詳細

栗原　新 （クリハラ　シン）

  • 生物理工学部 食品安全工学科　准教授
Last Updated :2024/05/19

コミュニケーション情報 byコメンテータガイド

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    細菌学、特に食品微生物学および腸内細菌学

研究者情報

学位

  • 博士（生命科学）（2006年03月 京都大学）

ホームページURL

J-Global ID

プロフィール

  • 1978年生まれ、京都府綾部市出身です。
    ヒト腸内常在菌の遺伝子機能を解明し、得られた知見を用いて腸内細菌の代謝産物を最適化することで腸内環境を改善し、ヒト健康寿命を延伸させたいと思っています。また、腸内細菌のプレバイオティクス利用能について実際に培養して解析すると同時にその利用に関わる遺伝子の同定を行い、有用腸内細菌のみを選択的に増殖させる次世代型プレバイオティクスの開発を行っています。

研究キーワード

  • 腸内常在菌叢   プレバイオティクス   Enterococcus faecalis   クオラムセンシング   Proteus mirabilis   ポリアミン   大腸菌   腸内細菌   

現在の研究分野（キーワード）

    細菌学、特に食品微生物学および腸内細菌学

研究分野

  • ライフサイエンス / 細菌学
  • ライフサイエンス / 応用微生物学

経歴

  • 2021年04月 - 現在  近畿大学生物理工学部 食品安全工学科准教授
  • 2019年04月 - 2021年03月  近畿大学生物理工学部 食品安全工学科講師
  • 2013年10月 - 2019年03月  石川県立大学腸内細菌共生機構学寄附講座寄附講座准教授
  • 2013年09月 - 2013年09月  石川県立大学生物資源工学研究所特別研究員
  • 2013年03月 - 2013年08月  米国テキサス大学ダラス校サウスウェスタンメディカルセンター薬学部訪問上席研究員
  • 2011年03月 - 2013年02月  米国エモリー大学医学部微生物免疫学科日本学術振興会海外特別研究員
  • 2010年04月 - 2011年02月  京都大学大学院生命科学研究科微生物細胞機構学分野特定助教
  • 2009年10月 - 2010年03月  京都工芸繊維大学大学院工芸化学研究科微生物工学研究室産学官連携研究員
  • 2009年04月 - 2009年09月  京都工芸繊維大学大学院工芸科学研究科微生物工学研究室日本学術振興会特別研究員 (PD)
  • 2007年04月 - 2009年03月  (独) 理化学研究所バイオリソースセンター微生物材料開発室日本学術振興会特別研究員 (PD)
  • 2006年04月 - 2006年04月  京都大学大学院生命科学研究科分子応答機構学分野有期雇用教職員（研究員）

学歴

  • 2003年04月 - 2006年03月   京都大学大学院   生命科学研究科   統合生命科学専攻 微生物細胞機構学研究室 博士課程
  • 2001年04月 - 2003年03月   京都大学大学院   生命科学研究科   統合生命科学専攻 微生物細胞機構学研究室 修士課程
  • 1997年04月 - 2001年03月   京都大学   農学部   生物機能科学科 応用生命科学専攻

研究活動情報

論文

  • Yuna Miyajima; Shigehiro Karashima; Ren Mizoguchi; Masaki Kawakami; Kohei Ogura; Kazuhiro Ogai; Aoi Koshida; Yasuo Ikagawa; Yuta Ami; Qiunan Zhu; Hiromasa Tsujiguchi; Akinori Hara; Shin Kurihara; Hiroshi Arakawa; Hiroyuki Nakamura; Ikumi Tamai; Hidetaka Nambo; Shigefumi Okamoto
    Scientific reports 14 1 9901 - 9901 2024年04月 [査読有り]
     
    Hyperuricemia (HUA) is a symptom of high blood uric acid (UA) levels, which causes disorders such as gout and renal urinary calculus. Prolonged HUA is often associated with hypertension, atherosclerosis, diabetes mellitus, and chronic kidney disease. Studies have shown that gut microbiota (GM) affect these chronic diseases. This study aimed to determine the relationship between HUA and GM. The microbiome of 224 men and 254 women aged 40 years was analyzed through next-generation sequencing and machine learning. We obtained GM data through 16S rRNA-based sequencing of the fecal samples, finding that alpha-diversity by Shannon index was significantly low in the HUA group. Linear discriminant effect size analysis detected a high abundance of the genera Collinsella and Faecalibacterium in the HUA and non-HUA groups. Based on light gradient boosting machine learning, we propose that HUA can be predicted with high AUC using four clinical characteristics and the relative abundance of nine bacterial genera, including Collinsella and Dorea. In addition, analysis of causal relationships using a direct linear non-Gaussian acyclic model indicated a positive effect of the relative abundance of the genus Collinsella on blood UA levels. Our results suggest abundant Collinsella in the gut can increase blood UA levels.
  • Bin Li; Jue Liang; Hamid R Baniasadi; Shin Kurihara; Margaret A Phillips; Anthony J Michael
    The Journal of biological chemistry 107281 - 107281 2024年04月 [査読有り]
     
    Spermine synthase is an aminopropyltransferase that adds an aminopropyl group to the essential polyamine spermidine to form tetraamine spermine, needed for normal human neural development, plant salt and drought resistance, and yeast CoA biosynthesis. We functionally identify for the first time bacterial spermine synthases, derived from phyla Bacillota, Rhodothermota, Thermodesulfobacteriota, Nitrospirota, Deinococcota and Pseudomonadota. We also identify bacterial aminopropyltransferases that synthesize the spermine same mass isomer thermospermine, from phyla Cyanobacteriota, Thermodesulfobacteriota, Nitrospirota, Dictyoglomota, Armatimonadota and Pseudomonadota, including the human opportunistic pathogen Pseudomonas aeruginosa. Most of these bacterial synthases were capable of synthesizing spermine or thermospermine from the diamine putrescine, and so possess also spermidine synthase activity. We found that most thermospermine synthases could synthesize tetraamine norspermine from triamine norspermidine, i.e., they are potential norspermine synthases. This finding could explain the enigmatic source of norspermine in bacteria. Some of the thermospermine synthases could synthesize norspermidine from diamine 1,3-diaminopropane, demonstrating that they are potential norspermidine synthases. Of 18 bacterial spermidine synthases identified, 17 were able to aminopropylate agmatine to form N1-aminopropylagmatine, including the spermidine synthase of Bacillus subtilis, a species known to be devoid of putrescine. This suggests that the N1-aminopropylagmatine pathway for spermidine biosynthesis, which bypasses putrescine, may be far more widespread than realized and may be the default pathway for spermidine biosynthesis in species encoding L-arginine decarboxylase for agmatine production. Some thermospermine synthases were able to aminopropylate N1-aminopropylagmatine to form N12-guanidinothermospermine. Our study reveals an unsuspected diversification of bacterial polyamine biosynthesis, and suggests a more prominent role for agmatine.
  • Rika Hirano; Izumi Nishita; Riho Nakai; Ayaka Bito; Ryunosuke Sasabe; Shin Kurihara
    Frontiers in Cellular and Infection Microbiology 13 1056866  2023年07月 [査読有り]
     
    In recent years, with the development of non-cultivation approaches, it has become evident that intestinal bacteria have a significant impact on human health. However, because one-third of the genes cannot be annotated, it is difficult to elucidate the function of all intestinal bacteria by in silico analysis, and it is necessary to study the intestinal bacteria by culturing them. In addition, various media recommended for each individual bacterium have been used for culturing intestinal bacteria; however, the preparation of each medium is complex. To simultaneously culture many bacteria and compare bacterial phenotypes under the same conditions, a medium capable of culturing a wide range of bacteria is needed. In this study, we developed GAM + blood medium (GB medium), which consists of Gifu anaerobic medium containing 5% (v/v) horse blood; it is easy to prepare and it allowed the successful cultivation of 85% of the available predominant species in the human intestinal microbiota.
  • Yuta Ami; Narumi Kodama; Masahiro Umeda; Hanae Nakamura; Hideto Shirasawa; Takashi Koyanagi; Shin Kurihara
    International Journal of Molecular Sciences 24 11 9668  2023年06月 [査読有り][招待有り]
     
    Polyamine intake has been reported to help extend the lifespan of animals. Fermented foods contain high concentrations of polyamines, produced by fermenting bacteria. Therefore, the bacteria, isolated from fermented foods that produce large amounts of polyamines, are potentially used as a source of polyamines for humans. In this study, the strain Levilactobacillus brevis FB215, which has the ability to accumulate approximately 200 µM of putrescine in the culture supernatant, was isolated from fermented foods, specifically the Blue Stilton cheese. Furthermore, L. brevis FB215 synthesized putrescine from agmatine and ornithine, which are known polyamine precursors. When cultured in the extract of Sakekasu, a byproduct obtained during the brewing of Japanese rice wine containing high levels of both agmatine and ornithine, L. brevis FB215 grew to OD600 = 1.7 after 83 h of cultivation and accumulated high concentrations (~1 mM) of putrescine in the culture supernatant. The fermentation product also did not contain histamine or tyramine. The Sakekasu-derived ingredient fermented by the food-derived lactic acid bacteria developed in this study could contribute to increasing polyamine intake in humans.
  • Hiromi Shimokawa; Mikiyasu Sakanaka; Yuki Fujisawa; Hirokazu Ohta; Yuta Sugiyama; Shin Kurihara
    Biomedicines 11 4 1123  2023年04月 [査読有り]
     
    Polyamines are bioactive amines that play a variety of roles, such as promoting cell proliferation and protein synthesis, and the intestinal lumen contains up to several mM polyamines derived from the gut microbiota. In the present study, we conducted genetic and biochemical analyses of the polyamine biosynthetic enzyme N-carbamoylputrescine amidohydrolase (NCPAH) that converts N-carbamoylputrescine to putrescine, a precursor of spermidine in Bacteroides thetaiotaomicron, which is one of the most dominant species in the human gut microbiota. First, ncpah gene deletion and complemented strains were generated, and the intracellular polyamines of these strains cultured in a polyamine-free minimal medium were analyzed using high-performance liquid chromatography. The results showed that spermidine detected in the parental and complemented strains was depleted in the gene deletion strain. Next, purified NCPAH-(His)6 was analyzed for enzymatic activity and found to be capable of converting N-carbamoylputrescine to putrescine, with a Michaelis constant (Km) and turnover number (kcat) of 730 µM and 0.8 s−1, respectively. Furthermore, the NCPAH activity was strongly (>80%) inhibited by agmatine and spermidine, and moderately (≈50%) inhibited by putrescine. This feedback inhibition regulates the reaction catalyzed by NCPAH and may play a role in intracellular polyamine homeostasis in B. thetaiotaomicron.
  • Hideto Shirasawa; Chisato Nishiyama; Rika Hirano; Takashi Koyanagi; Shujiro Okuda; Hiroki Takagi; Shin Kurihara
    Bioscience of Microbiota, Food and Health 42 1 24 - 33 2023年01月 [査読有り]
  • Yuta Sugiyama; Yumiko Mori; Misaki Nara; Yusuke Kotani; Emiko Nagai; Hiroki Kawada; Mayu Kitamura; Rika Hirano; Hiromi Shimokawa; Akira Nakagawa; Hiromichi Minami; Aina Gotoh; Mikiyasu Sakanaka; Noriho Iida; Takashi Koyanagi; Takane Katayama; Shigefumi Okamoto; Shin Kurihara
    Gut Microbes 14 2128605  2022年12月 [査読有り]
     
