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Last Updated :2024/10/23

研究者情報

マスター

アカウント（マスター）

  • 氏名

    吹谷　智(フキヤ　サトル), フキヤ　サトル

所属（マスター）

  • 農学研究院　基盤研究部門　生物機能化学分野

所属（マスター）

  • 農学研究院　基盤研究部門　生物機能化学分野

独自項目

syllabus

  • 2021, 食品安全・機能性開発学特論, Advanced Safety and Function of Food, 修士課程, 農学院, 食品安全性、予測微生物、食料確保と安全性、植物由来機能性食品、微生物由来機能性食品、腸内細菌叢の改変に基づいた機能性食品、食肉由来機能性食品
  • 2021, 食品安全・機能性開発学特論演習, Advanced Seminar on Safety and Function of Food, 修士課程, 農学院, 食品の安全、予測微生物学、食料確保と安全性、植物由来機能性食品、微生物由来機能性食品、腸内細菌叢の改変に基づいた機能性食品、食肉由来機能性食品
  • 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, 食品安全性、予測微生物、食料確保と安全性、植物由来機能性食品、微生物由来機能性食品、腸内細菌叢の改変に基づいた機能性食品、食肉由来機能性食品
  • 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, 腸内細菌，腸内細菌叢，ルーメン，プロバイオティクス，乳酸菌，ビフィズス菌、腸管免疫、疾病発症 Gut microbes, Gut microbiota, Rumen, Probiotics, Lactic acid bacteria, Bifidobacteria, Gut immunity, Disease development
  • 2021, 胃腸内圏微生物学特論, Advanced Gastrointestinal Microbiology, 修士課程, 農学院, 腸内細菌，腸内細菌叢，ルーメン，プロバイオティクス，乳酸菌，ビフィズス菌、腸管免疫、疾病発症 Gut microbes, Gut microbiota, Rumen, Probiotics, Lactic acid bacteria, Bifidobacteria, Gut immunity, Disease development
  • 2021, 生物機能化学演習Ⅱ, Seminar on Bioscience and Chemistry II, 学士課程, 農学部, 専門関連英文原書精読
  • 2021, 環境と人間, Environment and People, 学士課程, 全学教育, 共生、寄生、進化、生物生産、植物、哺乳動物、昆虫、微生物、ウィルス、分子生物学、生態学、環境保全
  • 2021, 応用菌学, Applied Microbiology, 学士課程, 農学部, 代謝制御、アミノ酸、核酸、醸造、発酵食品、酵素、抗生物質、微生物利用
  • 2021, 一般教育演習(ﾌﾚｯｼｭﾏﾝｾﾐﾅｰ), Freshman Seminar, 学士課程, 全学教育, 食料、安全、作物生産、放射能汚染、食品微生物、酵素利用技術、毒物、ガン、アレルギー、メタボリックシンドローム、腸内細菌叢、保健機能食品
  • 2021, 生物学実験Ⅱ, Laboratry Work on Biology II, 学士課程, 農学部, 微生物，微生物バイオテクノロジー，代謝制御発酵，フィードバック調節，アミノ酸発酵，遺伝子工学，組換えDNA実験
  • 2021, 基礎微生物学, General Microbiology, 学士課程, 農学部, 微生物、真性細菌、古細菌、真核微生物、増殖、代謝、生理、分類、多様性、生態、遺伝、ウイルス
  • 2021, 微生物化学, Microbial Chemistry, 学士課程, 農学部, 大腸菌，大腸菌の構造と機能，大腸菌のエネルギー代謝，プロトン駆動力，大腸菌細胞のアッセンブリー，エネルギーコスト

researchmap

プロフィール情報

学位

  • 博士（農学）（北海道大学）

プロフィール情報

  • 姓

    吹谷, フキヤ

  • 名

    智, サトル

  • ID各種

    200901056451421836

業績リスト

研究キーワード

  • 腸管定着   胆汁酸変換菌   トランスポゾン   ビフィズス菌   変異導入   デオキシコール酸   二次胆汁酸   コール酸   カルジオリピン   リン脂質   腸内生存   胆汁酸   乳酸菌   微生物遺伝・育種   Molecular Microbiology   Microbial genetics and physiology   

研究分野

  • ライフサイエンス / 応用微生物学

経歴

  • 2022年04月 - 現在 北海道大学 大学院農学研究院 基盤研究部門 生物機能化学分野 微生物生理学研究室 教授
  • 2021年07月 - 現在 北海道大学 大学院農学研究院 准教授
  • 2013年10月 - 2021年06月 北海道大学 (連合)農学研究科(研究院) 講師
  • 2007年04月 - 2013年09月 北海道大学 (連合)農学研究科(研究院) 助教
  • 2005年06月 - 2007年03月 北海道大学大学院・農学研究科 助手

学歴

  •         - 2001年   北海道大学大学院   農学研究科   農芸化学専攻 博士後期課程

委員歴

  • 2016年02月 - 2023年01月   日本農芸化学会   学術活動強化委員会 幹事・委員
  • 2015年07月 - 2021年07月   日本乳酸菌学会   セミナー担当理事
  • 2012年04月 - 2015年06月   日本乳酸菌学会   泊まり込みセミナー委員会 委員長
  • 2007年04月 - 2012年03月   日本生物工学会   乳酸菌・腸内細菌工学研究部会 庶務会計幹事

受賞

  • 2015年10月 公益財団法人 日本生物工学会 生物工学奨励賞（斎藤賞）
     ビフィズス菌における実用的な遺伝子変異導入系の開発 
    受賞者: 吹谷 智

論文

  • Hiroki Wakahara, Takuya Mizokoshi, Kotaro Yamagami, Satoru Fukiya, Atsushi Yokota, Tomoya Maeda
    Journal of bioscience and bioengineering 2024年09月07日 
    Gamma-aminobutyric acid (GABA), which is synthesized from l-glutamic acid via glutamate decarboxylase (Gad), is used as food, supplements, and biodegradable plastics. Our previous study demonstrated an Escherichia coli mutant (ΔΔ) strain, lacking type I NADH dehydrogenase (NDH-I) and cytochrome bo3 oxidase (Cytbo3), produced 7 g/L glutamic acid on MS1 glucose-minimal medium. In this study, the ΔΔ strain was used for improving GABA production. A plasmid (pMBL19-gadB') expressing a mutated E. coli GadB (Glu89Gln/Δ452-466), retaining activity at neutral pH, was introduced into the ΔΔ strain and its parent strain (W1485). The ΔΔ strain carrying pMBL19-gadB' exhibited a twofold increase in GABA production compared to the W1485 strain carrying pMBL19-gadB'. Deleting the C-terminal (Δ471-511) of GadC antiporter in the ΔΔ strain further improved GABA yield by 1.5 g/L when cultured in MS1 glucose-minimal medium. On the other hand, a large amount of glutamic acid produced by the ΔΔ strain was not fully converted to GABA, likely due to the inhibition of GadB activity by the accumulation of acetic acid. Although there is room for improvement, these results indicate the efficacy of the ΔNDH-IΔCytbo3 double mutation in augmenting GABA production.
  • Akinobu Shinmori, Zhen Guo, Tomoya Maeda, Satoru Fukiya, Masaru Wada, Atsushi Yokota
    Journal of bioscience and bioengineering 2024年06月27日 
    Anaplerotic reactions catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) have important roles in the production of l-lysine to replenish oxaloacetic acid (OAA) in Corynebacterium glutamicum. However, the relative contributions of these enzymes to l-lysine production in C. glutamicum are not fully understood. In this study, using a parent strain (P) carrying a feedback inhibition-resistant aspartokinase with the T311I mutation, we constructed a PC gene-deleted mutant strain (PΔPC) and a PEPC gene-deleted mutant strain (PΔPEPC). Although the growth of both mutant strains was comparable to the growth of strain P, the maximum l-lysine production in strains PΔPC and PΔPEPC decreased by 14% and 49%, respectively, indicating that PEPC strongly contributed to OAA supply. l-Lysine production in strain PΔPC slightly decreased during the logarithmic phase, while production during the early stationary phase was comparable to production in strain P. By contrast, strain PΔPEPC produced l-lysine in an amount comparable to the production of strain P during the logarithmic phase; l-lysine production after the early stationary phase was completely stopped in strain PΔPEPC. These results indicate that OAA is supplied by both PC and PEPC during the logarithmic phase, while only PEPC can continuously supply OAA after the logarithmic phase.
  • Siddabasave Gowda B. Gowda, Fengjue Hou, Divyavani Gowda, Hitoshi Chiba, Kentaro Kawakami, Satoru Fukiya, Atsushi Yokota, Shu-Ping Hui
    Analytica Chimica Acta 1288 342145 - 342145 2024年02月
  • Mina Shimada, Youhei Kawase, Kei Sonoyama, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota, Satoru Fukiya
    Bioscience of microbiota, food and health 43 2 110 - 119 2024年 [査読有り]
     
    How bifidobacteria colonize and survive in the intestine is not fully understood. The administration of bifidobacteria to conventional mice can be used to evaluate their ability to colonize the intestine in the presence of endogenous gut microbiota. However, human-derived bifidobacteria do not readily colonize the intestines of conventional mice, and although colonization by Bifidobacterium breve UCC2003 has been achieved, the viability of such populations requires improvement. Therefore, we aimed to establish a colonization system with human-derived bifidobacteria of high viability in conventional mice using Bifidobacterium longum subsp. longum 105-A. Lactose, raffinose, and 1-kestose were identified as the preferred carbohydrate sources for the growth of this strain in culture. The administration of B. longum 105-A to conventional BALB/c mice fed these carbohydrates showed that diets containing 6% (w/w) raffinose or 1-kestose facilitated colonization with >108 colony-forming units/g feces for 2 weeks. The population of this strain was more stable in the raffinose-fed group than in the 1-kestose-fed group. The ingestion of these prebiotics had a greater impact on the composition of the microbiota than the administration of B. longum 105-A. The ingestion of these prebiotics also increased the fecal concentrations of organic acids, which was indicative of greater intestinal fermentation. Collectively, we established a colonization system for B. longum 105-A with high viability in conventional mice by feeding the mice raffinose or 1-kestose. This system should be useful for elucidation of the mechanisms of colonization and survival of bifidobacteria in the intestines in the presence of the endogenous gut microbiota.
  • Atsushi Hisatomi, Ni Wayan Eka Putri Gayatri Kastawa, Isaiah Song, Moriya Ohkuma, Satoru Fukiya, Mitsuo Sakamoto
    International Journal of Systematic and Evolutionary Microbiology 73 9 2023年09月21日 [査読有り]
     
    Obligately anaerobic, Gram-stain-positive, bacilli, strains 12BBH14T, 9CFEGH4 and 10CPCBH12, were isolated from faecal samples of healthy Japanese people. Strain 12BBH14T showed the highest 16S rRNA gene sequence similarity to Sellimonas monacensis Cla-CZ-80T (97.5 %) and ‘Lachnoclostridium phocaeense’ Marseille-P3177T (97.2 %). Strain 12BBH14T was also closely related to Eubacterium sp. c-25 with 99.7 % 16S rRNA gene sequence similarity. The 16S rRNA gene sequence analysis showed that strains 12BBH14T, 9CFEGH4 and 10CPCBH12 formed a monophyletic cluster with Eubacterium sp. c-25. Near this monophyletic cluster, S. monacensis Cla-CZ-80T and ‘L. phocaeense’ Marseille-P3177T formed a cluster and did not form a cluster with other Sellimonas species. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strains 12BBH14T, 9CFEGH4, 10CPCBH12 and Eubacterium sp. c-25 were higher than the cut-off values of species demarcation (>88 % dDDH and >98 % ANI), indicating that these four strains are the same species. On the other hand, the dDDH and ANI values of these strains were lower than the cut-off values of species demarcation against other strains (<29 % dDDH and <76 % ANI). Moreover, the average amino acid identity values among these strains were higher than the genus boundary. These results indicate that the isolates should be considered to belong to a new genus of the family Lachnospiraceae. Based on the collected data, strains 12BBH14T, 9CFEGH4 and 10CPCBH12 represent a novel species of a novel genus, for which the name Claveliimonas bilis gen. nov., sp. nov. is proposed. The type strain of C. bilis is 12BBH14T (=JCM 35899T=DSM 115701T). Eubacterium sp. c-25 belongs to C. bilis. In addition, S. monacensis is transferred to the genus Claveliimonas as Claveliimonas monacensis comb. nov.
  • Miriam N Ojima, Yuya Asao, Aruto Nakajima, Toshihiko Katoh, Motomitsu Kitaoka, Aina Gotoh, Junko Hirose, Tadasu Urashima, Satoru Fukiya, Atsushi Yokota, Maher Abou Hachem, Mikiyasu Sakanaka, Takane Katayama
    Applied and environmental microbiology 88 2 e0143721  2022年01月25日 [査読有り]
     
    Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose [FL]), are associated with the formation of a Bifidobacterium-rich gut microbial community. Phylogenetic analysis revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subsp. kashiwanohense and Bifidobacterium pseudocatenulatum, both of which possess FL-binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO consumption analyses showed that the FL transporter type from B. catenulatum subsp. kashiwanohense JCM 15439T conferred a novel HMO uptake pattern that includes complex fucosylated HMOs (lacto-N-fucopentaose II and lacto-N-difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that the H-antigen- or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria in HMO-rich environments. IMPORTANCE The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breast milk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated host-microbe symbiosis. While the coevolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within Bifidobacterium. We have shown that the diversification of the solute-binding proteins of the FL transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allow bifidobacteria to consume different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.
  • Isaiah Song, Yasuhiro Gotoh, Yoshitoshi Ogura, Tetsuya Hayashi, Satoru Fukiya, Atsushi Yokota
    Microorganisms 9 11 2021年10月29日 [査読有り]
     
    The human gut houses bile acid 7α-dehydroxylating bacteria that produce secondary bile acids such as deoxycholic acid (DCA) from host-derived bile acids through enzymes encoded by the bai operon. While recent metagenomic studies suggest that these bacteria are highly diverse and abundant, very few DCA producers have been identified. Here, we investigated the physiology and determined the complete genome sequence of Eubacterium sp. c-25, a DCA producer that was isolated from human feces in the 1980s. Culture experiments showed a preference for neutral to slightly alkaline pH in both growth and DCA production. Genomic analyses revealed that c-25 is phylogenetically distinct from known DCA producers and possesses a multi-cluster arrangement of predicted bile-acid inducible (bai) genes that is considerably different from the typical bai operon structure. This arrangement is also found in other intestinal bacterial species, possibly indicative of unconfirmed 7α-dehydroxylation capabilities. Functionality of the predicted bai genes was supported by the induced expression of baiB, baiCD, and baiH in the presence of cholic acid substrate. Taken together, Eubacterium sp. c-25 is an atypical DCA producer with a novel bai gene cluster structure that may represent an unexplored genotype of DCA producers in the human gut.
  • 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 e1973835  2021年09月23日 [査読有り]
  • Kenta Maegawa, Haruka Koyama, Satoru Fukiya, Atsushi Yokota, Koichiro Ueda, Satoshi Ishizuka
    The British journal of nutrition 127 11 1 - 10 2021年07月14日 [査読有り]
     
    Enterohepatic circulation of 12α-hydroxylated (12αOH) bile acid (BA) is enhanced depending on the energy intake in high-fat diet-fed rats. Such BA metabolism can be reproduced using a diet supplemented with cholic acid (CA), which also induces simple steatosis, without inflammation and fibrosis, accompanied by some other symptoms that are frequently observed in the condition of non-alcoholic fatty liver in rats. We investigated whether supplementation of the diet with raffinose (Raf) improves hepatic lipid accumulation induced by the CA-fed condition in rats. After acclimation to the AIN-93-based control diet, male Wistar rats were fed diets supplemented with a combination of Raf (30 g/kg diet) and/or CA (0·5 g/kg diet) for 4 weeks. Dietary Raf normalised hepatic TAG levels (two-way ANOVA P < 0·001 for CA, P = 0·02 for Raf and P = 0·004 for interaction) in the CA-supplemented diet-fed rats. Dietary Raf supplementation reduced hepatic 12αOH BA concentration (two-way ANOVA P < 0·001 for CA, P = 0·003 for Raf and P = 0·03 for interaction). The concentration of 12αOH BA was reduced in the aortic and portal plasma. Raf supplementation increased acetic acid concentration in the caecal contents (two-way ANOVA P = 0·001 as a main effect). Multiple regression analysis revealed that concentrations of aortic 12αOH BA and caecal acetic acid could serve as predictors of hepatic TAG concentration (R2 = 0·55, P < 0·001). However, Raf did not decrease the secondary 12αOH BA concentration in the caecal contents as well as the transaminase activity in the CA diet-fed rats. These results imply that dietary Raf normalises hepatic lipid accumulation via suppression of enterohepatic 12αOH BA circulation.
  • Shota Hori, Minako Satake, Ohji Kohmoto, Ryo Takagi, Kazufumi Okada, Satoru Fukiya, Atsushi Yokota, Satoshi Ishizuka
    The Journal of nutrition 151 3 523 - 530 2021年03月11日 [査読有り]
     
    BACKGROUND: Primary 12α-hydroxylated bile acids (12αOH BAs) enhance intestinal iron uptake due to their ability ex vivo to chelate iron. However, no information is available on their role in vivo, especially in the liver. OBJECTIVES: To investigate the effects and mechanisms of primary 12αOH BAs on hepatic iron concentration in vivo. METHODS: Male Wistar King A Hokkaido male rats (WKAH/HkmSlc) rats aged 4-5 weeks were fed a control diet or a diet with cholic acid (CA; 0.5 g/kg diet), the primary 12αOH BA, for 2 weeks (Study 1) or 13 weeks (Study 2). In Study 3, rats fed the same diets were given drinking water either alone or containing vancomycin (200 mg/L) for 6 weeks. The variables measured included food intake (Studies 1-3), bile acid profiles (Studies 1 and 3), hepatic iron concentration (Studies 1-3), fecal iron excretion (Studies 1 and 2), iron-related liver gene expression (Studies 2 and 3), and plasma iron-related factors (Studies 2 and 3). RESULTS: In Study 1, CA feed reduced the hepatic iron concentration (-16%; P = 0.005) without changing food intake or fecal iron excretion. In Study 2, we found a significant increase in the aortic plasma concentration of lipocalin 2 (LCN2; +65%; P < 0.001), an iron-trafficking protein. In Study 3, we observed no effect of vancomycin treatment on the CA-induced reduction of hepatic iron concentration (-32%; P < 0.001), accompanied by increased plasma LCN2 concentration (+72%; P = 0.003), in the CA-fed rats despite a drastic reduction in the secondary 12αOH BA concentration (-94%; P < 0.001) in the aortic plasma. CONCLUSIONS: Primary 12αOH BAs reduced the hepatic iron concentration in rats. LCN2 may be responsible for the hepatic iron-lowering effect of primary 12αOH BAs by transporting iron out of the liver.
  • Masaru Wada, Satoru Fukiya, Azusa Suzuki, Nanae Matsumoto, Miki Matsuo, Atsushi Yokota
    Bioscience of microbiota, food and health 40 1 80 - 83 2021年 [査読有り]
     
    Although bifidobacteria are already widely used as beneficial microbes with health-promoting effects, their amino acid utilization and metabolism are not yet fully understood. Knowledge about the metabolism of sulfur-containing amino acids in bifidobacteria is especially limited. In this study, we tested the methionine utilization ability of several bifidobacterial strains when it was the sole available sulfur source. Although bifidobacteria have long been predominantly considered to be cysteine auxotrophs, we showed that this is not necessarily the case.
  • Ja-Young Lee, Hidehisa Shimizu, Masahito Hagio, Satoru Fukiya, Masamichi Watanabe, Yasutake Tanaka, Ga-Hyun Joe, Hitoshi Iwaya, Reika Yoshitsugu, Keidai Kikuchi, Misaki Tsuji, Nanako Baba, Takuma Nose, Koji Tada, Taketo Hanai, Shota Hori, Akari Takeuchi, Yumiko Furukawa, Bungo Shirouchi, Masao Sato, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota, Satoshi Ishizuka
    Biochimica et biophysica acta. Molecular and cell biology of lipids 1865 12 158811 - 158811 2020年12月 [査読有り]
     
    There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4β-hydroxycholesterol (4βOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4βOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4βOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.
  • Siddabasave Gowda B Gowda, Divyavani Gowda, Chongsheng Liang, Yonghan Li, Kentaro Kawakami, Satoru Fukiya, Atsushi Yokota, Hitoshi Chiba, Shu-Ping Hui
    Metabolites 10 10 2020年10月08日 [査読有り]
     
    Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are novel endogenous lipids with important physiological functions in mammals. We previously identified a new type of FAHFAs, named short-chain fatty acid esterified hydroxy fatty acids (SFAHFAs), with acetyl or propyl esters of hydroxy fatty acids of carbon chains, C ≥ 20. However, sensitive determination of SFAHFAs is still a challenge, due to their high structural similarity and low abundance in biological samples. This study employs one-step chemical derivatization following total lipid extraction using 2-dimethylaminoethylamine (DMED) for enhanced detection of SFAHFAs. The labeled extracts were subjected to ultrahigh performance liquid chromatography coupled to linear ion trap quadrupole-Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap MS). Our results demonstrated that the detection sensitivities of SFAHFAs increased after DMED labeling, and is highly helpful in discovering six additional novel SFAHFAs in the cecum and colon contents of WKAH/HKmSlc rats fed with normal and high-fat diet (HFD). The identified DMED labeled SFAHFAs were characterized by their detailed MS/MS analysis, and their plausible fragmentation patterns were proposed. The concentrations of SFAHFAs were significantly reduced in the cecum of HFD group compared to the control. Hence, the proposed method could be a promising tool to apply for the enhanced detection of SFAHFAs in various biological matrices, which in turn facilitate the understanding of their sources, and physiological functions of these novel lipids.
  • Siddabasave Gowda B Gowda, Chongsheng Liang, Divyavani Gowda, Fengjue Hou, Kentaro Kawakami, Satoru Fukiya, Atsushi Yokota, Hitoshi Chiba, Shu-Ping Hui
    Rapid communications in mass spectrometry : RCM 34 17 e8831  2020年09月15日 [査読有り]
     
    RATIONALE: Fatty acid esters of hydroxy fatty acids (FAHFAs) are recently discovered endogenous lipids with outstanding health benefits. FAHFAs are known to exhibit antioxidant, antidiabetic and anti-inflammatory properties. The number of known long-chain FAHFAs in mammalian tissues and dietary resources increased recently because of the latest developments in high-resolution tandem mass spectrometry techniques. However, there are no reports on the identification of short-chain fatty acid esterified hydroxy fatty acids (SFAHFAs). METHODS: Intestinal contents, tissues, and plasma of rats fed with high-fat diet (HFD) and normal diet (ND) were analyzed for fatty acids, hydroxy fatty acids, and FAHFAs using ultra-high-performance liquid chromatography (UHPLC) and linear trap quadrupole-Orbitrap mass spectrometry (LTQ Orbitrap MS) with negative heated electrospray ionization. RESULTS: Untargeted analysis of total lipid extracts from murine samples (male 13-week-old WKAH/HKmSlc rats) led to the identification of several new SFAHFAs of acetic acid or propanoic acid esterified long-chain (>C20)-hydroxy fatty acids. Furthermore, MS3 analysis revealed the position of the hydroxyl group in the long-chain fatty acid as C-2. The relative amounts of SFAHFAs were quantified in intestinal contents and their tissues (Cecum, small intestine, and large intestine), liver, and plasma of rats fed with HFD and ND. The large intestine showed the highest abundance of SFAHFAs with a concentration range from 0.84 to 57 pmol/mg followed by the cecum with a range of 0.66 to 28.6 pmol/mg. The SFAHFAs were significantly altered between the HFD and ND groups, with a strong decreasing tendency under HFD conditions. CONCLUSIONS: Identification of these novel SFAHFAs can contribute to a better understanding of the chemical and biological properties of individual SFAHFAs and their possible sources in the gut, which in turn helps us tackle the role of these lipids in various metabolic diseases.
  • Shota Hori, Takayuki Abe, Dong Geun Lee, Satoru Fukiya, Atsushi Yokota, Nao Aso, Bungo Shirouchi, Masao Sato, Satoshi Ishizuka
    The Journal of nutritional biochemistry 83 108412 - 108412 2020年05月20日 [査読有り][通常論文]
     
