Chlamydia trachomatis L2 434/Bu readily activates glycolysis under hypoxia for efficient metabolism
Ruiyu Li, Saicheng Zhang, Satoko Otsuguro, Manabu Nagao, Akira Matsuda, Jeewan Thapa, Torahiko Okubo, Katsumi Maenaka, Hideaki Higashi, Hiroyuki Yamaguchi
Biochemical and Biophysical Research Communications, 150461, 150461, Elsevier BV, Aug. 2024, [Peer-reviewed], [Corresponding author]
English, Scientific journal, 32491674

Differential impact of environmental factors on airborne live bacteria and inorganic particles in an underground walkway.
Hiroyuki Yamaguchi, Torahiko Okubo, Eriko Nozaki, Takako Osaki
PloS one, 19, 3, e0300920, 2024, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, We previously reported that variations in the number and type of bacteria found in public spaces are influenced by environmental factors. However, based on field survey data alone, whether the dynamics of bacteria in the air change as a result of a single environmental factor or multiple factors working together remains unclear. To address this, mathematical modeling may be applied. We therefore conducted a reanalysis of the previously acquired data using principal component analysis (PCA) in conjunction with a generalized linear model (Glm2) and a statistical analysis of variance (ANOVA) test employing the χ2 distribution. The data used for the analysis were reused from a previous public environmental survey conducted at 8:00-20:00 on May 2, June 1, and July 5, 2016 (regular sampling) and at 5:50-7:50 and 20:15-24:15 on July 17, 2017 (baseline sampling) in the Sapporo underground walking space, a 520-meter-long underground walkway. The dataset consisted of 60 samples (22 samples for "bacterial flora"), including variables such as "temperature (T)," "humidity (H)," "atmospheric pressure (A)," "traffic pedestrians (TP)," "number of inorganic particles (Δ5: 1-5 μm)," "number of live airborne bacteria," and "bacterial flora." Our PCA with these environmental factors (T, H, A, and TP) revealed that the 60 samples could be categorized into four groups (G1 to G4), primarily based on variations in PC1 [Loadings: T(-0.62), H(-0.647), TP(0.399), A(0.196)] and PC2 [Loadings: A(-0.825), TP(0.501), H(0.209), T(-0.155)]. Notably, the number of inorganic particles significantly increased from G4 to G1, but the count of live bacteria was highest in G2, with no other clear pattern. Further analysis with Glm2 indicated that changes in inorganic particles could largely be explained by two variables (H/TP), while live bacteria levels were influenced by all explanatory variables (TP/A/H/T). ANOVA tests confirmed that inorganic particles and live bacteria were influenced by different factors. Moreover, there were minimal changes in bacterial flora observed among the groups (G1-G4). In conclusion, our findings suggest that the dynamics of live bacteria in the underground walkway differ from those of inorganic particles and are regulated in a complex manner by multiple environmental factors. This discovery may contribute to improving public health in urban settings.

Human pathogenic bacteria on high-touch dry surfaces can be controlled by warming to human-skin temperature under moderate humidity.
Ayano Konno, Torahiko Okubo, Yoshiaki Enoeda, Tomoko Uno, Toyotaka Sato, Shin-Ichi Yokota, Rika Yano, Hiroyuki Yamaguchi
PloS one, 18, 9, e0291765, Sep. 2023, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, Healthcare-associated infections have become a major health issue worldwide. One route of transmission of pathogenic bacteria is through contact with "high-touch" dry surfaces, such as handrails. Regular cleaning of surfaces with disinfectant chemicals is insufficient against pathogenic bacteria and alternative control methods are therefore required. We previously showed that warming to human-skin temperature affected the survival of pathogenic bacteria on dry surfaces, but humidity was not considered in that study. Here, we investigated environmental factors that affect the number of live bacteria on dry surfaces in hospitals by principal component analysis of previously-collected data (n = 576, for CFU counts), and experimentally verified the effect of warming to human-skin temperature on the survival of pathogenic bacteria on dry surfaces under humidity control. The results revealed that PCA divided hospital dry surfaces into four groups (Group 1~4) and hospital dry surfaces at low temperature and low humidity (Group 3) had much higher bacterial counts as compared to the others (Group 1 and 4) (p<0.05). Experimentally, warming to human-skin temperature (37°C with 90% humidity) for 18~72h significantly suppressed the survival of pathogenic bacteria on dry surfaces, such as plastic surfaces [p<0.05 vs. 15°C (Escherichia coli DH5α, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and blaNDM-5 E. coli)] or handrails [p<0.05 vs. 15~25°C (E. coli DH5α, S. aureus, P. aeruginosa, A. baumannii)], under moderate 55% humidity. Furthermore, intermittent heating to human-skin temperature reduced the survival of spore-forming bacteria (Bacillus subtilis) (p<0.01 vs. continuous heating to human-skin temperature). NhaA, an Na+/H+ antiporter, was found to regulate the survival of bacteria on dry surfaces, and the inhibitor 2-aminoperimidine enhanced the effect of warming at human-skin temperature on the survival of pathogenic bacteria (E. coli DH5α, S. aureus, A. baumannii) on dry surfaces. Thus, warming to human-skin temperature under moderate humidity is a useful method for impairing live pathogenic bacteria on high-touch surfaces, thereby helping to prevent the spread of healthcare-associated infections.

Chlamydia trachomatis relies on the scavenger role of aryl hydrocarbon receptor with detyrosinated tubulin for its intracellular growth, but this is impaired by excess indole
Saicheng Zhang, Yuki Funahashi, Satoho Tanaka, Torahiko Okubo, Jeewan Thapa, Shinji Nakamura, Hideaki Higashi, Hiroyuki Yamaguchi
Microbes and Infection, 25, 5, 105097, 105097, Elsevier BV, Jun. 2023, [Peer-reviewed], [Corresponding author]
Scientific journal

Chlamydia trachomatis L2/434/Bu Favors Hypoxia for its Growth in Human Lymphoid Jurkat Cells While Maintaining Production of Proinflammatory Cytokines
Ryoya Tsujikawa, Jeewan Thapa, Torahiko Okubo, Shinji Nakamura, Saicheng Zhang, Yoshikazu Furuta, Hideaki Higashi, Hiroyuki Yamaguchi
Current Microbiology, 79, 9, Springer Science and Business Media LLC, Sep. 2022, [Peer-reviewed], [Corresponding author]
Scientific journal

Chlamydia trachomatis Requires Functional Host-Cell Mitochondria and NADPH Oxidase 4/p38MAPK Signaling for Growth in Normoxia
Jeewan Thapa, Gen Yoshiiri, Koki Ito, Torahiko Okubo, Shinji Nakamura, Yoshikazu Furuta, Hideaki Higashi, Hiroyuki Yamaguchi
Frontiers in Cellular and Infection Microbiology, 12, Frontiers Media SA, 26 May 2022, [Peer-reviewed], [Corresponding author]
Scientific journal, Chlamydia trachomatis (Ct) is an intracellular energy-parasitic bacterium that requires ATP derived from infected cells for its growth. Meanwhile, depending on the O₂ concentration, the host cells change their mode of ATP production between oxidative phosphorylation in mitochondria (Mt) and glycolysis; this change depends on signaling via reactive oxygen species (ROS) produced by NADPH oxidases (NOXs) as well as Mt. It has been proposed that Ct correspondingly switches its source of acquisition of ATP between host-cell Mt and glycolysis, but this has not been verified experimentally. In the present study, we assessed the roles of host-cell NOXs and Mt in the intracellular growth of CtL2 (L2 434/Bu) under normoxia (21% O₂) and hypoxia (2% O₂) by using several inhibitors of NOXs (or the downstream molecule) and Mt-dysfunctional (Mt^d) HEp-2 cells. Under normoxia, diphenyleneiodonium, an inhibitor of ROS diffusion, abolished the growth of CtL2 and other Chlamydiae (CtD and C. pneumoniae). Both ML171 (a pan-NOX inhibitor) and GLX351322 (a NOX4-specific inhibitor) impaired the growth of CtL2 under normoxia, but not hypoxia. NOX4-knockdown cells diminished the bacterial growth. SB203580, an inhibitor of the NOX4-downstream molecule p38MAPK, also inhibited the growth of CtL2 under normoxia but not hypoxia. Furthermore, CtL2 failed to grow in Mt^d cells under normoxia, but no effect was observed under hypoxia. We conclude that under normoxia, Ct requires functional Mt in its host cells as an ATP source, and that this process requires NOX4/p38MAPK signaling in the host cells. In contrast to hypoxia, crosstalk between NOX4 and Mt via p38MAPK may be crucial for the growth of Ct under normoxia.

Wild ciliates differ in susceptibility to Legionella pneumophila JR32.
Airi Kawashiro, Torahiko Okubo, Shinji Nakamura, Jeewan Thapa, Masaki Miyake, Hiroyuki Yamaguchi
Microbiology (Reading, England), 167, 8, Aug. 2021, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, We investigated how Legionella pneumophila (Lp) JR32 interacts with Anteglaucoma CS11A and Colpoda E6, two ciliates that we isolated from sewage and sink trap sludge, respectively, using a handmade maze device containing a 96-well crafting plate. Our 18S rDNA-based phylogenetic analysis showed that Anteglaucoma CS11A and Colpoda E6 formed distinct clades. Scanning electron microscopy showed that Anteglaucoma CS11A had a bigger-sized body than Colpoda E6 and, unlike Tetrahymena IB (the reference strain), neither ciliate produced pellets, which are extracellular vacuoles. Fluorescence microscopic observations revealed that although the intake amounts differed, all three ciliates rapidly ingested LpJR32 regardless of the presence or absence of the icm/dot virulence genes, indicating that they all interacted with LpJR32. In co-cultures with Anteglaucoma CS11A, the LpJR32 levels were maintained but fell dramatically when the co-culture contained the LpJR32 icm/dot deletion mutant instead. Anteglaucoma CS11A died within 2 days of co-culture with LpJR32, but survived co-culture with the deletion mutant. In co-cultures with Colpoda E6, LpJR32 levels were maintained but temporarily decreased independently of the virulence gene. Concurrently, the Colpoda E6 ciliates survived by forming cysts, which may enable them to resist harsh environments, and by diminishing the sensitivity of trophozoites to Lp. In the Tetrahymena IB co-cultures with LpJR32 or Δicm/dot, the Lp levels were maintained, albeit with temporal decreases, and the Tetrahymena IB levels were also maintained. We conclude that unlike Tetrahymena IB with pellet production, Anteglaucoma CS11A can be killed by LpJR32 infection, and Colpoda E6 can resist LpJR32 infection through cyst formation and the low sensitivity of trophozoites to Lp. Thus, the two ciliates that we isolated had different susceptibilities to LpJR32 infection.

Usefulness of a 3D-printing air sampler for capturing live airborne bacteria and exploring the environmental factors that can influence bacterial dynamics
Saaya Mori, Sakura Ishiguro, Satoru Miyazaki, Torahiko Okubo, Ryosuke Omori, Ayako Kai, Kyohei Sugiyama, Airi Kawashiro, Masato Sumi, Jeewan Thapa, Shinji Nakamura, Chietsugu Katoh, Hiroyuki Yamaguchi
Research in Microbiology, 103864, 103864, Elsevier BV, Jul. 2021, [Peer-reviewed], [Corresponding author]
Scientific journal

Distribution of amoebal endosymbiotic environmental chlamydia Neochlamydia S13 via amoebal cytokinesis
Miho Okude, Junji Matsuo, Tomohiro Yamazaki, Kentaro Saito, Yoshokazu Furuta, Shinji Nakamura, Jeewan Thapa, Torahiko Okubo, Hideaki Higashi, Hiroyuki Yamaguchi
Microbiology and Immunology, 65, 3, 115, 124, Wiley, 27 Dec. 2020, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Hypoxia promotes Chlamydia trachomatis L2/434/Bu growth in immortal human epithelial cells via activation of the PI3K-AKT pathway and maintenance of a balanced NAD+/NADH ratio.
Jeewan Thapa, Kent Hashimoto, Saori Sugawara, Ryoya Tsujikawa, Torahiko Okubo, Shinji Nakamura, Hiroyuki Yamaguchi
Microbes and infection, 22, 9, 441, 450, 19 May 2020, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, Chlamydia trachomatis LGV (CtL2) causes systemic infection and proliferates in lymph nodes as well as genital tract or rectum producing a robust inflammatory response, presumably leading to a low oxygen environment. We therefore assessed how CtL2 growth in immortal human epithelial cells adapts to hypoxic conditions. Assessment of inclusion forming units, the quantity of chlamydial 16S rDNA, and inclusion size showed that hypoxia promotes CtL2 growth. Under hypoxia, HIF-1α was stabilized and p53 was degraded in infected cells. Moreover, AKT was strongly phosphorylated at S473 by CtL2 infection. This activation was significantly diminished by LY-294002, a PI3K-AKT inhibitor, which decreased the number of CtL2 progeny. HIF-1α stabilizers (CoCl2, desferrioxamine) had no effect on increasing CtL2 growth, indicating no autocrine impact of growth factors produced by HIF-1α stabilization. Furthermore, in normoxia, CtL2 infection changed the NAD+/NADH ratio of cells with increased gapdh expression; in contrast, under hypoxia, the NAD+/NADH ratio was the same in infected and uninfected cells with high and stable expression of gapdh, suggesting that CtL2-infected cells adapted better to hypoxia. Together, these data indicate that hypoxia promotes CtL2 growth in immortal human epithelial cells by activating the PI3K-AKT pathway and maintaining the NAD+/NADH ratio with stably activated glycolysis.

Complete genome and bimodal genomic structure of the amoebal symbiont Neochlamydia strain S13 revealed by ultra-long reads obtained from MinION.
Junya Yamagishi, Kyoko Hayashida, Junji Matsuo, Torahiko Okubo, Makoto Kuroda, Hiroki Nagai, Tsuyoshi Sekizuka, Hiroyuki Yamaguchi, Chihiro Sugimoto
Journal of human genetics, 65, 1, 41, 48, Jan. 2020, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, Neochlamydia strain S13 is an amoebal symbiont of an Acanthamoeba sp. The symbiont confers resistance to Legionella pneumophila on its host; however, the molecular mechanism underlying this resistance is not completely understood. Genome analyses have been crucial for understanding the complicated host-symbiont relationship but segregating the host's genome DNA from the symbiont's DNA is often challenging. In this study, we successfully identified a bimodal genomic structure in Neochlamydia strain S13 using PacBio RS II supported by ultra-long reads derived from MinION. One mode consisted of circular sequences of 2,586,667 and 231,307 bp; the other was an integrated sequence of the two via long homologous regions. They encoded 2175 protein-coding regions, some of which were implied to be acquired via horizontal gene transfer. They were specifically conserved in the genus Neochlamydia and formed a cluster in the genome, presumably by multiplication through genome replication. Moreover, it was notable that the sequenced DNA was obtained without segregating the symbiont DNA from the host. This is an easy and versatile technique that facilitates the characterization of diverse hosts and symbionts in nature.