    Colonic luminal aromatic amines have been historically considered to be derived from dietary source, especially fermented foods; however, recent studies indicate that the gut microbiota serves as an alternative source of these amines. Herein, we show that five prominent genera of Firmicutes (Blautia, Clostridium, Enterococcus, Ruminococcus, and Tyzzerella) have the ability to abundantly produce aromatic amines through the action of aromatic amino acid decarboxylase (AADC). In vitro cultivation of human fecal samples revealed that a significant positive correlation between aadc copy number of Ruminococcus gnavus and phenylethylamine (PEA) production. Furthermore, using genetically engineered Enterococcus faecalis-colonized BALB/cCrSlc mouse model, we showed that the gut bacterial aadc stimulates the production of colonic serotonin, which is reportedly involved in osteoporosis and irritable bowel syndrome. Finally, we showed that human AADC inhibitors carbidopa and benserazide inhibit PEA production in En. faecalis.
  • Shin Kurihara
    Bioscience, Biotechnology, and Biochemistry 86 8 957 - 966 2022年06月 [査読有り][招待有り]
     
    Abstract Polyamines (putrescine, spermidine, and spermine) are compounds with amino groups at both ends of a hydrocarbon. Polyamines produced by intestinal bacteria suppress chronic inflammation and enhance the intestinal barrier in the colon, and are also transferred into the blood via the colonic epithelium, resulting in significant improvement of host cognitive performance and life extension in mice. Upregulation of polyamine production by gut microbes can help compensate for aging-associated decrease in polyamine content through the uptake of intestinal luminal polyamine, thereby extending healthy life expectancy of the host. This review summarizes recent advances in the study of polyamine metabolism and transport in gut microbes, with particular reference to Escherichia coli and the most predominant species of the gut microbiota. Furthermore, we describe polyamine production by a novel hybrid system comprised of multiple gut microbes, as well as from high-polyamine-producing lactic acid bacteria derived from fermented foods.
  • Rika Hirano; Aiko Kume; Chisato Nishiyama; Ryosuke Honda; Hideto Shirasawa; Yiwei Ling; Yuta Sugiyama; Misaki Nara; Hiromi Shimokawa; Hiroki Kawada; Takashi Koyanagi; Hisashi Ashida; Shujiro Okuda; Mitsuharu Matsumoto; Hiroki Takagi; Shin Kurihara
    Microorganisms 10 4 697 - 697 2022年03月 [査読有り]
     
    Polyamines are aliphatic hydrocarbons with terminal amino groups and are essential for biological activities. It has been reported that polyamines have health-promoting effects in animals, such as the extension of lifespan by polyamine intake. The identification of a high polyamine-producing bacterium from foods could lead to the development of a novel probiotic candidate. We aimed to identify high polyamine-producing bacteria from food, and isolated and collected bacteria from vegetables and fermented foods produced in Japan. We successfully acquired Latilactobacillus curvatus KP 3-4 isolated from Kabura-zushi as a putrescine producing lactic acid bacteria. Comparing the polyamine synthesis capability of L. curvatus KP 3-4 with that of typical probiotic lactic acid bacteria and L. curvatus strains available from the Japan Collection of Microorganisms, it was found that only L. curvatus KP 3-4 was capable of exporting high levels of putrescine into the culture supernatant. The enhancement of putrescine production by the addition of ornithine, and whole-genome analysis of L. curvatus KP 3-4, suggest that putrescine is synthesized via ornithine decarboxylase. The administration of L. curvatus KP 3-4 to germ-free mice increased the concentration of putrescine in the feces.
  • Riko Ishibashi; Yukihiro Furusawa; Hiroe Honda; Yasuharu Watanabe; Shiho Fujisaka; Miyu Nishikawa; Shinichi Ikushiro; Shin Kurihara; Yoshiaki Tabuchi; Kazuyuki Tobe; Kiyoshi Takatsu; Yoshinori Nagai
    Molecular nutrition & food research 66 10 e2101119  2022年03月 [査読有り]
     
    SCOPE: Isoliquiritigenin (ILG) has been reported to attenuate adipose tissue inflammation and metabolic disorder; however, the underlying mechanisms remain to be elucidated. The aim of this study is to elucidate whether ILG shows the anti-inflammatory and antimetabolic syndrome effects through gut microbiota modification. METHODS & RESULTS: Mice were fed a high-fat diet (HFD) with or without ILG for up to 12 weeks. The effect of ILG on body weight, blood glucose level, adipose tissue inflammation, gut barrier function, and gut microbiota composition were investigated. ILG supplementation alleviated HFD-induced obesity, glucose tolerance, and insulin resistance and suppressed inflammatory gene expression in epididymal white adipose tissue (eWAT). Moreover, ILG supplementation modified gut bacterial composition by increasing the abundance of antimetabolic disease-associated species (e.g., Parabacteroides goldsteinii and Akkemansia muciniphila) and up-regulated genes associated with gut barrier function. Fecal microbiome transplantation (FMT) from ILG-fed donors counteracted HFD-induced body and eWAT weight changes, inflammation-related gene expression, glucose tolerance, and insulin resistance, thereby suggesting that ILG-responsive gut bacteria exert anti-inflammatory and antimetabolic syndrome effects. CONCLUSION: Alterations in gut bacteria underly the beneficial effects of ILG against adipose tissue inflammation and metabolic disorders. ILG may be a promising prebiotic for the prevention and treatment of metabolic syndrome . This article is protected by copyright. All rights reserved.
  • Ayumu Horinouchi; Hirokazu Hirai; Rika Hirano; Shin Kurihara; Hiroki Takagi; Kenji Matsumoto
    Journal of Functional Foods 87 104759 - 104759 2021年12月 [査読有り]
  • Keisuke Yoshida; Rika Hirano; Yohei Sakai; Moonhak Choi; Mikiyasu Sakanaka; Shin Kurihara; Hisakazu Iino; Jin-zhong Xiao; Takane Katayama; Toshitaka Odamaki
    Communications Biology 4 1 541  2021年12月 [査読有り]
     
    AbstractThis study aims to understand the mechanistic basis underlying the response of Bifidobacterium to lactulose ingestion in guts of healthy Japanese subjects, with specific focus on a lactulose transporter. An in vitro assay using mutant strains of Bifidobacterium longum subsp. longum 105-A shows that a solute-binding protein with locus tag number BL105A_0502 (termed LT-SBP) is primarily involved in lactulose uptake. By quantifying faecal abundance of LT-SBP orthologues, which is defined by phylogenetic analysis, we find that subjects with 107 to 109 copies of the genes per gram of faeces before lactulose ingestion show a marked increase in Bifidobacterium after ingestion, suggesting the presence of thresholds between responders and non-responders to lactulose. These results help predict the prebiotics-responder and non-responder status and provide an insight into clinical interventions that test the efficacy of prebiotics.
  • Rika Hirano; Mikiyasu Sakanaka; Kazuto Yoshimi; Naohisa Sugimoto; Syogo Eguchi; Yuko Yamauchi; Misaki Nara; Shingo Maeda; Yuta Ami; Aina Gotoh; Takane Katayama; Noriho Iida; Tamotsu Kato; Hiroshi Ohno; Satoru Fukiya; Atsushi Yokota; Mamoru Nishimoto; Motomitsu Kitaoka; Hiroyuki Nakai; Shin Kurihara
    Gut microbes 13 1 1973835 - 1973835 2021年09月 [査読有り]
     
    Certain existing prebiotics meant to facilitate the growth of beneficial bacteria in the intestine also promote the growth of other prominent bacteria. Therefore, the growth-promoting effects of β-galactosides on intestinal bacteria were analyzed. Galactosyl-β1,4-l-rhamnose (Gal-β1,4-Rha) selectively promoted the growth of Bifidobacterium. Bifidobacterium longum subsp. longum 105-A (JCM 31944) has multiple solute-binding proteins belonging to ATP-binding cassette transporters for sugars. Each strain in the library of 11 B. longum subsp. longum mutants, in which each gene of the solute-binding protein was disrupted, was cultured in a medium containing Gal-β1,4-Rha as the sole carbon source, and only the BL105A_0502 gene-disruption mutant showed delayed and reduced growth compared to the wild-type strain. BL105A_0502 homolog is highly conserved in bifidobacteria. In a Gal-β1,4-Rha-containing medium, Bifidobacterium longum subsp. infantis JCM 1222T, which possesses BLIJ_2090, a homologous protein to BL105A_0502, suppressed the growth of enteric pathogen Clostridioides difficile, whereas the BLIJ_2090 gene-disrupted mutant did not. In vivo, administration of B. infantis and Gal-β1,4-Rha alleviated C. difficile infection-related weight loss in mice. We have successfully screened Gal-β1,4-Rha as a next-generation prebiotic candidate that specifically promotes the growth of beneficial bacteria without promoting the growth of prominent bacteria and pathogens.
  • Atsuo Nakamura; Shin Kurihara; Daisuke Takahashi; Wakana Ohashi; Yutaka Nakamura; Shunsuke Kimura; Masayoshi Onuki; Aiko Kume; Yukiko Sasazawa; Yukihiro Furusawa; Yuuki Obata; Shinji Fukuda; Shinji Saiki; Mitsuharu Matsumoto; Koji Hase
    Nature Communications 12 2105  2021年04月 [査読有り]
     
    AbstractIntestinal microbiota-derived metabolites have biological importance for the host. Polyamines, such as putrescine and spermidine, are produced by the intestinal microbiota and regulate multiple biological processes. Increased colonic luminal polyamines promote longevity in mice. However, no direct evidence has shown that microbial polyamines are incorporated into host cells to regulate cellular responses. Here, we show that microbial polyamines reinforce colonic epithelial proliferation and regulate macrophage differentiation. Colonisation by wild-type, but not polyamine biosynthesis-deficient, Escherichia coli in germ-free mice raises intracellular polyamine levels in colonocytes, accelerating epithelial renewal. Commensal bacterium-derived putrescine increases the abundance of anti-inflammatory macrophages in the colon. The bacterial polyamines ameliorate symptoms of dextran sulfate sodium-induced colitis in mice. These effects mainly result from enhanced hypusination of eukaryotic initiation translation factor. We conclude that bacterial putrescine functions as a substrate for symbiotic metabolism and is further absorbed and metabolised by the host, thus helping maintain mucosal homoeostasis in the intestine.
  • Rika Hirano; Hideto Shirasawa; Shin Kurihara
    Medical Sciences 9 1 8  2021年02月 [査読有り][招待有り]
     
    The purpose of this paper is to summarize the latest information on the various aspects of polyamines and their health benefits. In recent years, attempts to treat cancer by reducing elevated polyamines levels in cancer cells have been made, with some advancing to clinical trials. However, it has been reported since 2009 that polyamines extend the healthy life span of animals by inducing autophagy, protecting the kidneys and liver, improving cognitive function, and inhibiting the progression of heart diseases. As such, there is conflicting information regarding the relationship between polyamines and health. However, attempts to treat cancer by decreasing intracellular polyamines levels are a coping strategy to suppress the proliferation-promoting effects of polyamines, and a consensus is being reached that polyamine intake does not induce cancer in healthy individuals. To provide further scientific evidence for the health-promoting effects of polyamines, large-scale clinical studies involving multiple groups are expected in the future. It is also important to promote basic research on polyamine intake in animals, including elucidation of the polyamine balance between food, intestinal bacteria, and biosynthesis.
  • Takao Nagano; Rika Hirano; Shin Kurihara; Katsuyoshi Nishinari
    Bioscience, Biotechnology, and Biochemistry 84 7 1467 - 1474 2020年07月 [査読有り]
     
    Abstract Improving the physicochemical properties of okara for various applications in foods is of great importance. Here, okara and microcrystalline cellulose (MCC) were atomized using a water jet (WJ) system. The WJ-treated okara and MCC dispersed homogeneously in water, and their median sizes in particle size distribution were 6.6 μm and 9.5 μm, respectively. The dispersions of WJ-treated okara and MCC showed high apparent viscosity and shear thinning behavior. Moreover, the inhibition of α-amylase activities by WJ-treated okara was more effective than that by untreated MCC and cellulose. Furthermore, the production of short-chain fatty acids by 32 dominant species of human gut microbes was determined. An increase in butyrate production by Roseburia intestinalis was observed in the presence of WJ-treated okara, but not in untreated okara or WJ-treated MCC. These results demonstrate that WJ system can be used on okara to increase inhibited α-amylase activities and butyrate production by gut microbiota.
  • Takao Nagano; Yuya Arai; Hiromi Yano; Takafumi Aoki; Shin Kurihara; Rika Hirano; Katsuyoshi Nishinari
    Food Hydrocolloids 109 105964  2020年04月 [査読有り]
  • Yuta Sugiyama; Hirokazu Ohta; Rika Hirano; Hiromi Shimokawa; Mikiyasu Sakanaka; Takashi Koyanagi; Shin Kurihara
    Analytical Biochemistry 593 113607 - 113607 2020年03月 [査読有り]
     