    High-fat (HF) diet induces hepatic steatosis that is a risk factor for noncommunicable diseases such as obesity, type 2 diabetes and cardiovascular disease. Previously, we found that HF feeding in rats increases the excretion of fecal bile acids (BAs), specifically 12α-hydroxylated (12αOH) BAs. Although the liver is the metabolic center in our body, the association between hepatic steatosis and 12αOH BAs in HF-fed rats is unclear. Thus, we investigated extensively BA composition in HF-fed rats and evaluated the association between hepatic steatosis and 12αOH BAs. Acclimated male inbred WKAH/HkmSlc rats were divided into two groups and fed either control or HF diet for 8 weeks. Feeding HF diet increased hepatic triglyceride and total cholesterol concentrations, which correlated positively with 12αOH BAs concentrations but not with non-12αOH BAs in the feces, portal plasma and liver. Accompanied by the increase in 12αOH BAs, the rats fed HF diet showed increased fat absorption and higher mRNA expression of liver Cidea. The enhancement of 12αOH BA secretion may contribute to hepatic steatosis by the promotion of dietary fat absorption and hepatic Cidea mRNA expression. The increase in 12αOH BAs was associated with enhanced liver cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1) mRNA expression. There was a significant increase in 7α-hydroxycholesterol, a precursor of BAs, in the liver of HF-fed rats. Altogether, these data suggest that the HF diet increases preferentially 12αOH BAs synthesis by utilizing the accumulated hepatic cholesterol and enhancing mRNA expression of Cyp7a1 and Cyp8b1 in the liver.
  • Hiroka Koguchi, Natsumi Ishigami, Mikiyasu Sakanaka, Kako Yoshida, Sayaka Hiratou, Mina Shimada, Satoru Fukiya, Kei Sonoyama, Atsushi Yokota
    Microorganisms 8 3 2020年03月13日 [査読有り][通常論文]
     
    Bifidobacteria are one of the major components in human gut microbiota and well-known as beneficial microbes. However, clarification of commensal mechanisms of bifidobacteria in the intestines is still ongoing, especially in the presence of the gut microbiota. Here, we applied recombinase-based in vivo expression technology (R-IVET) using the bacteriophage P1 Cre/loxP system to Bifidobacterium longum subsp. longum 105-A (B. longum 105-A) to identify genes that are specifically expressed in the gastrointestinal tract of conventionally raised mice. Oral administration of the genomic DNA library of B. longum 105-A to conventionally raised mice resulted in the identification of 73 in vivo-induced genes. Four out of seven tested genes were verified in vivo-specific induction at least in the cecum by quantitative reverse transcription PCR. Although there is still room for improvement of the system, our findings can contribute to expanding our understanding of the commensal behavior of B. longum in the gut ecosystem.
  • Siddabasave Gowda B Gowda, Zi-Jun Gao, Zhen Chen, Takayuki Abe, Shota Hori, Satoru Fukiya, Satoshi Ishizuka, Atsushi Yokota, Hitoshi Chiba, Shu-Ping Hui
    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 36 7 821 - 828 2020年01月17日 [査読有り][通常論文]
     
    High-fat diet (HFD)-induced obesity is a primary risk factor for serious health problems. Although much research has been performed at the genomic level, lipidomic studies were limited. In this study, we aim to obtain a comprehensive profile of circulating plasma lipids, which are altered in rodent rat obesity by untargeted liquid chromatography-mass spectrometry. Rats fed with HFD for 8 weeks had increased body weight, liver and adipose tissue weight. The analysis results revealed that polyunsaturated fatty acids (PUFAs) and their corresponding phosphatidylcholine, phosphatidylinositol, and phosphatidylserine were significantly decreased in rats fed with HFD. In contrast, less unsaturated and ether type phosphatidylglycerols were increased. The triacylglycerides (TAGs) having saturated FA were increased in the HFD condition, whereas TAGs having PUFA were decreased. The levels of many plasma lipids were altered, and interestingly PUFA derived lipids were negatively associated with obesity. This signifies the importance of a PUFAs enriched diet to overwhelm obesity associated diseases.
  • Hiroko Yoshioka, Masamichi Watanabe, Fumio Nanba, Toshio Suzuki, Satoru Fukiya, Atsushi Yokota, Toshiya Toda
    Journal of nutritional science and vitaminology 66 6 571 - 576 2020年 [査読有り]
     
    Equol (Eq) is a metabolite of soy isoflavone daidzein (De) produced by the intestinal microbiota. The clinical effectiveness of soy isoflavone is considered to depend on the individual ability of Eq production. Previous studies have demonstrated that habitual dietary patterns may influence the production of Eq. For example, high Eq producers consumed less fat as a percentage of energy than low Eq producers. However, the inhibitory factors of Eq production are unknown. Recently, it was reported that bile acids induced by high-fat diet consumption may be a host-related factor controlling the composition of the intestinal microbiota. In this study, we investigated the effect of cholic acid (CA) administration, a mimic of the microbiota altered by a high-fat diet, on Eq production in mice. CA administration significantly decreased the levels of the De metabolites Eq, dihydrodaidzein, and O-desmethylangolensin in the serum of mice. However, CA administration did not affect the total molar concentration of De and its metabolites. Moreover, CA administration increased the levels of secondary bile acids, particularly deoxycholic acid (DCA), which has strong antibacterial activity in the cecum contents of mice. Thus, CA administration may increase the levels of DCA, a secondary bile acid, resulting in inhibition of Eq production. These findings may help to reveal the factors inhibiting Eq production and enhance the clinical effectiveness of isoflavone intake.
  • 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][通常論文]
     
    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.
  • Lee DG, Hori S, Kohmoto O, Kitta S, Yoshida R, Tanaka Y, Shimizu H, Takahashi K, Nagura T, Uchino H, Fukiya S, Yokota A, Ishizuka S
    Bioscience, biotechnology, and biochemistry 83 7 1329 - 1335 2019年07月 [査読有り][通常論文]
     
    Difructose anhydride III (DFAIII) is a prebiotic involved in the reduction of secondary bile acids (BAs). We investigated whether DFAIII modulates BA metabolism, including enterohepatic circulation, in the rats fed with a diet supplemented with cholic acid (CA), one of the 12α-hydroxylated BAs. After acclimation, the rats were fed with a control diet or a diet supplemented with DFAIII. After 2 weeks, each group was further divided into two groups and was fed diet with or without CA supplementation at 0.5 g/kg diet. BA levels were analyzed in aortic and portal plasma, liver, intestinal content, and feces. As a result, DFAIII ingestion reduced the fecal deoxycholic acid level via the partial suppression of deconjugation and 7α-dehydroxylation of BAs following CA supplementation. These results suggest that DFAIII suppresses production of deoxycholic acid in conditions of high concentrations of 12α-hydroxylated BAs in enterohepatic circulation, such as obesity or excess energy intake. Abbreviation: BA: bile acid; BSH: bile salt hydrolase; CA: cholic acid; DCA: deoxycholic acid; DFAIII: difructose anhydride III; MCA: muricholic acid; MS: mass spectrometry; NCDs: non-communicable diseases; LC: liquid chromatography; SCFA: short-chain fatty acid; TCA: taurocholic acid; TCDCA: taurochenodeoxycholic acid; TDCA: taurodeoxycholic acid; TUDCA: tauroursodeoxychlic acid; TαMCA: tauro-α-muricholic acid; TβMCA: tauro-β-muricholic acid; TωMCA: tauro-ω-muricholic acid.
  • Shinji Kato, Haruhi Tobe, Hiroki Matsubara, Mariko Sawada, Yasuko Sasaki, Satoru Fukiya, Naoki Morita, Atsushi Yokota
    Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids 1864 3 403 - 412 2019年03月 [査読有り][通常論文]
     
    © 2018 Elsevier B.V. Bile acids exhibit strong antimicrobial activity as natural detergents, and are involved in lipid digestion and absorption. We investigated the mechanism of bile acid adaptation in Lactobacillus gasseri JCM1131 T . Exposure to sublethal concentrations of cholic acid (CA), a major bile acid in humans, resulted in development of resistance to otherwise-lethal concentrations of CA by this intestinal lactic acid bacterium. As this adaptation was accompanied by decreased cell-membrane damage, we analyzed the membrane lipid composition of L. gasseri. Although there was no difference in the proportions of glycolipids (~70%) and phospholipids (~20%), adaptation resulted in an increased abundance of long-sugar-chain glycolipids and a 100% increase in cardiolipin (CL) content (to ~50% of phospholipids) at the expense of phosphatidylglycerol (PG). In model vesicles, the resistance of PG vesicles to solubilization by CA increased with increasing CL/PG ratio. Deletion of the two putative CL synthase genes, the products of which are responsible for CL synthesis from PG, decreased the CL content of the mutants, but did not affect their ability to adapt to CA. Exposure to CA restored the CL content of the two single-deletion mutants, likely due to the activities of the remaining CL synthase. In contrast, the CL content of the double-deletion mutant was not restored, and the lipid composition was modified such that PG predominated (~45% of total lipids) at the expense of glycolipids. Therefore, CL plays important roles in bile acid resistance and maintenance of the membrane lipid composition in L. gasseri.
  • Mikiyasu Sakanaka, Shingo Nakakawaji, Shin Nakajima, Satoru Fukiya, Arisa Abe, Wataru Saburi, Haruhide Mori, Atsushi Yokota
    Applied and environmental microbiology 84 17 2018年09月01日 [査読有り][通常論文]
     
    Bifidobacteria are a major component of the intestinal microbiota in humans, particularly breast-fed infants. Therefore, elucidation of the mechanisms by which these bacteria colonize the intestine is desired. One approach is transposon mutagenesis, a technique currently attracting much attention because, in combination with next-generation sequencing, it enables exhaustive identification of genes that contribute to microbial fitness. We now describe a transposon mutagenesis system for Bifidobacterium longum subsp. longum 105-A (JCM 31944) based on ISBlo11, a native IS3 family insertion sequence. To build this system, xylose-inducible or constitutive bifidobacterial promoters were tested to drive the expression of full-length or a truncated form at the N terminus of the ISBlo11 transposase. An artificial transposon plasmid, pBFS12, in which ISBlo11 terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS3 elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >103 CFU/μg DNA. The plasmid targets random 3- to 4-bp sequences, but with a preference for noncoding regions. This mutagenesis system also worked at least in B. longum NCC2705. Characterization of a transposon insertion mutant revealed that a putative α-glucosidase mediates palatinose and trehalose assimilation, demonstrating the suitability of transposon mutagenesis for loss-of-function analysis. We anticipate that this approach will accelerate functional genomic studies of B. longum subsp. longumIMPORTANCE Several hundred species of bacteria colonize the mammalian intestine. However, the genes that enable such bacteria to colonize and thrive in the intestine remain largely unexplored. Transposon mutagenesis, combined with next-generation sequencing, is a promising tool to comprehensively identify these genes but has so far been applied only to a small number of intestinal bacterial species. In this study, a transposon mutagenesis system was established for Bifidobacterium longum subsp. longum, a representative health-promoting Bifidobacterium species. The system enables the identification of genes that promote colonization and survival in the intestine and should help illuminate the physiology of this species.
  • T. Shiraishi, S. Yokota, Y. Sato, T. Ito, S. Fukiya, S. Yamamoto, T. Sato, A. Yokota
    Beneficial Microbes 9 4 653 - 662 2018年 [査読有り][通常論文]
     
    Lipoteichoic acid (LTA) is a cell surface molecule specific to Gram-positive bacteria. How LTA localises on the cell surface is a fundamental issue in view of recognition and immunomodulation in hosts. In the present study, we examined LTA localisation using strain JCM 1131T of Lactobacillus gasseri, which is a human intestinal lactic acid bacterium, during various growth phases by immunoelectron microscopy. We first evaluated the specificity of anti-LTA monoclonal antibody clone 55 used as a probe. The glycerophosphate backbone comprising almost intact size (20 to 30 repeating units) of LTA was required for binding. The antibody did not bind to other cellular components, including wall-teichoic acid. Immunoelectron microscopy indicated that LTA was embedded in the cell wall during the logarithmic phase, and was therefore not exposed on the cell surface. Similar results were observed for Lactobacillus fermentum ATCC 9338 and Lactobacillus rhamnosus ATCC 7469T. By contrast, membrane vesicles were observed in the logarithmic phase of L. gasseri with LTA exposed on their surface. In the stationary and death phases, LTA was exposed on cell wall-free cell membrane generated by autolysis. The dramatic alternation of localisation in different growth phases and exposure on the surface of membrane vesicles should relate with complicated interaction between bacteria and host.
  • Sarinya Tawthep, Satoru Fukiya, Ja-Young Lee, Masahito Hagio, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota
    Journal of bioscience and bioengineering 124 5 514 - 522 2017年11月 [査読有り][通常論文]
     