Chlamydia trachomatis isolated from cervicovaginal samples in Sapporo, Japan, reveals the circulation of genetically diverse strains　　　　　　　　　　　　　　　
BMC Infectious Diseases, 20, 53, Jan. 2020, [Peer-reviewed], [Corresponding author]
English

Screening of hospital-manhole sewages using MacConkey agar with cefotaxime reveals extended-spectrum β-lactamase-producing Escherichia coli.
Okubo T, Hasegawa T, Fukuda A, Thapa J, Usui M, Tamura Y, Yamaguchi H
International journal of antimicrobial agents, 54, 6, 831, 833, Aug. 2019, [Peer-reviewed], [Corresponding author], [International Magazine]
English

A simple and short microbiology practical improves undergraduate nursing students' awareness of bacterial traits and ability to avoid spreading infections.
Rika Yano, Torahiko Okubo, Tomoko Shimoda, Junji Matsuo, Hiroyuki Yamaguchi
BMC medical education, 19, 1, 53, 53, 11 Feb. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, BACKGROUND: Nurses are responsible for implementing appropriate measures to reduce hospital infections, especially with multidrug resistant bacteria, so nursing students should learn about microbiology. This helps them to understand bacterial dissemination and infectious disease control. Because of tight schedules, however, its teaching is limited in undergraduate nursing classes in Japan. We therefore tested whether a simple short practical session in a microbiology class could help to improve undergraduate nursing students' awareness of bacterial traits and how to prevent infections. METHODS: This study involved second-grade nursing students (n = 76). Two short practical sessions (a total of 3 h, across 2 days) were used to assess the effectiveness of washing or disinfection on hand bacteria in a 16-class microbiology course (total class time was 24 h, plus an exam). Hand bacteria were sampled on LB agar plates with orientation during the first half-day, and the plates examined for colonies with distinct color or morphological traits, and discussed, in the second session, a week later. Questionnaires before and after the exercise were used to assess changes in awareness of unseen bacteria inhabiting around us connecting bacterial traits and how to prevent infections. RESULTS: The results showed that the practical increased the nursing students' awareness of fomites (utensils) (p = 0.0115), fomites (contact-based) (p = 0.0016), habitats (body surface) (p = 0.0127), action facilitating hospital infection (p = 0.0166), and changes in physical condition caused by bacterial infections (p = 0.0136). There were no changes in word associations (p = 0.627) or habitats (inside body) (p = 0.308). Difficulty score, which is an element in questionnaire psychometric properties, tended to be close to the expected score through the practical, but not statistical significant. In addition, regardless of before or after practical, Cronbach α score, which is an indicator of the reliability among items of multi-choice questions, showed > 0.8, indicating validity of evaluation items. Thus, the student's awareness of unseen bacteria inhabiting around us was significantly increased as compared to those before practical in microbiology class. CONCLUSIONS: The simple short practical effectively improved nursing students' awareness of unseen bacteria inhabiting around us in microbiology course, useful for even tight teaching schedules.

Inhibition effect of flavophospholipol on conjugative transfer of the extended-spectrum β-lactamase and vanA genes.
Kudo H, Usui M, Nagafuji W, Oka K, Takahashi M, Yamaguchi H, Tamura Y
The Journal of antibiotics, 72, 2, 79, 85, Feb. 2019, [Peer-reviewed]

Effect of thermal control of dry fomites on regulating the survival of human pathogenic bacteria responsible for nosocomial infections.
Tomoko Shimoda, Torahiko Okubo, Yoshiki Enoeda, Rika Yano, Shinji Nakamura, Jeewan Thapa, Hiroyuki Yamaguchi
PloS one, 14, 12, e0226952, 2019, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, We monitored the survival of human pathogenic bacteria [Escherichia coli (ATCC), extended-spectrum β-lactamase-producing E. coli (Clinical isolate), New Delhi metallo-β-lactamase-producing E. coli (clinical isolate), Staphylococcus aureus (ATCC)] on dry materials (vinyl chloride, aluminum, plastic, stainless steel) at distinct temperatures (room temperature or 15°C-37°C). These bacteria favored a lower temperature for their prolonged survival on the dry fomites, regardless of the material type. Interestingly, when mixed with S. aureus, E. coli survived for a longer time at a lower temperature. Cardiolipin, which can promote the survival of S. aureus in harsh environments, had no effect on maintaining the survival of E. coli. Although the trends remained unchanged, adjusting the humidity from 40% to 60% affected the survival of bacteria on dry surfaces. Scanning electron microscopic analysis revealed no morphological differences in these bacteria immediately before or after one day of dry conditions. In addition, ATP assessment, a method used to visualize high-touch surfaces in hospitals, was not effective at monitoring bacterial dynamics. A specialized handrail device fitted with a heater, which was maintained at normal human body core temperature, successfully prohibited the prolonged survival of bacteria [Enterococcus faecalis (ATCC), E. coli (ATCC), Pseudomonas aeruginosa (ATCC), S. aureus (ATCC), Acinetobacter baumannii (clinical isolate), and Serratia marcescens (clinical isolate)], with the exception of spore-forming Bacillus subtilis (from our laboratory collection) and the yeast-like fungus Candida albicans (from our laboratory collection)] on dry surfaces. Taken together, we concluded that the tested bacteria favor lower temperatures for their survival in dry environments. Therefore, the thermal control of dry fomites has the potential to control bacterial survival on high-touch surfaces in hospitals.

Chlamydia pneumoniae enhances IL-8 production with reduced azithromycin sensitivity under hypoxia　　　　　　　　　　　　　　　
Matsuo J, Sakai K, Okubo T, Yamaguchi H
APMIS, in press, Dec. 2018, [Peer-reviewed]
English, Scientific journal, 低酸素状態(2%)にてHEp-2細胞内での肺炎クラミジアの感染動態を解析した。その結果、通常酸素分圧に比べ低酸素では、肺炎クラミジア増殖が促進し、それに伴い炎症性サイトカインIL-8の産生されアジスロマイシンへの効果が減弱した。これらの研究成果は、低酸素状態が肺炎クラミジアの病原性を規定する一つの因子であることを示唆している。

Activation of caspase-3 during Chlamydia trachomatis-induced apoptosis at a late stage.
Matsuo J, Haga S, Hashimoto K, Okubo T, Ozawa T, Ozaki M, Yamaguchi H
Canadian journal of microbiology, 65, 2, 135, 143, Oct. 2018, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, The obligate intracellular bacterium Chlamydia trachomatis activates the host cell apoptosis pathway at a late stage of its developmental cycle. However, whether caspase-3, which is a key enzyme of apoptosis, is activated in Chlamydia-infected cells remains unknown. Here, we established HEp-2 cells stably expressing cFluc-DEVD, which is a caspase-3 substrate sequence inserted into cyclic firefly luciferase, and then monitored the dynamics of caspase-3 activity in cells infected with Chlamydia. Transfected cells without infection showed a significant increase in luciferase activity due to stimulation with staurosporine, an inducer of apoptosis. Activation was significantly blocked by addition of caspase inhibitor z-VAD-fmk. Furthermore, as expected, Chlamydia infection caused a significant increase in luciferase activation at 36-48 h postinfection with a contrastive decrease at 24 h postinfection, which is already well known. Such activation caused by the infection was much stronger when the amount of bacteria was increased. Thus, caspase-3 activation was accurately monitored by the luciferase activity in HEp-2 cells constitutively expressing the cFluc-DEVD probe. Furthermore, our data showed that C. trachomatis activates caspase-3 in host cells at a late stage of infection.

Tetrahymena promotes interactive transfer of carbapenemase gene encoded in plasmid between fecal Escherichia coli and environmental Aeromonas caviae.
Matsushita M, Okubo T, Hasegawa T, Matsuo J, Watanabe T, Iwasaki S, Fukumoto T, Hayasaka K, Akizawa K, Shimizu C, Yamaguchi H
Microbiology and immunology, 62, 11, 720, 728, Oct. 2018, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, Tetrahymena can facilitate plasmid transfer among Escherichia coli or from E. coli to Salmonella Enteritidis via vesicle accumulation. In this study, whether ciliates promote the interactive transfer of plasmids encoding blaIMP-1 between fecal E. coli and environmental Aeromonas caviae was investigated. Both bacteria were mixed with or without ciliates and incubated overnight at 30°C. The frequency of plasmid-acquired bacteria was estimated by colony counts using an agar plate containing ceftazidim (CAZ) followed by determination of the minimum inhibitory concentration (MIC). Cultures containing ciliates interactively transferred the plasmid between E. coli and Aeromonas with a frequency of 10-4 to 10-5 . All plasmid-acquired bacteria showed a MIC against CAZ of >128 μg/mL and the plasmid transfer was confirmed by PCR amplification of the blaIMP-1 gene. Fluorescent observation showed that both bacteria accumulated in the same vesicle and that transwell sequestering significantly decreased the transfer frequency. Although ciliates preferentially ingested E. coli rather than A. caviae, both bacteria were co-localized into the same vesicles of ciliates, indicating that their meeting is associated with the gene transfer. Thus, ciliates interactively promote plasmid transfer between E. coli and A. caviae. The results of this study will facilitate control of the spread of multiple-antibiotic resistant bacteria.

Subtle changes in host cell density cause a serious error in monitoring of the intracellular growth of Chlamydia trachomatis in a low-oxygen environment: Proposal for a standardized culture method.
Sakai K, Matsuo J, Watanabe T, Okubo T, Nakamura S, Yamaguchi H
Journal of microbiological methods, 153, 84, 91, Sep. 2018, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, We monitored Chlamydia trachomatis growth in HeLa cells cultured with either DMEM or RPMI medium containing 10% FCS under 2% or 21% O2 conditions for 2 days. Bacterial numbers, host cell numbers, and fibrosis-related gene expression in the host cells were estimated by an inclusion forming unit assay, a cell counting assay, and a PCR array, respectively. In contrast to RPMI, bacterial growth under low oxygen conditions in DMEM rapidly decreased with increasing host cell density. The addition of supplements (glucose, glutamine, vitamin B12, D-biotin, non-essential amino acids, glutathione) to the media had no effect. The growth of host cells in DMEM under low oxygen conditions rapidly decreased, although the cells remained healthy morphologically. Furthermore, the downregulation of 17 genes was observed under low oxygen in DMEM. Whereas no effect on bacterial growth was observed when culturing in RPMI medium at low oxygen, and the downregulation of three genes (CTGF, SERPINE1, JUN) was observed following bacterial infection compared with the uninfected control cells. Thus, our findings indicate the need for carefully selected culture conditions when performing experiments with C. trachomatis under low-oxygen environments, and RPMI (rather than DMEM) is recommended when a low host cell density is to be used, proposing the major modification of cell culturing method of C. trachomatis in a low-oxygen environment.

Impact of bacterial traces belonging to the Enterobacteriaceae on the prevalence of Chlamydia trachomatis in women visiting a community hospital in Japan.
Taki K, Watanabe T, Matsuo J, Sakai K, Okubo T, Matsushita M, Abe K, Minami K, Yamaguchi H
Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy, 24, 10, 815, 821, Aug. 2018, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, We explored the bacteria present in the vaginal microbiota facilitating the prevalence of Chlamydia trachomatis in women visiting a community hospital in Sapporo, Japan, by amplicon sequencing. A total of 273 cervical swab samples were collected, and bacterial vaginosis was evaluated in all specimens by assessment of the Nugent score. In 16 of the samples, bacterial 16S rDNA could not be detected and they were therefore omitted from subsequent experiments (n = 257). A significant negative correlation was observed between the Nugent scores and the amount of Lactobacillus 16S rDNA. Among the 257 samples, chlamydial plasmid was detected in 20 samples and was used for amplicon sequencing. No significant association between the Nugent score and the prevalence of C. trachomatis was detected. Based on the results of chlamydial plasmid detection and the Nugent score, chlamydia-negative samples (n = 27) were randomly selected. Finally, the number of operational taxonomic units (OTUs) obtained from amplicon sequencing was compared between chlamydia-positive (n = 20) and -negative samples (n = 27), revealing that a significant difference was only detected for the OTU numbers of Enterobacteriaceae between the C. trachomatis-positive and -negative groups. However, almost all of the samples utilized for amplicon sequencing failed to grow on MacConkey agar plates and produce indole. Taken together, we concluded that traces of bacteria, not live bacteria, belonging to the Enterobacteriaceae indicated the flow of bacteria through the anogenital route along with gut indole, and the resulting impact on the prevalence of C. trachomatis in the cervicogenital tract of women in Japan.

Acanthamoeba S13WT relies on its bacterial endosymbiont to backpack human pathogenic bacteria and resist Legionella infection on solid media
Torahiko Okubo, Mizue Matsushita, Shinji Nakamura, Junji Matsuo, Hiroki Nagai, Hiroyuki Yamaguchi
Environmental Microbiology Reports, 10, 3, 344, 354, Wiley-Blackwell, 01 Jun. 2018, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Amoebal endosymbiont Neochlamydia protects host amoebae against Legionella pneumophila infection by preventing Legionella entry
Chinatsu Maita, Mizue Matsushita, Masahiro Miyoshi, Torahiko Okubo, Shinji Nakamura, Junji Matsuo, Masaharu Takemura, Masaki Miyake, Hiroki Nagai, Hiroyuki Yamaguchi
Microbes and Infection, 20, 4, 236, 244, Elsevier Masson SAS, 01 Apr. 2018, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Long-term survival of Naegleria polaris from Antarctica after 10 years of storage at 4 A degrees C
Junji Matsuo, Shinji Nakamura, Torahiko Okubo, Manabu Fukui, Hiroyuki Yamaguchi
PARASITOLOGY RESEARCH, 117, 3, 937, 941, Mar. 2018, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Impact of capsaicin, an active component of chili pepper, on pathogenic chlamydial growth (Chlamydia trachomatis and Chlamydia pneumoniae) in immortal human epithelial HeLa cells
Kazuya Yamakawa, Junji Matsuo, Torahiko Okubo, Shinji Nakamura, Hiroyuki Yamaguchi
Journal of Infection and Chemotherapy, 24, 2, 83, 87, Elsevier B.V., 01 Feb. 2018, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Lateral Gene Transfer Between Protozoa-Related Giant Viruses of Family Mimiviridae and Chlamydiae.
Watanabe T, Yamazaki S, Maita C, Matushita M, Matsuo J, Okubo T, Yamaguchi H
Evolutionary bioinformatics online, 14, 1176934318788337, 1176934318788337, 2018, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, Obligate intracellular chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. While pathogenic chlamydiae have adapted to a wide range of vertebrates, environmental chlamydiae inhabit unicellular amoebae, the free-living Acanthamoeba. However, how and why this divergence occurred remains unclear. Meanwhile, giant viruses consisting of protozoa-related and protozoa-unrelated viruses have been discovered, with the former group being suggested to have more influenced environmental chlamydiae during their evolution while cohabiting host amoebae. Against this background, we attempted to visualize genes of giant viruses in chlamydial genomes by bioinformatic analysis mainly with comparative genome and phylogenic analysis, seeking genes present in chlamydiae that are specifically shared with protozoa-related giant viruses. As a result, in contrast to protozoa-unrelated giant viruses, the genes of protozoa-related giant viruses were significantly shared in both the chlamydia genomes depending on the giant virus type. In particular, the prevalence of Mimiviridae genes among the protozoa-related giant virus genes in chlamydial genomes was significantly high. Meanwhile, the prevalence of protozoa-related giant virus genes in pathogenic chlamydia genomes was consistently higher than those of environmental chlamydiae; the actual number of sequences similar to giant virus was also significantly predominant compared with those in the environmental chlamydial genomes. Among them, the most prevalent of giant virus was in the case of chlamydiae with Megavirus chiliensis; total of 1338 genes of the chlamydiae were found to be shared with the virus (444 genes specific to environmental chlamydiae, 892 genes shared between both chlamydiae, only two genes in the pathogenic chlamydiae). Phylogenic analysis with most prevalent sets (Megavirus chiliensis and Protochlamydia EI2 or Chlamydia trachomatis L2 434Bu) showed the presence of orthologs between these with several clustered. In addition, Pearson's single regression analysis revealed that almost the prevalence of the genes from the giant viruses in chlamydial genomes was negatively and specifically correlated with the number of chlamydial open reading frames (ORFs). Thus, these results indicated the trace of lateral gene transfer between protozoa-related giant viruses of family Mimiviridae and chlamydiae. This is the first demonstration of a putative linkage between chlamydiae and the giant viruses, providing us with a hint to understand chlamydial evolution.

