Updated on 2022/07/23

写真a

 
GOTOH Kazuyoshi
 
Organization
Faculty of Medicine, Dentistry and Pharmaceutical Sciences Assistant Professor
Position
Assistant Professor
External link

Degree

  • 博士(医学)

Research Areas

  • Life Science / Bacteriology

Professional Memberships

 

Papers

  • Fecal microbiota transplantation as therapy for recurrent Clostridioides difficile infection is associated with amelioration of delirium and accompanied by changes in fecal microbiota and the metabolome. Reviewed

    Gotoh K, Sakaguchi Y, Kato H, Osaki H, Jodai Y, Wakuda M, Také A, Hayashi S, Morita E, Sugie T, Ito Y, Ohmiya N

    Anaerobe   73   102502   2021.12

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  • Detection of in-frame mutation by IS30-family insertion sequence in the phospholipid phosphatidylglycerol synthase gene (pgsA2) of high-level daptomycin-resistant Corynebacterium striatum Reviewed

    Kazuyoshi Gotoh, I. Putu Bayu Mayura, Yusaku Enomoto, Koji Iio, Osamu Matsushita, Fumio Otsuka, Hideharu Hagiya

    European Journal of Clinical Microbiology & Infectious Diseases   2021.10

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    DOI: 10.1007/s10096-021-04369-1

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    Other Link: https://link.springer.com/article/10.1007/s10096-021-04369-1/fulltext.html

  • In vitro effectiveness of biapenem against IMP-producing Enterobacteriaceae Reviewed

    Kazuyoshi Gotoh, Makoto Miyoshi, I Putu Bayu Mayura, Koji Iio, Osamu Matsushita, Fumio Otsuka, Hideharu Hagiya

    Journal of Medical Microbiology   70 ( 10 )   2021.10

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    The options available for treating infections with carbapenemase-producing <italic>
    <named-content content-type="family">
    <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3091" xlink:type="simple">Enterobacteriaceae</ext-link>
    </named-content>
    </italic> (CPE) are limited; with the increasing threat of these infections, new treatments are urgently needed. Biapenem (BIPM) is a carbapenem, and limited data confirming its <italic>in vitro</italic> killing effect against CPE are available. In this study, we examined the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of BIPM for 14 IMP-1-producing <italic>
    <named-content content-type="family">
    <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3091" xlink:type="simple">Enterobacteriaceae</ext-link>
    </named-content>
    </italic> strains isolated from the Okayama region in Japan. The MICs against almost all the isolates were lower than 0.5 µg ml−1, indicating susceptibility to BIPM, while approximately half of the isolates were confirmed to be bacteriostatic to BIPM. However, initial killing to a 99.9 % reduction was observed in seven out of eight strains in a time–kill assay. Despite the small data set, we concluded that the <italic>in vitro</italic> efficacy of BIPM suggests that the drug could be a new therapeutic option against infection with IMP-producing CPE.

    DOI: 10.1099/jmm.0.001430

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  • Elizabethkingia anophelis, an emerging pathogen, inhibits RAW 264.7 macrophage function

    I. Putu Bayu Mayura, Kazuyoshi Gotoh, Hayato Nishimura, Erina Nakai, Takehiko Mima, Yumiko Yamamoto, Kenji Yokota, Osamu Matsushita

    Microbiology and Immunology   65 ( 8 )   317 - 324   2021.8

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    Elizabethkingia anophelis is a pathogen that can cause a life-threatening infection in immunocompromised patients. The first case of E. anophelis infection was reported in 2013; subsequently, an increase in its incidence has been reported globally. Additionally, a mortality rate of more than 30% was observed in the US outbreak of 2015. To date, the pathogenic mechanisms underlying E. anophelis infection, such as toxin production, remain unclear. Since tissue macrophages act as the first line of defense against pathogens, in the present study the interactions between E. anophelis and a macrophage-like cell line RAW 264.7 were examined. Although E. anophelis showed no cytotoxicity toward RAW 264.7 macrophages, the infection inhibited LPS-induced morphological changes and activation of differentiation markers for the polarization of RAW 264.7 macrophages toward an M1-like phenotype. However, when the cell contact was restricted using Transwell inserts or bacterial culture supernatants were used instead of live bacteria, no such inhibition was observed. Moreover, it was shown that E. anophelis evaded phagocytosis. Overall, the results suggest that E. anophelis infection inhibits the differentiation of RAW 264.7 macrophages to a pro-inflammatory phenotype in a contact-dependent manner.

    DOI: 10.1111/1348-0421.12888

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  • Serodiagnosis and bacterial genome of helicobacter pylori infection

    Aina Ichihara, Hinako Ojima, Kazuyoshi Gotoh, Osamu Matsushita, Susumu Take, Hiroyuki Okada, Akari Watanabe, Kenji Yokota

    Toxins   13 ( 7 )   2021.7

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    The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.

    DOI: 10.3390/toxins13070467

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  • Septic pulmonary emboli caused by Tsukamurella inchonensis: A case report

    Kazuyoshi Gotoh, I. Putu Bayu Mayura, Hideharu Hagiya, Kyoichi Obata, Tatsuo Ogawa, Koji Iio, Takumi Fujimori, Fumio Otsuka, Osamu Matsushita

    Journal of Infection and Chemotherapy   27 ( 2 )   369 - 372   2021.2

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    The genus Tsukamurella is a fastidious, environmental organism that potentially causes various infections in humans. Due to the morphological and biochemical similarities to others pathogens, such as Gordona, Rhodococcus, Corynebacterium, Nocardia, and Mycobacterium, a molecular-based approach is indispensable to correctly identify them. Herein, we describe a case of Tsukamurella inchonensis bacteremia complicated with septic pulmonary emboli (SPE), which is the first in the literature. A 44-year-old Japanese woman diagnosed with tongue cancer had undergone partial tongue resection. While receiving chemotherapy and radiotherapy, she developed high fever. Chest computed tomography suggested multiple emboli at the bilateral, peripheral lungs, indicating SPE. Blood culture detected Gram-positive rods, to which matrix-assisted laser desorption/ionization-time of flight mass spectrometry failed to identify. Then, we attempted to characterize it by 16S rRNA sequence, which suggested the organism to be Tsukamurella species but resulted in low resonance of the species-level identification. Additionally, we employed a confidence gene targeting groEL, leading to 100% matching (753/753 bps) with T. inchonensis NCTC 10741 (GenBank accession no. LR131273.1), which has been incorrectly registered as wrong species name Tsukamurella paurometabola in the database. Under the diagnosis of T. inchonensis-associated SPE, we successfully treated the patient with imipenem/cilastatin administration for 4 weeks. Sequencing analysis of groEL was of great use in identifying the organism in this case. More clinical cases based on molecular diagnosis of the fastidious pathogens need to be accumulated to further understand the characteristics and appropriate treatment regimen.

    DOI: 10.1016/j.jiac.2020.09.024

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  • Antibacterial effects of disulfiram in helicobacter pylori

    Tomomi Kobatake, Keiki Ogino, Hiroyuki Sakae, Kazuyoshi Gotoh, Akari Watanabe, Osamu Matsushita, Hiroyuki Okada, Kenji Yokota

    Infection and Drug Resistance   14   1757 - 1764   2021

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    Background: Helicobacter pylori infection poses a risk of the occurrence of gastrointestinal diseases, such as gastric cancer. Its incidence rate is significantly reduced by eradication, and thereby, eradication therapy is generally performed. Disulfiram is an oral prescription drug mainly used for the treatment of alcohol dependence. In recent years, reports have been made on its anticancer and antibacterial effects, and thus, it has recently become an interesting subject. This study aimed to examine the antibacterial activity of disulfiram, investigate the presence or absence of its antibacterial activity on H. pylori, and determine whether it could be a new bactericidal drug against drug-resistant H. pylori. Materials and Methods: Drug-sensitive strains of H. pylori and amoxicillin-resistant, clarithromycin-resistant, and metronidazole-resistant strains were used, and a growth inhibition test of H. pylori using disulfiram was performed. Furthermore, the expression of urease, vacuolating cytotoxin A (VacA), and CagA, the virulence proteins of H. pylori, was quantitatively analyzed using the Western blotting method. In addition, for H. pylori used in this study, the 16SrDNA sequence, a ribosomal gene involved in protein production, was analyzed to examine the presence or absence of gene mutation. Results: Disulfiram suppressed the growth of 7 out of 12 H. pylori strains at 1 µg/mL, and no correlation was observed between their susceptibility/resistance to current eradication antimicrobial drugs and disulfiram resistance. Disulfiram reduced the expression levels of urease, VacA, and CagA proteins. H. pylori, which showed resistance to disulfiram, tended to have fewer gene deletions/insertions in the 16S rDNA sequence; however, no specific mutation was detected. Conclusion: Disulfiram has a bactericidal effect on H. pylori at low concentrations, suggesting that it can be used as a supplement for current H. pylori eradication drugs.