    Quantification of polyamines, including putrescine, is generally performed using high-performance liquid chromatography (HPLC) or gas chromatography. However, these methods are time-consuming because of sample derivatization and analytical reagent preparation. In this study, we developed a simple and high-throughput putrescine quantification method on a 96-well microtiter plate using putrescine oxidase from Rhodococcus erythropolis NCIMB 11540, peroxidase, 4-aminoantipyrine, and N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt. The developed method (named as PuO-POD-4AA-TOPS method) was applicable to bacterial culture supernatants. Furthermore, putrescine concentrations determined by the developed method roughly corresponded to the concentrations determined by HPLC.
  • Shin Kurihara
    Bioscience of Microbiota, Food and Health 40 1 19 - 26 2020年 [査読有り][招待有り]
  • Ashida Hisashi; Fujimoto Taku; Kurihara Shin; Nakamura Masayuki; Komeno Masahiro; Huang Yibo; Katayama Takane; Kinoshita Takashi; Takegawa Kaoru
    Journal of Applied Glycoscience 67 1 23 - 29 日本応用糖質科学会 2020年 [査読有り]
     

    Bifidobacterium longum subsp. infantis ATCC 15697 possesses five α-L-fucosidases, which have been previously characterized toward fucosylated human milk oligosaccharides containing α1,2/3/4-linked fucose [Sela et al.: Appl. Environ. Microbiol., 78, 795-803 (2012)]. In this study, two glycoside hydrolase family 29 α-L-fucosidases out of five (Blon_0426 and Blon_0248) were found to be 1,6-α-L-fucosidases acting on core α1,6-fucose on the N-glycan of glycoproteins. These enzymes readily hydrolyzed p-nitrophenyl-α-L-fucoside and Fucα1-6GlcNAc, but hardly hydrolyzed Fucα1-6(GlcNAcβ1-4)GlcNAc, suggesting that they de-fucosylate Fucα1-6GlcNAcβ1-Asn-peptides/proteins generated by the action of endo-β-N-acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from Cordyceps militaris. To generate homogenous non-fucosylated N-glycan-containing IgG with high antibody-dependent cellular cytotoxicity (ADCC) activity, the resulting GlcNAc-IgG has a potential to be a good acceptor substrate for the glycosynthase mutant of Endo-M from Mucor hiemalis. Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.

  • Miki Matsue; Yumiko Mori; Satoshi Nagase; Yuta Sugiyama; Rika Hirano; Kazuhiro Ogai; Kohei Ogura; Shin Kurihara; Shigefumi Okamoto
    Cell transplantation 28 12 1528 - 1541 2019年12月 [査読有り]
     
    Lauric acid (LA) has a broad spectrum of anti-microbiological activities against enveloped viruses and various bacteria, and might be useful to protect against microbial infection and control the balance and distribution of bacteria in human gut microbiota. It is not necessarily more difficult to measure antimicrobial activity the traditional way, but it is, however, more laborious. In the present study, we developed a new method to measure the antimicrobial activity of LA in multiple samples with a microplate reader. A "test complex" (TC) was produced consisting of 100 μL of agar medium with LA in the bottom layer and 300 μL of broth in the top layer in 96-well deep-well microplates. Afterward, analysis of the broth in the top layer showed that the antimicrobial activity was the same as that of the "control complex," (CC) which consisted of 100 μL of agar medium in the bottom layer and 300 μL of broth with LA in the top layer. Furthermore, evaluation of the antimicrobial effect of the TC when using a microplate reader was the same as that with the use of the colony counting method. The colony counting method has confirmed that the antimicrobial activity of LA when bacteria are inoculated into the broth was equivalent between CC and TC, and we validated this by correlating the number of bacteria with absorbance. In addition, the broth itself in TC was transparent enough that the turbidity of broth can be used as an index of the number of bacteria, which enabled the use of a microplate reader for multiple samples. For human gut microbes, LA was shown to have low antimicrobial activity against commensal lactic acid bacteria, but high antimicrobial activity against pathogenic Bacteroides and Clostridium, suggesting that LA might modulate intestinal health, as confirmed by the proposed method.
  • Bin Li; Shin Kurihara; Sok Ho Kim; Jue Liang; Anthony J Michael
    The Biochemical journal 476 18 2579 - 2594 2019年09月 [査読有り]
     
    The only known function of S-adenosylmethionine decarboxylase (AdoMetDC) is to supply, with its partner aminopropyltransferase enzymes such as spermidine synthase (SpdSyn), the aminopropyl donor for polyamine biosynthesis. Polyamine spermidine is probably essential for the growth of all eukaryotes, most archaea and many bacteria. Two classes of AdoMetDC exist, the prokaryotic class 1a and 1b forms, and the eukaryotic class 2 enzyme, which is derived from an ancient fusion of two prokaryotic class 1b genes. Herein, we show that 'eukaryotic' class 2 AdoMetDCs are found in bacteria and are enzymatically functional. However, the bacterial AdoMetDC class 2 genes are phylogenetically limited and were likely acquired from a eukaryotic source via transdomain horizontal gene transfer, consistent with the class 2 form of AdoMetDC being a eukaryotic invention. We found that some class 2 and thousands of class 1b AdoMetDC homologues are present in bacterial genomes that also encode a gene fusion of an N-terminal membrane protein of the Major Facilitator Superfamily (MFS) class of transporters and a C-terminal SpdSyn-like domain. Although these AdoMetDCs are enzymatically functional, spermidine is absent, and an entire fusion protein or its SpdSyn-like domain only, does not biochemically complement a SpdSyn deletion strain of E. coli This suggests that the fusion protein aminopropylates a substrate other than putrescine, and has a role outside of polyamine biosynthesis. Another integral membrane protein found clustered with these genes is DUF350, which is also found in other gene clusters containing a homologue of the glutathionylspermidine synthetase family and occasionally other polyamine biosynthetic enzymes.
  • Mikiyasu Sakanaka; Morten Ejby Hansen; Aina Gotoh; Toshihiko Katoh; Keisuke Yoshida; Toshitaka Odamaki; Hiroyuki Yachi; Yuta Sugiyama; Shin Kurihara; Junko Hirose; Tadasu Urashima; Jin-Zhong Xiao; Motomitsu Kitaoka; Satoru Fukiya; Atsushi Yokota; Leila Lo Leggio; Maher Abou Hachem; Takane Katayama
    Science advances 5 8 eaaw7696  2019年08月 [査読有り]
     
    The human gut microbiota established during infancy has persistent effects on health. In vitro studies have suggested that human milk oligosaccharides (HMOs) in breast milk promote the formation of a bifidobacteria-rich microbiota in infant guts; however, the underlying molecular mechanism remains elusive. Here, we characterized two functionally distinct but overlapping fucosyllactose transporters (FL transporter-1 and -2) from Bifidobacterium longum subspecies infantis. Fecal DNA and HMO consumption analyses, combined with deposited metagenome data mining, revealed that FL transporter-2 is primarily associated with the bifidobacteria-rich microbiota formation in breast-fed infant guts. Structural analyses of the solute-binding protein (SBP) of FL transporter-2 complexed with 2'-fucosyllactose and 3-fucosyllactose, together with phylogenetic analysis of SBP homologs of both FL transporters, highlight a unique adaptation strategy of Bifidobacterium to HMOs, in which the gain-of-function mutations enable FL transporter-2 to efficiently capture major fucosylated HMOs. Our results provide a molecular insight into HMO-mediated symbiosis and coevolution between bifidobacteria and humans.
  • Bin Li; Yukari Maezato; Sok Ho Kim; Shin Kurihara; Jue Liang; Anthony J Michael
    Molecular microbiology 111 1 159 - 175 2019年01月 [査読有り]
     
    Polyamines such as spermidine and spermine are primordial polycations that are ubiquitously present in the three domains of life. We have found that Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis have lost either all or most polyamine biosynthetic genes, respectively, and are devoid of any polyamine when grown in polyamine-free media. In contrast to bacteria such as Pseudomonas aeruginosa, Campylobacter jejuni and Agrobacterium tumefaciens, which absolutely require polyamines for growth, S. aureus and E. faecalis grow normally over multiple subcultures in the absence of polyamines. Furthermore, S. aureus and E. faecalis form biofilms normally without polyamines, and exogenous polyamines do not stimulate growth or biofilm formation. High levels of external polyamines, including norspermidine, eventually inhibit biofilm formation through inhibition of planktonic growth. We show that spermidine/spermine N-acetyltransferase (SSAT) homologues encoded by S. aureus USA300 and E. faecalis acetylate spermidine, spermine and norspermidine, that spermine is the more preferred substrate, and that E. faecalis SSAT is almost as efficient as human SSAT with spermine as substrate. The polyamine auxotrophy, polyamine-independent growth and biofilm formation, and presence of functional polyamine N-acetyltransferases in S. aureus and E. faecalis represent a new paradigm for bacterial polyamine biology.
  • Aina Gotoh; Toshihiko Katoh; Mikiyasu Sakanaka; Yiwei Ling; Chihaya Yamada; Sadaki Asakuma; Tadasu Urashima; Yusuke Tomabechi; Ayako Katayama-Ikegami; Shin Kurihara; Kenji Yamamoto; Gaku Harata; Fang He; Junko Hirose; Motomitsu Kitaoka; Shujiro Okuda; Takane Katayama
    Scientific reports 8 1 13958 - 13958 2018年09月 [査読有り]
     
    Gut microbiota of breast-fed infants are generally rich in bifidobacteria. Recent studies show that infant gut-associated bifidobacteria can assimilate human milk oligosaccharides (HMOs) specifically among the gut microbes. Nonetheless, little is known about how bifidobacterial-rich communities are shaped in the gut. Interestingly, HMOs assimilation ability is not related to the dominance of each species. Bifidobacterium longum susbp. longum and Bifidobacterium breve are commonly found as the dominant species in infant stools; however, they show limited HMOs assimilation ability in vitro. In contrast, avid in vitro HMOs consumers, Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, are less abundant in infant stools. In this study, we observed altruistic behaviour by B. bifidum when incubated in HMOs-containing faecal cultures. Four B. bifidum strains, all of which contained complete sets of HMO-degrading genes, commonly left HMOs degradants unconsumed during in vitro growth. These strains stimulated the growth of other Bifidobacterium species when added to faecal cultures supplemented with HMOs, thereby increasing the prevalence of bifidobacteria in faecal communities. Enhanced HMOs consumption by B. bifidum-supplemented cultures was also observed. We also determined the complete genome sequences of B. bifidum strains JCM7004 and TMC3115. Our results suggest B. bifidum-mediated cross-feeding of HMOs degradants within bifidobacterial communities.
  • Yuta Sugiyama; Misaki Nara; Mikiyasu Sakanaka; Aya Kitakata; Shujiro Okuda; Shin Kurihara
    Bioscience, biotechnology, and biochemistry 82 9 1606 - 1614 2018年09月 [査読有り]
     
    Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium. ABBREVIATIONS: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion.
  • Yusuke Kitada; Koji Muramatsu; Hirokazu Toju; Ryoko Kibe; Yoshimi Benno; Shin Kurihara; Mitsuharu Matsumoto
    Science advances 4 6 eaat0062  2018年06月 [査読有り]
     