Analysis of adult damselfly fecal material aids in the estimation of antibiotic-resistant Enterobacterales contamination of the local environment.
Yamaguchi Y, Okubo T, Matsushita M, Wataji M, Iwasaki S, Hayasaka K, Akizawa K, Matsuo J, Shimizu C, Yamaguchi H
PeerJ, 6, e5755, 2018, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, Because damselflies are ubiquitously but focally present in natural environments and play a critical role as predators of other insect species, the fecal matter of damselflies may be useful for investigating antibiotic-resistant bacterial populations, including human pathogens, in local environments. We therefore examined the prevalence of antibiotic-resistant bacteria, including Enterobacterales, in fecal material from 383 damselflies (adults and larvae) collected from seven locations around Sapporo City, Japan, in 2016 and 2017. Fecal samples were plated on soybean casein digest (SCD) agar plates with and without antibiotics (SCD-A and SCD-w/o, respectively) to identify environmental bacteria and gut bacteria, respectively, and on MacConkey agar plates with antibiotics (MacConkey-A) to select for Gram-negative bacteria, including human pathogenic Enterobacterales species. The prevalence of colonies on each of the plates was compared, and representative colonies on MacConkey-A plates were identified to the species level using an API 20E kit and the MALDI Biotyper system. Overall, SCD-w/o plates showed a gut bacterial load of approximately 108 colony-forming units per adult damselfly or larva. There was a significant difference between the prevalence of colonies on the SCD-A and MacConkey-A plates, and a significantly increased prevalence of antibiotic-resistant bacteria on MacConkey-A plates was observed in samples collected from Shinoroshinkawa. Cluster analysis based on minimum inhibitory concentration values of 59 representative isolates from MacConkey-A agar plates revealed that samples from Shinoroshinkawa contained a higher prevalence of Enterobacterales than those from other sampling locations. Thus, fecal materials discharged by adult damselflies could be used in future studies as a simple tool for estimating antibiotic-resistant bacteria, including Enterobacterales species, in the local environment.

Walker occupancy has an impact on changing airborne bacterial communities in an underground pedestrian space, as small-dust particles increased with raising both temperature and humidity
Torahiko Okubo, Takako Osaki, Eriko Nozaki, Akira Uemura, Kouhei Sakai, Mizue Matushita, Junji Matsuo, Shinji Nakamura, Shigeru Kamiya, Hiroyuki Yamaguchi
PLOS ONE, 12, 9, e0184980, Sep. 2017, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Diversity changes of microbial communities into hospital surface environments
Rika Yano, Tomoko Shimoda, Reina Watanabe, Yasutoshi Kuroki, Torahiko Okubo, Shinji Nakamura, Junji Matsuo, Sadako Yoshimura, Hiroyuki Yamaguchi
JOURNAL OF INFECTION AND CHEMOTHERAPY, 23, 7, 439, 445, Jul. 2017, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Ciliates promote the transfer of a plasmid encoding bla(NDM)-5 from Escherichia coli, isolated from a hospital in Japan, to other human pathogens
Torahiko Okubo, Mizue Matushita, Yukiko Ohara, Junji Matsuo, Satoshi Oguri, Tatsuya Fukumoto, Kasumi Hayasaka, Kouzi Akizawa, Hitoshi Shibuya, Chikara Shimizu, Hiroyuki Yamaguchi
INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS, 49, 3, 387, 388, Mar. 2017, [Peer-reviewed], [Corresponding author]
English

Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation
Tatsuya Fukumoto, Junji Matsuo, Torahiko Okubo, Shinji Nakamura, Kentaro Miyamoto, Kentaro Oka, Motomichi Takahashi, Kouji Akizawa, Hitoshi Shibuya, Chikara Shimizu, Hiroyuki Yamaguchi
BMC MICROBIOLOGY, 16, 1, 292, Dec. 2016, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Draft genome sequences of Legionella pneumophila JR32 and Lp01 laboratory strains domesticated in Japan
Chinatsu Maita, Mizue Matushita, Torahiko Okubo, Junji Matsuo, Masaki Miyake, Hiroki Nagai, Hiroyuki Yamaguchi
Genome Announcements, 4, 4, American Society for Microbiology, 2016, [Peer-reviewed], [Corresponding author]
English, Scientific journal

ATP bioluminescence values are significantly different depending upon material surface properties of the sampling location in hospitals.
Tomoko Shimoda, Rika Yano, Shinji Nakamura, Mitsutaka Yoshida, Junji Matsuo, Sadako Yoshimura, Hiroyuki Yamaguchi
BMC research notes, 8, 807, 807, 21 Dec. 2015, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, BACKGROUND: Our previous study into assessing hospital cleanliness in Japan by two common methods, ATP bioluminescence and the stamp agar method, revealed considerable variability in the data of both methods (BMC Research Notes, 7: 121, 2014). To investigate the reason(s) for the variability, we reanalyzed the data (n = 752) from the point of view of the material surface properties of sampling sites. METHODS: Data obtained from surfaces with unknown properties and different purposes such as floor were omitted, and the remaining data (n = 488) were used for this study. The material surface properties on sampling sites were divided into six categories: melamine coated (n = 216), vinyl chloride (n = 16), stainless steel (n = 144), wood (n = 63), and acrylonitrile-butadiene styrene resin coated (n = 48). The data between individual material properties were compared. RESULTS: The ATP values of high-touch places were significantly different depending on the type of surface, but no significant difference in stamp values between material properties was seen, indicating that in contrast to stamp values, ATP-accumulation more depends on the physical properties of the material surface such as electronic charges or roughness. To confirm this, we assessed a degree of roughness on vinyl chloride material surface (disutilized floor samples actually used for each of the hospitals) by observation with scanning electron microscope (SEM). As a result, SEM observation similarly revealed considerable roughness on the materials, which may allow microbes to contaminate the materials without noticing it. CONCLUSION: Material properties must be considered when evaluating hospital cleanliness with ATP values, and provide a strong warning into evaluating hospital cleanliness.

A characteristic of polymorphic membrane protein F of Chlamydia trachomatis isolated from male urogenital tracts in Japan
Tomohiro Yamazaki, Junji Matsuo, Satoshi Takahashi, Shouta Kumagai, Tomoko Shimoda, Kiyotaka Abe, Kunihiro Minami, Hiroyuki Yamaguchi
Journal of Infection and Chemotherapy, 21, 12, 842, 848, Elsevier, 01 Dec. 2015, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Synergistic Costimulatory Effect of Chlamydia pneumoniae with Carbon Nanoparticles on NLRP3 Inflammasome-Mediated Interleukin-1 beta Secretion in Macrophages
Junji Matsuo, Shinji Nakamura, Seiji Takeda, Kasumi Ishida, Tomohiro Yamazaki, Mitsutaka Yoshida, Hitoshi Chiba, Shu-Ping Hui, Hiroyuki Yamaguchi
INFECTION AND IMMUNITY, 83, 7, 2917, 2925, Jul. 2015, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Chlamydia pneumoniae CopNはアルドラーゼAを抑制して細菌の増殖に効果をもたらす(Chlamydia pneumoniae CopN sequesters aldolase A, providing a benefit to the bacterial growth)　　　　　　　　　　　　　　　
石田 香澄, 松尾 淳司, 中村 眞二, 山口 博之
日本細菌学雑誌, 70, 1, 221, 221, 日本細菌学会, Feb. 2015
English

Amoebal Endosymbiont Parachlamydia acanthamoebae Bn-9 Can Grow in Immortal Human Epithelial HEp-2 Cells at Low Temperature; An In Vitro Model System to Study Chlamydial Evolution
Chikayo Yamane, Tomohiro Yamazaki, Shinji Nakamura, Junji Matsuo, Kasumi Ishida, Sumire Yamazaki, Satoshi Oguri, Natsumi Shouji, Yasuhiro Hayashi, Mitsutaka Yoshida, Yimin, Hiroyuki Yamaguchi
PLOS ONE, 10, 2, e0116486, Feb. 2015, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Draft genome sequence of high-temperature-adapted Protochlamydia sp. HS-T3, an amoebal endosymbiotic bacterium found in Acanthamoeba isolated from a hot spring in Japan
Hiroyuki Yamaguchi, Junji Matsuo, Tomohiro Yamazaki, Kasumi Ishida, Kenji Yagita
Genome Announcements, 3, 1, American Society for Microbiology, 2015, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal

Draft genome sequence of Chlamydia trachomatis strain 54, isolated from the urogenital tract of a male in Japan
Tomohiro Yamazaki, Junji Matsuo, Momoka Kikuchi, Kentaro Miyamoto, Kentaro Oka, Motomichi Takahashi, Satoshi Takahashi, Torahiko Okubo, Hiroyuki Yamaguchi
Genome Announcements, 3, 5, American Society for Microbiology, 2015, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Protozoal ciliate promotes bacterial autoinducer-2 accumulation in mixed culture with Escherichia coli
Satoshi Oguri, Tomoko Hanawa, Junji Matsuo, Kasumi Ishida, Tomohiro Yamazaki, Shinji Nakamura, Torahiko Okubo, Tatsuya Fukumoto, Kouzi Akizawa, Chikara Shimizu, Shigeru Kamiya, Hiroyuki Yamaguchi
JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, 61, 5, 203, 210, 2015, [Peer-reviewed]
English, Scientific journal

Chlamydia pneumoniae effector chlamydial outer protein N sequesters fructose bisphosphate aldolase A, providing a benefit to bacterial growth
Kasumi Ishida, Junji Matsuo, Yoshimasa Yamamoto, Hiroyuki Yamaguchi
BMC MICROBIOLOGY, 14, 330, Dec. 2014, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Effect of Ureaplasma parvum co-incubation on Chlamydia trachomatis maturation in human epithelial HeLa cells treated with interferon-gamma
Tomohiro Yamazaki, Junji Matsuo, Shinji Nakamura, Satoshi Oguri, Hiroyuki Yamaguchi
JOURNAL OF INFECTION AND CHEMOTHERAPY, 20, 7-8, 460, 464, Jul. 2014, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Amoebal Endosymbiont Neochlamydia Genome Sequence Illuminates the Bacterial Role in the Defense of the Host Amoebae against Legionella pneumophila
Kasumi Ishida, Tsuyoshi Sekizuka, Kyoko Hayashida, Junji Matsuo, Fumihiko Takeuchi, Makoto Kuroda, Shinji Nakamura, Tomohiro Yamazaki, Mitsutaka Yoshida, Kaori Takahashi, Hiroki Nagai, Chihiro Sugimoto, Hiroyuki Yamaguchi
PLOS ONE, 9, 4, e95166, Apr. 2014, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Visualization of hospital cleanliness in three Japanese hospitals with a tendency toward long-term care.
Reina Watanabe, Tomoko Shimoda, Rika Yano, Yasuhiro Hayashi, Shinji Nakamura, Junji Matsuo, Hiroyuki Yamaguchi
BMC research notes, 7, 121, 121, 04 Mar. 2014, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, BACKGROUND: Hospital cleanliness in hospitals with a tendency toward long-term care in Japan remains unevaluated. We therefore visualized hospital cleanliness in Japan over a 2-month period by two distinct popular methods: ATP bioluminescence (ATP method) and the standard stamp agar method (stamp method). METHODS: The surfaces of 752 sites within nurse and patient areas in three hospitals located in a central area of Sapporo, Japan were evaluated by the ATP and stamp methods, and each surface was sampled 8 times in 2 months. These areas were located in different ward units (Internal Medicine, Surgery, and Obstetrics and Gynecology). Detection limits for the ATP and stamp methods were determined by spike experiments with a diluted bacterial solution and a wipe test on student tables not in use during winter vacation, respectively. Values were expressed as the fold change over the detection limit, and a sample with a value higher than the detection limit by either method was defined as positive. RESULTS: The detection limits were determined to be 127 relative light units (RLU) per 100 cm2 for the ATP method and 5.3 colony-forming units (CFU) per 10 cm2 for the stamp method. The positive frequency of the ATP and stamp methods was 59.8% (450/752) and 47.7% (359/752), respectively, although no significant difference in the positive frequency among the hospitals was seen. Both methods revealed the presence of a wide range of organic contamination spread via hand touching, including microbial contamination, with a preponderance on the entrance floor and in patient rooms. Interestingly, the data of both methods indicated considerable variability regardless of daily visual assessment with usual wiping, and positive surfaces were irregularly seen. Nurse areas were relatively cleaner than patient areas. Finally, there was no significant correlation between the number of patients or medical personnel in the hospital and organic or microbiological contamination. CONCLUSIONS: Ongoing daily hospital cleanliness is not sufficient in Japanese hospitals with a tendency toward long-term care.