    DOI: 10.2147/IDR.S299177

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  • Enhancement of intestinal epithelial barrier function by Weissella confusa F213 and Lactobacillus rhamnosus FBB81 probiotic candidates in an in vitro model of hydrogen peroxide-induced inflammatory bowel disease

    Ni Nengah Dwi Fatmawati, Kazuyoshi Gotoh, I. Putu Bayu Mayura, Komang Ayu Nocianitri, Gede Ngurah Rsi Suwardana, Ni Luh Gede Yoni Komalasari, Yan Ramona, Masakiyo Sakaguchi, Osamu Matsushita, I. Nengah Sujaya

    BMC Research Notes   13 ( 1 )   2020.12

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    Objective: Weissella confusa F213 (WCF213) and Lactobacillus rhamnosus FBB81 (LrFBB81) are two probiotic candidates isolated from humans in our previous study. Their functional activity on the mucosal barrier has not yet been adequately investigated. Therefore, the objective of this study was to investigate the effect of these strains on maintaining mucosal integrity in vitro. Caco-2 cell monolayers were pretreated with WCF213 and LrFBB81 before being exposed to hydrogen peroxide. The integrity of mucosal cells was evaluated by measuring the transepithelial resistance (TER), flux of FITC-labelled dextran, and ZO-1 protein distribution with the help of an immunofluorescence method. Results: WCF213 was found to significantly maintain the TER better than the control hydrogen peroxide-treated cells (p < 0.001), followed by the strain combination, and LrFBB81 alone (p < 0.05). The permeability of mucosa was also successfully maintained by the WCF213 strain. This was illustrated by the significant reduction in the flux of FITC-labelled dextran (p < 0.05), which was larger than that exhibited by the other groups. The ZO-1 distribution of strain-treated cells showed less disruption than hydrogen peroxide-treated cells, consistent with the TER and FITC experimental results. These findings indicate that WCF213 and LrFBB81 plays important roles in the maintenance of mucosal integrity in a strain-dependent manner.

    DOI: 10.1186/s13104-020-05338-1

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  • Analysis of a plasmid encoding botulinum neurotoxin type G gene in Clostridium argentinense

    Yoshihiko Sakaguchi, Jumpei Uchiyama, Akira Také, Kazuyoshi Gotoh, Masakiyo Sakaguchi, Tomonori Suzuki, Yumiko Yamamoto, Koji Hosomi, Tomoko Kohda, Masafumi Mukamoto, Shunji Kozaki, Shunji Hayashi, Keiji Oguma

    Anaerobe   66   2020.12

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    Clostridium argentinense produces botulinum neurotoxin type G (BoNT/G). We sequenced and analyzed the plasmid harboring the bont/G gene, designated pCAG, in C. argentinense strain 2740. The pCAG consisted of 140,070 bp containing the bont/G gene cluster. Although this gene cluster showed high similarities in its DNA sequence and ORF arrangement to those of other bont gene clusters, the other regions of the plasmid did not. A phylogenetic study suggested that pCAG had a unique evolutionary history compared with other clostridial bont-harboring plasmids. This suggests that pCAG is possibly a novel type of plasmid expressing the bont/G gene in C. argentinense.

    DOI: 10.1016/j.anaerobe.2020.102281

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  • Caco-2 cells monolayer as an in-vitro model for probiotic strain translocation

    Ni Nengah Dwi Fatmawati, Kazuyoshi Goto, I. Putu Bayu Mayura, Komang Ayu Nocianitri, Yan Ramona, Masakiyo Sakaguchi, Osamu Matsushita, I. Nengah Sujaya

    Bali Medical Journal   9 ( 1 )   137 - 142   2020

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    Background: Caco-2 cells monolayer is one of in vitro models to evaluate the translocation capacity of Lactobacillus spp probiotic strains. The translocation is influenced by mucosa permeability of enterocytes as shown by increasing transepithelial resistance (TER) and formation of tight junction proteins. The pore size of the supported permeable membrane used in in vitro assay was one of the crucial factors in performing bacterial translocation assay. Almost no study has been conducted using Caco-2 cells monolayer grown on 8-μm pore size polycarbonate membrane for evaluating probiotics translocation. Therefore this study aimed to determine whether the Caco-2 cells monolayer model was suitable as an in vitro translocation model. Methods: Caco-2 cells monolayer was seeded onto 8-μm collagen-coated polycarbonate membrane insert Transwell®. Differentiation of Caco-2 cells was detected by measuring the TER, while the ZO-1 protein (the tight junction proteins) was detected by immunofluorescence. H2 O2 was used as a tight junction disruptive agent. Data were analyzed using SPSS version 23 software to compare the mean of TER measurement between untreated and H2 O2-treated Caco-2 cells monolayer. Results: The result showed that the TER of Caco-2 cells monolayer was gradually increasing until day 14, reaching more than 800 ohm.cm2. Furthermore, the ZO-1 protein was successfully detected, indicated the tight junction formation. TER value of H2 O2-treated cells showed significantly lower than that of untreated cells (P<0.05), indicating a disturbance of cells monolayer integrity. Lactobacillus rhamnosus FBB81 was used for validating the translocation. There was no translocation observed; however, translocation was observed in H2 O2-treated cells. Conclusion: Altogether suggests that Caco-2 cells grown on 8 μm-pore size permeable filters could be considered as a suitable in vitro model for probiotics strains translocation.

    DOI: 10.15562/bmj.v9i1.1633

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  • Expression of Collagenase is Regulated by the VarS/VarA Two-Component Regulatory System in Vibrio alginolyticus

    Takehiko Mima, Kazuyoshi Gotoh, Yumiko Yamamoto, Keiko Maeda, Taku Shirakawa, Shunsuke Matsui, Yumi Murata, Takaki Koide, Hiroshi Tokumitsu, Osamu Matsushita

    Journal of Membrane Biology   251 ( 1 )   51 - 63   2018.2

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    Vibrio alginolyticus is an opportunistic pathogen in both humans and marine animals. Collagenase encoded by colA is considered to be one of the virulence factors. Expression of colA is regulated by multiple environmental factors, e.g., temperature, growth phase, and substrate. To elucidate the mechanism of regulation of colA expression, transposon mutagenesis was performed. VarS, a sensor histidine kinase of the two-component regulatory system, was demonstrated to regulate the expression of colA. VarA, a cognate response regulator of VarS, was also identified and shown to be involved in the regulation of colA expression. In vitro phosphorylation assays showed that phosphorylated VarS acted as a phosphoryl group donor to VarA. A site-directed mutagenesis study showed that the His300, Asp718 and His874 residues in VarS were essential for the phosphorylation of VarS, and the Asp54 residue in VarA was likely to receive the phosphoryl group from VarS. The results demonstrate that the VarS/VarA two-component regulatory system regulates the expression of collagenase in V. alginolyticus.

    DOI: 10.1007/s00232-017-9991-9

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  • Vibrio alginolyticus VepA induces lysosomal membrane permeability and cathepsin-independent cell death

    Agus Eka Darwinata, Kazuyoshi Gotoh, Takehiko Mima, Yumiko Yamamoto, Kenji Yokota, Osamu Matsushita

    Acta Medica Okayama   72 ( 3 )   231 - 239   2018

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    The bacterium Vibrio alginolyticus, an opportunistic pathogen in humans, has a type III secretion system (T3SS) that is responsible for its cytotoxicity toward eukaryotic cells. The effector of T3SS that is responsible for the cytotoxicity had not been identified. Here we demonstrate that VepA, a homolog of the T3SS effector in V. parahaemolyticus, is required for cytotoxicity in V. alginolyticus. VepA induces lysosomal membrane permeabilization, and it allows the leakage of only small molecules into the cytosol. Our findings revealed that VepA induces cathepsin-independent cell death in mammalian cells. The ferrous ion, one of the small molecules in the lysosome contents, appears to be involved in the cell death caused by V. alginolyticus VepA.

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  • DEC205 mediates local and systemic immune responses to Helicobacter pylori infection in humans

    Masahide Kita, Kenji Yokota, Chihiro Kageyama, Susumu Take, Kazuyoshi Goto, Yoshiro Kawahara, Osamu Matsushita, Hiroyuki Okada

    Oncotarget   9 ( 22 )   15828 - 15835   2018

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    Helicobacter pylori infections cause gastritis and affect systemic immune responses; however, no direct association between immune cells and stomach bacteria has yet been reported. The present study investigated DEC205-mediated phagocytosis of H. pylori and the role of DEC205-positive macrophages in the human gastric mucosa. DEC205 mediated phagocytosis of H. pylori was detected immunocytochemically in PMAstimulated macrophages differentiated from NOMO1 cells. Expression of DEC205 mRNA in peripheral blood mononuclear cells (PBMCs) from H. pylori-infected patients was analyzed following stimulation with H. pylori cell lysate. We found that anti-DEC205 antibodies inhibited phagocytosis of H. pylori. The number of cells double-positive for DEC205 and CD14 in human gastric mucosa was higher in H. pylori-infected patients. DEC205-positive macrophages invaded the extracellular space between epithelial cells within gastric pits. In addition, DEC205 mRNA expression was upregulated in human PBMCs stimulated with H. pylori lysate. These findings suggest DEC205-expressing macrophages are important for recognition of H. pylori in human gastric mucosa, which affects systemic immunity.