    Metabolites of the intestinal microbiota are thought to be generated through metabolic pathways spanning multiple taxa of intestinal bacteria. We have previously shown that the level of putrescine, a polyamine found abundantly in the human intestinal lumen, is increased in the colonic lumen following administration of arginine and the probiotic Bifidobacterium sp.; however, the underlying mechanism remained poorly understood. We report a novel pathway for putrescine production from arginine through agmatine involving the collaboration of two bacterial groups, and triggered by environmental acidification (drop in pH to below 6.5 from neutral). This pathway comprises the acid tolerance system of Escherichia coli, representing bacteria that have an arginine-dependent acid resistance system; the energy production system of Enterococcus faecalis, representing bacteria that have an agmatine deiminase system; and the acid production system of the acid-producing bacteria, represented by Bifidobacterium spp. This pathway is unique in that it represents a relationship between the independent survival strategies of multiple bacteria.
  • Mikiyasu Sakanaka; Yuta Sugiyama; Misaki Nara; Aya Kitakata; Shin Kurihara
    FEMS microbiology letters 365 4 fny003  2018年02月 [査読有り]
     
    Polyamine concentrations in the intestine are regulated by their biosynthesis by hundreds of gut microbial species and these polyamines are involved in host health and disease. However, polyamine biosynthesis has not been sufficiently analyzed in major members of the human gut microbiota, possibly owing to a lack of gene manipulation systems. In this study, we successfully performed markerless gene deletion in Bacteroides dorei, one of the major members of the human gut microbiota. The combination of a thymidine kinase gene (tdk) deletion mutant and a counter-selection marker tdk, which has been applied in other Bacteroides species, was used for the markerless gene deletion. Deletion of tdk in B. dorei caused 5-fluoro-2΄-deoxyuridine resistance, suggesting the utility of B. dorei Δtdk as the host for future markerless gene deletions. Compared to parental strains, an arginine decarboxylase gene (speA) deletion mutant generated in this system showed a severe growth defect and decreased concentration of spermidine in the cells and culture supernatant. Collectively, our results indicate the accessibility of gene deletion and the important role of speA in polyamine biosynthesis in B. dorei.
  • 栗原新
    腸内細菌学雑誌 32 4 175‐186(J‐STAGE) - 186 公益財団法人 日本ビフィズス菌センター 2018年 
    近年の精力的な解析により，多くの腸内細菌叢のデータが蓄積され，腸内環境制御への期待が高まっている．一方で，食餌を変化させた場合の腸内細菌叢とトランスクリプトームの変化の比較から，腸内環境制御に重要なのは腸内細菌の遺伝子機能の制御であることが示唆されている．しかしながら，ヒト腸内細菌叢のメタゲノムデータから得られる塩基配列の情報のうち，機能が予測可能なもの，明らかとなっているものはごく一部であり，遺伝子機能の制御は現状困難である．この状況を打破するためには腸内細菌の遺伝子機能の実験的な同定が重要であると考えられる．腸内細菌の菌体そのものは，大腸の免疫機構に阻まれてごく一部しかヒト組織に接触できない．これとは対照的に腸内細菌の代謝産物は，腸管上皮を通過して体内に取り込まれ，ヒト健康に対してより直接的な影響を与える．腸内細菌の代謝産物を制御するためにはその合成・輸送に関わる遺伝子機能の制御が必須となる．我々は，腸内細菌の遺伝子機能の解明を通じた腸内環境制御を最終目標として，ヒト腸内常在菌叢最優勢種のハイスループット培養系を構築した．この系を用いて腸内細菌のポリアミン合成・輸送をウェットに解析し，得られた解析結果を<i>in silico</i>解析結果と比較したところ，全く新規な配列を持つポリアミン合成酵素や輸送タンパクの存在が示唆された．次に，<i>Bacteroides</i>属細菌のポリアミン合成系についての遺伝子破壊株・遺伝子相補株を用いて解析を行った．腸内細菌は複雑な細菌叢を形成しているため，細菌叢を構成する細菌間で代謝産物の授受を行っていると考えられている．我々は，遺伝子操作した大腸菌および<i>Enterococcus faecalis</i>を共培養し，複数菌種にまたがるポリアミン合成系の存在を遺伝子レベルで証明した．さらに，国内外の遺伝操作した腸内細菌を用いた研究の動向と，腸内細菌の遺伝子レベルでの研究による波及効果について概説した．これまでの多くの腸内細菌研究は宿主側を中心に行われており，腸内細菌側の解析については菌叢解析を中心としており，腸内環境の制御については今後の課題である．腸内細菌の遺伝子レベルでの研究はその制御に必須であることから，今後，その重要性が高まると考えられる．</p>
  • 栗原 新
    日本微生物資源学会誌 = Microbial resources and systematics 33 2 75 - 80 日本微生物資源学会 2017年12月
  • Yuta Sugiyama; Misaki Nara; Mikiyasu Sakanaka; Aina Gotoh; Aya Kitakata; Shujiro Okuda; Shin Kurihara
    The international journal of biochemistry & cell biology 93 52 - 61 2017年12月 [査読有り]
     
    Recent studies have reported that polyamines in the colonic lumen might affect animal health and these polyamines are thought to be produced by gut bacteria. In the present study, we measured the concentrations of three polyamines (putrescine, spermidine, and spermine) in cells and culture supernatants of 32 dominant human gut bacterial species in their growing and stationary phases. Combining polyamine concentration analysis in culture supernatant and cells with available genomic information showed that novel polyamine biosynthetic proteins and transporters were present in dominant human gut bacteria. Based on these findings, we suggested strategies for optimizing polyamine concentrations in the human colonic lumen via regulation of genes responsible for polyamine biosynthesis and transport in the dominant human gut bacteria.
  • Hong-qi Xia; Yuki Kitazumi; Osamu Shirai; Hirokazu Ohta; Shin Kurihara; Kenji Kano
    JOURNAL OF ELECTROANALYTICAL CHEMISTRY 804 128 - 132 2017年11月 [査読有り]
     
    A mediator-less amperometric biosensor for putrescine detection was proposed to obtain a (pseudo) steady-state catalytic current. Putrescine oxidase (PuOD) and peroxidase (POD) were co-immobilized with glutaraldehyde on a Ketjen Black (KB)-based mesoporous electrode. A POD-catalyzed direct electron transfer-type reduction wave of H2O2 generated by the reaction of PuOD was observed at a PuOD/POD-immobilized and KB-modified rotating disk glassy carbon electrode with an onset potential of 0.60 V vs. Ag vertical bar AgCl. A PuOD/POD-immobilized and KB-modified microdisk electrode produced a spherical diffusion-controlled (pseudo) steady-state catalytic current under quiescent conditions in the presence of putrescine. The bienzyme mesoporous microelectrode exhibited a linear range from 17 mu M to 500 mu M with a sensitivity of 0.33 +/- 0.01 mA mM(-1) cm(-2) and a lower detection limit of 5 mu M (S/N > 3).
  • Aina Gotoh; Misaki Nara; Yuta Sugiyama; Mikiyasu Sakanaka; Hiroyuki Yachi; Aya Kitakata; Akira Nakagawa; Hiromichi Minami; Shujiro Okuda; Toshihiko Katoh; Takane Katayama; Shin Kurihara
    Bioscience, biotechnology, and biochemistry 81 10 2009 - 2017 2017年10月 [査読有り]
     
    Recently, a "human gut microbial gene catalogue," which ranks the dominance of microbe genus/species in human fecal samples, was published. Most of the bacteria ranked in the catalog are currently publicly available; however, the growth media recommended by the distributors vary among species, hampering physiological comparisons among the bacteria. To address this problem, we evaluated Gifu anaerobic medium (GAM) as a standard medium. Forty-four publicly available species of the top 56 species listed in the "human gut microbial gene catalogue" were cultured in GAM, and out of these, 32 (72%) were successfully cultured. Short-chain fatty acids from the bacterial culture supernatants were then quantified, and bacterial metabolic pathways were predicted based on in silico genomic sequence analysis. Our system provides a useful platform for assessing growth properties and analyzing metabolites of dominant human gut bacteria grown in GAM and supplemented with compounds of interest.
  • Chihaya Yamada; Aina Gotoh; Mikiyasu Sakanaka; Mitchell Hattie; Keith A Stubbs; Ayako Katayama-Ikegami; Junko Hirose; Shin Kurihara; Takatoshi Arakawa; Motomitsu Kitaoka; Shujiro Okuda; Takane Katayama; Shinya Fushinobu
    Cell chemical biology 24 4 515 - 524 2017年04月 [査読有り]
     
    Breast-fed infants generally have a bifidobacteria-rich microbiota with recent studies indicating that human milk oligosaccharides (HMOs) selectively promote bifidobacterial growth. Bifidobacterium bifidum possesses a glycoside hydrolase family 20 lacto-N-biosidase for liberating lacto-N-biose I from lacto-N-tetraose, an abundant HMO unique to human milk, while Bifidobacterium longum subsp. longum has a non-classified enzyme (LnbX). Here, we determined the crystal structure of the catalytic domain of LnbX and provide evidence for creation of a novel glycoside hydrolase family, GH136. The structure, in combination with inhibition and mutation studies, provides insight into the molecular mechanism and broader substrate specificity of this enzyme. Moreover, through genetic studies, we show that lnbX is indispensable for B. longum growth on lacto-N-tetraose and is a key genetic factor for persistence in the gut of breast-fed infants. Overall, this study reveals possible evolutionary routes for the emergence of symbiosis between humans and bifidobacterial species in the infant gut.
  • Sugiyama Y; Katoh T; Honda Y; Gotoh A; Ashida H; Kurihara S; Yamamoto K; Katayama T
    Bioscience, Biotechnology, and Biochemistry 81 2 283 - 291 2017年02月 [査読有り]
     
    We have recently generated a highly efficient 1,2-α-l-fucosynthase (BbAfcA N423H mutant) by protein engineering of 1,2-α-l-fucosidase from Bifidobacterium bifidum JCM 1254. This synthase could specifically introduce H-antigens (Fucα1-2Gal) into the non-reducing ends of oligosaccharides and in O-linked glycans in mucin glycoprotein. In the present study, we show an extended application of the engineered 1,2-α-l-fucosynthase by demonstrating its ability to insert Fuc residues into N- and O-glycans in fetuin glycoproteins, GM1 ganglioside, and a plant-derived xyloglucan nonasaccharide. This application study broadens the feasibility of this novel H-antigen synthesis technique in functional glycomics.
  • Yuta Sugiyama; Atsuo Nakamura; Mitsuharu Matsumoto; Ayaka Kanbe; Mikiyasu Sakanaka; Kyohei Higashi; Kazuei Igarashi; Takane Katayama; Hideyuki Suzuki; Shin Kurihara
    The Journal of biological chemistry 291 51 26343 - 26351 2016年12月 [査読有り]
     
    Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intestinal tract impact the health of animals either negatively or positively. The concentration of polyamines in the intestinal tract results from the balance of uptake and export of the intestinal bacteria. However, the mechanism of polyamine export from bacterial cells to the intestinal lumen is still unclear. In Escherichia coli, PotE was previously identified as a transporter responsible for putrescine excretion in an acidic growth environment. We observed putrescine concentration in the culture supernatant was increased from 0 to 50 μm during growth of E. coli under neutral conditions. Screening for the unidentified putrescine exporter was performed using a gene knock-out collection of E. coli, and deletion of sapBCDF significantly decreased putrescine levels in the culture supernatant. Complementation of the deletion mutant with the sapBCDF genes restored putrescine levels in the culture supernatant. Additionally, the ΔsapBCDF strain did not facilitate uptake of putrescine from the culture supernatant. Quantification of stable isotope-labeled putrescine derived from stable isotope-labeled arginine supplemented in the medium revealed that SapBCDF exported putrescine from E. coli cells to the culture supernatant. It was previously reported that SapABCDF of Salmonella enterica sv. typhimurium and Haemophilus influenzae conferred resistance toantimicrobial peptides; however, the E. coli ΔsapBCDF strain did not affect resistance to antimicrobial peptide LL-37. These results strongly suggest that the natural function of the SapBCDF proteins is the export of putrescine.
  • Sugiyama Y; Gotoh A; Katoh T; Honda Y; Yoshida E; Kurihara S; Ashida H; Kumagai H; Yamamoto K; Kitaoka M; Katayama T
    Glycobiology 26 11 1235 - 1247 2016年11月 [査読有り]
     