High-temperature adapted primitive Protochlamydia found in Acanthamoeba isolated from a hot spring can grow in immortalized human epithelial HEp-2 cells
Aya Sampo, Junji Matsuo, Chikayo Yamane, Kenji Yagita, Shinji Nakamura, Natsumi Shouji, Yasuhiro Hayashi, Tomohiro Yamazaki, Mitsutaka Yoshida, Miho Kobayashi, Kasumi Ishida, Hiroyuki Yamaguchi
ENVIRONMENTAL MICROBIOLOGY, 16, 2, 486, 497, Feb. 2014, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Chlamydophila pneumoniae in human immortal Jurkat cells and primary lymphocytes uncontrolled by interferon-gamma
Kasumi Ishida, Takeru Kubo, Ayumi Saeki, Chikayo Yamane, Junji Matsuo, Yimin, Shinji Nakamura, Yasuhiro Hayashi, Miyuki Kunichika, Mitsutaka Yoshida, Kaori Takahashi, Itara Hirai, Yoshimasa Yamamoto, Ken-ichiro Shibata, Hiroyuki Yamaguchi
MICROBES AND INFECTION, 15, 3, 192, 200, Mar. 2013, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Protochlamydia Induces Apoptosis of Human HEp-2 Cells through Mitochondrial Dysfunction Mediated by Chlamydial Protease-Like Activity Factor
Junji Matsuo, Shinji Nakamura, Atsushi Ito, Tomohiro Yamazaki, Kasumi Ishida, Yasuhiro Hayashi, Mitsutaka Yoshida, Kaori Takahashi, Tsuyoshi Sekizuka, Fumihiko Takeuchi, Makoto Kuroda, Hiroki Nagai, Kyoko Hayashida, Chihiro Sugimoto, Hiroyuki Yamaguchi
PLOS ONE, 8, 2, e56005, Feb. 2013, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Impact of free-living amoebae on presence of Parachlamydia acanthamoebae in the hospital environment and its survival in vitro without requirement for amoebae
Tatsuya Fukumoto, Junji Matsuo, Yasuhiro Hayashi, Satoshi Oguri, Shinji Nakamura, Yoshihiko Mizutani, Takashi Yao, Kouzi Akizawa, Haruki Suzuki, Chikara Shimizu, Kazuhiko Matsuno, Hiroyuki Yamaguchi
Journal of Clinical Microbiology, 51, 1, 385, Jan. 2013, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Environmental Chlamydiae Alter the Growth Speed and Motility of Host Acanthamoebae
Miho Okude, Junji Matsuo, Shinji Nakamura, Kouhei Kawaguchi, Yasuhiro Hayashi, Haruna Sakai, Mitsutaka Yoshida, Kaori Takahashi, Hiroyuki Yamaguchi
MICROBES AND ENVIRONMENTS, 27, 4, 423, 429, Dec. 2012, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Ciliates Expel Environmental Legionella-Laden Pellets To Stockpile Food
Fuhito Hojo, Daisuke Sato, Junji Matsuo, Masaki Miyake, Shinji Nakamura, Miyuki Kunichika, Yasuhiro Hayashi, Mitsutaka Yoshida, Kaori Takahashi, Hiromu Takemura, Shigeru Kamiya, Hiroyuki Yamaguchi
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 78, 15, 5247, 5257, Aug. 2012, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Chlamydia trachomatis serovar L2 infection model using human lymphoid Jurkat cells
Takeru Kubo, Kasumi Ishida, Junji Matsuo, Shinji Nakamura, Yasuhiro Hayashi, Haruna Sakai, Mitsutaka Yoshida, Kaori Takahashi, Itaru Hirai, Yoshimasa Yamamoto, Hiroyuki Yamaguchi
MICROBIAL PATHOGENESIS, 53, 1, 1, 11, Jul. 2012, [Peer-reviewed], [Corresponding author]
English, Scientific journal

A domino-like chlamydial attachment process: concurrent Parachlamydia acanthamoebae attachment to amoebae is required for several amoebal released molecules and serine protease activity
Yasuhiro Hayashi, Yimin, Junji Matsuo, Shinji Nakamura, Miyuki Kunichika, Mitsutaka Yoshida, Kaori Takahashi, Hiroyuki Yamaguchi
MICROBIOLOGY-SGM, 158, 1607, 1614, Jun. 2012, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Frequency of Chlamydia trachomatis in Ureaplasma-positive healthy women attending their first prenatal visit in a community hospital in Sapporo, Japan
Tomohiro Yamazaki, Megumi Matsumoto, Junji Matsuo, Kiyotaka Abe, Kunihiro Minami, Hiroyuki Yamaguchi
BMC INFECTIOUS DISEASES, 12, 82, Apr. 2012, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Effect of the steroid receptor antagonist RU486 (mifepristone) on an IFN gamma-induced persistent Chlamydophila pneumoniae infection model in epithelial HEp-2 cells
Kasumi Ishida, Tomohiro Yamazaki, Kazuki Motohashi, Miho Kobayashi, Junji Matsuo, Takako Osaki, Tomoko Hanawa, Shigeru Kamiya, Yoshimasa Yamamoto, Hiroyuki Yamaguchi
JOURNAL OF INFECTION AND CHEMOTHERAPY, 18, 1, 22, 29, Feb. 2012, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Amoebal Endosymbiont Protochlamydia Induces Apoptosis to Human Immortal HEp-2 Cells
Atsushi Ito, Junji Matsuo, Shinji Nakamura, Asahi Yoshida, Miho Okude, Yasuhiro Hayashi, Haruna Sakai, Mitsutaka Yoshida, Kaori Takahashi, Hiroyuki Yamaguchi
PLOS ONE, 7, 1, e30270, Jan. 2012, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Chlamydophila pneumoniae attachment and infection in low proteoglycan expressing human lymphoid Jurkat cells
Miho Kobayashi, Kasumi Ishida, Junji Matsuo, Shinji Nakamura, Ayumi Nagasawa, Kazuki Motohashi, Takashi Yao, Itaru Hirai, Yoshimasa Yamamoto, Haruki Suzuki, Chikara Shimizu, Kazuhiko Matsuno, Hiroyuki Yamaguchi
MICROBIAL PATHOGENESIS, 51, 3, 209, 216, Sep. 2011, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Impact of anaerobic and oligotrophic conditions on survival of Alloiococcus otitidis, implicated as a cause of otitis media
Junji Matsuo, Atsushi Harimaya, Tatsuya Fukumoto, Shinji Nakamura, Mitsutaka Yoshida, Kaori Takahashi, Masahiro Iida, Nobuhiro Fujii, Hiroyuki Yamaguchi
JOURNAL OF INFECTION AND CHEMOTHERAPY, 17, 4, 478, 482, Aug. 2011, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Increased concentrations of antibody against heat shock protein in patients with myeloperoxidase anti- neutrophil cytoplasmic autoantibody positive microscopic polyangiitis
Ikuko Komiya, Yoshihiro Arimura, Kimimasa Nakabayashi, Akira Yamada, Takako Osaki, Hiroyuki Yamaguchi, Shigeru Kamiya
MICROBIOLOGY AND IMMUNOLOGY, 55, 8, 531, 538, Aug. 2011, [Peer-reviewed]
English, Scientific journal

Ciliates promote the transfer of the gene encoding the extended-spectrum beta-lactamase CTX-M-27 between Escherichia coli strains
Satoshi Oguri, Junji Matsuo, Yasuhiro Hayashi, Shinji Nakamura, Tomoko Hanawa, Tatsuya Fukumoto, Yoshihiko Mizutani, Takashi Yao, Kouzi Akizawa, Haruki Suzuki, Chikara Shimizu, Kazuhiko Matsuno, Shigeru Kamiya, Hiroyuki Yamaguchi
JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, 66, 3, 527, 530, Mar. 2011, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Host range of obligate intracellular bacterium Parachlamydia acanthamoebae
Yasuhiro Hayashi, Shinji Nakamura, Junji Matsuo, Tatsuya Fukumoto, Mitsutaka Yoshida, Kaori Takahashi, Yoshihiko Mizutani, Takashi Yao, Hiroyuki Yamaguchi
MICROBIOLOGY AND IMMUNOLOGY, 54, 11, 707, 713, Nov. 2010, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Ciliates rapidly enhance the frequency of conjugation between Escherichia coli strains through bacterial accumulation in vesicles
Junji Matsuo, Satoshi Oguri, Shinji Nakamura, Tomoko Hanawa, Tatsuya Fukumoto, Yasuhiro Hayashi, Kouhei Kawaguchi, Yoshihiko Mizutani, Takashi Yao, Kouzi Akizawa, Haruki Suzuki, Chikara Simizu, Kazuhiko Matsuno, Shigeru Kamiya, Hiroyuki Yamaguchi
RESEARCH IN MICROBIOLOGY, 161, 8, 711, 719, Oct. 2010, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Impact of Free-Living Amoebae on Presence of Parachlamydia acanthamoebae in the Hospital Environment and Its Survival In Vitro without Requirement for Amoebae
Tatsuya Fukumoto, Junji Matsuo, Masahiro Hayashi, Satoshi Oguri, Shinji Nakamura, Yoshihiko Mizutani, Takashi Yao, Kouzi Akizawa, Haruki Suzuki, Chikara Shimizu, Kazuhiko Matsuno, Hiroyuki Yamaguchi
JOURNAL OF CLINICAL MICROBIOLOGY, 48, 9, 3360, 3365, Sep. 2010, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Survival and transfer ability of phylogenetically diverse bacterial endosymbionts in environmental Acanthamoeba isolates
Junji Matsuo, Kouhei Kawaguchi, Shinji Nakamura, Yasuhiro Hayashi, Mitsutaka Yoshida, Kaori Takahashi, Yoshihiko Mizutani, Takashi Yao, Hiroyuki Yamaguchi
ENVIRONMENTAL MICROBIOLOGY REPORTS, 2, 4, 524, 533, Aug. 2010, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Endosymbiotic bacterium Protochlamydia can survive in acanthamoebae following encystation
Shinji Nakamura, Junji Matsuo, Yasuhiro Hayashi, Kouhei Kawaguchi, Mitsutaka Yoshida, Kaori Takahashi, Yoshihiko Mizutani, Takashi Yao, Hiroyuki Yamaguchi
ENVIRONMENTAL MICROBIOLOGY REPORTS, 2, 4, 611, 618, Aug. 2010, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Chlamydia pneumoniae infection suppresses Staphylococcus enterotoxin B-induced proliferation associated with down-expression of CD25 in lymphocytes
Itaru Hirai, Maki Utsumi, Hiroyuki Yamaguchi, Yoshimasa Yamamoto
CANADIAN JOURNAL OF MICROBIOLOGY, 56, 4, 289, 294, Apr. 2010, [Peer-reviewed]
English, Scientific journal

Stability of Chlamydophila pneumoniae in a harsh environment without a requirement for acanthamoebae
Junji Matsuo, Miho Kobayashi, Shinji Nakamura, Yoshihiko Mizutani, Takashi Yao, Itaru Hirai, Yoshimasa Yamamoto, Hiroyuki Yamaguchi
MICROBIOLOGY AND IMMUNOLOGY, 54, 2, 63, 73, Feb. 2010, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Prevalence of Helicobacter and Acanthamoeba in natural environment
K. Kawaguchi, J. Matsuo, T. Osaki, S. Kamiya, H. Yamaguchi
LETTERS IN APPLIED MICROBIOLOGY, 48, 4, 465, 471, Apr. 2009, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Inhibition of lymphocyte CD3 expression by Chlamydophila pneumoniae infection
Hiroyuki Yamaguchi, Junji Matsuo, Shigehiro Sugimoto, Maki Utsumi, Yoshimasa Yamamoto
MICROBIAL PATHOGENESIS, 45, 4, 290, 296, Oct. 2008, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal

Novel Parachlamydia acanthamoebae quantification method based on coculture with amoebae
Junji Matsuo, Yasuhiro Hayashi, Shinji Nakamura, Marie Sato, Yoshihiko Mizutani, Masahiro Asaka, Hiroyuki Yamaguchi
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 74, 20, 6397, 6404, Oct. 2008, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Chlamydia pneumoniae growth inhibition in cells by the steroid receptor antagonist RU486 (mifepristone)
Hiroyuki Yamaguchi, Shigerul Kamiya, Tomonori Uruma, Takako Osaki, Haruhiko Taguchi, Tomoko Hanawa, Minoru Fukuda, Hayato Kawakami, Hajime Goto, Herman Friedman, Yoshimasa Yamamoto
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 52, 6, 1991, 1998, Jun. 2008, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal

Mutation of luxS affects motility and infectivity of Helicobacter pylori in gastric mucosa of a Mongolian gerbil model
Takako Osaki, Tomoko Hanawa, Taki Manzoku, Minoru Fukuda, Hayato Kawakami, Hidekazu Suzuki, Hiroyuki Yamaguchi, Xu Yan, Haruhiko Taguchi, Satoshi Kurata, Shigeru Kamiya
JOURNAL OF MEDICAL MICROBIOLOGY, 55, 11, 1477, 1485, Nov. 2006, [Peer-reviewed]
English, Scientific journal

Development of diabetes in non-obese diabetic mice promotes Chlamydia pneumoniae dissemination from lung to peripheral blood
H Yamaguchi, Oshio, I, T Osaki, S Kurata, Y Yamamoto, S Kamiya
INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, 87, 2, 121, 129, Apr. 2006, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Chlamydia pneumoniae growth inhibition in human monocytic THP-1 cells and human epithelial HEp-2 cells by a novel phenoxazine derivative
T Uruma, H Yamaguchi, M Fukuda, H Kawakami, H Goto, T Kishimoto, Y Yamamoto, A Tomoda, S Kamiya
JOURNAL OF MEDICAL MICROBIOLOGY, 54, 12, 1143, 1149, Dec. 2005, [Peer-reviewed]
English, Scientific journal

Cytokine response of lymphocytes persistently infected with Chlamydia pneumoniae
R Takano, H Yamaguchi, S Sugimoto, S Nakamura, H Friedman, Y Yamamoto
CURRENT MICROBIOLOGY, 50, 3, 160, 166, Mar. 2005, [Peer-reviewed]
English, Scientific journal

Long-term infection of mongolian gerbils with Helicobacter pylori: Microbiological, histopathological, and serological analyses
S Nakagawa, T Osaki, Y Fujioka, H Yamaguchi, S Kamiya
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, 12, 2, 347, 353, Feb. 2005, [Peer-reviewed]
English, Scientific journal

Effect of Helicobacter pylori on DNA synthesis of human epithelial cells
Atsushi Toyoda, Takako Osaki, Hiroyuki Yamaguchi, Tomoko Hanawa, Haruhiko Taguchi, Makoto Hasegawa, Shigeru Kamiya
Journal of Infection and Chemotherapy, 11, 3, 129, 135, Springer Japan, 2005, [Peer-reviewed]
English, Scientific journal

The effect of probiotic treatment with Clostridium butyricum on enterohemorrhagic Escherichia coli O157 : H7 infection in mice
M Takahashi, H Taguchi, H Yamaguchi, T Osaki, A Komatsu, S Kamiya
FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY, 41, 3, 219, 226, Jul. 2004, [Peer-reviewed]
English, Scientific journal

Prevalence of viable Chlamydia pneumoniae in peripheral blood mononuclear cells of healthy blood donors
H Yamaguchi, M Yamada, T Uruma, M Kanamori, H Goto, Y Yamamoto, S Kamiya
TRANSFUSION, 44, 7, 1072, 1078, Jul. 2004, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal

Legionella pneumophila suppresses macrophage interleukin-12 production by activating the p42/44 mitogen-activated protein kinase cascade
K Matsunaga, H Yamaguchi, TW Klein, H Friedman, Y Yamamoto
INFECTION AND IMMUNITY, 71, 11, 6672, 6675, Nov. 2003, [Peer-reviewed]
English, Scientific journal

Effect of bacterial flora on postimmunization gastritis following oral vaccination of mice with Helicobacter pylori heat shock protein 60
H Yamaguchi, T Osaki, H Taguchi, N Sato, A Toyoda, M Takahashi, M Kai, N Nakata, A Komatsu, Y Atomi, S Kamiya
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, 10, 5, 808, 812, Sep. 2003, [Peer-reviewed], [Lead author]
English, Scientific journal

Chlamydia pneumoniae resists antibiotics in lymphocytes
H Yamaguchi, H Friedman, M Yamamoto, K Yasuda, Y Yamamoto
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 47, 6, 1972, 1975, Jun. 2003, [Peer-reviewed], [Lead author]
English, Scientific journal

Involvement of nicotinic acetylcholine receptors in controlling Chlamydia pneumoniae growth in epithelial HEp-2 cells
H Yamaguchi, H Friedman, Y Yamamoto
INFECTION AND IMMUNITY, 71, 6, 3645, 3647, Jun. 2003, [Peer-reviewed], [Lead author]
English, Scientific journal

Chlamydia pneumoniae infection of alveolar macrophages: A model
S Haranaga, H Yamaguchi, H Ikejima, H Friedman, Y Yamamoto
JOURNAL OF INFECTIOUS DISEASES, 187, 7, 1107, 1115, Apr. 2003, [Peer-reviewed]
English, Scientific journal