    DOI: 10.18632/oncotarget.24574

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  • Fungal ITS1 deep-sequencing strategies to reconstruct the composition of a 26-species community and evaluation of the gut mycobiota of healthy Japanese individuals

    Daisuke Motooka, Kosuke Fujimoto, Reiko Tanaka, Takashi Yaguchi, Kazuyoshi Gotoh, Yuichi Maeda, Yoki Furuta, Takashi Kurakawa, Naohisa Goto, Teruo Yasunaga, Masashi Narazaki, Atsushi Kumanogoh, Toshihiro Horii, Tetsuya Iida, Kiyoshi Takeda, Shota Nakamura

    Frontiers in Microbiology   8 ( FEB )   2017.2

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    The study of mycobiota remains relatively unexplored due to the lack of sufficient available reference strains and databases compared to those of bacterial microbiome studies. Deep sequencing of Internal Transcribed Spacer (ITS) regions is the de facto standard for fungal diversity analysis. However, results are often biased because of the wide variety of sequence lengths in the ITS regions and the complexity of high-throughput sequencing (HTS) technologies. In this study, a curated ITS database, ntF-ITS1, was constructed. This database can be utilized for the taxonomic assignment of fungal community members. We evaluated the efficacy of strategies for mycobiome analysis by using this database and characterizing a mock fungal community consisting of 26 species representing 15 genera using ITS1 sequencing with three HTS platforms: Illumina MiSeq (MiSeq), Ion Torrent Personal Genome Machine (IonPGM), and Pacific Biosciences (PacBio). Our evaluation demonstrated that PacBio's circular consensus sequencing with greater than 8 full-passes most accurately reconstructed the composition of the mock community. Using this strategy for deep-sequencing analysis of the gut mycobiota in healthy Japanese individuals revealed two major mycobiota types: a single-species type composed of Candida albicans or Saccharomyces cerevisiae and a multi-species type. In this study, we proposed the best possible processing strategies for the three sequencing platforms, of which, the PacBio platform allowed for the most accurate estimation of the fungal community. The database and methodology described here provide critical tools for the emerging field of mycobiome studies.

    DOI: 10.3389/fmicb.2017.00238

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  • Dysbiosis Contributes to Arthritis Development via Activation of Autoreactive T Cells in the Intestine

    Yuichi Maeda, Takashi Kurakawa, Eiji Umemoto, Daisuke Motooka, Yoshinaga Ito, Kazuyoshi Gotoh, Keiji Hirota, Masato Matsushita, Yoki Furuta, Masashi Narazaki, Noriko Sakaguchi, Hisako Kayama, Shota Nakamura, Tetsuya Iida, Yukihiko Saeki, Atsushi Kumanogoh, Shimon Sakaguchi, Kiyoshi Takeda

    Arthritis and Rheumatology   68 ( 11 )   2646 - 2661   2016.11

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    Objective: The intestinal microbiota is involved in the pathogenesis of arthritis. Altered microbiota composition has been demonstrated in patients with rheumatoid arthritis (RA). However, it remains unclear how dysbiosis contributes to the development of arthritis. The aim of this study was to investigate whether altered composition of human intestinal microbiota in RA patients contributes to the development of arthritis. Methods: We analyzed the fecal microbiota of patients with early RA and healthy controls, using 16S ribosomal RNA−based deep sequencing. We inoculated fecal samples from RA patients and healthy controls into germ-free arthritis-prone SKG mice and evaluated the immune responses. We also analyzed whether the lymphocytes of SKG mice harboring microbiota from RA patients react with the arthritis-related autoantigen 60S ribosomal protein L23a (RPL23A). Results: A subpopulation of patients with early RA harbored intestinal microbiota dominated by Prevotella copri; SKG mice harboring microbiota from RA patients had an increased number of intestinal Th17 cells and developed severe arthritis when treated with zymosan. Lymphocytes in regional lymph nodes and the colon, but not the spleen, of these mice showed enhanced interleukin-17 (IL-17) responses to RPL23A. Naive SKG mouse T cells cocultured with P copri−stimulated dendritic cells produced IL-17 in response to RPL23A and rapidly induced arthritis. Conclusion: We demonstrated that dysbiosis increases sensitivity to arthritis via activation of autoreactive T cells in the intestine. Autoreactive SKG mouse T cells are activated by dysbiotic microbiota in the intestine, causing joint inflammation. Dysbiosis is an environmental factor that triggers arthritis development in genetically susceptible mice.

    DOI: 10.1002/art.39783

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  • Temporal dynamics of bacterial microbiota in the human oral cavity determined using an in situ model of dental biofilms

    Nanako Wake, Yoko Asahi, Yuichiro Noiri, Mikako Hayashi, Daisuke Motooka, Shota Nakamura, Kazuyoshi Gotoh, Jiro Miura, Hiroyuki Machi, Tetsuya Iida, Shigeyuki Ebisu

    npj Biofilms and Microbiomes   2   2016.8

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    Numerous studies on oral biofilms have been performed in vitro, although it is difficult to mimic the oral environment. Here we used an in situ model to conduct a quantitative analysis and comprehensive identification of bacterial communities over time by performing deep sequencing of 16S rRNA genes. We show here that the number of viable bacteria in supragingival biofilms increased in two steps. Using scanning and transmission electron microscopy, as well as confocal laser scanning microscopy, we detected gram-positive cocci during the first 8 h. The biofilm was subsequently covered with a thick matrix-like structure composed of different bacterial morphotypes that diversified as the number of bacteria increased. Streptococcus accounted for 420% of the population until 16 h, and obligate anaerobes such as Fusobacterium, Prevotella and Porphyromonas predominated after 48 h, and this increase was statistically significant after 96 h (Po0.05). Together, our data demonstrate that an initial population of facultative anaerobic bacteria was replaced with a population of gram-negative anaerobic bacteria during oral biofilm formation. This study, therefore, contributes to a comprehensive understanding of the composition of the bacterial microbiota involved in the health of the human oral cavity.

    DOI: 10.1038/npjbiofilms.2016.18

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  • Metagenomic Analysis Reveals Dynamic Changes of Whole Gut Microbiota in the Acute Phase of Intensive Care Unit Patients

    Masahiro Ojima, Daisuke Motooka, Kentaro Shimizu, Kazuyoshi Gotoh, Ayumi Shintani, Kazuhisa Yoshiya, Shota Nakamura, Hiroshi Ogura, Tetsuya Iida, Takeshi Shimazu

    Digestive Diseases and Sciences   61 ( 6 )   1628 - 1634   2016.6

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    Background: Metagenomic analysis targeting the 16S rRNA gene has made it possible to characterize the vast array of microorganisms contained in the gut. Aim: The purpose of this study was to evaluate how gut microbiota change in intensive care unit (ICU) patients in the acute phase after admission. Methods: This prospective observational cohort study investigated 12 patients admitted to a single ICU of a large urban tertiary referral hospital. All patients were mechanically ventilated on admission. Fecal samples were collected from patients on days 1–2, 2–4, 5–8, and 7–10 after admission. DNA was extracted from fecal samples, and 16S rRNA deep sequencing was performed to monitor gut changes. Results: Bacteria belonging to the phyla Firmicutes or Bacteroidetes were predominant in each sample. We observed serial dynamic changes in the percentages of Bacteroidetes and Firmicutes that were significantly altered during study period (p < 0.05). A ratio of Bacteroidetes to Firmicutes (B/F ratio) of >10 was seen in four of the six patients who died, whereas a B/F ratio of <0.10 was seen in only one of the six deaths. None of the survivors had a B/F ratio of >10 or <0.10. There was a statistical difference in the B/F ratio between the dead patients and survivors (p = 0.022). Conclusions: Dynamic changes in gut microbiota at the phylum level of ICU patients during the acute phase were identified by high-throughput DNA sequencing. An extreme imbalance in gut microbiota may be associated with prognosis in critically ill patients.

    DOI: 10.1007/s10620-015-4011-3

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  • Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia

    Ryu Okumura, Takashi Kurakawa, Takashi Nakano, Hisako Kayama, Makoto Kinoshita, Daisuke Motooka, Kazuyoshi Gotoh, Taishi Kimura, Naganori Kamiyama, Takashi Kusu, Yoshiyasu Ueda, Hong Wu, Hideki Iijima, Soumik Barman, Hideki Osawa, Hiroshi Matsuno, Junichi Nishimura, Yusuke Ohba, Shota Nakamura, Tetsuya Iida, Masahiro Yamamoto, Eiji Umemoto, Koichi Sano, Kiyoshi Takeda

    Nature   532 ( 7597 )   117 - 121   2016.4

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    Colonic epithelial cells are covered by thick inner and outer mucus layers. The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis. In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified7-11. Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8-/- mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8-/- mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.

    DOI: 10.1038/nature17406

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  • Functional specialization in regulation and quality control in thermal adaptive evolution

    Kazuma Yama, Yuki Matsumoto, Yoshie Murakami, Shigeto Seno, Hideo Matsuda, Kazuyoshi Gotoh, Daisuke Motooka, Shota Nakamura, Bei Wen Ying, Tetsuya Yomo

    Genes to Cells   20 ( 11 )   943 - 955   2015.11

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    Distinctive survival strategies, specialized in regulation and in quality control, were observed in thermal adaptive evolution with a laboratory Escherichia coli strain. The two specialists carried a single mutation either within rpoH or upstream of groESL, which led to the activated global regulation by sigma factor 32 or an increased amount of GroEL/ES chaperonins, respectively. Although both specialists succeeded in thermal adaptation, the common winner of the evolution was the specialist in quality control, that is, the strategy of chaperonin-mediated protein folding. To understand this evolutionary consequence, multilevel analyses of cellular status, for example, transcriptome, protein and growth fitness, were carried out. The specialist in quality control showed less change in transcriptional reorganization responding to temperature increase, which was consistent with the finding of that the two specialists showed the biased expression of molecular chaperones. Such repressed changes in gene expression seemed to be advantageous for long-term sustainability because a specific increase in chaperonins not only facilitated the folding of essential gene products but also saved cost in gene expression compared with the overall transcriptional increase induced by rpoH regulation. Functional specialization offered two strategies for successful thermal adaptation, whereas the evolutionary advantageous was more at the points of cost-saving in gene expression and the essentiality in protein folding.