    Understanding the dynamics of secondary bile acid (SBA) formation in the gut by SBA-producing bacteria is important for host health, as SBAs have been shown to affect host pathophysiology and gut microbiota composition. However, our knowledge of SBA producers is limited in light of the diversity of gut microbes. Here, we isolated six novel SBA-producing bacteria from rat cecal contents, all of which were members of known species of gut microbes. Anaerostipes caccae D10, Bacteroides nordii C5, Clostridioides difficile D7, and Clostridium cadaveris G11 were capable of oxidizing cholic acid and chenodeoxycholic acid into 7-oxo-derivatives with varying yields. B. nordii C5 and its type strain JCM 12987T had the highest molar yield, ∼90%. Clostridium disporicum F4 and Clostridium subterminale C4 epimerized cholic acid into ursocholic acid with yields of ∼85%; the corresponding type strains lacked epimerization activity. Furthermore, although not novel as an SBA producer, Clostridium scindens G10 that produced deoxycholic acid from cholic acid was isolated for the first time from rodents. These findings will contribute to elucidation of SBA formation in the gut.
  • Keita Nishiyama, Yuji Yamamoto, Makoto Sugiyama, Takashi Takaki, Tadasu Urashima, Satoru Fukiya, Atsushi Yokota, Nobuhiko Okada, Takao Mukai
    mBio 8 5 2017年10月03日 [査読有り][通常論文]
     
    Bifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. We studied the role of the extracellular sialidase (SiaBb2, 835 amino acids [aa]) from Bifidobacterium bifidum ATCC 15696 in mucosal surface adhesion and carbohydrate catabolism. Human milk oligosaccharides (HMOs) or porcine mucin oligosaccharides as the sole carbon source enhanced B. bifidum growth. This was impaired in a B. bifidum ATCC 15696 strain harboring a mutation in the siabb2 gene. Mutant cells in early to late exponential growth phase also showed decreased adhesion to human epithelial cells and porcine mucin relative to the wild-type strain. These results indicate that SiaBb2 removes sialic acid from HMOs and mucin for metabolic purposes and may promote bifidobacterial adhesion to the mucosal surface. To further characterize SiaBb2-mediated bacterial adhesion, we examined the binding of His-tagged recombinant SiaBb2 peptide to colonic mucins and found that His-SiaBb2 as well as a conserved sialidase domain peptide (aa 187 to 553, His-Sia) bound to porcine mucin and murine colonic sections. A glycoarray assay revealed that His-Sia bound to the α2,6-linked but not to the α2,3-linked sialic acid on sialyloligosaccharide and blood type A antigen [GalNAcα1-3(Fucα1-2)Galβ] at the nonreducing termini of sugar chains. These results suggest that the sialidase domain of SiaBb2 is responsible for this interaction and that the protein recognizes two distinct carbohydrate structures. Thus, SiaBb2 may be involved in Bifidobacterium-mucosal surface interactions as well as in the assimilation of a variety of sialylated carbohydrates.IMPORTANCE Adhesion to the host mucosal surface and carbohydrate assimilation are important for bifidobacterium colonization and survival in the host gastrointestinal tract. In this study, we investigated the mechanistic basis for B. bifidum extracellular sialidase (SiaBb2)-mediated adhesion. SiaBb2 cleaved sialyl-human milk oligosaccharides and mucin glycans to produce oligosaccharides that supported B. bifidum growth. Moreover, SiaBb2 enhanced B. bifidum adhesion to mucosal surfaces via specific interactions with the α2,6 linkage of sialyloligosaccharide and blood type A antigen on mucin carbohydrates. These findings provide insight into the bifunctional role of SiaBb2 and the adhesion properties of B. bifidum strains.
  • Masamichi Watanabe, Satoru Fukiya, Atsushi Yokota
    Journal of lipid research 58 6 1143 - 1152 2017年06月 [査読有り][通常論文]
     
    In addition to functioning as detergents that aid digestion of dietary lipids in the intestine, some bile acids have been shown to exhibit antimicrobial activity. However, detailed information on the bactericidal activities of the diverse molecular species of bile acid in humans and rodents is largely unknown. Here, we investigated the toxicity of 14 typical human and rodent free bile acids (FBAs) by monitoring intracellular pH, membrane integrity, and viability of a human intestinal bacterium, Bifidobacterium breve Japan Collection of Microorganisms (JCM) 1192T, upon exposure to these FBAs. Of all FBAs evaluated, deoxycholic acid (DCA) and chenodeoxycholic acid displayed the highest toxicities. Nine FBAs common to humans and rodents demonstrated that α-hydroxy-type bile acids are more toxic than their oxo-derivatives and β-hydroxy-type epimers. In five rodent-specific FBAs, β-muricholic acid and hyodeoxycholic acid showed comparable toxicities at a level close to DCA. Similar trends were observed for the membrane-damaging effects and bactericidal activities to Blautia coccoides JCM 1395T and Bacteroides thetaiotaomicron DSM 2079T, commonly represented in the human and rodent gut microbiota. These findings will help us to determine the fundamental properties of FBAs and better understand the role of FBAs in the regulation of gut microbiota composition.
  • Soya Maeda, Kumiko Shimizu, Chie Kihira, Yuki Iwabu, Ryuichi Kato, Makoto Sugimoto, Satoru Fukiya, Masaru Wada, Atsushi Yokota
    Journal of bioscience and bioengineering 123 4 437 - 443 2017年04月 [査読有り][通常論文]
     
    Pyruvate dehydrogenase complex regulator (PdhR) is a transcriptional regulator that negatively regulates formation of pyruvate dehydrogenase complex (PDHc), NADH dehydrogenase (NDH)-2, and cytochrome bo3 oxidase in Escherichia coli. To investigate the effects of a PdhR defect on glucose metabolism, a pdhR deletion mutant was derived from the wild-type E. coli W1485 strain by λ Red-mediated recombination. While no difference in the fermentation profiles was observed between the two strains under oxygen-sufficient conditions, under oxygen-limited conditions, the growth level of the wild-type strain was significantly decreased with retarded glucose consumption accompanied by by-production of substantial amounts of pyruvic acid and acetic acid. In contrast, the mutant grew and consumed glucose more efficiently than did the wild-type strain with enhanced respiration, little by-production of pyruvic acid, less production yield and rates of acetic acid, thus displaying robust metabolic activity. As expected, increased activities of PDHc and NDH-2 were observed in the mutant. The increased activity of PDHc may explain the loss of pyruvic acid by-production, probably leading to decreased acetic acid formation, and the increased activity of NDH-2 may explain the enhanced respiration. Measurement of the intracellular NAD+/NADH ratio in the mutant revealed more oxidative or more reductive intracellular environments than those in the wild-type strain under oxygen-sufficient and -limited conditions, respectively, suggesting another role of PdhR: maintaining redox balance in E. coli. The overall results demonstrate the biotechnological advantages of pdhR deletion in boosting glucose metabolism and also improve our understanding of the role of PdhR in bacterial physiology.
  • 白石 宗, 横田 伸一, 吹谷 智, 横田 篤
    JATAFFジャーナル = JATAFF journal : 農林水産技術 5 3 26 - 32 農林水産・食品産業技術振興協会 2017年03月 [査読無し][通常論文]
  • 吹谷 智
    生物工学会誌 94 3 110 - 116 公益社団法人日本生物工学会 2016年03月25日 [査読無し][招待有り]
  • A. Aoki-Yoshida, S. Saito, S. Fukiya, R. Aoki, Y. Takayama, C. Suzuki, K. Sonoyama
    BENEFICIAL MICROBES 7 3 421 - 429 2016年 [査読有り][通常論文]
     
    Administration of Lactobacillus rhamnosus GG (LGG) has been reported to be therapeutically effective against acute secretory diarrhoea resulting from the structural and functional intestinal mucosal lesions induced by rotavirus infection; however, the underlying mechanisms remain to be completely elucidated. Because Toll-like receptor 3 (TLR3) plays a key role in the innate immune responses following the recognition of rotavirus, the present study examined whether LGG influences TLR3 gene expression in murine small intestine ex vivo and in vivo. We employed cultured intestinal organoids derived from small intestinal crypts as an ex vivo tissue model. LGG supplementation increased TLR3 mRNA levels in the intestinal organoids, as estimated by quantitative real-time polymerase chain reaction. Likewise, single and 7-day consecutive daily administrations of LGG increased TLR3 mRNA levels in the small intestine of C57BL/6N mice. The mRNA levels of other TLRs were not substantially altered both ex vivo and in vivo. In addition, LGG supplementation increased the mRNA levels of an antiviral type 1 interferon, interferon-alpha (IFN-alpha), and a neutrophil chemokine, CXCL1, upon stimulation with a synthetic TLR3 ligand, poly(I:C) in the intestinal organoids. LGG administration did not alter IFN-alpha and CXCL1 mRNA levels in the small intestine in vivo. Supplementation of other bacterial strains, Bifidobacterium bifidum and Lactobacillus paracasei, failed to increase TLR3 and poly(I: C)-stimulated CXCL1 mRNA levels ex vivo. We propose that upregulation of TLR3 gene expression may play a pivotal role in the therapeutic efficacy of LGG against rotavirus-associated diarrhoea. In addition, we demonstrated that intestinal organoids may be a promising ex vivo tissue model for investigating host-pathogen interactions and the antiviral action of probiotics in the intestinal epithelium.
  • Tsukasa Shiraishi, Shinichi Yokota, Satoru Fukiya, Atsushi Yokota
    Bioscience of microbiota, food and health 35 4 147 - 161 2016年 [査読有り][通常論文]
     
    Bacterial cell surface molecules are at the forefront of host-bacterium interactions. Teichoic acids are observed only in Gram-positive bacteria, and they are one of the main cell surface components. Teichoic acids play important physiological roles and contribute to the bacterial interaction with their host. In particular, lipoteichoic acid (LTA) anchored to the cell membrane has attracted attention as a host immunomodulator. Chemical and biological characteristics of LTA from various bacteria have been described. However, most of the information concerns pathogenic bacteria, and information on beneficial bacteria, including probiotic lactic acid bacteria, is insufficient. LTA is structurally diverse. Strain-level structural diversity of LTA is suggested to underpin its immunomodulatory activities. Thus, the structural information on LTA in probiotics, in particular strain-associated diversity, is important for understanding its beneficial roles associated with the modulation of immune response. Continued accumulation of structural information is necessary to elucidate the detailed physiological roles and significance of LTA. In this review article, we summarize the current state of knowledge on LTA structure, in particular the structure of LTA from lactic acid bacteria. We also describe the significance of structural diversity and biological roles of LTA.
  • Mikiyasu Sakanaka, Satoru Fukiya, Ryoko Kobayashi, Arisa Abe, Yosuke Hirayama, Yasunobu Kano, Atsushi Yokota
    FEMS microbiology letters 362 7 2015年04月 [査読有り][通常論文]
     
    Transposon mutagenesis systems are still under development in bifidobacteria, partly because intrinsic active insertion sequences are not well characterized in bifidobacteria. Here, we isolated an active insertion sequence, ISBlo11, from Bifidobacterium longum 105-A using a sacB-based counterselection system, which is generally used to screen for active insertion sequences from bacterial genomes. ISBlo11 is 1432 bp long and belongs to the IS3 family. It has a single ORF encoding a transposase and 25-bp inverted repeats at its termini. Full-length copies of ISBlo11 are specifically conserved among certain B. longum genomes and exist in different sites. Transposition analysis of an artificial ISBlo11 transposon using an Escherichia coli conjugation system revealed that ISBlo11 has adequate transposition activity, comparable to the reported activity of IS629, another IS3 family element initially isolated from Shigella sonnei. ISBlo11 also showed low transposition selectivity for non-conserved 3- or 4-bp target sequences. These characteristics of ISBlo11 seem suitable for the development of a new transposon mutagenesis system in bifidobacteria.
  • Hidenori Matsui, Satoru Fukiya, Chie Kodama-Akaboshi, Masahiro Eguchi, Tomoko Yamamoto
    Journal of medical microbiology 64 Pt 3 295 - 302 2015年03月 [査読有り][通常論文]
     
    In BALB/c mouse models of Salmonella enterica serovar Typhimurium infection, a single oral immunization with a mutant strain with an insertion of the chloramphenicol resistance gene into the ATP-dependent protease clpP or lon gene decreased the number of salmonellae in each tissue sample 5 days after oral challenge with virulent S. Typhimurium at weeks 26 and 54 post-immunization. These data suggested that an oral immunization with the ClpP- or Lon-disrupted S. Typhimurium strain could provide long-term protection against oral challenge with virulent S. Typhimurium. Accordingly, recombinant oral non-typhoidal Salmonella (NTS) vaccines were constructed by incorporating mutants of both S. Typhimurium and S. enterica serovar Enteritidis harbouring stable in-frame markerless deletions of the clpP-lon-sulA (suppressor of lon), lon-sulA or lon-msbB (acyltransferase) genes. Amongst these orally administered vaccine candidates, those with the lon-sulA gene deletion mutants of S. Typhimurium and S. Enteritidis protected BALB/c and C57BL/6J mice against oral challenge with both virulent S. Typhimurium and virulent S. Enteritidis. Therefore, the in-frame markerless lon-sulA gene deletion mutant of S. Typhimurium or S. Enteritidis could be a promising cross-protective NTS live vaccine candidate for practical use in humans.
  • Hagio M, Shimizu H, Joe GH, Takatsuki M, Shiwaku M, Xu H, Lee JY, Fujii N, Fukiya S, Hara H, Yokota A, Ishizuka S
    Toxicology letters 232 1 246 - 52 2015年01月05日 [査読有り][通常論文]
     