Immune response to heat shock protein of Helicobacter pylori - A candidate as a vaccine component
Shigeru Kamiya, Takako Osaki, Haruhiko Taguchi, Hiroyuki Yamaguchi
Keio Journal of Medicine, 51, 2, 24, 25, Dec. 2002, [Peer-reviewed]
English, International conference proceedings

A Chlamydia pneumoniae infection model using established human lymphocyte cell lines
H Yamaguchi, S Haranaga, H Friedman, JA Moor, KE Muffly, Y Yamamoto
FEMS MICROBIOLOGY LETTERS, 216, 2, 229, 234, Nov. 2002, [Peer-reviewed], [Lead author]
English, Scientific journal

Chlamydia pneumoniae infection induces differentiation of monocytes into macrophages
H Yamaguchi, S Haranaga, R Widen, H Friedman, Y Yamamoto
INFECTION AND IMMUNITY, 70, 5, 2392, 2398, May 2002, [Peer-reviewed], [Lead author]
English, Scientific journal

Interleukin-8 induction and adhesion of the coccoid form of Helicobacter pylori
T Osaki, H Yamaguchi, H Taguchi, M Fukada, H Kawakami, H Hirano, S Kamiya
JOURNAL OF MEDICAL MICROBIOLOGY, 51, 4, 295, 299, Apr. 2002, [Peer-reviewed]
English, Scientific journal

Experimental infection of germ-free mice with hyper-toxigenic enterohaemorrhagic Escherichia coli O157 : H7, strain 6
H Taguchi, M Takahashi, H Yamaguchi, T Osaki, A Komatsu, Y Fujioka, S Kamiya
JOURNAL OF MEDICAL MICROBIOLOGY, 51, 4, 336, 343, Apr. 2002, [Peer-reviewed]
English, Scientific journal

Analysis of Chlamydia pneumoniae growth in cells by reverse transcription-PCR targeted to bacterial gene transcripts
S Haranaga, H Ikejima, Yamaguchi, I, H Friedman, Y Yamamoto
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, 9, 2, 313, 319, Mar. 2002, [Peer-reviewed]
English, Scientific journal

Chlamydia pneumoniae infects and multiplies in lymphocytes in vitro
S Haranaga, H Yamaguchi, H Friedman, SI Izumi, Y Yamamoto
INFECTION AND IMMUNITY, 69, 12, 7753, 7759, Dec. 2001, [Peer-reviewed]
English, Scientific journal

Detection of Chlamydia pneumoniae antigen in PBMNCs of healthy blood donors
S Haranaga, H Yamaguchi, GF Leparc, H Friedman, Y Yamamoto
TRANSFUSION, 41, 9, 1114, 1119, Sep. 2001, [Peer-reviewed]
English, Scientific journal

Induction of secretion of interleukin-8 from human gastric epithelial cells by heat-shock protein 60 homologue of Helicobacter pylori
H Yamaguchi, T Osaki, N Kurihara, M Kitajima, M Kai, M Takahashi, H Taguchi, S Kamiya
JOURNAL OF MEDICAL MICROBIOLOGY, 48, 10, 927, 933, Oct. 1999, [Peer-reviewed], [Lead author]
English, Scientific journal

Colony formation by Helicobacter pylori after long-term incubation under anaerobic conditions
H Yamaguchi, T Osaki, M Takahashi, H Taguchi, S Kamiya
FEMS MICROBIOLOGY LETTERS, 175, 1, 107, 111, Jun. 1999, [Peer-reviewed], [Lead author]
English, Scientific journal

Relationship between expression of HSP60, urease activity, production of vacuolating toxin, and adherence activity of Helicobacter pylori
H Yamaguchi, T Osaki, H Taguchi, T Hanawa, T Yamamoto, S Kamiya
JOURNAL OF GASTROENTEROLOGY, 33, Suppl 10, 6, 9, Nov. 1998, [Peer-reviewed]
English, Scientific journal

Production and characterisation of monoclonal antibodies to heat-shock protein 60 of Helicobacter pylori
H Yamaguchi, T Osaki, H Taguchi, T Hanawa, T Yamamoto, S Kamiya
JOURNAL OF MEDICAL MICROBIOLOGY, 46, 10, 819, 824, Oct. 1997, [Peer-reviewed]
English, Scientific journal

Heat-shock protein 60 homologue of Helicobacter pylori is associated with adhesion of H-pylori to human gastric epithelial cells
H Yamaguchi, T Osaki, N Kurihara, H Taguchi, T Hanawa, T Yamamoto, S Kamiya
JOURNAL OF MEDICAL MICROBIOLOGY, 46, 10, 825, 831, Oct. 1997, [Peer-reviewed]
English, Scientific journal

Studies on the relationship between adhesive activity and haemagglutination by Helicobacter pylori
T Osaki, H Yamaguchi, H Taguchi, J Kumada, S Ogata, S Kamiya
JOURNAL OF MEDICAL MICROBIOLOGY, 46, 2, 117, 121, Feb. 1997, [Peer-reviewed]
English, Scientific journal

Induction and epitope analysis of Helicobacter pylori heat shock protein
H Yamaguchi, T Osaki, H Taguchi, T Hanawa, T Yamamoto, S Kamiya
JOURNAL OF GASTROENTEROLOGY, 31, Suppl 9, 12, 15, Nov. 1996, [Peer-reviewed]
English, Scientific journal

Characteristics of monoclonal antibody neutralizing vacuolation of cultured cells by cytotoxin produced by Helicobacter pylori
H Yamaguchi, H Taguchi, S Kamiya
JAPANESE JOURNAL OF MEDICAL SCIENCE & BIOLOGY, 49, 5-6, 262, 263, Oct. 1996, [Peer-reviewed]
English, Scientific journal

Flow cytometric analysis of the heat shock protein 60 expressed on the cell surface of Helicobacter pylori
H Yamaguchi, T Osaki, H Taguchi, T Hanawa, T Yamamoto, S Kamiya
JOURNAL OF MEDICAL MICROBIOLOGY, 45, 4, 270, 277, Oct. 1996, [Peer-reviewed]
English, Scientific journal

Analysis of the epitopes recognized by mouse monoclonal antibodies directed to Yersinia enterocolitica heat-shock protein 60
H Yamaguchi, H Miura, K Ohsumi, T Osaki, H Taguchi, T Yamamoto, T Hanawa, S Ogata, S Kamiya
MICROBIOLOGY AND IMMUNOLOGY, 40, 1, 77, 80, 1996, [Peer-reviewed]
English, Scientific journal

EPITOPE HOMOLOGY BETWEEN BACTERIAL HEAT-SHOCK PROTEIN AND SELF-PROTEINS IN THE HOST-CELL
H YAMAGUCHI, T YAMAMOTO, Y KONOEDA, H TAGUCHI, S OGATA
APMIS, 100, 11, 957, 962, Nov. 1992, [Peer-reviewed], [Lead author]
English, Scientific journal

Properties of a protein antigen common to Yersinia enterocolitica and other gram-negative bacteria.
S. Ogata, M. Kanamori, H. Yamaguchi, H. Taguchi
Contributions to microbiology and immunology, 9, 201, 212, 1987
English, Scientific journal

The characterization of cross-reacting protein antigen in gram-negative bacilli.
YAMAGUCHI Hiroyuki, TAGUCHI Haruhiko, ISHIYAMA Narihiro, KANAMORI Masato, OGATA Sachio
Zeitschrift der Japanischen Mikrobiologische Gesellschaft, 41, 4, 701, 707, JAPANESE SOCIETY FOR BACTERIOLOGY, 1986, [Lead author]
Japanese, Distribution of cross-reacting protein antigen (CRPA) among 11 bacterial species was examined by immunoelectrophoresis. CRPA was detected in the sonicates of Shigella sonnei, Proteus mirabilis, Salmonella enteritidis, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Yersinia enterocolitica, Pseudomonas aeruginosa and Vibrio cholerae but not in those of Neisseria gonorrhoeae or Staphylococcus aureus. A major protein with a molecular weight of 60 kilodaltons was found to be shared by the above nine species by SDS-PAGE and immunoblotting. CRPAs of V. cholerae and V. enterocolitica were partially purified by a combination of starch gel electrophoresis and gel filtration. The molecular weights of both CRPAs were estimated at about 500 kilodaltons by gel filtration. The 60 kilodalton protein was found also in both CRPAs by SDS-PAGE and immunoblotting, therefore, it is a major antigenic component of CRPAs of the above nine gram-negative rods. On the other hand, the major protein was not found in the outer membrane preparation obtained from V. cholerae. The present study suggests that CRPA is distinct from such previously reported common antigens as the outer membrane protein of V. cholerae and Kunin's antigen of enteric bacteria in the immunological and physico-chemical properties.

Pathogenic Chlamydia L2 requires aryl hydrocarbon receptors and detyrosinated tubulin for its growth
ZHANG Saicheng, 大久保寅彦, 中村眞二, 東秀明, 山口博之, 日本細菌学雑誌(Web), 78, 1, 2023

マイクローブトランスファー 身の回りの環境に潜む病原細菌の生態deja vuと健康リスク 病院での環境清浄度評価の盲点と院内細菌叢の実態　　　　　　　　　　　　　　　
矢野 理香, 山口 博之, 松尾 淳司, 大久保 寅彦, 良村 貞子, 下田 智子, 日本細菌学雑誌, 71, 1, 56, 56, Feb. 2016
日本細菌学会, Japanese

Enhanced survival of Helicobacter pylori in co-culture with Acanthamoeba castellanii and the expressions of bacterial genes
北条 史, 大﨑 敬子, 米澤 英雄, 花輪 智子, 蔵田 訓, 山口 博之, 神谷 茂, 無菌生物, 46, 2, 62, 64, 2016
日本無菌生物ノートバイオロジー学会, Japanese

SURVIVAL STRATEGY OF HELICOBACTER PYLORI IN ENVIRONMENTS BY CO-EXISTENCE IN ACANTHAMOEBA CASTELLANII
F. Hojo, T. Osaki, H. Yonezawa, S. Kurata, T. Hanawa, H. Yamaguchi, S. Kamiya, HELICOBACTER, 19, 103, 103, Sep. 2014
English, Summary international conference

Comparative genomics of primitive chlamydiae showing distinct mutualistic symbiosis with amoebae
YAMAGUCHI HIROYUKI, MATSUO JUNJI, SEKIZUKA TSUYOSHI, KURODA MAKOTO, NAGAI HIROKI, SUGIMOTO CHIHIRO, 日本細菌学雑誌, 68, 1, 186, 25 Feb. 2013
English

医療施設内での感染拡散防止策の向上を目的とした一つの取り組み 病棟内の人の流れや動きが微生物汚染度に与える影響　　　　　　　　　　　　　　　
渡辺 玲奈, 矢野 理香, 下田 智子, 山口 博之, 感染症学雑誌, 86, 臨増, 261, 261, Mar. 2012
(一社)日本感染症学会, Japanese

自然環境に生息するアカントアメーバの分離・遺伝子型別法とその中に見いだされる難培養性共生細菌の検出法について　　　　　　　　　　　　　　　
松尾 淳司, 林 泰弘, 中村 眞二, 山口 博之, 寄生虫学研究：材料と方法 2012年版, 1, 2, 2012

Symbiosis between amoebae and endosymbiotic bacteria : axenic amoebae and its phenotypic alternation
YAMAGUCHI HIROYUKI, Journal of germfree life and gnotobiology, 41, 1, 54, 58, 01 Sep. 2011
日本無菌生物ノートバイオロジー学会, Japanese

Diagnosis of pneumonia with Mycoplasma pneumoniae by PCR in children in Kenya
KAMIYA Shigeru, TAGUCHI Haruhiko, YAMAGUCHI Hiroyuki, CHAKAYA James M, BII Christine C, Japanese journal of mycoplasmology, 37, 75, 77, 31 Mar. 2011
日本マイコプラズマ学会事務局, Japanese

【環境、生態の変動と感染症】 環境クラミジアの生態　　　　　　　　　　　　　　　
松尾 淳司, 林 泰弘, 福元 達也, 中村 眞二, 山口 博之, 化学療法の領域, 26, 12, 2416, 2424, Nov. 2010
(株)医薬ジャーナル社, Japanese

土壌環境からの新興再興感染症起因菌の遺伝子検出頻度について
松尾 淳司, 山口 博之, 日本細菌学雑誌, 62, 1, 79, 79, 25 Feb. 2007
Japanese

土壌環境からの新興再興感染症起因菌の遺伝子検出頻度について
松尾 淳司, 山口 博之, 日本細菌学雑誌, 62, 1, 58, 58, 25 Feb. 2007
Japanese

月見草種子ポリフェノールの抗Helicobacter Pylori作用
島田 太一, 大崎 敬子, 山口 博之, Food function, 1, 1, 40, 47, Mar. 2005
国際食品機能学会, Japanese

Salmonella enterica serovar Enteritidisに対するClostridium butyricumの感染防御効果
高橋志達, 田口晴彦, 山口博之, 花輪智子, 大崎敬子, 神谷茂, 日本臨床腸内微生物学会誌, 6, 1, 2004

Protective effect of oral vaccination of mice by Helicobacter pylori heat-shock protein 60 and the following post immunization gastritis
YAMAGUCHI HIROYUKI, OSAKI TAKAKO, TAGUCHI HARUHIKO, KAMIYA SHIGERU, Journal of germfree life and gnotobiology, 33, 2, 84, 86, 01 Dec. 2003
日本無菌生物ノートバイオロジー学会, Japanese

Cytokine inducibility of Mycoplasma pneumoniae in mycoplasmal pneumonia model using germfree mice
TAGUCHI HARUHIKO, SEKINE HIDEAKI, HAYAKAWA MASAYUKI, OSAKI TAKAKO, HANAWA TOMOKO, YAMAGUCHI HIROYUKI, KAMIYA SHIGERU, Journal of germfree life and gnotobiology, 33, 2, 87, 90, 01 Dec. 2003
日本無菌生物ノートバイオロジー学会, Japanese

D-5 Helicobacter pylori熱ショック蛋白の経口ワクチンとしての有用性とその問題点(一般口演,第31回杏林医学会総会)
山口 博之, 大崎 敬子, 田口 晴彦, 神谷 茂, 杏林医学会雑誌, 34, 1, 62, 62, 30 Mar. 2003
杏林医学会, Japanese

D-6 ヘリコバクターピロリのスナネズミヘの感染とアポトーシスの誘導(一般口演,第31回杏林医学会総会)
大崎 敬子, 花輪 智子, 田口 晴彦, 山口 博之, 豊田 篤, 神谷 茂, 杏林医学会雑誌, 34, 1, 62, 62, 30 Mar. 2003
杏林医学会, Japanese

Chlamydia pneumoniae activation in lymphocytes of healthy donors by immunomodulators.
Y Yamamoto, H Ikejima, H Yamaguchi, G Leparc, H Friedman, CLINICAL IMMUNOLOGY, 103, 3, S5, S5, Jun. 2002
English, Summary international conference

Detection of Chlamydia pneumoniae DNA in frozenhuman blood clots by touchdown PCR.
H Yamaguchi, H Ikejima, GF Leparc, SE Phillips, CA Weideman, H Friedman, Y Yamamoto, FASEB JOURNAL, 16, 4, A218, A218, Mar. 2002
English, Summary international conference