    Fucα1-2 Gal linkages, or H-antigens, constitute histo-blood group antigens and are involved in various physiological processes. In addition, recent studies have shown that the H-antigen-containing glycans play an important role, not only in establishing harmonious relationship between gut microbes and the host, but also in preventing gut dysbiosis-related diseases. Therefore, development of an efficient method for introducing Fuc residue at Gal residue at the nonreducing end of glycans via α-(1→2) linkage is desired for research as well as medicinal purposes. In this study, we succeeded in derivatizing inverting 1,2-α-l-fucosidase (AfcA) into a highly efficient 1,2-α-l-fucosynthase. The synthase specifically synthesized H type 1-, type 2-, type 3- and type 4-chain-containing oligosaccharides with yields of 57-75% based on acceptor depletion. The synthase was also able to specifically introduce Fuc residues into Lewis a/x antigens to produce Lewis b/y antigens, with yields of 43% and 62%, respectively. In addition, the enzyme efficiently introduced H-antigens into sugar chains of porcine gastric mucins, as revealed by lectin blotting and mass spectroscopy analysis of the sugars. Detailed acceptor specificity analysis using various monosaccharides and oligosaccharides unraveled unique substrate recognition feature of this synthase at the subsite (+1), which can be explained by our previous X-ray crystallographic study of AfcA. These results show that the synthase developed in this study could serve as an alternative to other H-antigen synthesis methods involving α-1,2-fucosyltransferases and retaining α-fucosidase.
  • Bin Li; Tiffany Lowe-Power; Shin Kurihara; Stephen Gonzales; Jacinth Naidoo; John B MacMillan; Caitilyn Allen; Anthony J Michael
    ACS chemical biology 11 10 2782 - 2789 2016年10月 [査読有り]
     
    The small polyamine putrescine (1,4-diaminobutane) is ubiquitously and abundantly found in all three domains of life. It is a precursor, through N-aminopropylation or N-aminobutylation, for biosynthesis of the longer polyamines spermidine, sym-homospermidine, spermine, and thermospermine and longer and branched chain polyamines. Putrescine is also biochemically modified for purposes of metabolic regulation and catabolism, e.g. N-acetylation and N-glutamylation, and for incorporation into specialized metabolites, e.g. N-methylation, N-citrylation, N-palmitoylation, N-hydroxylation, and N-hydroxycinnamoylation. Only one example is known where putrescine is modified on a methylene carbon: the formation of 2-hydroxyputrescine by an unknown C-hydroxylase. Here, we report the functional identification of a previously undescribed putrescine 2-hydroxylase, a Rieske-type nonheme iron sulfur protein from the β-proteobacteria Bordetella bronchiseptica and Ralstonia solanacearum. Identification of the putrescine 2-hydroxylase will facilitate investigation of the physiological functions of 2-hydroxyputrescine. One known role of 2-hydroxyputrescine has direct biomedical relevance: its role in the biosynthesis of the cyclic hydroxamate siderophore alcaligin, a potential virulence factor of the causative agent of whooping cough, Bordetella pertussis. We also report the functional identification of a putrescine N-hydroxylase from the γ-proteobacterium Shewanella oneidensis, which is homologous to FAD- and NADPH-dependent ornithine and lysine N-monooxygenases involved in siderophore biosynthesis. Heterologous expression of the putrescine N-hydroxylase in E. coli produced free N-hydroxyputrescine, never detected previously in a biological system. Furthermore, the putrescine C- and N-hydroxylases identified here could contribute new functionality to polyamine structural scaffolds, including C-H bond functionalization in synthetic biology strategies.
  • Mikiyasu Sakanaka; Yuta Sugiyama; Aya Kitakata; Takane Katayama; Shin Kurihara
    Amino acids 48 10 2443 - 51 2016年10月 [査読有り]
     
    Recent studies have indicated that polyamines produced by gut microbes significantly influence host health; however, little is known about the microbial polyamine biosynthetic pathway except for that in Escherichia coli, a minor component of the gastrointestinal microbiota. Here, we investigated the polyamine biosynthetic ability of Bacteroides thetaiotaomicron, a predominant gastrointestinal bacterial species in humans. High-performance liquid chromatography analysis revealed that B. thetaiotaomicron cultured in polyamine-free minimal medium accumulated spermidine intracellularly at least during the mid-log and stationary phases. Deletion of the gene encoding a putative carboxyspermidine decarboxylase (casdc), which converts carboxyspermidine to spermidine, resulted in the depletion of spermidine and loss of decarboxylase activity in B. thetaiotaomicron. The Δcasdc strain also showed growth defects in polyamine-free growth medium. The complemented Δcasdc strain restored the spermidine biosynthetic ability, decarboxylase activity, and growth. These results indicate that carboxyspermidine decarboxylase is essential for synthesizing spermidine in B. thetaiotaomicron and contributes to the growth of this species.
  • 栗原新
    化学と生物 53 11 756 - 762 公益社団法人 日本農芸化学会 2015年10月 
    細菌は環境変化に応じて細胞間でコミュニケーションを行う．この結果，病原性の獲得やバイオフィルムの形成といった問題が生じる．細菌細胞間のコミュニケーションに必要なシグナル分子としては<i>N</i>-アシル-L-ホモセリンラクトンなどが有名であるが，2004年以降新たなシグナル物質として，ポリアミンが注目を集めている．これまでに多くの細菌において，運動性細胞への分化，バイオフィルムの形成および分解，病原性の獲得がポリアミンによって仲介される細胞間コミュニケーションによって引き起こされることが報告されている．本稿では，近年の研究の進展をさまざまな細菌におけるポリアミン代謝系・輸送系・センサーについての遺伝学的・生化学的知見とともに概説する．
  • Gotoh A; Katoh T; Sugiyama Y; Kurihara S; Honda Y; Sakurama H; Kambe T; Ashida H; Kitaoka M; Yamamoto K; Katayama T
    Carbohydrate Research 408 18 - 24 2015年05月 [査読有り]
     
    We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo- and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Galβ1-3GalNAc) and 2'-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of β-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids.
  • Shimada Y; Watanabe Y; Wakinaka T; Funeno Y; Kubota M; Chaiwangsri T; Kurihara S; Yamamoto K; Katayama T; Ashida H
    Applied Microbiology and Biotechnology 99 9 3941 - 8 2015年05月 [査読有り]
     
    α-Linked N-acetylglucosamine is one of the major glyco-epitopes in O-glycan of gastroduodenal mucin. Here, we identified glycoside hydrolase (GH) family 89 α-N-acetylglucosaminidase, termed AgnB, from Bifidobacterium bifidum JCM 1254, which is essentially specific to GlcNAcα1-4Gal structure. AgnB is a membrane-anchored extracellular enzyme consisting of a GH89 domain and four carbohydrate-binding module (CBM) 32 domains. Among four CBM32 domains, three tandem ones at C-terminus showed to bind porcine gastric mucin, suggesting that these domains enhance the enzyme activity by increasing affinity for multivalent substrates. AgnB might be important for assimilation of gastroduodenal mucin by B. bifidum and also applicable to production of prebiotic oligosaccharides from porcine gastric mucin.
  • Shin Kurihara; Hideyuki Suzuki
    Polyamines: A Universal Molecular Nexus for Growth, Survival, and Specialized Metabolism 171 - 178 2015年01月 [査読有り][招待有り]
     
    Many bacteria take up external polyamines to optimize their growth and adaptation to the environment. Furthermore, cell-to-cell communication using polyamines recently reported requires export of polyamines and uptake or recognition of polyamine outside the cell. At physiological pH, polyamines are positively charged and hydrophilic and therefore cannot pass through hydrophobic cytoplasmic membranes. Consequently, a polyamine transporter is required for their uptake and export in bacteria. Seven polyamine transporters, that is, PotABCD, PotE, PotFGHI, PuuP, PlaP, CadB and MdtJI, have been reported in Escherichia coli, in which polyamine transporters have been well studied since the 1990s. Recently, a growing body of research on polyamine transporters of other bacteria, especially pathogenic bacteria such as Proteus mirabilis, Vibrio cholerae, Aggregatibacter actinomycetemcomitans, and Streptococcus pneumoniae, is underway guided by current understanding about polyamine transporters of E. coli. In this chapter, recent understanding in bacterial polyamine transport is outlined in addition to the overview of polyamine transporters in E. coli.
  • Hideyuki Suzuki; Shin Kurihara
    Polyamines: A Universal Molecular Nexus for Growth, Survival, and Specialized Metabolism 47 - 60 2015年01月 
    Polyamines play important roles in cell growth and proliferation. In particular, these biogenic compounds are involved in the regulation of transcription and translation processes required for bacterial proliferation. Consequently, intracellular polyamine content is strictly regulated at several levels, including biosynthesis, degradation, and uptake from and excretion into the environment. In this chapter, we discuss polyamine catabolism in prokaryotes, focusing on the well-studied polyamine catabolism pathway in Escherichia coli. E. coli catabolizes putrescine to succinate via γaminobutyraldehyde (GABA) through the aminotransferase pathway or the γglutamylate pathway (the Puu pathway). Excess spermidine is acetylated to yield acetylspermidine, but whether this metabolite is then excreted from cells, as it is in eukaryotes, is not clear. Pseudomonas aeruginosa POA1, in contrast to E. coli, has expanded catabolic pathways to salvage cadaverine and spermidine as carbon and nitrogen sources.
  • Ryoko Kibe; Shin Kurihara; Yumi Sakai; Hideyuki Suzuki; Takushi Ooga; Emiko Sawaki; Koji Muramatsu; Atsuo Nakamura; Ayano Yamashita; Yusuke Kitada; Masaki Kakeyama; Yoshimi Benno; Mitsuharu Matsumoto
    Scientific reports 4 4548 - 4548 2014年04月 [査読有り]
     
    Prevention of quality of life (QOL) deterioration is associated with the inhibition of geriatric diseases and the regulation of brain function. However, no substance is known that prevents the aging of both body and brain. It is known that polyamine concentrations in somatic tissues (including the brain) decrease with increasing age, and polyamine-rich foods enhance longevity in yeast, worms, flies, and mice, and protect flies from age-induced memory impairment. A main source of exogenous polyamines is the intestinal lumen, where they are produced by intestinal bacteria. We found that arginine intake increased the concentration of putrescine in the colon and increased levels of spermidine and spermine in the blood. Mice orally administered with arginine in combination with the probiotic bifidobacteria LKM512 long-term showed suppressed inflammation, improved longevity, and protection from age-induced memory impairment. This study shows that intake of arginine and LKM512 may prevent aging-dependent declines in QOL via the upregulation of polyamines.
  • Yusuke Terui; Sunil D Saroj; Akihiko Sakamoto; Taketo Yoshida; Kyohei Higashi; Shin Kurihara; Hideyuki Suzuki; Toshihiko Toida; Keiko Kashiwagi; Kazuei Igarashi
    Amino acids 46 3 661 - 70 2014年03月 [査読有り]
     
    Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance were studied using a polyamine biosynthesis and uptake deficient Escherichia coli KK3131 transformed with pACYC184 containing potFGHI or puuP. Putrescine uptake activity of E. coli KK3131 transformed with pACYC184-PotFGHI was higher than that of E. coli 3131 transformed with pACYC-PuuP when cells were cultured in the absence of putrescine. Putrescine uptake by PotFGHI was both ATP and membrane potential dependent, while that by PuuP was membrane potential dependent. Feedback inhibition by polyamines occurred at the PotFGHI uptake system but not at the PuuP uptake system. Expression of PuuP was reduced in the presence of PuuR, a negative regulator for PuuP, and expression of PuuR was positively regulated by glucose, which reduces the level of cAMP. The complex of cAMP and CRP (cAMP receptor protein) inhibited the expression of PuuR in the absence of glucose. Thus, the growth rate of E. coli KK3131 in the presence of both 0.4% (22.2 mM) glucose and 10 mM putrescine was in the order of cells transformed with pACYC-PotFGHI > pACYC-PuuP > pACYC-PuuP + PuuR, which was parallel with the polyamine content in cells. The results indicate that PotFGHI is necessary for rapid cell growth in the presence of glucose as an energy source. When glucose in medium was depleted, however, PuuP was absolutely necessary for cell growth in the presence of putrescine, because accumulation of putrescine to a high level by PuuP was necessary for utilization of putrescine as an energy source.
  • 栗原新
    アンチ・エイジング医学 9 5 710 - 716 メディカルレビュー社 2013年10月
  • Shin Kurihara; Yumi Sakai; Hideyuki Suzuki; Aaron Muth; Otto Phanstiel 4th; Philip N Rather
    The Journal of biological chemistry 288 22 15668 - 76 2013年05月 [査読有り]
     
    Previously, we reported that the speA gene, encoding arginine decarboxylase, is required for swarming in the urinary tract pathogen Proteus mirabilis. In addition, this previous study suggested that putrescine may act as a cell-to-cell signaling molecule (Sturgill, G., and Rather, P. N. (2004) Mol. Microbiol. 51, 437-446). In this new study, PlaP, a putative putrescine importer, was characterized in P. mirabilis. In a wild-type background, a plaP null mutation resulted in a modest swarming defect and slightly decreased levels of intracellular putrescine. In a P. mirabilis speA mutant with greatly reduced levels of intracellular putrescine, plaP was required for the putrescine-dependent rescue of swarming motility. When a speA/plaP double mutant was grown in the presence of extracellular putrescine, the intracellular levels of putrescine were greatly reduced compared with the speA mutant alone, indicating that PlaP functioned as the primary putrescine importer. In urothelial cell invasion assays, a speA mutant exhibited a 50% reduction in invasion when compared with wild type, and this defect could be restored by putrescine in a PlaP-dependent manner. The putrescine analog Triamide-44 partially inhibited the uptake of putrescine by PlaP and decreased both putrescine stimulated swarming and urothelial cell invasion in a speA mutant.
  • Wakinaka T; Kiyohara M; Kurihara S; Hirata A; Chaiwangsri T; Ohnuma T; Fukamizo T; Katayama T; Ashida H; Yamamoto K
    Glycobiology 23 2 232 - 40 2013年02月 [査読有り]
     
    Bifidobacterium bifidum is one of the most frequently found bifidobacteria in the intestines of newborn infants. We previously reported that B. bifidum possesses unique metabolic pathways for O-linked glycans on gastrointestinal mucin (Yoshida E, Sakurama H, Kiyohara M, Nakajima M, Kitaoka M, Ashida H, Hirose J, Katayama T, Yamamoto K, Kumagai H. 2012. Bifidobacterium longum subsp. infantis uses two different β-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides. Glycobiology. 22:361-368). The nonreducing termini of O-linked glycans on mucin are frequently covered with histo-blood group antigens. Here, we identified a gene agabb from B. bifidum JCM 1254, which encodes glycoside hydrolase (GH) family 110 α-galactosidase. AgaBb is a 1289-amino acid polypeptide containing an N-terminal signal sequence, a GH110 domain, a carbohydrate-binding module (CBM) 51 domain, a bacterial Ig-like (Big) 2 domain and a C-terminal transmembrane region, in this order. The recombinant enzyme expressed in Escherichia coli hydrolyzed α1,3-linked Gal in branched blood group B antigen [Galα1-3(Fucα1-2)Galβ1-R], but not in a linear xenotransplantation antigen (Galα1-3Galβ1-R). The enzyme also acted on group B human salivary mucin and erythrocytes. We also revealed that CBM51 specifically bound blood group B antigen using both isothermal titration calorimetry and a solid-phase binding assay, and it enhanced the affinity of the enzyme toward substrates with multivalent B antigens. We suggest that this enzyme plays an important role in degrading B antigens to acquire nutrients from mucin oligosaccharides in the gastrointestinal tracts.
  • Naoki Nemoto; Shin Kurihara; Yuzuru Kitahara; Kei Asada; Kenji Kato; Hideyuki Suzuki
    Journal of bacteriology 194 13 3437 - 47 2012年07月 [査読有り]
     
    In Escherichia coli, putrescine is metabolized to succinate for use as a carbon and nitrogen source by the putrescine utilization pathway (Puu pathway). One gene in the puu gene cluster encodes a transcription factor, PuuR, which has a helix-turn-helix DNA-binding motif. DNA microarray analysis of an E. coli puuR mutant, in which three amino acid residues in the helix-turn-helix DNA binding motif of PuuR were mutated to alanine to eliminate DNA binding of PuuR, suggested that PuuR is a negative regulator of puu genes. Results of gel shift and DNase I footprint analyses suggested that PuuR binds to the promoter regions of puuA and puuD. The binding of wild-type PuuR to a DNA probe containing PuuR recognition sites was diminished with increasing putrescine concentrations in vitro. These results suggest that PuuR regulates the intracellular putrescine concentration by the transcriptional regulation of genes in the Puu pathway, including puuR itself. The puu gene cluster is found in E. coli and closely related enterobacteria, but this gene cluster is uncommon in other bacterial groups. E. coli and related enterobacteria may have gained the Puu pathway as an adaptation for survival in the mammalian intestine, an environment in which polyamines exist at relatively high concentrations.
  • Kiyohara M; Nakatomi T; Kurihara S; Fushinobu S; Suzuki H; Tanaka T; Shoda S; Kitaoka M; Katayama T; Yamamoto K; Ashida H
    The Journal of Biological Chemistry 287 1 693 - 700 2012年01月 [査読有り]
     
    Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAcβ1-3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galβ1-3/4GlcNAcβ1-3(Neu5Acα2-6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), β-galactosidase (BbgIII), and β-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.
  • Mitsuharu Matsumoto; Ryoko Kibe; Takushi Ooga; Yuji Aiba; Shin Kurihara; Emiko Sawaki; Yasuhiro Koga; Yoshimi Benno
    Scientific reports 2 233 - 233 2012年 [査読有り]
     
    Low-molecular-weight metabolites produced by intestinal microbiota play a direct role in health and disease. In this study, we analyzed the colonic luminal metabolome using capillary electrophoresis mass spectrometry with time-of-flight (CE-TOFMS) -a novel technique for analyzing and differentially displaying metabolic profiles- in order to clarify the metabolite profiles in the intestinal lumen. CE-TOFMS identified 179 metabolites from the colonic luminal metabolome and 48 metabolites were present in significantly higher concentrations and/or incidence in the germ-free (GF) mice than in the Ex-GF mice (p < 0.05), 77 metabolites were present in significantly lower concentrations and/or incidence in the GF mice than in the Ex-GF mice (p < 0.05), and 56 metabolites showed no differences in the concentration or incidence between GF and Ex-GF mice. These indicate that intestinal microbiota highly influenced the colonic luminal metabolome and a comprehensive understanding of intestinal luminal metabolome is critical for clarifying host-intestinal bacterial interactions.
  • Mitsuharu Matsumoto; Shin Kurihara
    Medical hypotheses 77 4 469 - 72 2011年10月 [査読有り]
     
    Many mechanisms contribute to senescence, such as telomere shortening in replicative cells, cumulative damage to DNA leading to genomic instability, and oxidative damage to molecules by reactive oxygen species (ROS). These include chronic low-grade inflammation (inflammageing), a major risk factor for ageing and age-related diseases, such as Alzheimer's disease and type II diabetes. Furthermore, the prevention of inflammageing seems to be one of the most effective approaches to increase longevity. Here, I discuss the rationale and recent evidence for probiotic-induced upregulation of intestinal luminal polyamine (PA) production in the extension of lifespan by preventing inflammageing.
  • 栗原新
    生物工学会誌 89 9 555 - 555 日本生物工学会 2011年09月
  • Shin Kurihara; Hideyuki Suzuki; Mayu Oshida; Yoshimi Benno
    The Journal of biological chemistry 286 12 10185 - 92 2011年03月 [査読有り]
     
    Recently, many studies have reported that polyamines play a role in bacterial cell-to-cell signaling processes. The present study describes a novel putrescine importer required for induction of type 1 pili-driven surface motility. The surface motility of the Escherichia coli ΔspeAB ΔspeC ΔpotABCD strain, which cannot produce putrescine and cannot import spermidine from the medium, was induced by extracellular putrescine. Introduction of the gene deletions for known polyamine importers (ΔpotE, ΔpotFGHI, and ΔpuuP) or a putative polyamine importer (ΔydcSTUV) into the ΔspeAB ΔspeC ΔpotABCD strain did not affect putrescine-induced surface motility. The deletion of yeeF, an annotated putative putrescine importer, in the ΔspeAB ΔspeC ΔpotABCD ΔydcSTUV strain abolished surface motility in putrescine-supplemented medium. Complementation of yeeF by a plasmid vector restored surface motility. The surface motility observed in the present study was abolished by the deletion of fimA, suggesting that the surface motility is type 1 pili-driven. A transport assay using the yeeF(+) or ΔyeeF strains revealed that YeeF is a novel putrescine importer. The K(m) of YeeF (155 μM) is 40 to 300 times higher than that of other importers reported previously. On the other hand, the V(max) of YeeF (9.3 nmol/min/mg) is comparable to that of PotABCD, PotFGHI, and PuuP. The low affinity of YeeF for putrescine may allow E. coli to sense the cell density depending on the concentration of extracellular putrescine.
  • Mitsuharu Matsumoto; Shin Kurihara; Ryoko Kibe; Hisashi Ashida; Yoshimi Benno
    PloS one 6 8 e23652  2011年 [査読有り]
     
    BACKGROUND: Chronic low-grade inflammation is recognized as an important factor contributing to senescence and age-related diseases. In mammals, levels of polyamines (PAs) decrease during the ageing process; PAs are known to decrease systemic inflammation by inhibiting inflammatory cytokine synthesis in macrophages. Reductions in intestinal luminal PAs levels have been associated with intestinal barrier dysfunction. The probiotic strain Bifidobacterium animalis subsp. lactis LKM512 is known to increase intestinal luminal PA concentrations. METHODOLOGY/PRINCIPAL FINDINGS: We supplemented the diet of 10-month-old Crj:CD-1 female mice with LKM512 for 11 months, while the controls received no supplementation. Survival rates were compared using Kaplan-Meier survival curves. LKM512-treated mice survived significantly longer than controls (P<0.001); moreover, skin ulcers and tumors were more common in the control mice. We then analyzed inflammatory and intestinal conditions by measuring several markers using HPLC, ELISA, reverse transcription-quantitative PCR, and histological slices. LKM512 mice showed altered 16S rRNA gene expression of several predominant intestinal bacterial groups. The fecal concentrations of PAs, but not of short-chain fatty acids, were significantly higher in LKM512-treated mice (P<0.05). Colonic mucosal function was also better in LKM512 mice, with increased mucus secretion and better maintenance of tight junctions. Changes in gene expression levels were evaluated using the NimbleGen mouse DNA microarray. LKM512 administration also downregulated the expression of ageing-associated and inflammation-associated genes and gene expression levels in 21-month-old LKM512-treated mice resembled those in 10-month-old untreated (younger) mice. CONCLUSION/SIGNIFICANCE: Our study demonstrated increased longevity in mice following probiotic treatment with LKM512, possibly due to the suppression of chronic low-grade inflammation in the colon induced by higher PA levels. This indicates that ingestion of specific probiotics may be an easy approach for improving intestinal health and increasing lifespan. Further studies are required to clarify its effectiveness in humans.
  • 栗原新; 鈴木秀之
    化学と生物 48 10 664 - 666 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2010年10月
  • Shin Kurihara; Kenji Kato; Kei Asada; Hidehiko Kumagai; Hideyuki Suzuki
    Journal of bacteriology 192 18 4582 - 91 2010年09月 [査読有り]
     