    Consumption of a high-fat diet increases some secondary bile acids (BAs) such as deoxycholic acid (DCA) in feces. DCA is derived from cholic acid (CA), a primary BA. We evaluated intestinal epithelial proliferation and BA metabolism in response to oral administration of cholic acid (CA) in rats to determine the influence of a CA diet on the responses of gut epithelia to γ-rays. WKAH/HkmSlc rats were divided into two dietary groups: control diet or CA-supplemented (2g/kg diet) diet. Some of the rats from each group were irradiated with γ-rays, and epithelial cell proliferation in the colon was analyzed histochemically. Unirradiated CA-fed rats had high levels of DCA and CA in the sera, as well as the presence of taurocholic acid in their feces. Significant increases were observed in both epithelial proliferation and the number of epithelial cells in the colon of the CA-fed rats, and this effect was observed at 8 weeks after γ-ray exposure. Furthermore, extracts from both cecal contents and sera of the unirradiated CA-fed rats promoted proliferation of IEC-6 cells. These results indicate that BAs in enterohepatic circulation promote proliferation and survival of the intestinal epithelium after receiving DNA damage.
  • Hidehisa Shimizu, Nanako Baba, Takuma Nose, Ryoko Taguchi, Shinya Tanaka, Ga-Hyun Joe, Hideaki Maseda, Nobuhiko Nomura, Masahito Hagio, Ja-Young Lee, Satoru Fukiya, Atsushi Yokota, Satoshi Ishizuka, Hitoshi Miyazaki
    Bioscience, biotechnology, and biochemistry 79 6 937 - 42 2015年 [査読有り][通常論文]
     
    The signal molecule, 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), is similar to a mammalian hormone in bacteria. Although most studies have examined the effects of high 3-oxo-C12-HSL concentrations (>200 μM) on mammalian cellular functions because ~600 μM 3-oxo-C12-HSL can be secreted in biofilms of Pseudomonas aeruginosa grown in vitro, we previously showed that a low 3-oxo-C12-HSL concentration (30 μM) induces the apoptosis of undifferentiated Caco-2 cells through suppressing Akt activity. Here, we found that a low concentration of 3-oxo-C12-HSL-activated ERK1/2 in undifferentiated Caco-2 cells. Incubating cells with the ERK pathway inhibitor U0126 for 30 min alleviated the mucin 3 (MUC3) expression suppressed by 3-oxo-C12-HSL, and the upregulation of MUC3 expression induced by a 48-h incubation with U0126-reduced cell death. Thus, altered MUC3 expression caused by long-term attenuated ERK1/2 activity might correlate with the death of undifferentiated Caco-2 cells induced by 3-oxo-C12-HSL.
  • Yu Kanesaki, Hisayoshi Masutani, Mikiyasu Sakanaka, Yuh Shiwa, Takatomo Fujisawa, Yasukazu Nakamura, Atsushi Yokota, Satoru Fukiya, Tohru Suzuki, Hirofumi Yoshikawa
    Genome announcements 2 6 2014年12月18日 [査読有り][通常論文]
     
    Bifidobacterium longum 105-A shows high transformation efficiency and allows for the generation of gene knockout mutants through homologous recombination. Here, we report the complete genome sequence of strain 105-A. Genes encoding at least four putative restriction-modification systems were found in this genome, which might contribute to its transformation efficiency.
  • Sakanaka M, Tamai S, Hirayama Y, Onodera A, Koguchi H, Kano Y, Yokota A, Fukiya S
    Journal of bioscience and bioengineering 118 5 489 - 95 2014年11月 [査読有り][通常論文]
     
    Heterologous gene expression in bifidobacteria requires weak, strong, and inducible promoters depending on the objectives of different expression studies. Weak promoters in Escherichia coli can also be desirable for stable heterologous gene cloning. Here, we developed a reporter system using the Bifidobacterium longum α-galactosidase gene and investigated the activity and inducibility of seven bifidobacterial promoters in B. longum and their activities in E. coli. These studies revealed diverse promoter activities. Three promoters were highly active in B. longum, but only slightly active in E. coli. Among these, two phosphoketolase gene (xfp) promoters exhibited strong activity in B. longum cells grown on glucose. In contrast, the promoter activity of the fructose transporter operon (fruEKFG) was strongly induced by carbohydrates other than glucose, including fructose, xylose, and ribose. These promoters will allow strong or highly inducible expression in bifidobacteria and stable gene cloning in E. coli. In contrast to the functions of these promoters, the promoter of sucrose-utilization operon cscBA showed very high activity in E. coli but low activity in B. longum. Other three promoters were functional in both B. longum and E. coli. In particular, two sucrose phosphorylase gene (scrP) promoters showed inducible activity by sucrose and raffinose in B. longum, indicating their applicability for regulated expression studies. The diverse promoter functions revealed in this study will contribute to enabling the regulated expression of heterologous genes in bifidobacteria research.
  • Hidehisa Shimizu, Masahito Hagio, Hitoshi Iwaya, Ikuya Tsuneki, Ja-Young Lee, Satoru Fukiya, Atsushi Yokota, Hitoshi Miyazaki, Hiroshi Hara, Satoshi Ishizuka
    Journal of nutritional science and vitaminology 60 6 450 - 4 2014年 [査読有り][通常論文]
     
    Obesity is increasingly becoming associated with increased risk of atherosclerosis. Serum levels of the bile acid deoxycholic acid (DCA) are elevated in mice with obesity induced by a high-fat (HF) diet. Therefore, we investigated the influence of DCA on the functions of vascular smooth muscle cells (VSMCs) because the initiation and progression of atherosclerosis are associated with VSMC proliferation and migration. DCA induced c-jun N-terminal kinase (JNK) activation whereas a JNK inhibitor prevented DCA-induced VSMC proliferation and migration. Based on these findings, we examined whether DCA promotes the expression of platelet-derived growth factor β-receptor (PDGFRβ) that has a c-Jun binding site in its promoter region. The mRNA and protein expression levels of PDGFRβ were upregulated in VSMCs after a 24- and 48-h incubation with DCA, respectively. The effects of PDGF such as proliferation and migration of VSMCs were promoted after a 48-h incubation with DCA despite the absence of DCA during PDGF stimulation. These findings suggest that elevated serum concentrations of DCA are involved in the pathogenesis of atherosclerosis in HF-induced obesity.
  • Tsukasa Shiraishi, Shin-ichi Yokota, Naoki Morita, Satoru Fukiya, Satoru Tomita, Naoto Tanaka, Sanae Okada, Atsushi Yokota
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 79 24 7931 - 7931 2013年12月 [査読有り][通常論文]
  • Lee JY, Arai H, Nakamura Y, Fukiya S, Wada M, Yokota A
    Journal of lipid research 54 11 3062 - 9 11 2013年11月 [査読有り][通常論文]
     
    Bile acid composition in the colon is determined by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes. Ursodeoxycholic acid (UDCA) is regarded as a chemopreventive beneficial bile acid due to its low hydrophobicity. However, it is a minor constituent of human bile acids. Here, we characterized an UDCA-producing bacterium, N53, isolated from human feces. 16S rDNA sequence analysis identified this isolate as Ruminococcus gnavus, a novel UDCA-producer. The forward reaction that produces UDCA from 7-oxo-lithocholic acid was observed to have a growth-dependent conversion rate of 90-100% after culture in GAM broth containing 1 mM 7-oxo-lithocholic acid, while the reverse reaction was undetectable. The gene encoding 7β-hydroxysteroid dehydrogenase (7β-HSDH), which facilitates the UDCA-producing reaction, was cloned and overexpressed in Escherichia coli. Characterization of the purified 7β-HSDH revealed that the kcat/Km value was about 55-fold higher for the forward reaction than for the reverse reaction, indicating that the enzyme favors the UDCA-producing reaction. As R. gnavus is a common, core bacterium of the human gut microbiota, these results suggest that this bacterium plays a pivotal role in UDCA formation in the colon.
  • Haruko Sakurama, Masashi Kiyohara, Jun Wada, Yuji Honda, Masanori Yamaguchi, Satoru Fukiya, Atsushi Yokota, Hisashi Ashida, Hidehiko Kumagai, Motomitsu Kitaoka, Kenji Yamamoto, Takane Katayama
    The Journal of biological chemistry 288 35 25194 - 206 2013年08月30日 [査読有り][通常論文]
     
    Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and sialyllacto-N-tetraose a (Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1-3GlcNAcβ-pNP) and GalNAcβ1-3GlcNAcβ-pNP. GalNAcβ1-3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca(2+) and Mg(2+)) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.
  • Tsukasa Shiraishi, Shin-ichi Yokota, Naoki Morita, Satoru Fukiya, Satoru Tomita, Naoto Tanaka, Sanae Okada, Atsushi Yokota
    Applied and environmental microbiology 79 10 3315 - 8 2013年05月 [査読有り][通常論文]
     
    We determined the chemical structure of lipoteichoic acid (LTA) from Lactobacillus gasseri JCM 1131(T). The repeating unit was comprised of glycerolphosphate and 2-alanylglycerolphosphate. The glycolipid anchor was tetrahexosylglycerol with two or three acyl groups. To our knowledge, this is the first demonstration of a tetrahexose structure in an LTA glycolipid anchor.
  • Kazuaki Ninomiya, Ryuji Yamada, Masami Matsumoto, Satoru Fukiya, Takane Katayama, Chiaki Ogino, Nobuaki Shimizu
    Journal of bioscience and bioengineering 115 2 196 - 9 2013年02月 [査読有り][通常論文]
     
    An image analyzing method was developed to evaluate in situ bioluminescence expression, without exposing the culture sample to the ambient oxygen atmosphere. Using this method, we investigated the effect of dissolved oxygen concentration on bioluminescence from an obligate anaerobe Bifidobacterium longum expressing bacterial luciferase which catalyzes an oxygen-requiring bioluminescent reaction.
  • Hirayama Y, Sakanaka M, Fukuma H, Murayama H, Kano Y, Fukiya S, Yokota A
    Applied and environmental microbiology 78 14 4984 - 94 14 2012年07月 [査読有り][通常論文]
     
    Functional analysis of Bifidobacterium genes is essential for understanding host-Bifidobacterium interactions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover in Bifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-ΔrepA, which lacks the plasmid replication gene repA, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harbors repA, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected in B. longum 105-A using the chromosomal full-length β-galactosidase gene as a target. Markerless gene deletion was tested using the aga gene, which encodes α-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion of aga. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based aga-complemented strain. These functional analyses revealed that aga is the only gene encoding a functional α-galactosidase enzyme in B. longum 105-A.
  • Chie Kihira, Yukari Hayashi, Naoki Azuma, Sakiko Noda, Soya Maeda, Satoru Fukiya, Masaru Wada, Kazunobu Matsushita, Atsushi Yokota
    Journal of biotechnology 158 4 215 - 23 2012年04月30日 [査読有り][通常論文]
     
    The effects of reduced efficiency of proton-motive force (pmf) generation on glucose metabolism were investigated in Escherichia coli respiratory-chain mutants. The respiratory chain of E. coli consists of two NADH dehydrogenases and three terminal oxidases, all with different abilities to generate a pmf. The genes for isozymes with the highest pmf-generating capacity (NADH dehydrogenase-1 and cytochrome bo₃ oxidase) were knocked out singly or in combination, using a wild-type strain as the parent. Analyses of glucose metabolism by jar-fermentation revealed that the glucose consumption rate per cell increased with decreasing efficiency of pmf generation, as determined from the growth parameters of the mutants. The highest rate of glucose metabolism was observed in the double mutant, and the lowest was observed in the wild-type strain. The respiration rates of the single-knockout mutants were comparable to that of the wild-type strain, and that of the double mutant was higher, apparently as a result of the upregulation of the remaining respiratory chain enzymes. All of the strains excreted 2-oxoglutaric acid as a product of glucose metabolism. Additionally, all of the mutants excreted pyruvic acid and/or acetic acid. Interestingly, the double mutant excreted L-glutamic acid. Alterations of the fermentation profiles provide clues regarding the metabolic regulation in each mutant.
  • Satoru Fukiya, Yosuke Hirayama, Mikiyasu Sakanaka, Yasunobu Kano, Atsushi Yokota
    Bioscience of microbiota, food and health 31 2 15 - 25 2012年 [査読有り][通常論文]
     