HSP60リコンビナント蛋白の経口投与によるH.pylori感染防御効果
神谷 茂, 山口 博之, 大崎 敬子, 豊田 篤, 山西 愼吾, 田口 晴彦, 日本細菌学雑誌, 57, 1, 322, 322, Feb. 2002
日本細菌学会, Japanese

耳鼻科疾患患者咽頭ぬぐい液からのHelicobacter pyloriの検出
神谷茂, 大崎敬子, 山口博之, 田口晴彦, 花輪智子, 大畑敦, 豊田篤, 長谷川誠, Bacterial Adherence & Biofilm, 15, 2001

Protective effect of cross-reactive region(189-203rd) of Helicobacter pylori heat shock protein 60 (HSP60) on H-pylori infection.
H Yamaguchi, A Toyoda, M Takahashi, H Taguchi, S Kamiya, GUT, 47, A60, A61, Oct. 2000
English, Summary international conference

Studies of the effect of Clostridium butyricum on Helicobacter pylori in several test models including gnotobiotic mice
M Takahashi, H Taguchi, H Yamaguchi, T Osaki, S Kamiya, JOURNAL OF MEDICAL MICROBIOLOGY, 49, 7, 635, 642, Jul. 2000
English

Immune response against a cross-reactive epitope on the heat shock protein 60 homologue of Helicobacter pylori
H Yamaguchi, T Osaki, M Kai, H Taguchi, S Kamiya, INFECTION AND IMMUNITY, 68, 6, 3448, 3454, Jun. 2000
English

Experimental Infection with entherohemorrhagic Escherichia coli in germfree mice
TAGUCHI Haruhiko, TAKAHASHI Motomiti, YAMAGUCHI Hiroyuki, OSAKI Takako, KAMIYA Shigeru, Journal of germfree life and gnotobiology., 29, 2, 93, 95, 01 Dec. 1999
日本無菌生物ノ-トバイオロジ-学会, Japanese

Reactivity of monoclonal antibody to HSP60 homologue of Helicobacter pylori with human gastric epithelial cells and induction of IL-8 from these cells by purified H-pylori HSP60
H Yamaguchi, T Osaki, N Kurihara, H Taguchi, S Kamiya, JOURNAL OF GASTROENTEROLOGY, 34, Supplement 11, 1, 5, Dec. 1999
English

Cross-reactive epitope on Helicobacter pylori heat-shock protein 60 (HSP60) is involved in protection against H-pylori infection
H Yamaguchi, T Osaki, M Takahashi, H Taguchi, S Kamiya, GUT, 45, Supple111, A36, A36, Sep. 1999
English, Summary international conference

Antibacterial Effects of Cacao Mass on Enterohemorrhagic Escherichia coli O157 : H7
TAKAHASHI Toshio, TAGUCHI Haruhiko, YAMAGUCHI Hiroyuki, OSAKI Takako, SATO Susumu, KAMEI Masanori, HASHIZUME Shuichi, KAMIYA Shigeru, 感染症学雑誌 : 日本伝染病学会機関誌 : the journal of the Japanese Association for Infectious Diseases, 73, 7, 694, 701, 20 Jul. 1999
日本感染症学会, Japanese

Production of monoclonal antibodies neutralizing vacuolation of cultured cells by Helicobacter pylori cytotoxin
H Yamaguchi, T Osaki, H Taguchi, S Kamiya, FEMS MICROBIOLOGY LETTERS, 168, 2, 277, 282, Nov. 1998
English

Characteristics of Helicobacter pylori strains isolated from patients with different gastric diseases
N Kurihara, S Kamiya, H Yamaguchi, T Osaki, H Shinohara, T Kitahora, H Ishida, A Ozawa, Y Otani, T Kubota, K Kumai, M Kitajima, JOURNAL OF GASTROENTEROLOGY, 33, Suppl10, 10, 13, Nov. 1998
English

Induction of interleukin-8 from primary human gastric epithelial cell by heat-shock protein 60 of Helicobacter pylori
H Yamaguchi, T Osaki, N Kurihara, H Taguchi, S Kamiya, GUT, 43, A33, A34, Sep. 1998
English, Summary international conference

Establishment and characterisation of a monoclonal antibody to inhibit adhesion of Helicobacter pylori to gastric epithelial cells
T Osaki, H Yamaguchi, H Taguchi, M Fukuda, H Kawakami, H Hirano, S Watanabe, A Takagi, S Kamiya, JOURNAL OF MEDICAL MICROBIOLOGY, 47, 6, 505, 512, Jun. 1998
English

The Role of Heat Shock Protein 60 (HSP60) of Helicobacter pylori in Adhesion of H. pylori to Human Gastric Epithelial Cell
YAMAGUCHI Hiroyuki, OSAKI Takako, KURIHARA Naoto, TAGUCHI Haruhiko, KAMIYA Shigeru, 感染症学雑誌 : 日本伝染病学会機関誌 : the journal of the Japanese Association for Infectious Diseases, 72, 5, 487, 492, 20 May 1998
日本感染症学会, Japanese

Current Statsu of Acute Respiratoly Infections in Children under Five Years of Age in Nairobi, Kenya
TAGUCHI Haruhiko, YAMAGUCHI Hiroyuki, KAMIYA Shigeru, KOBAYASHI Hiroyuki, TSUTSUMI Hiroyuki, CHIBA Shu-zoh, KAMIYA Yasuhiko, Journal of the Japanese Association for Infectious Diseases, 72, 12, 29, 34, 1998
Two hundreds and twenty-six children under five years of age with pneumonia were recruited from an urban poor area in Nairobi, Kenya, and examined for pathogens for 1 year from February 1997.
One hundred and twenty-eight of the 226 patients were pathogen-positive cases. The patients under 1-year-old were 61.8% of the pathogen-positive cases. A total 192 organisms were isolated from 128 pathogen-positive patients. Streptococcus pneumoniae had the highest prevalence rate of 31.3%, followed by respiratory syncytial virus with 10.4%, Candida albicans with 9.9%, Moraxella (B) catarrhalis with 7.8%.
In S. pneumoniae, 66.7% of the orgamism was resistant to oxacillin. It was also shown that 51.1% and 65.1% of the S. pneumoniae strains were resistant to gentamicin and trimethoprim/sulfam, respectively. From these results, it is clear that a lot of multi-drug resistant S. pneumoniae strains including penicillin-resistant S. pneumoniae were frequently detected in an urban poor area., The Japanese Association for Infectious Diseases

Detection of Helicobacter pylori in fecal samples of gnotobiotic mice infected with H-pylori by an immunomagnetic-bead separation technique
T Osaki, H Taguchi, H Yamaguchi, S Kamiya, JOURNAL OF CLINICAL MICROBIOLOGY, 36, 1, 321, 323, Jan. 1998
English

Association between heat shock protein 60 of Helicobacter pylori and adhesion of its bacteria to human gastric epithelial cells
YAMAGUCHI Hiroyuki, OSAKI Takako, TAGUCHI Haruhiko, KAMIYA Shigeru, Cytometry research : 日本サイトメトリー学会機関誌, 7, 2, 51, 55, 12 Dec. 1997
Japanese

Role of heat-shock protein 60 homologue of Helicobacter pylori for human chronic gastric inflammation following H-pylori infection
H Yamaguchi, T Osaki, N Kurihara, S Kamiya, GUT, 41, A24, A24, Sep. 1997
English, Summary international conference

Mouse enterocolitis induced by heat-shock protein 60 of Yersinia enterocolitica
YAMAGUCHI Hiroyuki, TAGUCHI Haruhiko, OSAKI Takako, HANAWA Tomoko, YAMAMOTO Tomoko, KAMIYA Shigeru, Journal of germfree life and gnotobiology., 27, 1, 68, 71, Jun. 1997
日本無菌生物ノ-トバイオロジ-学会, Japanese

Effect of Clostridium butyricum MIYAIRI on adhesion and colonization of Helicobacter pylori in germfree mice
TAKAHASHI Motomichi, TAGUCHI Haruhiko, YAMAGUCHI Hiroyuki, OSAKI Takako, KAMIYA Shigeru, Journal of germfree life and gnotobiology., 27, 1, 40, 43, Jun. 1997
日本無菌生物ノ-トバイオロジ-学会, Japanese

Prevention of Clostridium difficile induced hemorrhagic enterocolitis in gnotobiotic mice by Clostridium butyricum
TAGUCHI Haruhiko, YAMAGUCHI Hiroyuki, OSAKI Takako, TAKAHASHI Motomichi, KAMIYA Shigeru, Journal of germfree life and gnotobiology., 27, 1, 65, 67, Jun. 1997
日本無菌生物ノ-トバイオロジ-学会, Japanese

Growth inhibition of Helicobacter pylori by monoclonal antibody to heat-shock protein 60
H Yamaguchi, T Osaki, H Taguchi, T Hanawa, T Yamamoto, M Fukuda, H Kawakami, H Hirano, S Kamiya, MICROBIOLOGY AND IMMUNOLOGY, 41, 12, 909, 916, 1997
English

Epitope analysis of monoclonal antibody 3C8 recognizing intestinal tissue antigen of patients with bacteria HSP60 and inflammatory enteric disease.
山口博之, 助川寧, 八木田旭邦, 大崎敬子, 田口晴彦, 花輪智子, 山本友子, 神谷茂, JJPEN, 18, 12, 1996

Rapid detection of Mycoplasma pneumoniae by polymerase chain reaction
KAMIYA S., YAMAGUCHI H., TAGUCHI H., KAI M., SHIOZAWA K., OGATA S., 日本マイコプラズマ学会雑誌 = Japanese Journal of Mycoplasmology, 21, 162, 165, 01 Oct. 1995
Japanese

FLOW CYTOMETRIC ANALYSIS USING LIPOPHILIC DYE PKH-2 FOR ADHESION OF VIBRIO-CHOLERAE TO INTESTINE-407-CELLS
H TAGUCHI, T OSAKI, H YAMAGUCHI, S KAMIYA, MICROBIOLOGY AND IMMUNOLOGY, 39, 11, 891, 894, 1995
English, Introduction scientific journal

DETECTION AND CHARACTERIZATION OF ANTIBODIES TO BACTERIAL HEAT-SHOCK PROTEIN-60 IN SERA OF PATIENTS WITH PRIMARY BILIARY-CIRRHOSIS
H YAMAGUCHI, H MIURA, K OHSUMI, N ISHIMI, H TAGUCHI, N ISHIYAMA, Y SHIRAISHI, T YAMAMOTO, S OGATA, MICROBIOLOGY AND IMMUNOLOGY, 38, 6, 483, 487, 1994
English, Introduction scientific journal

YERSINIA-ENTEROCOLITICA IMMUNODOMINANT 60-KDA ANTIGEN, COMMON TO A BROAD RANGE OF BACTERIA, IS A HEAT-SHOCK PROTEIN
H YAMAGUCHI, T YAMAMOTO, H TAGUCHI, S OGATA, JOURNAL OF GENERAL MICROBIOLOGY, 136, 1091, 1097, Jun. 1990
English

Search for new target molecules used by Chlamydia trachomatis (L2 434/Bu strain) in infected cells by screening approved drug libraries　　　　　　　　　　　　　　　
Saicheng Zhang, Ruiyu Li, Torahiko Okubo, Hiroyuki Yamaguchi
第97回日本細菌学会総会(札幌), 08 Aug. 2024, Japanese

乾燥面での付着菌数を低減させる温度条件とその応用：加温便座の有効性の検討　　　　　　　　　　　　　　　
栗城琴華, 大久保寅彦, 山口博之
第97回日本細菌学会総会(札幌), 07 Aug. 2024, Japanese

Chlamydia trachomatisが感染細胞内で利用するMAPKおよび PI3K-AKT経路に付随する新たな標的分子の探索　　　　　　　　　　　　　　　
黒岩青空, 大久保寅彦, 山口博之
第97回日本細菌学会総会(札幌), 07 Aug. 2024, Japanese, Poster presentation

選抜WS3-8: クラミジア・トラコマティスはメチオニン関連代謝が抑制される低酸素環境を好む　　　　　　　　　　　　　　　
Hiroyuki Yamaguchi, Ruiyu Li, Saicheng Zhang, Sora Kuroiwa, Jeewan Thapa, Torahiko Okubo, Hideaki Higashi
第97回日本細菌学会総会(札幌), 07 Aug. 2024, Japanese, Public symposium

偏性細胞内寄生性細菌クラミジアのヒト細胞内への適応機構　　　　　　　　　　　　　　　
山口博之
第72回日本感染症学会東日本地方会学術集会 (教育講演12), 26 Oct. 2023, Japanese, Public discourse
[Invited]

Chlamydia trachomatis感染時における細胞増殖を促す受容体のアダプター分子 GAB2の影響
黒岩青空, 高橋小夏, 中村穂香, 大久保寅彦, 山口博之
第88回日本細菌学会北海道支部総会 (帯広), 26 Aug. 2023, Japanese, Oral presentation
32491674

低酸素環境における感染細胞内での病原性クラミジア(L2 434/Bu)の増殖はメチオニン代謝産物量の変化に依存する
李睿語, 張賽成, タパ・ジーワン, 大久保寅彦, 東秀明, 山口博之
第88回日本細菌学会北海道支部総会 (帯広), 26 Aug. 2023, Japanese, Oral presentation
32491674

Soil bacteria release and migrate into the air in response to change in environmental factors: an experimental field study
Torahiko OKUBO, Hiroyuki YAMAGUCHI
アメリカ微生物学会 (ヒューストン) ASM Microbe, 19 Jun. 2023, English, Poster presentation
30469620

Exploration for novel signal transduction pathways utilized by Chlamydia trachomatis L2 through screening approved drug libraries　　　　　　　　　　　　　　　
Hiroyuki Yamaguchi, Ruiyu Li, Saicheng Zhang, Torahiko Okubo
アメリカ微生物学会 (ヒューストン) ASM Microbe, 18 Jun. 2023, English, Poster presentation

繊毛虫の殺滅現象を利用したLegionella pneumophilaの新規病原因子の探索　　　　　　　　　　　　　　　
大久保寅彦, 山口博之
第96回日本細菌学会総会, 16 Mar. 2023, Japanese, Poster presentation

病原性クラミジアL2はその細胞内増殖に芳香族炭化水素受容体と脱チロシン化チューブリンを要求する
張 賽成, 大久保寅彦, 山口博之
第96回日本細菌学会総会, 16 Mar. 2023, Japanese, Poster presentation
32491674

偏性細胞内寄生性のChlamydia trachomatis (L2 434/Bu)は低酸素で培養した宿主細胞環境を好む
李睿語, 張賽成, 大久保寅彦, 山口博之
第96回日本細菌学会総会, 16 Mar. 2023, Japanese, Poster presentation
32491674

インドールによるChlamydia trachomatis のヒト上皮系株化細胞HEp-2細胞内での増殖抑制機構について: 芳香族炭化水素受容体AhRの関与について
張賽成, 船橋悠希, 大久保寅彦, 中村眞二, 山口博之
第16回細菌学若手コロッセウム (札幌市), Japanese, Oral presentation
25 Aug. 2022 - 26 Aug. 2022, 32491674