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  • Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

    Tetsuya Hirata, Morihisa Fujita, Shota Nakamura, Kazuyoshi Gotoh, Daisuke Motooka, Yoshiko Murakami, Yusuke Maeda, Taroh Kinoshita

    Molecular Biology of the Cell   26 ( 17 )   3071 - 3084   2015.9

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    The importance of endosome-to-trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51-VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP-and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport.

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  • Baseline assessment of mesophotic reefs of the Vitória-Trindade Seamount Chain based on water quality, microbial diversity, benthic cover and fish biomass data

    Pedro M. Meirelles, Gilberto M. Amado-Filho, Guilherme H. Pereira-Filho, Hudson T. Pinheiro, Rodrigo L. De Moura, Jean Christophe Joyeux, Eric F. Mazzei, Alex C. Bastos, Robert A. Edwards, Elizabeth Dinsdale, Rodolfo Paranhos, Eidy O. Santos, Tetsuya Iida, Kazuyoshi Gotoh, Shota Nakamura, Tomoo Sawabe, Carlos E. Rezende, Luiz M.R. Gadelha, Ronaldo B. Francini-Filho, Cristiane Thompson, Fabiano L. Thompson

    PLoS ONE   10 ( 6 )   2015.6

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    Seamounts are considered important sources of biodiversity and minerals. However, their biodiversity and health status are not well understood; therefore, potential conservation problems are unknown. The mesophotic reefs of the Vitória-Trindade Seamount Chain (VTC) were investigated via benthic community and fish surveys, metagenomic and water chemistry analyses, and water microbial abundance estimations. The VTC is a mosaic of reef systems and includes fleshy algae dominated rhodolith beds, crustose coralline algae (CCA) reefs, and turf algae dominated rocky reefs of varying health levels. Macro-carnivores and larger fish presented higher biomass at the CCA reefs (4.4 kg per frame) than in the rhodolith beds and rocky reefs (0.0 to 0.1 kg per frame). A larger number of metagenomic sequences identified as primary producers (e.g., Chlorophyta and Streptophyta) were found at the CCA reefs. However, the rocky reefs contained more diseased corals (>90%) than the CCA reefs (∼40%) and rhodolith beds (∼10%). Metagenomic analyses indicated a heterotrophic and fast-growing microbiome in rocky reef corals that may possibly lead to unhealthy conditions possibly enhanced by environmental features (e.g. light stress and high loads of labile dissolved organic carbon). VTC mounts represent important hotspots of biodiversity that deserve further conservation actions.

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  • Interaction between the Type III Effector VopO and GEF-H1 Activates the RhoA-ROCK Pathway

    Hirotaka Hiyoshi, Ryu Okada, Shigeaki Matsuda, Kazuyoshi Gotoh, Yukihiro Akeda, Tetsuya Iida, Toshio Kodama

    PLoS Pathogens   11 ( 3 )   1 - 19   2015.3

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    Vibrio parahaemolyticus is an important pathogen that causes food-borne gastroenteritis in humans. The type III secretion system encoded on chromosome 2 (T3SS2) plays a critical role in the enterotoxic activity of V. parahaemolyticus. Previous studies have demonstrated that T3SS2 induces actin stress fibers in various epithelial cell lines during infection. This stress fiber formation is strongly related to pathogenicity, but the mechanisms that underlie T3SS2-dependent actin stress fiber formation and the main effector have not been elucidated. In this study, we identified VopO as a critical T3SS2 effector protein that activates the RhoA-ROCK pathway, which is an essential pathway for the induction of the T3SS2-dependent stress fiber formation. We also determined that GEF-H1, a RhoA guanine nucleotide exchange factor (GEF), directly binds VopO and is necessary for T3SS2-dependent stress fiber formation. The GEF-H1-binding activity of VopO via an alpha helix region correlated well with its stress fiber-inducing capacity. Furthermore, we showed that VopO is involved in the T3SS2-dependent disruption of the epithelial barrier. Thus, VopO hijacks the RhoA-ROCK pathway in a different manner compared with previously reported bacterial toxins and effectors that modulate the Rho GTPase signaling pathway.

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  • Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan

    Koji Hosomi, Yoshihiko Sakaguchi, Tomoko Kohda, Kazuyoshi Gotoh, Daisuke Motooka, Shota Nakamura, Kaoru Umeda, Tetsuya Iida, Shunji Kozaki, Masafumi Mukamoto

    Molecular Genetics and Genomics   289 ( 6 )   1267 - 1274   2014.11

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    Botulinum neurotoxins (BoNTs) are highly potent toxins that are produced by Clostridiumbotulinum. We determined the complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in C.botulinum type B strain 111 in order to obtain an insight into the toxigenicity and evolution of the bont gene in C.botulinum. Group I C.botulinum type B strain 111 was isolated from the first case of infant botulism in Japan in 1995. In previous studies, botulinum neurotoxin subtype B2 (BoNT/B2) produced by strain 111 exhibited different antigenic properties from those of authentic BoNT/B1 produced by strain Okra. We have recently shown that the isolates of strain 111 that lost toxigenicity were cured of the plasmid containing the bont/B2 gene. In the present study, the plasmid (named pCB111) was circular 265,575 bp double-stranded DNA and contained 332 predicted open reading frames (ORFs). 85 gene products of these ORFs could be functionally assigned on the basis of sequence homology to known proteins. The bont/B2 complex genes were located on pCB111 and some gene products may be involved in the conjugative plasmid transfer and horizontal transfer of bont genes. pCB111 was similar to previously identified plasmids containing bont/B1, /B5, or/A3 complex genes in other group I C.botulinum strains. It was suggested that these plasmids had been derived from a common ancestor and had played important roles for the bont gene transfer between C.botulinum.

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  • Endosomes-to-TGN retrograde transport mediated by GARP is required for post-Golgi anterograde transport and glycosylation Reviewed

    Tetsuya Hirata, Morihisa Fujita, Shota Nakamura, Kazuyoshi Gotoh, Daisuke Motooka, Yoshiko Murakami, Yusuke Maeda, Taroh Kinoshita

    GLYCOBIOLOGY   24 ( 11 )   1188 - 1189   2014.11

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  • Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes

    Mari Miyamoto, Daisuke Motooka, Kazuyoshi Gotoh, Takamasa Imai, Kazutoshi Yoshitake, Naohisa Goto, Tetsuya Iida, Teruo Yasunaga, Toshihiro Horii, Kazuharu Arakawa, Masahiro Kasahara, Shota Nakamura

    BMC Genomics   15 ( 1 )   2014.8

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    Background: The availability of diverse second- and third-generation sequencing technologies enables the rapid determination of the sequences of bacterial genomes. However, identifying the sequencing technology most suitable for producing a finished genome with multiple chromosomes remains a challenge. We evaluated the abilities of the following three second-generation sequencers: Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq) and a third-generation sequencer, the Pacific Biosciences RS sequencer (PacBio), by sequencing and assembling the genome of Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes.Results: We sequenced the genome of V. parahaemolyticus with GS Jr, Ion PGM, MiSeq, and PacBio and performed de novo assembly with several genome assemblers. Although GS Jr generated the longest mean read length of 418 bp among the second-generation sequencers, the maximum contig length of the best assembly from GS Jr was 165 kbp, and the number of contigs was 309. Single runs of Ion PGM and MiSeq produced data of considerably greater sequencing coverage, 279× and 1,927×, respectively. The optimized result for Ion PGM contained 61 contigs assembled from reads of 77× coverage, and the longest contig was 895 kbp in size. Those for MiSeq were 34 contigs, 58× coverage, and 733 kbp, respectively. These results suggest that higher coverage depth is unnecessary for a better assembly result. We observed that multiple rRNA coding regions were fragmented in the assemblies from the second-generation sequencers, whereas PacBio generated two exceptionally long contigs of 3,288,561 and 1,875,537 bps, each of which was from a single chromosome, with 73× coverage and mean read length 3,119 bp, allowing us to determine the absolute positions of all rRNA operons.Conclusions: PacBio outperformed the other sequencers in terms of the length of contigs and reconstructed the greatest portion of the genome, achieving a genome assembly of " finished grade" because of its long reads. It showed the potential to assemble more complex genomes with multiple chromosomes containing more repetitive sequences.

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  • BEC, a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks

    Shinya Yonogi, Shigeaki Matsuda, Takao Kawai, Tomoko Yoda, Tetsuya Harada, Yuko Kumeda, Kazuyoshi Gotoh, Hirotaka Hiyoshi, Shota Nakamura, Toshio Kodama, Tetsuya Iida

    INFECTION AND IMMUNITY   82 ( 6 )   2390 - 2399   2014.6

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    Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.