    Bifidobacteria are well known as beneficial intestinal bacteria that exert health-promoting effects in humans. In addition to physiological and immunological investigations, molecular genetic technologies have been developed and have recently started to be applied to clarify the molecular bases of host-Bifidobacterium interactions. These technologies include transformation technologies and Escherichia coli-Bifidobacterium shuttle vectors that enable heterologous gene expression. In this context, a plasmid artificial modification method that protects the introduced plasmid from the restriction system in host bifidobacteria has recently been developed to increase transformation efficiency. On the other hand, targeted gene inactivation systems, which are vital for functional genomics, seemed far from being practically applicable in bifidobacteria. However, remarkable progress in this technology has recently been achieved, enabling functional genomics in bifidobacteria. Integrated use of these molecular genetic technologies with omics-based analyses will surely boost characterization of the molecular basis underlying beneficial effects of bifidobacteria. Applications of recombinant bifidobacteria to medical treatments have also progressed.
  • Atsushi Yokota, Satoru Fukiya, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Satoshi Ishizuka
    Gut Microbes 3 5 455 - 459 2012年 [査読有り][通常論文]
     
    Recently, we discovered that bile acid, a main component of bile, is a host factor that regulates the composition of the cecal microbiota in rats. Because bile secretion increases on a high-fat diet and bile acids generally have strong antimicrobial activity, we speculated that bile acids would be a determinant of the gut microbiota in response to a high-fat diet. The observed changes in the rat cecal microbiota triggered by cholic acid (the most abundant bile acid in human biliary bile) administration resemble those found in animals fed high-fat diets. Here, we discuss the rationale for this hypothesis by evaluating reported diet-induced gut microbiota alterations based on the postulate that bile acids worked as an underlying determinant. The identification of host factors determining the gut microbiota greatly contributes to understanding the causal relationships between changes in the gut microbiota and disease development, which remain to be elucidated. © 2012 Landes Bioscience.
  • K B M Saiful Islam, Satoru Fukiya, Masahito Hagio, Nobuyuki Fujii, Satoshi Ishizuka, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota
    Gastroenterology 141 5 1773 - 81 2011年11月 [査読有り][通常論文]
     
    BACKGROUND & AIMS: Alterations in the gastrointestinal microbiota have been associated with metabolic diseases. However, little is known about host factors that induce changes in gastrointestinal bacterial populations. We investigated the role of bile acids in this process because of their strong antimicrobial activities, specifically the effects of cholic acid administration on the composition of the gut microbiota in a rat model. METHODS: Rats were fed diets supplemented with different concentrations of cholic acid for 10 days. We used 16S ribosomal RNA gene clone library sequencing and fluorescence in situ hybridization to characterize the composition of the cecal microbiota of the different diet groups. Bile acids in feces, organic acids in cecal contents, and some blood parameters were also analyzed. RESULTS: Administration of cholic acid induced phylum-level alterations in the composition of the gut microbiota; Firmicutes predominated at the expense of Bacteroidetes. Cholic acid feeding simplified the composition of the microbiota, with outgrowth of several bacteria in the classes Clostridia and Erysipelotrichi. Externally administered cholic acid was efficiently transformed into deoxycholic acid by a bacterial 7α-dehydroxylation reaction. Serum levels of adiponectin decreased significantly in rats given the cholic acid diet. CONCLUSIONS: Cholic acid regulates the composition of gut microbiota in rats, inducing similar changes to those induced by high-fat diets. These findings improve our understanding of the relationship between metabolic diseases and the composition of the gastrointestinal microbiota.
  • Takuya Suzuki, Megumi Nishimukai, Aki Shinoki, Hidenori Taguchi, Satoru Fukiya, Atsushi Yokota, Wataru Saburi, Takeshi Yamamoto, Hiroshi Hara, Hirokazu Matsui
    Journal of agricultural and food chemistry 58 19 10787 - 92 2010年10月13日 [査読有り][通常論文]
     
    Gastrectomy often results in osteopenia and anemia because of calcium (Ca) and iron (Fe) malabsorption. Here, we investigated the effects of feeding epilactose, a non-digestible disaccharide, on gastrectomy-induced osteopenia, anemia, and Ca and Fe malabsorption in male Sprague-Dawley rats. Totally gastrectomized or sham-operated rats were fed the control or epilactose (50 g/kg) diets for 30 days. Gastrectomy severely decreased intestinal Ca and Fe absorption, femoral bone strength, Ca content, hemoglobin concentration, and hematocrit. These decreases were partly or totally restored by feeding epilactose. Feeding epilactose increased the cecal tissue weight and the soluble Ca concentration and short-chain fatty acid pools of the cecal contents. Collectively, the increases in cecal mucosal area and/or soluble Ca concentration of the cecal contents, resulting from short-chain fatty acid production by intestinal microbes, are thought to be responsible for the epilactose-mediated promotion of Ca and Fe absorption in the gastrectomized rats.
  • Satoru Fukiya, Tomohiko Sugiyama, Yasunobu Kano, Atsushi Yokota
    Journal of bioscience and bioengineering 110 2 141 - 6 2010年08月 [査読有り][通常論文]
     
    The characteristics of mobile genetic elements in bifidobacteria are not well understood. We characterized an insertion sequence-like element of the IS200/IS605 family found in a size-increased cryptic plasmid in Bifidobacterium longum. During a plasmid profile analysis of B. longum BK strains, we encountered a 6.5-kbp cryptic plasmid pBK283 in B. longum BK28, the size of which has not been identified in bifidobacteria. Nucleotide sequence analysis indicated that an insertion sequence-like element was inserted into the 5.0-kbp pKJ50-like plasmid and resulted in a size increase of pBK283. The element, named ISBlo15, was 1593 bp in length and contained a single ORF encoding a putative transposase, which is similar to the transposase OrfB encoded by IS200/IS605 family elements. Several sequence characteristics, including conserved transposase motifs in OrfB and terminal palindromic sequences that differ from the typical terminal inverted repeats, strongly suggested that ISBlo15 is a member of the IS200/IS605 family. Sequences similar to ISBlo15 were widely distributed among the nine Bifidobacterium species tested, and those of highly homologous sequences were detected only in Bifidobacterium gallicum JCM8224(T).
  • Shinsuke Miki, Kotaro Matsui, Hideki Kito, Keisuke Otsuka, Taketo Ashizawa, Nobuko Yasuda, Satoru Fukiya, Junko Sato, Kazuyuki Hirayae, Yoshikatsu Fujita, Toshihiko Nakajima, Fusao Tomita, Teruo Sone
    Molecular plant pathology 10 3 361 - 74 2009年05月 [査読有り][通常論文]
     
    In order to clone and analyse the avirulence gene AVR-Pia from Japanese field isolates of Magnaporthe oryzae, a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95-1, gained virulence towards the rice cultivar Aichi-asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co-segregated with avirulence towards Aichi-asahi was isolated. Three cosmid clones that included the regions that flanked PM01 were isolated from a genomic DNA library. One of these clones (46F3) complemented the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR-Pia. Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I-VI, and these were cloned individually into a hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR-Pia. By deletion analysis of fragment V, AVR-Pia was localized to an 1199-bp DNA fragment, which included a 255-bp open reading frame with weak homology to a bacterial cytochrome-c-like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co-segregated with the AVR-Pia locus in a genetic map that was constructed using Chinese isolates.
  • Satoru Fukiya, Miki Arata, Hiroko Kawashima, Daisuke Yoshida, Maki Kaneko, Kimiko Minamida, Jun Watanabe, Yoshio Ogura, Kiyohisa Uchida, Kikuji Itoh, Masaru Wada, Susumu Ito, Atsushi Yokota
    FEMS microbiology letters 293 2 263 - 70 2009年04月 [査読有り][通常論文]
     
    Secondary bile acid-producing bacteria were isolated from human feces to improve our appreciation of the functional diversity and redundancy of the intestinal microbiota. In total, 619 bacterial colonies were isolated using a nutrient-poor agar medium and the level of secondary bile acid formation was examined in each by a liquid culture, followed by thin-layer chromatography. Of five strains analyzed by 16S rRNA gene sequencing and biochemical testing, one was identified as Bacteroides intestinalis AM-1, which was not previously recognized as a secondary bile-acid producer. GC-MS revealed that B. intestinalis AM-1 converts cholic acid (CA) and chenodeoxycholic acid into their 7-oxo derivatives, 7-oxo-deoxycholic acid (7-oxo-DCA) and 7-oxo-lithocholic acid, respectively. Thus, B. intestinalis AM-1 possesses 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH) activity. In liquid culture, B. intestinalis AM-1 showed a relatively higher productivity of 7-oxo-DCA than Escherichia coli HB101 and Bacteroides fragilis JCM11019(T), which are known to possess 7alpha-HSDH activity. The level of 7alpha-HSDH activity was higher in B. intestinalis AM-1 than in the other two strains under the conditions tested. The 7alpha-HSDH activity in each of the three strains is not induced by CA; instead, it is regulated in a growth phase-dependent manner.
  • Takeshi Senoura, Hidenori Taguchi, Shigeaki Ito, Shigeki Hamada, Hirokazu Matsui, Satoru Fukiya, Atsushi Yokota, Jun Watanabe, Jun Wasaki, Susumu Ito
    Bioscience, biotechnology, and biochemistry 73 2 400 - 6 2009年02月 [査読有り][通常論文]
     
    Cellobiose 2-epimerase (CE, EC 5.1.3.11) catalyzes the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose. In this study, we found a CE gene in the genome sequence of non-cellulolytic Bacteroides fragilis NCTC 9343. The recombinant enzyme, expressed in Escherichia coli cells, catalyzed a hydroxyl stereoisomerism at the C-2 positions of the reducing terminal glucose and at the mannose moiety of cello-oligosaccharides, lactose, beta-mannobiose (4-O-beta-D-mannopyranosyl-D-mannose), and globotriose [O-alpha-D-galactopyranosyl-(1-->4)-O-beta-D-galactopyranosyl-(1-->4)-D-glucose]. The CE from B. fragilis showed less than 40% identity to reported functional CEs. It exhibited 44-63% identities to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins of unknown function in bacterial genome sequences of the phyla Firmicutes, Bacteroidetes, Proteobacteria, Chloroflexi, and Verrucomicrobia. On the other hand, it showed less than 26% identity to functional N-acyl-D-glucosamine 2-epimerases. Based on the amino acid homology and phylogenetic positions of the functional epimerases, we emphasize that many genes for putative N-acyl-D-glucosamine 2-epimerases and related hypothetical proteins of unknown function reported to date in the bacterial genomes should be annotated as CE-like proteins or putative CEs.
  • Satoru Fukiya, Atsushi Yokota
    Seikagaku. The Journal of Japanese Biochemical Society 80 5 421 - 5 2008年05月 [査読無し][招待有り]
  • Achmad Dinoto, Tatiana M Marques, Kanta Sakamoto, Satoru Fukiya, Jun Watanabe, Susumu Ito, Atsushi Yokota
    Applied and environmental microbiology 72 12 7739 - 47 2006年12月 [査読有り][通常論文]
     
    The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.
  • ディノト アハマド, 吹谷 智, 横田 篤
    日本乳酸菌学会誌 = Journal of Japan Society for Lactic Acid Bacteria 17 2 110 - 117 2006年12月01日 [査読無し][招待有り]
  • Satoru Fukiya, Hiroshi Mizoguchi, Toru Tobe, Hideo Mori
    Journal of bacteriology 186 12 3911 - 21 2004年06月 [査読有り][通常論文]
     
    Escherichia coli, including the closely related genus Shigella, is a highly diverse species in terms of genome structure. Comparative genomic hybridization (CGH) microarray analysis was used to compare the gene content of E. coli K-12 with the gene contents of pathogenic strains. Missing genes in a pathogen were detected on a microarray slide spotted with 4,071 open reading frames (ORFs) of W3110, a commonly used wild-type K-12 strain. For 22 strains subjected to the CGH microarray analyses 1,424 ORFs were found to be absent in at least one strain. The common backbone of the E. coli genome was estimated to contain about 2,800 ORFs. The mosaic distribution of absent regions indicated that the genomes of pathogenic strains were highly diversified because of insertions and deletions. Prophages, cell envelope genes, transporter genes, and regulator genes in the K-12 genome often were not present in pathogens. The gene contents of the strains tested were recognized as a matrix for a neighbor-joining analysis. The phylogenic tree obtained was consistent with the results of previous studies. However, unique relationships between enteroinvasive strains and Shigella, uropathogenic, and some enteropathogenic strains were suggested by the results of this study. The data demonstrated that the CGH microarray technique is useful not only for genomic comparisons but also for phylogenic analysis of E. coli at the strain level.
  • Satoru Fukiya, Hiroshi Mizoguchi, Hideo Mori
    FEMS microbiology letters 234 2 325 - 31 2004年05月15日 [査読有り][通常論文]
     