    Gamma-aminobutyrate (GABA) is metabolized to succinic semialdehyde by GABA aminotransferase (GABA-AT), and the succinic semialdehyde is subsequently oxidized to succinate by succinic semialdehyde dehydrogenase (SSADH). In Escherichia coli, there are duplicate GABA-ATs (GabT and PuuE) and duplicate SSADHs (GabD and YneI). While GabT and GabD have been well studied previously, the characterization and expression analysis of PuuE and YneI are yet to be investigated. By analyzing the amino acid profiles in cells of DeltapuuE and/or DeltagabT mutants, this study demonstrated that PuuE plays an important role in GABA metabolism in E. coli cells. The similarity of the amino acid sequences of PuuE and GabT is 67.4%, and it was biochemically demonstrated that the catalytic center of GabT is conserved as an amino acid residue important for the enzymatic activity in PuuE as Lys-247. However, the regulation of expression of PuuE is significantly different from that of GabT. PuuE is induced by the addition of putrescine to the medium and is repressed by succinate and low aeration conditions; in contrast, GabT is almost constitutive. Similarly, YneI is induced by putrescine, while GabD is not. For E. coli, PuuE is important for utilization of putrescine as a sole nitrogen source and both PuuE and YneI are important for utilization of putrescine as a sole carbon source. The results demonstrate that the PuuE-YneI pathway was a putrescine-inducible GABA degradation pathway for utilizing putrescine as a nutrient source.
  • Shin Kurihara; Hideyuki Suzuki; Mayu Oshida; Yuichi Tsuboi; Yuma Ogasa; Yoshimi Benno
    AMINO ACIDS 37 1 113 - 113 2009年07月
  • Mayu Oshida; Shin Kurihara; Hideyuki Suzuki
    AMINO ACIDS 37 1 113 - 113 2009年07月
  • Shin Kurihara; Hideyuki Suzuki; Yuichi Tsuboi; Yoshimi Benno
    FEMS microbiology letters 294 1 97 - 101 2009年05月 [査読有り]
     
    In a previous work, it was observed that the swarming of polyamine-deficient Proteus mirabilis (speB::sm) was severely inhibited on Luria-Bertani (LB) swarming plates (LBSw) (LB, 0.5% glucose, 0.5% agar), and it was clarified that extracellular putrescine was important as a signaling molecule for the induction of swarming in P. mirabilis. However, a polyamine-deficient strain (delta-speAB delta-speC) of Escherichia coli swarmed as well as the parental strain on LBSw plates. We report that the swarming phenotype of a polyamine-deficient E. coli strain is dependent on spermidine and PotABCD, a spermidine importer.
  • Shin Kurihara; Yuichi Tsuboi; Shinpei Oda; Hyeon Guk Kim; Hidehiko Kumagai; Hideyuki Suzuki
    Journal of bacteriology 191 8 2776 - 82 2009年04月 [査読有り]
     
    The Puu pathway is a putrescine utilization pathway involving gamma-glutamyl intermediates. The genes encoding the enzymes of the Puu pathway form a gene cluster, the puu gene cluster, and puuP is one of the genes in this cluster. In Escherichia coli, three putrescine importers, PotFGHI, PotABCD, and PotE, were discovered in the 1990s and have been studied; however, PuuP had not been discovered previously. This paper shows that PuuP is a novel putrescine importer whose kinetic parameters are equivalent to those of the polyamine importers discovered previously. A puuP(+) strain absorbed up to 5 mM putrescine from the medium, but a DeltapuuP strain did not. E. coli strain MA261 has been used in previous studies of polyamine transporters, but PuuP had not been identified previously. It was revealed that the puuP gene of MA261 was inactivated by a point mutation. When E. coli was grown on minimal medium supplemented with putrescine as the sole carbon or nitrogen source, only PuuP among the polyamine importers was required. puuP was expressed strongly when putrescine was added to the medium or when the puuR gene, which encodes a putative repressor, was deleted. When E. coli was grown in M9-tryptone medium, PuuP was expressed mainly in the exponential growth phase, and PotFGHI was expressed independently of the growth phase.
  • Shin Kurihara; Shinpei Oda; Yuichi Tsuboi; Hyeon Guk Kim; Mayu Oshida; Hidehiko Kumagai; Hideyuki Suzuki
    The Journal of biological chemistry 283 29 19981 - 90 2008年07月 [査読有り]
     
    Glutamate-putrescine ligase (gamma-glutamylputrescine synthetase, PuuA, EC 6.3.1.11) catalyzes the gamma-glutamylation of putrescine, the first step in a novel putrescine utilization pathway involving gamma-glutamylated intermediates, the Puu pathway, in Escherichia coli. In this report, the character and physiological importance of PuuA are described. Purified non-tagged PuuA catalyzed the ATP-dependent gamma-glutamylation of putrescine. The K(m) values for glutamate, ATP, and putrescine are 2.07, 2.35, and 44.6 mm, respectively. There are two putrescine utilization pathways in E. coli: the Puu pathway and the pathway without gamma-glutamylation. Gene deletion experiments of puuA, however, indicated that the Puu pathway was more critical in utilizing putrescine as a sole carbon or nitrogen source. The transcription of puuA was induced by putrescine and in a puuR-deleted strain. The amino acid sequences of PuuA and glutamine synthetase (GS) show high similarity. The molecular weights of the monomers of the two enzymes are quite similar, and PuuA exists as a dodecamer as does GS. Moreover the two amino acid residues of E. coli GS that are important for the metal-catalyzed oxidation of the enzyme molecule involved in protein turnover are conserved in PuuA, and it was experimentally shown that the corresponding amino acid residues in PuuA were involved in the metal-catalyzed oxidation similarly to GS. It is suggested that the intracellular concentration of putrescine is optimized by PuuA transcriptionally and posttranslationally and that excess putrescine is converted to a nutrient source by the Puu pathway.
  • Shin Kurihara; Shinpei Oda; Hidehiko Kumagai; Hideyuki Suzuki
    FEMS microbiology letters 256 2 318 - 23 2006年03月 [査読有り]
     
    gamma-Glutamyl-gamma-aminobutyrate hydrolase (PuuD) was purified and the properties of the enzyme were characterized. The active center of PuuD was identified as Cys-114 by site-directed mutagenesis. The expression of PuuD was induced by putrescine and O2 (substrates of the Puu pathway), while the addition of succinate or NH4Cl (products of the Puu pathway) to the medium reduced the expression of PuuD. The findings that the puuD-deficient strain accumulated gamma-glutamyl-gamma-aminobutyrate (gamma-Glu-GABA) and could not grow on putrescine as a sole nitrogen source indicate that PuuD is physiologically important as a gamma-Glu-GABA hydrolase.
  • Shin Kurihara; Shinpei Oda; Kenji Kato; Hyeon Guk Kim; Takashi Koyanagi; Hidehiko Kumagai; Hideyuki Suzuki
    The Journal of biological chemistry 280 6 4602 - 8 2005年02月 [査読有り]
     
    A novel bacterial putrescine utilization pathway was discovered. Seven genes, the functions of whose products were not known, are involved in this novel pathway. Five of them encode enzymes that catabolize putrescine; one encodes a putrescine importer, and the other encodes a transcriptional regulator. This novel pathway involves six sequential steps as follows: 1) import of putrescine; 2) ATP-dependent gamma-glutamylation of putrescine; 3) oxidization of gamma-glutamylputrescine; 4) dehydrogenation of gamma-glutamyl-gamma-aminobutyraldehyde; 5) hydrolysis of the gamma-glutamyl linkage of gamma-glutamyl-gamma-aminobutyrate; and 6) transamination of gamma-aminobutyrate to form the final product of this pathway, succinate semialdehyde, which is the precursor of succinate.

書籍

  • 大熊, 盛也; 石田, 達也; 乙黒, 美彩; 飯田, 敏也 エヌ・ティー・エス 2023年09月 ISBN: 9784860438586 4, 12, 521, 12p, 図版22p
  • 北本, 勝ひこ; 大矢, 禎一; 後藤, 奈美; 五味, 勝也; 高木, 博史 朝倉書店 2021年06月 ISBN: 9784254431254 1 online resource (xii, 483, 11p, 図版 [4] p)

講演・口頭発表等

MISC

産業財産権

  • 特許7321495:末梢セロトニンまたは腸内芳香族アミンの増加を伴う疾患の予防または治療用医薬組成物および末梢セロトニンまたは腸内芳香族アミンの増加を伴う疾患の予防または治療用薬剤のスクリーニング方法    2023年08月07日
    栗原 新, 杉山 友太, 岡本 成史
  • 栗原 新  学校法人近畿大学
  • 特許7203403:タマリンド分解物、酪酸産生菌増殖促進剤、腸内酪酸産生用組成物、並びに、タマリンド分解物の製造方法  
    山西 健太, 栗原 新
  • 特願2022-205105:腸内ポリアミン産生剤  2022年12月22日
    栗原新, 平野啓太, 齋藤 努  不二製油グループ本社株式会社, 学校法人近畿大学
  • 特願2022-72662:サポニンを含む酪酸産生菌増殖能向上組成物、腸内常在菌増殖能向上組成物、乳酸菌増殖能向上組成物及びビフィズス菌増殖能向上組成物  2022年04月26日
    長野 隆男, 栗原 新  石川県公立大学法人、学校法人近畿大学
  • 特願2022-014261:腸内細菌改善剤  2022年02月01日
    栗原 新, 平垣内 一子, 齋藤 努, 片瀬 満
  • 特許6886142:腸内細菌叢改善剤及びその使用  
    中井 博之, 栗原 新, 片山 高嶺, 北岡 本光

受賞

  • 2023年07月 一般社団法人バイオインダストリー協会 バイオインダストリー奨励賞
     日欧ヒト腸内常在菌叢における最優勢種のハイスループット培養・解析法の開発と応用 
    受賞者: 栗原 新
  • 2023年04月 日本農芸化学会 トピックス賞（発表責任者）
     腸内細菌は大豆由来難消化性ペプチド中のアルギニンをポリアミンに変換できる 
    受賞者: 網 優太;平野 啓太;齋藤 努;栗原 新
  • 2020年01月 日本農芸化学会 2020年度大会 トピックス演題選出（発表責任者）
     次世代型プレバイオティクス・ガラクトシル-β-1,4-ラムノースのビフィズス菌特異的な増殖促進機構の解析 
    受賞者: 平野里佳;阪中幹祥;杉本直久;江口省吾;奈良未沙希;片山高嶺;北岡本光;中井博之;栗原新
  • 2019年03月 日本農芸化学会 農芸化学奨励賞
     腸内細菌のポリアミン代謝・輸送機構の解明 
    受賞者: 栗原 新
  • 2018年03月 日本農芸化学会 BBB論文賞（責任著者）
     Use of Gifu Anaerobic Medium for culturing 32 dominant species of human gut microbes and its evaluation based on short-chain fatty acids fermentation profiles. 
    受賞者: Gotoh A;Nara M;Sugiyama Y;Sakanaka M;Yachi H;Kitakata A;Nakagawa A;Minami H;Okuda S;Katoh T;Katayama T;Kurihara S
  • 2017年04月 日本農芸化学会 大会トピックス賞（発表責任者）
     プロバイオティクス細菌を特異的に増殖させる次世代型プレバイオティクス・ガラクトシル-β-1,4-ラムノースの開発と偽膜性腸炎原因菌Clostridium difficileの生育抑制 
    受賞者: 栗原 新
  • 2016年04月 日本農芸化学会 大会トピックス賞（共同発表者）
     腸内環境の酸性化は腸内細菌由来の生理活性物質であるポリアミンを増加させる 
    受賞者: 栗原 新
  • 2014年10月 バイオインダストリー協会 発酵と代謝研究奨励賞
     ポリアミンの新規代謝・輸送経路の発見とそのヒト健康への展開 
    受賞者: 栗原 新
  • 2010年03月 日本農芸化学会 大会トピックス賞（筆頭発表者）
     大腸菌の細胞外プトレッシンによって誘導される遊走に必要な新規プトレッシンインポーターYeeF japan_society 
    受賞者: 栗原 新
  • 2009年06月 Biochemical Journal ポリアミンに関するゴードン会議 ポスター賞佳作
     international_society 
    受賞者: 栗原 新
  • 2008年05月 日本農芸化学会関西支部 日本農芸化学会関西支部若手優秀発表賞
     japan_society 
    受賞者: 栗原 新