Chlamydia trachomatis L2 (434 / Bu)は、通常酸素分圧下において細胞内で増殖するためにNOX4/p38MAPK経路と健全なミトコンドリアを要求する　　　　　　　　　　　　　　　
山口博之, タパジーワン, 中村眞二, 大久保寅彦, 古田芳一, 東秀明
第16回細菌学若手コロッセウム (札幌市), Japanese, Oral presentation
25 Aug. 2022 - 26 Aug. 2022

既存薬ライブラリーのスクリーニングによる病原性クラミジアが利用する細胞内シグナル伝達系の探索　　　　　　　　　　　　　　　
李睿語, 張賽成, タパジーワン, 大久保寅彦, 山口博之
第16回細菌学若手コロッセウム (札幌市), Japanese, Oral presentation
25 Aug. 2022 - 26 Aug. 2022

インドールによるChlamydia trachomatis のヒト上皮系株化細胞HEp-2細胞内での増殖抑制機構について: 芳香族炭化水素受容体の関与について
張賽成, 大久保寅彦, 中村眞二, 山口博之
第16回日本臨床検査学教育学会学術大会, 18 Aug. 2022, Japanese, Oral presentation
32491674

既存薬ライブラリーのスクリーニングから探る病原性クラミジアが感染細胞内で利用する新たなシグナル伝達系について
李睿語, 張賽成, タパジーワン, 大久保寅彦, 山口博之
第16回日本臨床検査学教育学会学術大会, 18 Aug. 2022, Japanese, Oral presentation
32491674

Soil bacteria float in the air according to the changes of environmental factors: a field study　　　　　　　　　　　　　　　
Mori Saaya, Torahiko Okubo, Hiroyuki Yamaguchi
アメリカ微生物学会 (ワシントンDC) ASM Microbe (オンライン参加) Rapid fireに採択, 13 Jun. 2022, English, Public symposium

Molecular mechanism by which Legionella pneumophila JR32 instantly kills Anteglaucoma harbinensis CS11A, a ciliate isolated from sewage　　　　　　　　　　　　　　　
Torahiko Okubo, Hiroyuki Yamaguchi
アメリカ微生物学会 (ワシントンDC) ASM Microbe (オンライン参加) Rapid fireに採択, 11 Jun. 2022, English, Public symposium

Combined effect of temperature and humidity on the survival of Escherichia coli DH5α on dry surfaces
Ayano Konno, Torahiko Okubo, Hiroyuki Yamaguchi
アメリカ微生物学会 (ワシントンDC) ASM Microbe (オンライン参加) Rapid fireに採択, 11 Jun. 2022, English, Public symposium
30469620

Damselfly’s fecal materials can work as an indicator for revealing multidrug-resistant bacteria in local environments　　　　　　　　　　　　　　　
Torahiko Okubo, Yuyu Yamaguchi, Masashi Wataji, Sumio Iwasaki, Kasumi Hayasaka, Kouzi Akizawa, Hiroyuki Yamaguchi
Jun. 2019, English
[International presentation]

Genetic diversity of Chlamydia trachomatis isolates collected from Sapporo, Japan　　　　　　　　　　　　　　　
Jeewan Thapa, Takanori Watanabe, Torahiko Okubo, Hiroyuki Yamaguchi
アメリカ微生物学会 asm2019 (サンフランシスコ), Jun. 2019, English
[International presentation]

Hypoxia prompts Chlamydia trachomatis L2 growth in immortal human epithelial cells by stabilizing HIF-1α and disrupting p53　　　　　　　　　　　　　　　
Kento Hashimoto, Jeewan Thapa, Torahiko Okubo, Hiroyuki Yamaguchi
アメリカ微生物学会 asm2019 (サンフランシスコ), Jun. 2019, English
[International presentation]

Multilocus Sequence Typingによる性器クラミジアの分子疫学: 札幌での動向調査　　　　　　　　　　　　　　　
Jeewan Thapa, Takanori Watanabe, Keisuke Taki, Junji Matsuo, Torahiko Okubo, Hiroyuki Yamaguchi
第92回日本細菌学会総会, Apr. 2019, Japanese, Poster presentation
[Domestic Conference]

マンホール下水からのESBL産生菌分離と下水由来繊毛虫を介したESBL遺伝子伝達の検証　　　　　　　　　　　　　　　
長谷川貴生, 大久保寅彦, 山口博之
第92回日本細菌学会総会, Apr. 2019, Japanese, Poster presentation
[Domestic Conference]

レジオネラの感染に抵抗性を示す環境由来アメーバによる細菌運搬現象　　　　　　　　　　　　　　　
大久保寅彦, 松尾淳司, 山口博之
第92回日本細菌学会総会, Apr. 2019, Japanese, Nominated symposium
[Domestic Conference]

インピンジャー法を用いたエアサンプルからの浮遊細菌の分離・同定の試み　　　　　　　　　　　　　　　
鷲見優斗, 大久保寅彦, 山口博之
第92回日本細菌学会総会, Apr. 2019, Japanese, Poster presentation
[Domestic Conference]

低酸素環境はChlamydia trachomatis L2の細胞内増殖を促進する　　　　　　　　　　　　　　　
橋本拳人, 大久保寅彦, 山口博之
第92回日本細菌学会総会, Apr. 2019, Japanese, Poster presentation
[Domestic Conference]

微生物のシンプルな演習は看護学部学生の感染症や微生物そのものへの意識を改善する　　　　　　　　　　　　　　　
山口博之, 矢野理香, 下田智子, 大久保寅彦
第13回日本臨床検査学教育学会(札幌), Aug. 2018, Japanese, Oral presentation
[Domestic Conference]

Subtle change of host-cell density causes fatal error on monitoring the intracellular growth of Chlamydia trachomatis in a low-oxygen environment　　　　　　　　　　　　　　　
Kouhai Sakai, Junji Matsuo, Torahiko Okubo, Shinji Nakamura, Hiroyuki Yamaguchi
アメリカ微生物学会 asm microbe2020 (アトランタ) Poster, Jun. 2018, English, Oral presentation
[International presentation]

Ciliates promote the interactive transfer of plasmid encoding blaNDM-5 between human pathogenic Escherichia coli and environmental Aeromonas caviae　　　　　　　　　　　　　　　
Matsuchita Matsushita, Torahiko Okubo, Junji Matsuo, Sinji Nakamura, Hiroyuki Yamaguchi
アメリカ微生物学会 asm microbe2019 (アトランタ) Poster, Jun. 2018, English, Oral presentation
[International presentation]

Cyclic Luciferase Probe Reveals Caspase-3 Activation in Chlamydia-Infected Cells At Late Times During Infection　　　　　　　　　　　　　　　
Matsuo J, Haga S, Okubo T, Nakamura S, Ozawa T, Ozaki M, Yamaguchi H
アメリカ微生物学会 asm microbe2018 (アトランタ) RapidFire Talk (Oral selected), Jun. 2018
We proved that caspase-3 activation is accurately monitored using the luciferase activity in the HEp-2 cells constitutively expressing cFluc-DEVD probe that we established, promising the probe to accurately trace chlamydial dynamics controlling apoptosis.Furthermore, we for the first time demonstrated that C. trachomatis can activate caspase-3 in the host cells at late time infection, followed by apoptosis. The finding give us a crucial hint for understanding complicated chlamydial dynamics in host cells.
FireTalkに選抜されoral発表(松尾の代理で山口が発表)。

Staphylococcus aureus prompts Escherichia coli survival under dry conditions: A potential threat from the viewpoint of nosocomial infection　　　　　　　　　　　　　　　
Torahiko Okubo, Tomoko Shimoda, Rika Yano, Shinji Nakamura, Junji Matsuo, Hiroyuki Yamaguchi
アメリカ微生物学会 asm microbe2018 (アトランタ) Poster Talk (Oral selected), Jun. 2018, English, Public symposium
乾燥環境に置かれた大腸菌が黄色ブドウ球菌と共存することでより長く生存することを突き止めた。またこの生存性が温度により制御可能であることも見いだした。AES12: Microbiology in the Built Environmentのトラックoral発表に選抜された。, [International presentation]

札幌地下歩行空間における空気中浮遊細菌叢の解析: 通行人は浮遊菌叢に影響を与える　　　　　　　　　　　　　　　
渡辺宜典, 大久保寅彦, 大崎敬子, 松尾淳司, 神谷茂, 山口博之
第91回日本細菌学会総会(福岡), Mar. 2018, Japanese, Oral presentation
[Domestic Conference]

病院で用いられる乾燥床材上における細菌間の鬩ぎ合い: 残存生菌数とATP量のギャップから紐解く細菌の生存戦略　　　　　　　　　　　　　　　
大久保寅彦, 中村眞二, 松尾淳司, 山口博之
第91回日本細菌学会総会(福岡), Mar. 2018, Japanese, Oral presentation
[Domestic Conference]

低酸素環境においてChlamydia trachomatisの感染動態を修飾する要因　　　　　　　　　　　　　　　
酒井昴平, 松尾淳司, 渡辺宣典, 大久保寅彦, 中村眞二, 山口博之
第91回日本細菌学会総会(福岡), Mar. 2018, Japanese, Oral presentation
[Domestic Conference]

繊毛虫による水系環境菌への薬剤耐性プラスミド伝達の促進作用　　　　　　　　　　　　　　　
松下瑞江, 松尾淳司, 大久保寅彦, 山口博之
第91回日本細菌学会総会(福岡), Mar. 2018, Japanese, Oral presentation
[Domestic Conference]

鬩ぎ合う原生生物と細菌から学ぶ　　　　　　　　　　　　　　　
山口 博之
国立感染症研究所 文化祭学友会企画 ランチョンシンポジウム 招待講演, Dec. 2017, Japanese, Public discourse
[Domestic Conference]

Interaction between protozoa and bacteria evokes a novel paradigm on understanding unseen life　　　　　　　　　　　　　　　
Hiroyuki Yamaguchi
TMU Medical Laboratory Forum (台北医科大学検査医学フォーラム) 招待講演, Dec. 2017, Invited oral presentation
[International presentation]

関係法規(感染症法)　　　　　　　　　　　　　　　
北海道大学

保健微生物学　　　　　　　　　　　　　　　
北海道大学

医療安全管理学(検体採取)　　　　　　　　　　　　　　　
北海道大学

微生物学実習　　　　　　　　　　　　　　　
北海道大学

臨床微生物学　　　　　　　　　　　　　　　
北海道大学

微生物学　　　　　　　　　　　　　　　
北海道大学

日常生活動作レベルが高頻度接触面の細菌動態に与える影響と感染リスク判断への応用
科学研究費助成事業
01 Apr. 2024 - 31 Mar. 2028
宇野 智子, 渡辺 玲奈, 山口 博之, 大久保 寅彦, 矢野 理香
日本学術振興会, 基盤研究(C), 札幌医科大学, 24K13649

Functional elucidation of novel effectors of Legionella pneumophila approached through interactions with unique ciliate
Grants-in-Aid for Scientific Research
01 Apr. 2022 - 31 Mar. 2025
大久保 寅彦, 中村 眞二, 山口 博之
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 22K07051

New development of research to elucidate the intracellular adaptation mechanism of genital chlamydia unraveled from its similarity to cancer cells
Grants-in-Aid for Scientific Research
Apr. 2021 - Mar. 2025
山口 博之 Thapa Jeewan 中村眞二 大久保寅彦
性感染症の主な原因である細胞内寄生性細菌クラミジア(Chlamydia trachomatis: Ct)の細胞内への適応機構は、未だ遺伝子改変ができないこともあって明らかになっていない。そこでCt感染細胞と癌細胞との類似性[1. PI3K-AKT経路を活性化、2. 通常酸素分圧下でミトコンドリアを要求する、3. 癌抑制因子として知られる芳香族炭素水素受容体転写因子(AHR)のリガンドであるインドールにてその発育が抑制される、4. 北大既存薬(3200剤)/オリジナル(2640剤)ライブラリーのスクリーニングで、癌の進行を促進するG蛋白共役胆汁酸受容体(TGR5)やHIVインテグラーゼの阻害剤などがクラミジアの増殖を完全に阻止した]から、Ctが細胞内で利用している新たな細胞内情報伝達経路とそれら修飾に関わる分泌エフェクター分子を同定すると共に、それら分子の中から癌治療の標的となりうる分子を探ることを目的とする。その結果、初年度の実験にて、以下の成果を得た。
1. Ct感染細胞では低酸素状態にて感染後30分でAKT(Ser473)リン酸化が起こり、持続することを見いだした。
2. インドールがCtが細胞内で増殖を抑制し、その機構として芳香族炭化水素受容体AhR分子が関与する可能性を示唆する結果を得た。
3. Ctの通常酸素下でのミトコンドリアの要求性は、NOX4/p38MAPKと関連していることを明らかにした。4. 北大既存薬ライブラリーでヒットした12薬剤についてその有効性について検証し11薬剤がCtの細胞内発育を有意に抑制することを確認し、KEGG解析にてCtが利用すると予想される新規の22の情報伝達系を同定した。
5. 北大オリジナルライブラリーでヒットした1薬剤についてその有効性について検証し、KEGG解析にてCtが利用すると予想される新規標的分子候補を同定した。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, Principal investigator, 21H02726

入院患者の日常生活動作レベルがベッド周辺環境の微生物汚染に及ぼす影響
科学研究費助成事業
01 Apr. 2020 - 31 Mar. 2024
宇野 智子, 矢野 理香, 山口 博之, 大久保 寅彦
本研究は、患者の周辺環境への接触とその接触面での細菌の生存を具体的な患者の日常生活動作レベルを精査し、適切な看護実践（環境整備）へ応用することを目指す。
医療施設において、病原体は手指やその他の媒介物による接触を介して、感受性のある人に移動する可能性があるため病原体で汚染された環境表面の接触伝播経路に着目した。特に患者や医療従事者の手指が接触する機会が多い、ベッド柵やオーバーベッドテーブル等の患者周辺環境に着目した環境の調査研究を計画した。
2020年度は、新型コロナウイルス感染症の影響を受け、患者が療養している環境での調査が困難となった。そこで、2021年度は新型コロナウイルス感染症が発生する前に調査した結果から交絡因子を検討し、患者周辺の環境微生物汚染度と日常生活動作レベルの差異との関連について、準実験研究を用いて検討する計画へ変更した。
準実験研究の目的は、模擬病室内の患者周辺環境において、日常生活動作レベルの差異と模擬患者の手が触れる接触面積やアデノシン３リン酸（ATP）値との関連を検討した。模擬患者（12名）に先行研究で看護師の動作が分析されている車いすへの移乗を依頼した。その際のベッド周辺環境への接触面積やATP値を測定した。日常生活動作レベルの差異と模擬患者の手が触れる接触面積には相関関係がなかった。しかし、ATPで評価した環境微生物汚染度は、日常生活動作レベルが高くなるほど高値を示した。本結果は、模擬患者の日常生活動作レベルの差異とベッド周辺環境の汚染度に関連がある可能性を示唆したと考える。
日本学術振興会, 基盤研究(C), 札幌医科大学, 20K10626