    We have established an improved large deletion method in Escherichia coli genome using a combination of two different recombination systems, lambda Red and Cre/loxP. The loxP site could be rapidly and efficiently integrated in the genome by lambda Red and large deletions of both 117- and 165-kbp regions could be generated in 100% efficiency by Cre/loxP. Comparative genomic hybridization microarray experiments of deletion strains indicated that deletions were generated only in expected regions of the genome. These results have demonstrated that the method is useful for genome engineering in E. coli.
  • Hideki Kito, Yosuke Takahashi, Junko Sato, Satoru Fukiya, Teruo Sone, Fusao Tomita
    Current genetics 42 6 322 - 31 2003年03月 [査読有り][通常論文]
     
    We investigated a DNA fragment and its flanking region deleted in the spontaneous Pi-a virulent mutant of Magnaporthe grisea Ina168. A new transposon-like sequence was identified from a region adjacent to the deleted fragment and was named Occan. Occan contained a 2,259-bp ORF interrupted by one 63-bp intron and had both a TA dinucleotide and 77 bp of perfect inverted repeats at both termini, without direct repeats. These features indicated that Occan is a member of the Fot1 family. RT-PCR analysis confirmed the expression of the putative transposase and the presence of an intron. Southern analysis of pulse-field gel electrophoresis-separated chromosomes indicated that Occan was dispersed in all chromosomes of the rice pathogen, Ina168. Copy numbers of Occan were also preserved in a host-specific manner amongst M. grisea isolates. In particular, rice pathogens contained a large number of the element inserted into their genome. Phylogenetic analysis with other known members of the Fot1 family revealed that Occan was dissimilar to any other known elements and it is thus proposed that Occan be separated to a new subfamily.
  • Satoru Fukiya, Takao Kuge, Tomomi Tanishima, Teruo Sone, Takashi Kamakura, Isamu Yamaguchi, Fusao Tomita
    Bioscience, biotechnology, and biochemistry 66 3 663 - 6 2002年03月 [査読有り][通常論文]
     
    We identified and cloned a gene designated SPM1, encoding a serine protease from the rice blast fungus Magnaporthe grisea. SPM1 is a single-copy gene, encoding a subtilisin-like serine protease with 536 amino acids. Analyses of the deduced amino acid sequence of SPM1 suggested that SPM1 would be localized in a vacuole, an important organelle in pathogenicity.
  • S Fukiya, M Kodama, H Kito, T Sone, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 7 1464 - 1473 2001年07月 [査読有り][通常論文]
     
    Mating experiments between Magnaporthe grisea Japanese rice pathogens and Guy11, a hermaphroditic fertile rice pathogen, were done aimed at identification of avirulence genes. A cross named cross 2107 with thirty-six random progenies was obtained. Segregation analyses of genetic markers found that the cross was less suitable for genetic analysis. Backcrosses with cross 2107 progenies and Guy11 were done and another cross named cross 5307 with sixty-five progenies was obtained. A locus controlling kasuciamycin resistance named Ksg1R was identified and used for a model case of genetic mapping. Bulked segregant analysis was done to find adjacent RAPD markers for mapping of the gene. Three adjacent markers to Ksg1R were obtained and a genetic map around the Ksg1R was made, but these markers were not located on a single chromosome. These results suggest that genetic analysis to identify a gene locus is available in cross 5307. Infection assay of parental strains of cross 5307 to Japanese differential rice cultivars suggested the possibility of genetic analysis of cultivar specificity toward four rice cultivars: Aichiasahi, Kusabue, Tsuyuake, and K59.
  • T Sone, S Fukiya, M Kodama, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 64 8 1733 - 1736 2000年08月 [査読有り][通常論文]
     
    The 8-kb repeat unit of M, grisea rRNA-encoding DNA (rDNA) was cloned as three subclones, pM50, pM21, and pM86. Nucleotide sequencing of these subclones uncovered the structure of an rDNA repeat unit similar to those of other ascomycetes. The intergenic spacer (IGS) of the rDNA cistron contained a repetitive (R) region, which was rich in two hinds of short tandemly repeated elements.

MISC

書籍等出版物

  • Mattarelli, Paola, Biavati, Bruno, Holzapfel, W. H., Wood, Brian J. B. (担当:分担執筆範囲:15. Genetic Manipulation and Gene Modification Technologies in Bifidobacteria)
    Academic Press, an imprint of Elsevier 2018年 (ISBN: 9780128050606) xii, 312 p.
  • Lactic Acid Bacteria: Current Progress in Advanced Research
    Horizon Scientific Press 2011年
  • 乳酸菌とビフィズス菌のサイエンス
    京都大学学術出版会 2010年

講演・口頭発表等

所属学協会

  • アメリカ微生物学会   腸内細菌学会   日本ゲノム微生物学会   日本農芸化学会   日本乳酸菌学会   日本生物工学会   

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 : 2024年04月 -2028年03月 
    代表者 : 吹谷 智, 高須賀 太一
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2021年04月 -2025年03月 
    代表者 : 清水 英寿, 石塚 敏, 橋口 亜由未, 吹谷 智, 吉清 恵介, 田中 愛健
     
    本研究では、「腸内細菌代謝産物に焦点を当てた高食肉摂取による健康増進と病態発症の分岐点の解明」に向け、腸内で産生されるインドール系化合物の一種、インドール酢酸とその代謝産物であるスカトールを中心に、肝臓および腸管への影響について解析を行っている。 培養肝がん細胞を用いた解析では、スカトールは、その受容体として報告されているAryl hydrocarbon receptor （AhR）の活性化を介するだけでなく、ある核内受容体型転写因子の活性化も介して細胞増殖を導くことが明らかとなった。また我々は、スカトールによって発現増加する長寿遺伝子の1つであるSirt1が、培養肝がん細胞の増殖を導く結果を得た。 培養大腸がん細胞を用いた解析では、スカトールは、Nuclear factor-kappa B（NF-kappaB）の活性化を介してInterleukin-6（IL-6）の発現増加を導いた。またスカトールは、Tumor necrosis factor alpha（TNFalpha）を発現増加させる一方、インドール酢酸に関しては、その発現低下を導くことを我々は報告している。このTNFalphaの発現低下メカニズムとして、TLR4が関与していることが示唆された。さらにインドール酢酸は、AhRの活性化を介して、腸管上皮内腔膜の塩化物-炭酸水素交換体としてNa+/H+交換体とともに腸管での電気陰性NaCl吸収に関与しているsolute-linked carrier 26（SLC26A3）の発現増加を誘導することが明らかとなった。SLC26A3の発現低下は、炎症性腸疾患や大腸癌で観察されることから、インドール酢酸はこれら病態に対する予防および改善に寄与すると考えられる。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 吹谷 智, 後藤 恭宏
     
    1. 細胞表層にペプチドタグを提示したBifidobacterium longum 105-A株の構築 ビフィズス菌の糖質ABCトランスポーターの基質結合タンパク質であるGltAのC末端側（細胞外へと露出する）に，選択的な回収に必要な4種類の候補ペプチドタグ（Strep-TagII, FLAG, HA, Myc）を付加したタグ融合GltAを発現可能なプラスミドを構築した．これらをB. longum 105-A株に導入し，4種類の表層タグ発現株を構築した． 2. 表層タグ発現105-A株の培養菌体を用いた回収方法の検討 ①RNA固定化の処理方法の検討とRNAの品質評価：市販のRNA固定化処理試薬を始めとした4種のRNA固定化溶液をテストした．培養菌体に固定化処理を施したのち，Total RNAを抽出し，バイオアナライザー(Agilent) を用いてRNAの品質（分解の度合）を評価した．その結果，RNAprotect Bacteria Reagent (QIAGEN) またはBacterial isolation buffer (Nobori et al., PNAS, 2018)を用いることで，RNAの品質を高く保つことができることが明らかになった． ②表層タグ発現株におけるタグ発現の検証：培養した表層タグ発現株菌体の全タンパク質に対して，各タグに特異的に結合する分子または抗体を用いて，ウェスタンブロット解析を行った．その結果，4種類のタグ全てがGltAとの融合タンパク質として発現していることが確認された．次に，表層タグ発現株を培養し，免疫蛍光染色を行い，蛍光顕微鏡を用いて細胞表層へのタグの提示を評価した．その結果，Strep-TagII発現株が最も高いタグ検出率を示したことから，4種の中でStrep-TagIIが最も高い割合で細胞表層へ提示されていることが明らかになった．
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 和田 大, 吹谷 智
     
    ビフィズス菌は広くCys要求性であると考えられてきたが，これまでの研究から，Bifidobacterium lomgum subsp. longum 105-A（105-A株）をはじめとする複数の菌株のCys要求性は，Metで代替可能であることを明らかにしている．これらの菌株はMetからCysへ代謝する逆流硫黄経路を有することが推定される．本経路は，(I) Metからホモシステイン(Hcy) へ代謝するS-Adenosyl methionine回路，(II) Hcyからシスタチオニン (Cth)，さらにCysへと代謝する経路により構成されると考えられる．(II) の経路の解明を目的として，CthからCysへと代謝するCystathionine-γ-lyase (CGL), HcyからCthへと代謝するCystathionine-β-synthase (CBS) の機能を有する酵素遺伝子について，105-A株のゲノム配列からの同定を進めてきた．そこで本研究では，それらの機能について，遺伝子欠損株を用いた解析を行った． CGLおよびCBSの候補遺伝子について，二重相同組み換え法を用いて遺伝子欠損株を構築した（ΔBL105A_0509，ΔBL105A_0510）．また，ビフィズス菌最少培地 (BMM)に単一有機硫黄源としてCys / Met / Cthを添加した培地を用いた生育試験を行い，野生株とΔBL105A_0509，ΔBL105A_0510の間で生育を比較した． BMM+Cys培地ではどちらの欠損株も野生株と同様の生育が観察されたが，BMM+Met培地では両変異株ともに生育の低下が観察された．さらに，BMM+Cth培地ではΔBL105A_0509は野生株と同様に生育したが，ΔBL105A_0510は生育の低下が観察された．これらの結果から，BL105A_0509とBL105A_0510はそれぞれCBS, CGLの機能を持つ遺伝子であると考えられた．
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 清水 英寿, 石塚 敏, 吹谷 智, 岡野 邦宏, 吉清 恵介
     
    本年度では、これまでに培養腸管細胞に対して機能が未解明であったスカトールが、細胞死を導く作用メカニズムを明らかにすることに焦点を当てた。スカトールによって活性化されるMAPキナーゼファミリーのERK、p38、JNK、そしてスカトールの受容体であるAhRのうち、p38の活性化を介して、TNFαおよびその受容体であるTNFR1の発現増加が導かれた。そこで、TNFαに対する中和抗体およびTNFR1のアンタゴニストを培養腸管細胞に処理したところ、スカトールによって導かれる細胞死が一部抑制された。さらにスカトールは、p38の活性化を介してIL-6とCOX-2の発現増加も導くことを確認した。以上から、高食肉摂取を起因とする炎症性腸疾患や大腸癌の発症・進展に、腸内細菌代謝産物であるスカトールが関与する可能性が示された。 培養肝細胞に対するスカトールの効果として、NF-κBのタンパク量増加を導くことを新たに確認した。NF-κBは種々のサイトカインの発現誘導に対して中心的な役割を担っている転写因子であることから、スカトールは肝細胞における炎症反応の亢進に寄与することが示唆された。 スカトールは腸内細菌による代謝を介してインドール酢酸から産生されるため、スカトールとインドール酢酸の作用効果の違いについても検討を試みた。健常ラットにインドール酢酸含有食を摂取させたところ、活動期における血糖値上昇が抑えられた。また、当研究グループより開発された胆汁酸誘導性脂肪肝発症モデルラットを用いてインドール酢酸の効果を調べたところ、インドール酢酸含有食摂取ラットにおいて肝臓での脂質蓄積が一部抑制された。以上から、インドール酢酸とその代謝産物であるスカトールは、臓器機能に対して異なる作用を有していることが明らかとなった。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 吹谷 智, 小椋 義俊, 園山 慶
     