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  • Oral pathobiont induces systemic inflammation and metabolic changes associated with alteration of gut microbiota

    Kei Arimatsu, Hitomi Yamada, Haruna Miyazawa, Takayoshi Minagawa, Mayuka Nakajima, Mark I. Ryder, Kazuyoshi Gotoh, Daisuke Motooka, Shota Nakamura, Tetsuya Iida, Kazuhisa Yamazaki

    Scientific Reports   4   2014.5

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    Periodontitis has been implicated as a risk factor for metabolic disorders such as type 2 diabetes, atherosclerotic vascular diseases, and non-alcoholic fatty liver disease. Although bacteremias from dental plaque and/or elevated circulating inflammatory cytokines emanating from the inflamed gingiva are suspected mechanisms linking periodontitis and these diseases, direct evidence is lacking. We hypothesize that disturbances of the gut microbiota by swallowed bacteria induce a metabolic endotoxemia leading metabolic disorders. To investigate this hypothesis, changes in the gut microbiota, insulin and glucose intolerance, and levels of tissue inflammation were analysed in mice after oral administration of Porphyromonas gingivalis, a representative periodontopathogens. Pyrosequencing revealed that the population belonging to Bacteroidales was significantly elevated in P. gingivalis-administered mice which coincided with increases in insulin resistance and systemic inflammation. In P. gingivalis-administered mice blood endotoxin levels tended to be higher, whereas gene expression of tight junction proteins in the ileum was significantly decreased. These results provide a new paradigm for the interrelationship between periodontitis and systemic diseases.

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  • Peyerʼs Patches Play a Protective Role in Nonsteroidal Anti-inflammatory Drug-induced Enteropathy in Mice

    Satoshi Hiyama, Hideki Iijima, Shinichiro Shinzaki, Takahiro Inoue, Eri Shiraishi, Shoichiro Kawai, Manabu Araki, Motohiko Kato, Yoshito Hayashi, Tsutomu Nishida, Hironobu Fujii, Akira Mukai, Naoko Shibata, Shintaro Sato, Hiroshi Kiyono, Kazuyoshi Gotoh, Daisuke Motooka, Shota Nakamura, Tetsuya Iida, Masahiko Tsujii, Tetsuo Takehara

    Inflammatory Bowel Diseases   20 ( 5 )   790 - 799   2014.5

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  • Comprehensive metagenomic approach for detecting causative microorganisms in culture-negative infective endocarditis

    Atsuko Imai, Kazuyoshi Gotoh, Yoshihiro Asano, Noriaki Yamada, Daisuke Motooka, Masaki Fukushima, Machiko Kanzaki, Tomohito Ohtani, Yasushi Sakata, Hiroyuki Nishi, Koichi Toda, Yoshiki Sawa, Issei Komuro, Toshihiro Horii, Tetsuya Iida, Shota Nakamura, Seiji Takashima

    International Journal of Cardiology   172 ( 2 )   2014.3

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  • Allergic bronchopulmonary mycosis due to co-infection with Aspergillus fumigatus and Schizophyllum commune Reviewed

    Masafumi Seki, Hideaki Ohno, Kazuyoshi Gotoh, Daisuke Motooka, Shota Nakamura, Tetsuya Iida, Yoshitsugu Miyazaki, Kazunori Tomono

    ID Cases   1 ( 1 )   5 - 8   2014.2

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  • Physiologic and metagenomic attributes of the rhodoliths forming the largest CaCO<inf>3</inf> bed in the South Atlantic Ocean

    Giselle S. Cavalcanti, Gustavo B. Gregoracci, Eidy O. Dos Santos, Cynthia B. Silveira, Pedro M. Meirelles, Leila Longo, Kazuyoshi Gotoh, Shota Nakamura, Tetsuya Iida, Tomoo Sawabe, Carlos E. Rezende, Ronaldo B. Francini-Filho, Rodrigo L. Moura, Gilberto M. Amado-Filho, Fabiano L. Thompson

    ISME Journal   8 ( 1 )   52 - 62   2014.1

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    Rhodoliths are free-living coralline algae (Rhodophyta, Corallinales) that are ecologically important for the functioning of marine environments. They form extensive beds distributed worldwide, providing a habitat and nursery for benthic organisms and space for fisheries, and are an important source of calcium carbonate. The Abrolhos Bank, off eastern Brazil, harbors the world's largest continuous rhodolith bed (of ∼21 000 km 2) and has one of the largest marine CaCO 3 deposits (producing 25 megatons of CaCO 3 per year). Nevertheless, there is a lack of information about the microbial diversity, photosynthetic potential and ecological interactions within the rhodolith holobiont. Herein, we performed an ecophysiologic and metagenomic analysis of the Abrolhos rhodoliths to understand their microbial composition and functional components. Rhodoliths contained a specific microbiome that displayed a significant enrichment in aerobic ammonia-oxidizing betaproteobacteria and dissimilative sulfate-reducing deltaproteobacteria. We also observed a significant contribution of bacterial guilds (that is, photolithoautotrophs, anaerobic heterotrophs, sulfide oxidizers, anoxygenic phototrophs and methanogens) in the rhodolith metagenome, suggested to have important roles in biomineralization. The increased hits in aromatic compounds, fatty acid and secondary metabolism subsystems hint at an important chemically mediated interaction in which a functional job partition among eukaryal, archaeal and bacterial groups allows the rhodolith holobiont to thrive in the global ocean. High rates of photosynthesis were measured for Abrolhos rhodoliths (52.16 μmol carbon m -2 s -1), allowing the entire Abrolhos rhodolith bed to produce 5.65 × 10 5 tons C per day. This estimate illustrates the great importance of the Abrolhos rhodolith beds for dissolved carbon production in the South Atlantic Ocean. © 2014 International Society for Microbial Ecology All rights reserved.

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  • Allergic bronchopulmonary mycosis due to co-infection with Aspergillus fumigatus and Schizophyllum commune. International journal

    Masafumi Seki, Hideaki Ohno, Kazuyoshi Gotoh, Daisuke Motooka, Shota Nakamura, Tetsuya Iida, Yoshitsugu Miyazaki, Kazunori Tomono

    IDCases   1 ( 1 )   5 - 8   2014

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    A 61-year-old female presented with eosinophilic pneumonia accompanied by bronchial asthma. She was finally diagnosed with allergic bronchopulmonary mycosis (ABPM) due to co-infection with Aspergillus fumigatus and Schizophyllum commune detected by genetic analysis of the plug and from cultures.

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  • Mutation accumulation in bacteria exposed to UV radiation

    Atsushi Shibai, Saburo Tsuru, Bei Wen Ying, Daisuke Motooka, Kazuyoshi Gotoh, Shota Nakamura, Tetsuya Yomo

    Artificial Life 14 - Proceedings of the 14th International Conference on the Synthesis and Simulation of Living Systems, ALIFE 2014   757 - 758   2014

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    Reducing native complexity from living organisms has significance in several aspects such as academic application as model organism and industrial application as factory of useful material. In particular, reduced complexity is also interesting in the field investigating the minimal form of "life-as-we-knowit." However, subtracting or inactivating genes from the genome without growth defects is difficult due to the complexity of gene network and error proofing functions. In this study, using model bacteria Escherichia coli (E. coli), we designed a culture system using ultraviolet as a mutagen in order to select possible mutants growing rapidly with genomic inactivation. Here, we demonstrated that the culture system could accumulation of many mutations and preservation of growth ability. These results suggest that our method is effective to obtain functionally reduced genome of E. coli.

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  • Severe respiratory failure due to co-infection with human metapneumovirus and Streptococcus pneumoniae. International journal

    Masafumi Seki, Hisao Yoshida, Kazuyoshi Gotoh, Nobuyuki Hamada, Daisuke Motooka, Shota Nakamura, Norihisa Yamamoto, Shigeto Hamaguchi, Yukihiro Akeda, Hiroshi Watanabe, Tetsuya Iida, Kazunori Tomono

    Respiratory medicine case reports   12   13 - 5   2014

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    A 64-year-old male patient was admitted with respiratory failure, although chest X-rays revealed only mild bronchiolitis. Streptococcus pneumoniae, which usually presents as massive lobular pneumonia, was isolated from sputum, however, pan-pathogen screening using a next-generation sequencer also detected human metapneumovirus genome fragments.

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  • Generation of colonic IgA-secreting cells in the caecal patch

    Kazunori Masahata, Eiji Umemoto, Hisako Kayama, Manato Kotani, Shota Nakamura, Takashi Kurakawa, Junichi Kikuta, Kazuyoshi Gotoh, Daisuke Motooka, Shintaro Sato, Tomonori Higuchi, Yoshihiro Baba, Tomohiro Kurosaki, Makoto Kinoshita, Yosuke Shimada, Taishi Kimura, Ryu Okumura, Akira Takeda, Masaru Tajima, Osamu Yoshie, Masahiro Fukuzawa, Hiroshi Kiyono, Sidonia Fagarasan, Tetsuya Iida, Masaru Ishii, Kiyoshi Takeda

    Nature communications   5   3704   2014

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    Gut-associated lymphoid tissues are responsible for the generation of IgA-secreting cells. However, the function of the caecal patch, a lymphoid tissue in the appendix, remains unknown. Here we analyse the role of the caecal patch using germ-free mice colonized with intestinal bacteria after appendectomy. Appendectomized mice show delayed accumulation of IgA(+) cells in the large intestine, but not the small intestine, after colonization. Decreased colonic IgA(+) cells correlate with altered faecal microbiota composition. Experiments using photoconvertible Kaede-expressing mice or adoptive transfer show that the caecal patch IgA(+) cells migrate to the large and small intestines, whereas Peyer's patch cells are preferentially recruited to the small intestine. IgA(+) cells in the caecal patch express higher levels of CCR10. Dendritic cells in the caecal patch, but not Peyer's patches, induce CCR10 on cocultured B cells. Thus, the caecal patch is a major site for generation of IgA-secreting cells that migrate to the large intestine.