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2023年06月 -2027年03月 
    代表者 : 松本 光晴; 斉木 臣二; 栗原 新; 坪井 貴司; 日暮 琢磨
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2023年04月 -2026年03月 
    代表者 : 栗原 新; 宮本 潤基; 松本 光晴
  • 大豆由来難消化性ペプチドを用いたヒト腸内常在菌叢の改善技術の開発と健康への応用
    公益財団法人不二たん白質研究振興財団：2023年度（第27期）研究助成 特定研究
    研究期間 : 2023年04月 -2026年03月 
    代表者 : 栗原 新; 金 倫基
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2023年06月 -2025年03月 
    代表者 : 栗原 新; 松岡 悠美; 芦田 久; 岡本 成史
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 生城 真一; 栗原 新; 西川 美宇
     
    食品中の機能性成分であるフラボノイドなどはグルクロン酸や硫酸基が付加された抱合代謝物として生体内に留まり、一部は脱抱合過程を通して活性体（アグリコン）に変換される。従って、体内における抱合代謝物への変換、輸送及び排泄過程がフラボノイドの生理機能発現を規定する。近年では宿主腸内細菌叢における脱抱合による再吸収過程を通して代謝物の体内動態に関与する可能性が示唆される。本申請では、（課題１）生体に取り込まれた機能性成分の代謝物を同定するために、異物代謝酵素発現酵母を用いた代謝物の網羅的な合成プラットフォームを確立する。さらに、課題１で得られた代謝物標準品を用いて、（課題２）腸内常在細菌優勢種における脱抱合能評価により機能性成分動態に対する腸肝循環の寄与を明らかにすることにより、生体における腸内細菌叢が関与する食品中機能性成分の抱合代謝物動態の総合的理解を通して食品中の機能性成分の真の作用メカニズム解明を目指した。ケルセチンのヘテロ抱合体合成においては、ヒト由来異物代謝酵素（UDP-グルクロン酸転移酵素および硫酸転移酵素）発現酵母株を用いた。出発基質としてグルクロン酸抱合あるいは硫酸抱合化ケルセチンを異なる抱合酵素を発現する酵母菌体に添加し、最適条件下において抱合変換を行った。また、メチル化ケルセチンであるイソラムネチンのヘテロ抱合体についても同様に合成した。以下に示すように４種のケルセチンヘテロ抱合体および３種のイソラムネチンヘテロ抱合体を調製した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年07月 -2023年03月 
    代表者 : 倉石 貴透; 栗原 新
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年07月 -2023年03月 
    代表者 : 栗原 新; 金 倫基
     
    腸内常在菌叢は様々な疾患と深く関連することが報告されており、その制御法を開発すれば人類の健康寿命は大きく伸長すると考えられる。現在、腸内細菌制御を目的として経口摂取される難消化性オリゴ糖（プレバイオティクス）が数多く上市され、2022年には60億ドルの市場に成長すると見込まれている。しかし、三大栄養素の残り２種に対応する難消化性脂質、難消化性ペプチドは腸内細菌制御剤として非常に有望であるにも関わらず、ほとんど研究されていない。 本研究では、大豆由来難消化性ペプチドを培地に添加して、ヒト腸内常在菌叢最優勢32種の生育に与える影響について試験した。この結果、ヒト腸内常在菌叢最優勢32種に含まれるBacteroides属細菌11菌種の内、腸管バリアを破壊するBacteroides caccae (Cell 167:1339-1353. (2016))、大腸がん発生を促進する可能性があるBacteroides fragilis (Science 359:592-597. (2018)) 等、健康に悪影響を及ぼす可能性のある菌種を含む9菌種の生育が抑制された。次に、糞便由来細菌の共存下における大豆由来難消化性ペプチドによるBacteroides属細菌の生育抑制効果を次世代シーケンサーにより解析したところ、大豆由来難消化性ペプチド添加により Bacteroides属細菌が糞便培養液中に占める割合が減少した (n = 7、48.4％→24.7％、p = 0.0032)。一方で、他の腸内細菌属の占有率には統計学的に有意な変動は観察されなかった。以上を総合すると、大豆由来難消化性ペプチドは、ヒト腸管内において悪玉菌である可能性が示唆されているBacteroides属細菌を「狙い撃ち」で減少させる素材であることが期待される。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 栗原 新; 小柳 喬; 芦田 久; 松本 光晴
     
    ポリアミンは、末端にアミノ基を持つ脂肪族炭化水素で、生命活動に不可欠な物質である。ポリアミンの摂取により寿命が延びるなど、動物においてポリアミンが健康増進効果を持つことが報告されている。食品からの高ポリアミン産生菌の同定は、新規プロバイオティクス候補の開発につながる可能性がある。 本研究では、食品からの高ポリアミン産生菌の同定を目指し、日本国内で生産される野菜や発酵食品から菌を分離・収集した。この結果、ポリアミンの一種であるプトレッシンを約200 μMの濃度で培養上清中に蓄積する乳酸菌として、石川県の伝統発酵食品である「かぶらずし」から分離したLatilactobacillus curvatus KP 3-4を取得することに成功した。L. curvatus KP 3-4のポリアミン合成能を、Japan Collection of Microorganismsから入手可能な代表的なプロバイオティクス乳酸菌およびL. curvatus株と比較したところ、L. curvatus KP 3-4のみが培養上清に多量のプトレッシンを放出できることが判明した。さらに本菌の全ゲノム解析から、プトレッシンはオルニチン脱炭酸酵素を介して合成されることが示唆された。L. curvatus KP 3-4 を無菌マウスに投与したところ、糞便中のプトレッシン濃度が上昇した。 同様の手法により、培養上清に高濃度（約500 μM）のプトレッシンを蓄積する能力を持つStaphylococcus epidermidis FB146を日本の発酵食品である味噌から単離した。S. epidermidis FB146に加え、代表的なブドウ球菌21種の基準株の培養上清と細胞中のポリアミンの存在を解析したところ、S. epidermidis FB146のみが高いプトレッシン生産性を有していた。
  • 大豆由来難消化性ペプチドが腸内常在菌のポリアミン産生に及ぼす影響の解析
    公益財団法人不二たん白質研究振興財団：令和3年度（第25期）研究助成 一般研究
    研究期間 : 2021年04月 -2022年03月 
    代表者 : 栗原 新
  • 日本学術振興会：科学研究費 挑戦的研究(萌芽)
    研究期間 : 2019年06月 -2021年03月 
    代表者 : 栗原 新; 倉石 貴透
  • 日本学術振興会：若手研究（A)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 栗原 新
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2017年04月 -2019年03月 
    代表者 : 小柳 喬; 栗原 新
     
    ドーパミン(DA)は、宿主-腸内細菌相互作用の有力な候補物質の一つである。本研究では、腸内細菌優勢種のうちGAM培地で培養可能な32種についてDA産生能を精査し、腸管内において実際にDA産生を担う菌種の特定を試みた。その結果、DAを著量産生できる菌種は腸球菌Enterococcus faecalisのみであることが明らかになったほか、芳香族アミノ酸脱炭酸酵素ホモログを保持する他の4菌種も少量のDA産生能を示したものの、他の芳香族アミン群と遜色ないDA産生能を示したのはE. faecalisのみであった。このことから、本菌種の腸管内におけるDA産生への高い寄与が窺われた。
  • キヤノン財団：研究助成プログラム「理想の追求」（2016年採択）
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 栗原 新; 岡本 成史; 小栁 喬
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 片山 高嶺; 日高 將文; 栗原 新; 廣瀬 潤子
     
    母乳栄養児の腸管ではビフィズスフローラが形成されることが知られていたが、その分子機構は解明されていなかった。我々は、ビフィズス菌が母乳中で3番目に多く含まれる成分であるオリゴ糖（母乳オリゴ糖）を資化する特異的な酵素群を有していることを見出し、それら酵素について解析を行った。その結果、母乳オリゴ糖の主要成分であるラクト-N-テトラオースを分解する酵素ラクト-N-ビオシダーゼがビフィズスフローラ形成に重要であることを明らかとした。また、酵素を改変することでラクト-N-テトラオース合成法を開発した。ビフィズス因子として人工乳への添加が期待される。
  • 公益財団法人 旭硝子財団：自然科学系「研究奨励」（平成28年度採択）
    研究期間 : 2016年04月 -2018年03月 
    代表者 : 栗原 新
  • 日本学術振興会：科学研究費 挑戦的萌芽研究
    研究期間 : 2016年04月 -2018年03月 
    代表者 : 栗原 新; 川原 圭博; 加納 健司
  • ノボザイムズジャパン（株）：2016年度研究ファンドプロジェクト
    研究期間 : 2016年01月 -2016年12月 
    代表者 : 栗原 新
  • 公益財団法人 三谷研究開発支援財団：助成金
    研究期間 : 2015年07月 -2016年08月 
    代表者 : 栗原 新
  • 日本学術振興会：科学研究費 挑戦的萌芽研究
    研究期間 : 2014年04月 -2016年03月 
    代表者 : 栗原 新; 松尾 道憲; 辨野 義己; 片山 高嶺
  • 日本学術振興会：海外特別研究員
    研究期間 : 2011年02月 -2013年02月 
    代表者 : 栗原 新
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2007年 -2009年 
    代表者 : 栗原 新
     
    大腸内でポリアミン濃度を上昇させるためには、腸内細菌の持つポリアミン放出系とポリアミン吸収系の双方を制御する必要がある。そこで、今年度はモデル生物大腸菌を用い、ポリアミン放出系とポリアミン吸収系の同定を進めた。その結果、吸収系と放出系共に新規のものをほぼ同定した。吸収系すなわちインポーターについては、YeeFが新規プトレッシンインポーターであることが確認された。YdcSTUVについては嫌気条件で作用するインポーターであることを示すデータを得ており、腸内環境下での作用も期待でき、現在詳細に解析中である。プトレッシン放出系については、大腸菌網羅的遺伝子破壊株コレクションから輸送系遺伝子破壊株について158株を取得し実験を試みた。その結果、細胞プトレッシン濃度が他と比較して著しく低いsapD破壊株、sapF破壊株を見出した。sapDとsapFはsapBCDFオペロンを形成していたので、現在sapBCDFオペロンに注目して、遺伝子破壊株、遺伝子相補株を作製し、細胞外ポリアミン濃度を測定したところ、遺伝子破壊株では細胞外プトレッシン濃度が低く、野生株、遺伝子相補株においては細胞外プトレッシン濃度が高かった。また、大腸菌の主要なプトレッシン分解系の抑制因子である調節タンパク質PuuRがpuuDの上流域に結合することを明らかとした。このことは、PuuRのpuuDの上流域への結合を活性化する成分が重要なポリアミン濃度制御物質(腸管内でポリアミン濃度を上昇させる成分)となりうる可能性を示唆している。

委員歴

  • 2023年07月 - 現在   日本乳酸菌学会   理事
  • 2018年12月 - 現在   日本ポリアミン学会   評議員
  • 2017年05月 - 現在   日本ポリアミン学会   企画運営委員

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