and the China Postdoctoral Science Foundation (2021M701547).

Funding

National Natural Science Foundation of China, 31872893, Haijin Mou, China Postdoctoral Science Foundation, 2021M701547, Xiaodan Fu.

Author information

Author notes

  1. Wei Gao and Mengshi Xiao have contributed equally to this work.

Authors and Affiliations

  1. College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, Shandong Province, People’s Republic of China

    Wei Gao, Mengshi Xiao, Ziqiang Gu, Xinmiao Ren, Ying Yu, Zhemin Liu, Changliang Zhu, Qing Kong & Haijin Mou

  2. State Key Laboratory of Food Science and Technology, China-Canada Joint Laboratory of Food Science and Technology (Nanchang), Key Laboratory of Bioactive Polysaccharides of Jiangxi Province, Nanchang University, Nanchang, 330047, Jiangxi Province, People’s Republic of China

    Xiaodan Fu

Authors
  1. Wei Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mengshi Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ziqiang Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaodan Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xinmiao Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ying Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhemin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Changliang Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Qing Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Haijin Mou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Haijin Mou.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest. This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal.

Human and animal rights

This article does not contain any studies with human or animal subjects performed by the any of the authors.

Additional information

Communicated by Martine Collart.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 6156 KB)

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gao, W., Xiao, M., Gu, Z. et al. Genome analysis and 2’-fucosyllactose utilization characteristics of a new Akkermansia muciniphila strain isolated from mice feces. Mol Genet Genomics 297, 1515–1528 (2022). https://doi.org/10.1007/s00438-022-01937-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-022-01937-8

Keywords

Search

Navigation