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  • Metagenomic Analysis of Healthy and White Plague-Affected Mussismilia braziliensis Corals

    Gizele D. Garcia, Gustavo B. Gregoracci, Eidy de O. Santos, Pedro M. Meirelles, Genivaldo G.Z. Silva, Rob Edwards, Tomoo Sawabe, Kazuyoshi Gotoh, Shota Nakamura, Tetsuya Iida, Rodrigo L. de Moura, Fabiano L. Thompson

    Microbial Ecology   65 ( 4 )   1076 - 1086   2013.5

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    Coral health is under threat throughout the world due to regional and global stressors. White plague disease (WP) is one of the most important threats affecting the major reef builder of the Abrolhos Bank in Brazil, the endemic coral Mussismilia braziliensis. We performed a metagenomic analysis of healthy and WP-affected M. braziliensis in order to determine the types of microbes associated with this coral species. We also optimized a protocol for DNA extraction from coral tissues. Our taxonomic analysis revealed Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, and Actinomycetes as the main groups in all healthy and WP-affected corals. Vibrionales, members of the Cytophaga-Flavobacterium-Bacteroides complex, Rickettsiales, and Neisseriales were more abundant in the WP-affected corals. Diseased corals also had more eukaryotic metagenomic sequences identified as Alveolata and Apicomplexa. Our results suggest that WP disease in M. braziliensis is caused by a polymicrobial consortium. © 2013 Springer Science+Business Media New York.

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  • Fatal sepsis caused by an unusual Klebsiella species that was misidentified by an automated identification system. International journal

    Masafumi Seki, Kazuyoshi Gotoh, Shota Nakamura, Yukihiro Akeda, Tadashi Yoshii, Shinichi Miyaguchi, Hidenori Inohara, Toshihiro Horii, Kazunori Oishi, Tetsuya Iida, Kazunori Tomono

    Journal of medical microbiology   62 ( Pt 5 )   801 - 803   2013.5

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    This is a description of fatal sepsis caused by infection with Klebsiella variicola, which is an isolate genetically related to Klebsiella pneumoniae. The patient's condition was incorrectly diagnosed as common sepsis caused by K. pneumoniae, which was identified using an automated identification system, but next-generation sequencing and the non-fermentation of adonitol finally identified the cause of sepsis as K. variicola.

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  • B型ボツリヌス菌の神経毒素遺伝子をコードするプラスミドの全ゲノム解析(The complete sequence of plasmid encoding neurotoxin gene in Clostridium botulinum type B strain)

    細見 晃司, 阪口 義彦, 後藤 和義, 中村 佳司, 幸田 知子, 向本 雅郁, 飯田 哲也, 小崎 俊司

    日本細菌学雑誌   68 ( 1 )   187 - 187   2013.2

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  • Metagenomic profile of gut microbiota in children during cholera and recovery

    Shirajum Monira, Shota Nakamura, Kazuyoshi Gotoh, Kaori Izutsu, Haruo Watanabe, Nur Haque Alam, Takaaki Nakaya, Toshihiro Horii, Sk Imran Ali, Tetsuya Iida, Munirul Alam

    Gut Pathogens   5 ( 1 )   2013

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    Background: The diverse bacterial communities colonizing the gut (gastrointestinal tract) of infants as commensal flora, which play an important role in nutrient absorption and determining the state of health, are known to alter due to diarrhea. Method. Bacterial community dynamics in children suffering from cholera and during recovery period were examined in the present study by employing metagenomic tool, followed by DNA sequencing and analysis. For this, bacterial community DNA was extracted from fecal samples of nine clinically confirmed cholera children (age 2-3 years) at day 0 (acute cholera), day 2 (antibiotic therapy), day 7 and, and day 28, and the variable region of 16S rRNA genes were amplified by universal primer PCR. Results: 454 parallel sequencing of the amplified DNA followed by similarity search of the sequenced data against an rRNA database allowed us to identify V. cholerae, the cause of cholera, in all nine children at day 0, and as predominant species in six children, accounting for 35% of the total gut microbiota on an average in all the nine children. The relative abundance (mean ± sem %) of bacteria belonging to phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, was 55 ± 7, 18 ± 4, 13 ± 4, and 8 ± 4, respectively, at day 0, while these values were 12 ± 4, 43 ± 4, 33 ± 3, and 12 ± 2, respectively, at day 28. As antibiotic therapy began, V. cholerae count declined significantly (p< 0.001) and was found only in four children at day 2 and two children at day 7 with the relative abundance of 3.7% and 0.01%, respectively, which continued up to day 28 in the two children. Compared to acute cholera condition (day 0), the relative abundance of Escherichia coli, Enterococcus, and Veillonella increased at day 2 (antibiotic therapy) while Bifidobacterium, Bacteroides, and Ruminococcus decreased. Conclusion: Cholera results expulsion of major commensal bacteria of phyla Bacteroidetes, Firmicutes, and Actinobacteria, and increase of harmful Proteobacteria to colonize the gut during acute and convalescence states. The observed microbiota disruption might explain the prevalent malnutrition in children of Bangladesh where diarrheal diseases are endemic. © 2013 Monira et al.; licensee BioMed Central Ltd.

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  • VopV, an F-actin-binding type III secretion effector, is required for vibrio parahaemolyticus-induced enterotoxicity

    Hirotaka Hiyoshi, Toshio Kodama, Kazunobu Saito, Kazuyoshi Gotoh, Shigeaki Matsuda, Yukihiro Akeda, Takeshi Honda, Tetsuya Iida

    Cell Host and Microbe   10 ( 4 )   401 - 409   2011.10

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    Vibrio parahaemolyticus, a Gram-negative halophilic bacterium that causes acute gastroenteritis in humans, is characterized by two type III secretion systems (T3SS), namely T3SS1 and T3SS2. T3SS2 is indispensable for enterotoxicity but the effector(s) involved are unknown. Here, we identify VopV as a critical effector that is required to mediate V. parahaemolyticus T3SS2-dependent enterotoxicity. VopV was found to possess multiple F-actin-binding domains and the enterotoxicity caused by VopV correlated with its F-actin-binding activity. Furthermore, a T3SS2-related secretion system and a vopV homologous gene were also involved in the enterotoxicity of a non-O1/non-O139 V. cholerae strain. These results indicate that the F-actin-targeting effector VopV is involved in enterotoxic activity of T3SS2-possessing bacterial pathogens. © 2011 Elsevier Inc.

    DOI: 10.1016/j.chom.2011.08.014

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  • Gut microbiota of healthy and malnourished children in Bangladesh

    Shirajum Monira, Shota Nakamura, Kazuyoshi Gotoh, Kaori Izutsu, Haruo Watanabe, Nur Haque Alam, Hubert Ph Endtz, Alejandro Cravioto, Sk Imran Ali, Takaaki Nakaya, Toshihiro Horii, Tetsuya Iida, Munirul Alam

    Frontiers in Microbiology   2 ( NOV )   2011

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    Poor health and malnutrition in preschool children are longstanding problems in Bangladesh. Gut microbiota plays a tremendous role in nutrient absorption and determining the state of health. In this study, metagenomic tool was employed to assess the gut microbiota composition of healthy and malnourished children. DNA was extracted from fecal samples of seven healthy and seven malnourished children (n= 14; age 2-3years) were analyzed for the variable region of 16S rRNA genes by universal primer PCR followed by high-throughput 454 parallel sequencing to identify the bacterial phyla and genera. Our results reveal that the healthy children had a significantly higher number of operational taxonomic unit in their gut than that of the malnourished children (healthy vs. malnourished: 546 vs. 310). In malnourished children, bacterial population of the phyla Proteobacteria and Bacteroidetes accounted for 46 and 18%, respectively. Conversely, in healthy children, Proteobacteria and Bacteroidetes accounted for 5% and 44, respectively (p < 0.001). In malnourished children, the phylum Proteobacteria included pathogenic genera, namely Klebsiella and Escherichia, which were 174-fold and 9-fold higher, respectively, than their healthy counterpart. The predominance of potentially pathogenic Proteobacteria and minimal level of Bacteroidetes as commensal microbiota might be associated to the ill health of malnourished children in Bangladesh. © 2011 Monira, Nakamura, Gotoh, Izutsu, Watanabe, Alam, Endtz, Cravioto, Ali, Nakaya, Horii, Iida and Alam.