    健康に有用な腸内細菌として知られるビフィズス菌が腸内でどのように活動しているのかについて，分子レベルでの機構を明らかにするために，まず通常飼育マウス腸内でヒト由来ビフィズス菌の長期定着が可能な飼育試験系を確立した．この系を用いて，腸内特異的に発現する遺伝子を薬剤耐性の変化により検出するR-IVET法による解析を行い，腸内特異的に発現する遺伝子を同定した．さらに，48,000株からなるビフィズス菌のトランスポゾン変異株集団を構築して無菌マウスに投与し，各変異株の腸内での増減をINSeq法により包括的に評価した結果，腸内での生存と定着に重要と考えられる442個の遺伝子を同定することができた．
  • 腸内細菌叢存在下でのビフィズス菌の腸内活動に重要な遺伝子の同定と機能検証
    文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 吹谷 智
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 清水 英寿, 萩尾 真人, 吹谷 智, 岡野 邦宏, 宮崎 均, 石塚 敏
     
    本研究では、腸内細菌代謝産物であるスカトールが、ラット個体、培養腸管細胞、培養肝ガン細胞、それぞれに与える影響について解析を行った。結果としてスカトールは、ラット個体においては、胆汁酸代謝の撹乱を誘発させ、肝臓や回腸での遺伝子発現、そして腸内細菌叢を変動させる事が明らかとなった。また、腸管細胞を用いた解析では、スカトールがAhRを介して細胞死を導く事が確認された。さらに培養肝ガン細胞では、スカトールはERKの活性化を介して細胞増殖を導く事が示唆された。以上の本研究結果から、腸内におけるスカトールの産生は、消化管機能の異常を誘発させ、さらに消化管疾患の発症・進展へも関与する可能性が示された。
  • 日本学術振興会：科学研究費助成事業 研究活動スタート支援
    研究期間 : 2015年08月 -2017年03月 
    代表者 : 西山 啓太, 岡田 信彦, 向井 孝夫, 山本 裕司, 横田 篤, 吹谷 智, 浦島 匡
     
    ビフィズス菌は，ヒト消化管に生息する共生細菌である。本研究では，Bifidobacterium bifidumのシアリダーゼ（SiaBb2）に着目し，付着因子と栄養獲得因子の両面からその役割について解析した。本研究から，SiaBb2がHMOに含まれるシアル化糖鎖を切断することで効率的な糖の資化を可能にしていること，さらにSiaBb2がB. bifidumのムチンに対する付着性を促進することを見出した。これらは，シアリダーゼが腸管付着性と栄養獲得というビフィズス菌の生存に重要な役割を担う二機能性の因子であることを示しており，SiaBb2を介した新たな定着機構の存在を示すものと考えられる。
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2015年04月 -2017年03月 
    代表者 : 清水 英寿, 杉浦 則夫, 宮崎 均, 石塚 敏, 吹谷 智, 清水 和哉, 岡野 邦宏
     
    湖沼の富栄養化によって発生するアオコは、ミクロシスチンと呼ばれる毒素を産生する。本研究では、培養肝細胞のHepG2細胞で、Microcystin-LR（MC-LR）の処理濃度の違いによるAMPKの活性化の有無で、細胞死に対して異なる作用が存在する事が示された。また、培養腸管細胞のCaco-2細胞では、p38とJNKの活性化を介した細胞増殖や、原がん遺伝子の発現誘導が観察された。ラットを用いた解析では、ミクロシスチン投与による影響は、摂食量、体重、各種臓器重量（肝臓・腎臓・脂肪）で見られなかった。今後、各種臓器の遺伝子発現量、血中パラメーター、肝脂質、胆汁酸、腸内細菌叢の解析なども行っていく。
  • 文部科学省：科学研究費補助金(基盤研究(C))
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 吹谷 智
     
    ビフィズス菌が腸内で生存するための分子機構については，ビフィズス菌の遺伝子変異導入系が十分確立されていないため，不明な部分が多い．本研究では，ビフィズス菌のトランスポゾン変異導入法と次世代シークエンサー解析を組み合わせて，ビフィズス菌の腸内での生存に寄与する遺伝子を同定することを目的とした． 研究の成果として，まず変異導入の宿主となるビフィズス菌株の完全長ゲノム配列を決定した．さらに転移因子ISBlo11およびHimar1C9を用いて，2種類のトランスポゾン変異導入系を確立した．本研究により，ビフィズス菌が腸内で生存する機構を分子レベルで解明するための基盤技術を整備することができた．
  • 文部科学省：科学研究費補助金(若手研究(B))
    研究期間 : 2011年04月 -2013年03月 
    代表者 : 吹谷 智
     
    平成23年度はR-IVET法の確立に向けた予備検討とビフィズス菌染色体への遺伝子導入法の確立を行った．1. 野生株Bifidobacterium longum 105-A株のマウス腸内での生存試験：野生株 B. longum 105-Aがマウス腸内で生存可能であるかを明らかにするため，BALB/cマウスへ105-A株を経口投与し，糞便中の105-A株の生菌数を経時的にモニターした．10の 9乗cfuの105-A株を経口投与したところ，105-A株は投与後6時間目に最も多く糞便中に検出され，その後24時間目まで糞便中に継続して検出された．この結果から，105-A株はマウス腸内で死滅せずに腸内を通過できることが明らかとなった．2. 相同組換えによるビフィズス菌染色体上への遺伝子導入法の確立：R-IVET法の確立に向けて，親株であるB. longum 105-A株の染色体上へloxP-スペクチノマイシン耐性遺伝子 (SpR)-loxP配列を導入する必要がある．そのため，二重相同組換え法による染色体上への遺伝子導入法を検討した．実際に2種類のベクターを用いて，染色体上の遺伝子に欠損変異を導入できたことから，105-A株の染色体上への遺伝子導入が可能であることが示された．3. 新たな薬剤耐性マーカーを持つシャトルベクターの構築と評価：R-IVET法の確立に必要なCreリコンビナーゼ発現プラスミドを構築するためには，前述のSpR以外の薬剤耐性遺伝子が選択マーカーとして必要である．そのため，クロラムフェニコール耐性遺伝子 (CmR) およびエリスロマイシン耐性遺伝子 (EmR) を保持するシャトルベクター2種を新たに構築した．構築したベクターを105-A株に導入し，薬剤耐性を評価したところ，どちらの薬剤耐性遺伝子も105-A株にそれぞれの薬剤に対する耐性を付与できることが明らかになった．
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2009年 -2011年 
    代表者 : 横田 篤, 吹谷 智, 森田 直樹, 横田 伸一
     
    遊離胆汁酸はその細胞膜損傷作用により腸内細菌にとって最も過酷な環境ストレス因子になっている.これまでの研究で,研究代表者らはLactobacillus gasseri JCM1131Tにヒトの主要な胆汁酸であるコール酸に対する適応現象を見出している.これは,対数増殖期の細胞を非致死濃度である4mMのコール酸に30分間暴露した後,致死濃度の15mMのコール酸で処理したところ,7時間後でも生残率がほぼ100%であったのに対し,何も処理しない細胞は生残率が千分の一あるいは一万分の一に急速に低下したというものである.この際の適応細胞では,致死濃度のコール酸による細胞膜の損傷が軽減されていて,適応に伴う細胞膜脂質組成の変化が示唆された.そこで適応細胞と非適応細胞から全脂質を抽出し,それらを中性脂質,糖脂質,リン脂質の3区分に分画した.それぞれの画分の比率は適応前後で変化なく,それぞれの画分の脂肪酸組成についても変化がなかった.しかし,糖脂質とリン脂質の組成に有意な変動があった.糖脂質では糖鎖の鎖長が長くなった.またリン脂質ではカルジオリピンの比率が有意に増大した.コール酸適応におけるカルジオリピンの役割について調べるため,ベシクル実験を行った.その結果,カルジオリピンはコール酸による攻撃に対してベシクルに抵抗性を付与することが分かり,カルジオリピンが適応機構に深く関わっていることが示唆された.本菌のゲノム上には,カルジオリピンの合成に作用すると考えられる2つのカルジオリピン合成酵素遺伝子が存在する.この遺伝子を単独又は両方欠損させた変異株を構築し,それらの株のコール酸適応能を調べた.単独欠損株ではいずれの株でもリン脂質画分におけるカルジオリピンの存在比が低下したが,コール酸適応により野生株並みに増大し,野生株と変わらないコール酸適応を示した.二重変異株ではカルジオリピンが殆ど検出されなかったが,それにも拘わらず,コール酸適応を示した.したがって,カルジオリピンはコール酸適応に必須ではないことが分かった.しかしこの二重変異株では,糖脂質含量の有意な減少とリン脂質含量の有意な増大が見られ,その変化は適応後により顕著になった.このことはカルジオリピンが細胞膜の脂質組成を決定する重要な役割を担っていることを示している.
  • 文部科学省：科学研究費補助金(若手研究(B))
    研究期間 : 2006年 -2007年 
    代表者 : 吹谷 智
     
    平成19年度はビフィズス菌からの挿入配列の単離および単離された挿入配列の詳細な解析を行った.1.新規挿入配列様因子TLS16の解析昨年度同定されたBifidobacterium longum BK28株由来の挿入配列様因子TLS16について塩基配列の解析および各種ビフィズス菌における保存性について解析を行った.TLS16は1.59kbpの挿入配列であり,その内部には転位に関与するタンパク質であるトランスポゼースをコードする単一の遺伝子が存在していた.アミノ酸レベルの相同性解析,および因子末端に存在する特徴的なステム・ループ構造などの特徴から,TLS16は挿入配列IS605ファミリーに属する因子と考えられた.またサザンハイブリダイゼーション解析により,TLS16はB.longum,B. breve,B.adolescentis,B.gallicumの基準株にそのホモログが保存されている事を明らかにした.2.転位活性を持つ挿入配列様因子TLS143の解析咋年度構築した転位因子のスクリーニング系を用いて,B.longum 105-A株を用いて転位活性を持つ因子を探索した.その結果,1.43kbpの長さの因子がスクリーニングベクターに挿入されていたため,本因子をTLS143と命名した.TLS143は末端に逆方向反復配列を持つ典型的なIS因子であり,その相同性からからIS3ファミリーに属する因子であると考えられた.1.と同様に各種ビフィズス菌における保存性を調べたところ,TLS16に比べて多くのビフィズス菌種壕存在する普遍性の高い因子であることが明らかになった.ビフィズス菌の転位因子の研究はほとんど行われていなかったが,本研究により二つの因子が同定された.特に,TLS143は転位活性を持つことが証明されたビフィズス菌由来の初めての因子であり,今後更なる応用が期待される.
  • Identification and functional analysis of the bile acid transforming intestinal bacteria
    Grant-in-Aid for Scientific Research
    研究期間 : 2007年
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2004年 -2006年 
    代表者 : 横田 篤, 和田 大, 湯本 勳, 吹谷 智
     
    糞便サンプルから,一次胆汁酸であるコール酸(CA)とケノデオキシコール酸(CDCA)をそれぞれ大腸ガンのプロモーター活性を持つ二次胆汁酸,デオキシコール酸(DCA)とリトコール酸(LCA),に変換する7α-脱水酸化活性を持つ未知の腸内細菌の分離を目的として研究を行った.嫌気チャンバー内で糞便サンプルから合計619株の腸内細菌を分離し,これらのCAあるいはCDCAからの二次胆汁酸生成活性をTLC,HPLC,GC-MS分析により検出した.その結果,8株の二次胆汁酸生成菌を取得した.16SrRNA遺伝子の塩基配列の決定により,DCAやLCA生成菌株としてClostridum scindensとC.leptumを得たが,これらは既知の変換菌であった。他の6株はCDCAからLCAとは異なる代謝産物として7-オキソリトコール酸(3α-hydroxy-7-oxo-5β-cholanoic acid,7-keto-LCA)を生成していた.これら6株は7-keto-LCA生成菌としては既知のEscherichia coli(3株)やBacteroides fragilis(2株)に加えて,今まで報告のなかったBeacteroides intestinalsを含むことが判明した.本株をB.intestinalis AM-1と命名した.AM-1株はCAからは7-keto-DCAを生成した.基準株JCM13265^Tでも同様の活性が確認された.変換反応を既知の変換株E.coli HB101およびB.fragilis JCM11019^Tと比較したところ,AM-1株は変換反応培養時の生育量は他株に比べて低いが,変換率は90%以上であり,菌体当たりの変換活性が格段に高いことが分かった.本反応を触媒する酵素7α-hydroxysteroid dehydrogenase活性はこれら3株間に大差を認めなかった.
  • Development of mutagenesis system in bifidobacterial genome
    Grant-in-Aid for Scientific Research
    研究期間 : 2006年
  • Mechanisms for adaptation and torelance toward bile acids in lactic acid bacteria
    研究期間 : 2005年

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