    DOI: 10.3389/fmicb.2011.00228

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  • Two Regulators of Vibrio parahaemolyticus Play Important Roles in Enterotoxicity by Controlling the Expression of Genes in the Vp-PAI Region Reviewed

    Toshio Kodama, Kazuyoshi Gotoh, Hirotaka Hiyoshi, Mikiharu Morita, Kaori Izutsu, Yukihiro Akeda, Kwon-Sam Park, Vlademir V. Cantarelli, Rikard Dryselius, Tetsuya Iida, Takeshi Honda

    PLOS ONE   5 ( 1 )   e8678   2010.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:PUBLIC LIBRARY SCIENCE  

    Vibrio parahaemolyticus is an important pathogen causing food-borne disease worldwide. An 80-kb pathogenicity island (Vp-PAI), which contains two tdh (thermostable direct hemolysin) genes and a set of genes for the type III secretion system (T3SS2), is closely related to the pathogenicity of this bacterium. However, the regulatory mechanisms of Vp-PAI's gene expression are poorly understood. Here we report that two novel ToxR-like transcriptional regulatory proteins (VtrA and VtrB) regulate the expression of the genes encoded within the Vp-PAI region, including those for TDH and T3SS2-related proteins. Expression of vtrB was under control of the VtrA, as vector-expressed vtrB was able to recover a functional protein secretory capacity for T3SS2, independent of VtrA. Moreover, these regulatory proteins were essential for T3SS2-dependent biological activities, such as in vitro cytotoxicity and in vivo enterotoxicity. Enterotoxic activities of vtrA and/or vtrB deletion strains derived from the wild-type strain were almost absent, showing fluid accumulation similar to non-infected control. Whole genome transcriptional profiling of vtrA or vtrB deletion strains revealed that the expression levels of over 60 genes were downregulated significantly in these deletion mutant strains and that such genes were almost exclusively located in the Vp-PAI region. These results strongly suggest that VtrA and VtrB are master regulators for virulence gene expression in the Vp-PAI and play critical roles in the pathogenicity of this bacterium.

    DOI: 10.1371/journal.pone.0008678

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  • Identification of two translocon proteins of Vibrio parahaemolyticus type III secretion system 2

    Toshio Kodama, Hirotaka Hiyoshi, Kazuyoshi Gotoh, Yukihiro Akeda, Shigeaki Matsuda, Kwon-Sam Park, Vlademir V. Cantarelli, Tetsuya Iida, Takeshi Honda

    INFECTION AND IMMUNITY   76 ( 9 )   4282 - 4289   2008.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC MICROBIOLOGY  

    The type III secretion system (T3SS) translocon complex is composed of several associated proteins, which form a translocation channel through the host cell plasma membrane. These proteins are key molecules that are involved in the pathogenicity of many T3SS-positive bacteria, because they are necessary to deliver effector proteins into host cells. A T3SS designated T3SS2 of Vibrio parahaemolyticus is thought to be related to the enterotoxicity of this bacterium in humans, but the effector translocation mechanism of T3SS2 is unclear because there is only one gene (the VPA1362 gene) in the T3SS2 region that is homologous to other translocon protein genes. It is also not known whether the VPA1362 protein is functional in the translocon of T3SS2 or whether it is sufficient to form the translocation channel of T3SS2. In this study, we identified both VPA1362 (designated VopB2) and VPA1361 (designated VopD2) as T3SS2-dependent secretion proteins. Functional analysis of these proteins showed that they are essential for T3SS2-dependent cytotoxicity, for the translocation of one of the T3SS2 effector proteins (VopT), and for the contact-dependent activity of pore formation in infected cells in vitro. Their targeting to the host cell membrane depends on T3SS2, and furthermore, they are necessary for T3SS2-dependent enterotoxicity in vivo. These results indicate that VopB2 and VopD2 act as translocon proteins of V. parahaemolyticus T3SS2 and hence have a critical role in the T3SS2-dependent enterotoxicity of this bacterium.

    DOI: 10.1128/IAI.01738-07

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MISC

  • Export of a Vibrio parahaemolyticus toxin by the Sec and type III secretion machineries in tandem

    Shigeaki Matsuda, Ryu Okada, Sarunporn Tandhavanant, Hirotaka Hiyoshi, Kazuyoshi Gotoh, Tetsuya Iida, Toshio Kodama

    Nature Microbiology   4 ( 5 )   781 - 788   2019.5

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    Publishing type:Rapid communication, short report, research note, etc. (scientific journal)  

    Many Gram-negative pathogens utilize dedicated secretion systems to export virulence factors such as exotoxins and effectors 1–4 . Several exotoxins are synthesized as precursors containing amino-terminal Sec signal peptides and are exported through the inner-membrane-bound Sec machinery to the periplasm, followed by secretion across the outer membrane to the exterior using a type II secretion system (T2SS) 3,5 . Here, we report that thermostable direct haemolysin (TDH), an exotoxin of the food-borne pathogen Vibrio parahaemolyticus, can be exported through the type III secretion system (T3SS), which engages in one-step secretion of effectors 4 , despite possessing a Sec signal peptide and being mainly secreted via the T2SS. Although the precursor of TDH is targeted to the Sec pathway, a fraction of mature TDH was observed to re-enter the bacterial cytoplasm. The N terminus of mature TDH comprises a T3SS signal sequence, allowing it to be loaded into the T3SS. We also show that T3SS-delivered TDH as an effector contributes to intestinal fluid accumulation in a rabbit diarrhoeal model of V. parahaemolyticus infection. Thus, our results show that an unconventional export mechanism for a bacterial toxin via the T3SS in tandem with the Sec machinery facilitates the virulence trait of V. parahaemolyticus.

    DOI: 10.1038/s41564-019-0368-y

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  • C型とD型ボツリヌス毒素変換ファージの解析(Analysis of botulinum neurotoxin-converting phages in Clostridium botulinum types C and D)

    阪口 義彦, 内山 淳平, 小椋 義俊, 後藤 和義, 山本 由弥子, 松崎 茂展, 山口 明日美, 林 哲也, 小熊 惠二, 林 俊治

    日本細菌学雑誌   73 ( 1 )   94 - 94   2018.2

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  • C型とD型ボツリヌス毒素変換ファージの解析(Analysis of botulinum neurotoxin-converting phages in Clostridium botulinum types C and D)

    阪口 義彦, 内山 淳平, 小椋 義俊, 後藤 和義, 山本 由弥子, 松崎 茂展, 山口 明日美, 林 哲也, 小熊 惠二, 林 俊治

    日本細菌学雑誌   73 ( 1 )   94 - 94   2018.2

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  • Regulation of Vibrio parahaemolyticus T3SS2 gene expression and function of T3SS2 effectors that modulate actin cytoskeleton

    Toshio Kodama, Hirotaka Hiyoshi, Ryu Okada, Shigeaki Matsuda, Kazuyoshi Gotoh, Tetsuya Iida

    Cellular Microbiology   17 ( 2 )   183 - 190   2015.2

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    Publishing type:Book review, literature introduction, etc.  

    Vibrio parahaemolyticus is a leading causative agent of seafood-borne gastroenteritis worldwide. Most clinical isolates from patients with diarrhoea possess two sets of genes for the type III secretion system (T3SS) on each chromosome (T3SS1 and T3SS2). T3SS is a protein secretion system that delivers effector proteins directly into eukaryotic cells. The injected effectors modify the normal cell functions by altering or disrupting the normal cell signalling pathways. Of the two sets of T3SS genes present in V.parahaemolyticus, T3SS2 is essential for enterotoxicity in several animal models. Recent studies have elucidated the biological activities of several T3SS2 effectors and their roles in virulence. This review focuses on the regulation of T3SS2 gene expression and T3SS2 effectors that specifically target the actin cytoskeleton.

    DOI: 10.1111/cmi.12408

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Research Projects

  • ピロリ菌除菌療法における腸内エコシステム破綻のメカニズムと制御

    Grant number:19K08395  2019.04 - 2022.03

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    岡田 裕之, 後藤 和義, 横田 憲治, 松下 治, 田中 健大, 岡上 昇太郎

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    Grant amount:\4160000 ( Direct expense: \3200000 、 Indirect expense:\960000 )

    大学新入生において標準的なピロリ菌除菌レジメンの下で下痢・軟便を主とした副作用を起こす患者の腸内細菌叢に共通するファクターを見出すことを目的とする。さらにはメタゲノムデータと常在細菌叢の抗菌薬感受性を融合させることで、なぜ特定の菌叢を持つ(または持たない)ことでDysbiosisが起こるのか、そのメカニズムを説明する。さらに内視鏡的な胃炎、組織学的胃炎評価も行い、最終的に腸内細菌叢解析データと融合する。平成31年度(2019年度)から3年間にわたり岡山大学医学部・歯学部新入生(医学科・保健学科・歯学部)に対してピロリ菌検診を例年通り実施し、H.pylori-IgG抗体陽性例を本研究の対象とする。
    2019年度は新入生314人中17人が抗体陽性であった。抗体陽性者14人に内視鏡検査を行い、内視鏡的胃炎、組織学的胃炎の評価および菌株培養を行った。組織学的陽性例は現感染と診断した。組織学的胃炎、菌株培養陰性例に対しては、さらに尿素呼気試験も行い、それら3検査とも陰性の場合は抗体検査が偽陽性と判断し、未感染と診断した。その結果、現感染11名、未感染3名であった。現感染者には除菌治療を行い、除菌前、除菌1週後、2ケ月後の糞便採取を行うとともに、除菌前後2週間の排便回数も含めた消化管症状についてアンケートを実施した。未感染者に対しても1回糞便採取とアンケートを実施した。
    得られた糞便の核酸抽出を実施した。
    2020年度、2021年度新入生に対しても同様に進めていく予定である。

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  • Study for a molecular machinery of the host recognition by Clostridioides difficile phage to develop the advanced therapy

    Grant number:19K07560  2019.04 - 2022.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    阪口 義彦, 加藤 はる, 大宮 直木, 内山 淳平, 後藤 和義, 妹尾 充敏, 林 俊治

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    Grant amount:\4290000 ( Direct expense: \3300000 、 Indirect expense:\990000 )

    クロストリディオイデス(クロストリジウム)・ディフィシル感染症(CDI)は、抗菌薬関連下痢症・腸炎の1つであり、抗菌薬の使用等により正常な腸内微生物叢が乱れ、ディフィシル菌が過剰に増殖することにより発症する。CDI治療では、バンコマイシンやメトロニダゾールなどの抗菌薬が有効であるが、抗菌薬は選択性に乏しく結局は有益な腸内微生物叢を撹乱させるため、再びCDIが引き起こされることがある。従って、腸内微生物叢を撹乱せず、原因となるディフィシル菌のみを特異的に殺菌するCDIの新規治療法の確立が喫緊である。本研究課題では、新規治療法の候補の1つとして、バクテリオファージ(ファージ)に着目した。ファージは、宿主である細菌に対して特異的に結合し、自己の溶菌酵素により殺菌する活性を示す。このようなファージの特性を有効に利用したCDIの新規治療法の確立を目指し研究開発を行っている。そこで、まず、溶菌性ファージの単離を試みたが、分離株からは困難であったことから、次に、ファージが有する溶菌酵素について解析を行った。ディフィシル菌ファージのゲノム情報を基に、ゲノム上の個々の遺伝子について他のファージの溶菌酵素との相同性解析を行ったところ、2つのファージ由来溶菌酵素と予想される遺伝子(cdph33およびcdph34)を特定した。そこで、これらの遺伝子を基にHis融合タンパク質(CDph33およびCDph34)として、大腸菌で発現・精製した。精製した2つのタンパク質について、ディフィシル菌に対する溶菌活性を測定したところ、CDph34は活性を示したが、CDph33は高濃度下でも活性が認められなかった。CDph33とCDph34を組み合わせた場合では、CDph34の溶菌活性と比較してほぼ同等の活性であったが、両タンパク質を組み合わせた方が時間依存的に高い活性を示した。現在、詳細な解析を進めている。

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  • Mechanism of ferroptosis-like cell death induced by Vibrio

    Grant number:18K15147  2018.04 - 2020.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Early-Career Scientists  Grant-in-Aid for Early-Career Scientists

    Gotoh Kazuyoshi

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    Grant amount:\4030000 ( Direct expense: \3100000 、 Indirect expense:\930000 )

    Opportunistic pathogen, Vibrio alginolticus induce cell death via effector VepA in type III secretion system. I found this cell death depends on intracellular ferrous ion, calling this type of cell death ferroptosis. When I transfected recombinant VepA into HeLa cell, I observed cell death that is similar with that by V. alginolyticus infection. In this condition, I treated the cell with ferrous chelater in the cell death induction by VepA-transfection. Unlike the infection experiment, that cell death didn't inhibit by ferrous chelataing. Hence, I deny correlation between VepA-dependent cell death and intracellular ferrous ion. I also examined other possible mechanism of the cell death such as cathepsin, calcium ion, proton and ER stress. However, these factor didn't relate to this cell death. I concluded that novel mechanism hides in VepA-dependent cell death.

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  • 歯周病細菌が産生するプロテアーゼによる歯周組織破壊機構の解明

    Grant number:17K11668  2017.04 - 2021.03

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    中山 真彰, 大森 一弘, 後藤 和義, 大原 直也

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    Grant amount:\4550000 ( Direct expense: \3500000 、 Indirect expense:\1050000 )

    本研究の目的は,「歯周病菌と宿主細胞間で起こる感染現象を細胞・分子生物学的アプローチにより,歯周病の発症機序および歯周病関連疾患の増悪への関与を解明する」ことである。これまでに申請者らは、歯周病菌Porphyromonas gingivalis (Pg)による宿主細胞に影響を与え、細胞内シグナル伝達経路の撹乱による歯周組織の炎症とその破壊に関連する分枝解析を行なってきた。歯周病の発症におけるジンジパイン(Gps)の役割を調べるために、Gps完全欠損株とその野生株を用いた感染実験による比較解析を行ない、歯周炎でみられる炎症性メディエーターであるCOX-2発現やPGE2産生へのGpsの重要性を明らかにした。さらに我々の研究結果から、本菌が産生するGpsは、他の病原因子であるLPSや線毛よりも強くCOX-2発現やPGE2産生を誘導することがわかった。GpsによるCOX-2発現の細胞内応答経路を調べたところ、ERK/AP-1(c-Jun/c-Fos)およびIKK/NF-kBp65の2つのシグナル伝達経路と転写因子の活性化が重要であることを示した。2019年度の研究では、GpsによるCOX-2発現につながる上流因子の分子解析を行なった。Pg野生株の感染における各種カルシウムイオンの挙動に関わる阻害剤・アンタゴニストを用いて、COX-2発現に関与するカルシウムの供給経路について調べた。その結果、COX-2の発現にはセカンドメッセンジャーである細胞内カルシウムが極めて重要であり、カルシウムの細胞質内への供給は細胞内貯蔵からの供給ではなく、細胞外からの流入であることが示唆された。2020年度の研究計画では、細胞外からのカルシウムイオンの流入に関わるチャネルや膜タンパク質を解析し、Gpsが作用する細胞表面のターゲットタンパク質とCOX-2発現との関連性について明らかにしていく。

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  • Posttranscriptional regulation and mutual regulation of small RNA

    Grant number:17K08830  2017.04 - 2020.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Mima Takehiko

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    Grant amount:\4680000 ( Direct expense: \3600000 、 Indirect expense:\1080000 )

    VarS/VarA two-component system controls the expression of target genes thorough regulating the expression of small RNAs (sRNAs). In other bacteria, the expression of sRNAs is considered to be regulated only by VarS/VarA system. Vibrio alginolyticus has four sRNAs. In this study, we found that different proteins bound to the promoter region of each sRNA. We also revealed that ArcA, a response regulator of ArcB/ArcA two-component regulatory system, bound only to the promoter region of sRNA1, but not to those of other sRNAs. Furthermore, we revealed that ArcB/ArcA system regulates the expression of sRNA1 when cells were grown under anaerobic conditions. It seems likely that expression of VarS/VarA-controlled sRNAs were controlled not only by VarS/VarA, but also by multiple regulators. From these results, we propose that sRNAs is the arithmetic system to optimize the expression of many target genes by integrating multiple environmental signals.

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  • Expression mechanism of three CsrB family regulatory RNA in Vibrio alginolyticus

    Grant number:15K19093  2015.04 - 2018.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)  Grant-in-Aid for Young Scientists (B)

    Gotoh Kazuyoshi

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    Grant amount:\3510000 ( Direct expense: \2700000 、 Indirect expense:\810000 )

    Vibrio alginolyticus posses three CsrB family regulatory RNA. In this study, we investigated the mechanism of these RNAs expression system in transcriptional and post-transcriptional level. First, we checked stability among these RNAs using rifampicin RNA-chase assay. As a result, no any difference of RNA stability in three RNAs was shown. Next, we analyzed the possibility that ORF regions affect transcriptional activity. However, reporter assay indicated that the no difference of the activity was shown in ORF containing promoter. These resultu indicated that unknown mechanism produce the non-redundancy among three CsrB family regulatory RNAs.

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  • Development of new enteropathogen detection system target at IgA

    Grant number:25670272  2013.04 - 2015.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    GOTOH Kazuyoshi

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    Grant amount:\3380000 ( Direct expense: \2600000 、 Indirect expense:\780000 )

    When we detect nucleic acids of enteric pathogens from diarrheal stool normal gut microbiota, a dominant in fecal DNA pools, masks the low abundance of pathogens. To solve this problem we employed IgA-coated bacterial sorting technologies. First, we examined that IgA-coated cells were separated by a cell sorter from healthy human stool. DNA was extracted from sorting fraction. However, undetectable level DNA by PCR was obtained from low sorted cell count with our equipment. Because it was difficult to find other optimal conditions, I tried magnetic beads-based separation against IgA. Analysis of 16S rDNA deep sequencing using beads bound showed that no any significant differences was revealed between total and IgA-positive fractions despite the direct observation of IgA-positive bacteria by fluorescent microscopy.
    We proposed that IgA-targeted separation should be carefully validated using microscopy although that technique has been published as successful experiences.

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  • Combat against Infectious Diseases (2020academic year) Second semester  - 木1,木2

  • Research Projects and Practicals: Bacteriology I (2020academic year) special  - その他

  • Lecture and Research Projects: Bacteriology I (2020academic year) special  - その他

  • Research Projects and Practicals: Bacteriology II (2020academic year) special  - その他

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