Understanding and application of microbiota in building environment: creation of pathogen control theory by temperature control
Grants-in-Aid for Scientific Research Challenging Research (Pioneering)
Jul. 2020 - Mar. 2024
山口 博之, 矢野 理香, 大森 亮介, 大久保 寅彦
消毒剤や抗菌剤に頼らない温度による新たな病原体制御理論を創成し、空間や高頻度接触面の温度を制御することで、感染予防へと応用を目指すために、2年目は以下の研究を実施しいくつかの成果を得た。
1. 乾燥表面の温度調節による大腸菌の生存性の制御に湿度変化が与える影響: 私達は乾燥面を37℃付近に温めることでその乾燥麺に塗抹した細菌の生存性が顕著に低下することを見いだした。その一方で環境温度は、熱を奪う空気中の水蒸気量すなわち湿度の影響も受ける。そこで恒温恒湿機を使用し，温度(25-37℃)と共に湿度(45-90%)が乾燥表面の大腸菌の生存性に与える影響を調査した。その結果湿度と大腸菌の生存率には逆相関関係(r=-0.241)があり，湿度が上がるほど生存率は有意に低下した(p=0.04)。このように、乾燥面での温度制御において湿度によるネガティブな効果は最小限であることを見いだした。
2. 温度制御手摺デバイス上での細菌の生存性の可視化法の開発: バイク用ハンドヒーターを改良し作成したデバイスの効果を振れ幅の大きい培養に頼らず正確かつ簡便に確認する方法を、無蛍光の透明テープとLIVE/DEAD染色による測定系とキーエンス画像解析ソフトを組み合わせることで実現した。具体的には、ヒーターより距離が離れるほど手摺上の生存菌数は有意に低下し、その効果は温度ヒートマップと一致した。
3. 土壌細菌の空間移動に環境要因の変化が及ぼす影響について: 新型コロナ感染症の影響で公共の閉鎖環境での採材ができなかったので、その代替えとして３Dプリンターを用いて空気中に浮遊する細菌を効率よく生け捕りにできるエアサンプラーを用いて北大農場にて実施した。その結果、環境因子(気圧、蒸気圧、湿度、風向き)が連動し変化することにより、空気中に巻き上げられ浮遊し移動することが明らかになった。
Japan Society for the Promotion of Science, Challenging Research (Pioneering), Hokkaido University, Principal investigator, 20K20613

Host DNA damage responses and the regulations in chlamydial infections
Grants-in-Aid for Scientific Research
01 Apr. 2019 - 31 Mar. 2022
MATSUO Junji
To understand host cell responses induced by an obligate intracellular bacterium, Chlamydia, we tried to investigate Chlamydia-induced host DNA damages and the regulations. As a result, host DNA damages were detected at late stage of Chlamydia infections. Although we also assessed host DNA damage inductions induced by Chlamydia using knockdown cells, obvious differences were not seen in these cells. On the other hands, when expressions of genes on DNA damage responses were analyzed in Chlamydia-infected cells using qPCR array, upregulated or downregulated genes were identified. Thus, host DNA damage responses may be regulated in Chlamydia infections.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Health Sciences University of Hokkaido, 19K08934

第51回内藤記念科学奨励金研究助成　　　　　　　　　　　　　　　
Oct. 2019 - Oct. 2020
山口 博之
クラミジアの感染細胞内での修飾機構の解明研究を通して癌細胞制御の新たな創薬に関わる分子基盤を明らかにすることを目的とする。具体的には低酸素での侵襲型クラミジアの細胞内増殖促進を統御する分泌エフェクターを同定し、感染細胞を生存に導くPI3K-AKT経路に関わる分子群との相互作用を明らかにする。さらにドラッグライブラリーからクラミジアの増殖抑制としてPI3K-AKTを標的とする新規抗癌剤候補を探索する。
Principal investigator, Competitive research funding

Methods for the analysis and control of biomolecules in living cells based on molecular imaging
Grants-in-Aid for Scientific Research
30 May 2014 - 31 Mar. 2019
Ozawa Takeaki
In order to understand the processes of molecules in living cells as a network, we have established a new analytical method for visualizing and manipulating biomolecules in living cells. We have developed 1) a method for visualizing and quantifying a small number of biomolecules, 2) a method for controlling enzyme activity by external light, and 3) a photomanipulation method for controlling G protein-coupled receptor activity. The developed method will be a basic technology for wide fields of life science and an innovative technology in the fields of medicine, agriculture and drug discovery.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (S), The University of Tokyo, 26220805

アメーバ共生細菌原始クラミジアのレジオネラ撃退に関わる分子マシナリーの探索 研究課題　　　　　　　　　　　　　　　
Apr. 2016 - Mar. 2019
山口 博之
私達が札幌の土壌より独自に株化した原始的なクラミジア(難培養性細菌ネオクラミジア: Neochlamydia S13株)が共生するアメーバは、アメーバの天敵レジオネラ(Legionella pneumophila JR32やLp01株)を撃退しその感染を許さない。これ迄の検討からこの撃退現象には、共生細菌ゲノム上の一つのキメラ様遺伝子peg2639 (セリン・スレオニンキナーゼ: C末端約260アミノ酸残基のキナーゼ部分とNCBIデータベースに全くヒットしないN末端約400アミノ酸残基をコードする)の発現増加と、宿主アメーバのアクチン重合の安定化が必要であることを見つけた。その一方で、これらの知見は、レジオネラ撃退現象そのものにまだ直接結びついていない。一体、ネオクラミジアは、このキナーゼを組み込んだどのような分子マシナリーを駆使し、宿主アメーバからの天敵レジオネラを排除しているのだろうか。そこでネオクラミジアpeg2639遺伝子の機能解析を手掛かりとし、レジオネラ撃退機構に関わる分子マシナリーの全容解明を3年間の予定で行う。本年度は、さまざまな発現宿主(大腸菌)を試しpeg2639遺伝子組換えタンパク発現系を構築した。またネオクラミジア共生アメーバでは共生細菌依存的にアクチンの局在が変化する可能性を示唆するデータを共焦点レーザー観察を介して見いだした。さらにこの共生アメーバが大腸菌やサルモネラといったヒト病原細菌を背負い寒天上を運ぶことを生物学的な解析とSEMにて発見した。
文部科学省(日本学術振興会)(挑戦的萌芽研究), Principal investigator, Competitive research funding

腟菌叢とメタボローム: クラミジア卵管線維化機構とPID診断バイオマーカーの探索　　　　　　　　　　　　　　　
科学研究補助金(基盤研究B)
Apr. 2016 - Mar. 2019
山口 博之
本邦における健康女性の子宮頸管部へのクラミジア(Chlamydia trachomatis)の感染率は、性的行動が活発な20歳台に限ってみると約10%と極めて高い。多くのケースは無症候性であり、無治療患者の約半数で、卵管へと上向性に感染は拡大し、骨盤内炎症性疾患(PID)を発症する。さらに20%程度で線維化に伴い卵管は閉塞し不妊となる。そこで本研究では、これまでの基盤研究を踏まえ、クラミジア感染に伴う不妊を未然に防ぐことを最終目的とし、クラミジア感染時に線維化を促進する菌側・宿主側因子と腟・尿・唾液からのPID診断バイオマーカーの探索を行うこととした。初年度(平成28年度)は、臨床材料(腟スワブ)からクラミジアの検出を行うと共に腟症の程度をNugent Scoreにて評価した。さらに菌叢解析も実施した。その結果、273検体を精査した結果、21検体でクラミジアが陽性であった。そのscoreと検体中の乳酸菌数との関連性について検討した結果、スコアの上昇と共に乳酸菌数は有意に減少したので、算定されたスコアの妥当性が確認できた。そこでNugent Scoreに沿って3グループに分け、クラミジアの検出頻度との関連性を検討した。しかしながらクラミジアの感染頻度と腟症との関連性は見出せなかった。一方、48検体について菌叢解析を行った。その結果、腟症がほとんど認められなかった検体において、クラミジア陽性検体にて、有意に腸内細菌科のOTU数が増加していることを見つけた。この結果は、腟症が認められない健常者において、肛門経由で膣内にインドール産生性の腸内細菌科が混入することで、クラミジアの生存性が高まることで、感染頻度を押し上げている可能性を示唆している。また線維化を加速する低酸素条件下でのクラミジアの培養系の構築がほぼ完了した。
日本学術振興会, Principal investigator, Competitive research funding

Effect of bacterial signal molecule AI-2 on the metabolism and pathogenicity of protozoa
Grants-in-Aid for Scientific Research
01 Apr. 2016 - 31 Mar. 2018
Okubo Torahiko, YAMAGUCHI Hiroyuki, MATSUO Junji, NAKAMURA Shinji, MATSUSHITA Mizue, AKIZAWA Kouji, HAYASAKA Kasumi, FUKUMOTO Tatsuya, IWASAKI Sumio
AI-2 is a bacterial signal molecule which is used for bacterial closs talk. In our previous studies, we reported that co-cultivation of bacteria and protozoa causes the raise of AI-2 concentration in the solution. However, the effect of AI-2 on protozoa was not reported. In this study, we compared phenotypic characteristics of protozoa under the presence or absence of AI-2. Our results showed that AI-2 causes the raise of mobility of amoeba cells in a concentraion dependent manner on solid agar surfaces. This suggests that amoeba can detect AI-2 concentration and control its mobility. We showed cross-Kingdom effect of AI-2 on protozoa for the first time.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, 16K19113

Regulations of inflammatory responses induced by Chlamydia
Grants-in-Aid for Scientific Research
01 Apr. 2015 - 31 Mar. 2018
Matsuo Junji
To understand host inflammatory responses induced by Chlamydia, we tried to identify a novel receptor of host cells and chlamydial factors, which are conserved in the Chlamydiales. As a result, a non-TLR receptor and a heat-labile chlamydial factor may be involved in host inflammatory responses. In addition, the responses were enhanced in low oxygen environments. On the other hands, host caspase-3 dynamics was revealed using a cyclic fire luciferase probe. We also showed that miRNA regulation may be involved in the host inflammatory responses induced by Chlamydia.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 15K09561

Molecular basis of the intracellular life of bacteria within eukaryotic cells
Grants-in-Aid for Scientific Research
01 Apr. 2011 - 31 Mar. 2016
NAGAI Hiroki, YAMAGUCHI Hiroyuki, MARUYAMA Fumito, Xuan Thanh Bui
Primary endosymbiosis, in which bacteria enter (pre-)eukaryotic cells and finally became mitochondria and chloroplasts, plays a critical role in eukaryogenesis. Even today, bacterial invasion into eukaryotic cells takes place every day at sites of infection. In this study, we focused on the relationship between various kinds of intracellular pathogenic/environmental bacteria and their eukaryotic host cells including free-living Acanthamoeba and human cells. We concluded several mechanistic principles which drive the first step of matryoshka-type evolution.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Osaka University, 23117002

原始クラミジアが共生するアメーバは何故レジオネラの感染から回避できるのか　　　　　　　　　　　　　　　
Apr. 2014 - Mar. 2016
山口 博之
自然環境に広く生息するアカントアメーバ(以下アメーバ)の約10%程度に難培養性細菌が共生する。私達は、レジオネラ(Legionella)の感染を阻止する難培養性細菌が共生するアメーバを見つけ、その共生基盤を明らかにするために本研究を行った。その結果、この共生細菌は、レジオレラの分泌装置(T4ASS)分子群を感知し、宿主アメーバの貪食機構への修飾作用が、この撃退現象に関与することを発見した。また共生細菌の責任分子候補としてセリンスレオニンキナーゼをコードするキメラ様遺伝子(peg2639)を同定した。
文部科学省(日本学術振興会)(挑戦的萌芽研究), Principal investigator, Competitive research funding

Search of the predictive diagnosis marker of the Chlamydia infection, using a pathological experiment.
Grants-in-Aid for Scientific Research
01 Apr. 2013 - 31 Mar. 2015
NAKAMURA Shinji, MATSUO Junji, ISHIZU Akihiro, YAMAGUCHI Hiroyuki, ITO Shin
Chlamydia trachomatis is the most common sexually transmitted pathogen that the approximately 10% of infection are occurred. Leaving the chlamydia infection untreated, leading to complication such as tubal obstruction and fibrosis in part infected person is known, but the reason is unclear. In this study, we investigated the expression of inflammatory mediators including IL-1, IL-Ra, IL-6 and PGE2 to find a cause of complications, especially fibrosis by chlamydial infections. The positive cells of IL-1, IL-1Ra, and PGE2 were appeared in interstitial infiltrating cells, but there were no signification differences between infected patients with chlamydial and not infected patients. However, in chlamydia infected patient, squamous epithelium and columnar epithelium epithelium of the cervix. and glandular epithelia was celled IL-1 alpha and strong expressions of PGE2. The results suggest that PGE2 plays an important role in the precipitating factor of complication by chlamydia infection.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Juntendo University, 25670278

原始クラミジアエフェクターの機能解析から紐解く病原体の新規制御システムの探索
科学研究費助成事業
01 Apr. 2013 - 31 Mar. 2014
山口 博之
日本学術振興会, 基盤研究(B), 北海道大学, 25293103

アメーバに共生する難培養性細菌のゲノム解析から紐解くマトリョーシカ進化原理
科学研究費補助金(新学術領域研究(研究領域提案型))
2012 - 2013
山口 博之
微生物間の共生機構の解明研究は、個々の微生物の進化や生態さらに病原体と私達の体を構成する細胞とのせめぎ合いを通して起こる病態形成機構を知る上で極めて重要である。そこで私達は土壌や水系環境に普遍的に生息するアカント・アメーバ(アメーバ)に共生する難培養性細菌に着目し、自然環境から難培養性細菌が持続的に感染するアメーバ株の樹立し共生細菌の特徴について検討してきた。特に興味深い難培養性細菌は原始的な姿を留めたクラミジアであり、それらに焦点を絞り研究を進めている。平成24年度は株化したアメーバに共生する原始クラミジア(Neochlamydia S13)のゲノム解読を次世代シークエンサーにより行い、そのドラフトゲノムより共生や宿主細胞の修飾に関わる遺伝子やそのユニークな構造を探索し詳細に解析した。その結果、ゲノム構造は既に知られている原始クラミジアProtochlamydia UWE25(Science, 2004)と大きく異なり、Neochlamydia S13のゲノム構造は極めてユニークであり、TCAサイクルが欠落しそれに伴い呼吸鎖の多くの遺伝子が脱落してることを見つけた。興味深いことに原始クラミジアは通常IV型分泌装置をコードする遺伝子クラスターを保持しているが、Neochlamydia S13にはその構造が認められなかった。また私達はこのNeochlamydia S13ゲノム上に多数の蛋白蛋白相互作用に関わると予想されるロイシンrichリピートやアンキリンドメインを持つ新規機能候補分子や膜貫通領域を複数持つエフェクター分子候補を見つけた。さらに通常クラミジアには認められない多数のトランスポゾンをコードする遺伝子を発見した。現在DNAマイクロアレイ解析により発現遺伝子のプロファイリングを行っている。これらの研究成果は現在論文化するとともに投稿準備を進めている。
文部科学省, 新学術領域研究(研究領域提案型), 北海道大学, Principal investigator, Competitive research funding, 24117501

アメーバ共生細菌プロトクラミジアアンキリンエフェクターの機能解析
科学研究費補助金(挑戦的萌芽研究)
2012 - 2013
山口 博之
以下の2点についてこれ迄に明らかにした。
1. 札幌の河川水より分離・株化したアメーバに共生する偏性細胞内寄生性細菌Protochlamydia R18株のドラフトゲノムを決定し、比較ゲノム解析より蛋白蛋白相互作用に深く関わるアンキリン(Ank)モチーフやロイシンリッチリピート(LRR)を持つ分子をコードする計38つの遺伝子を同定した。
2. 同定したAnkやLRRを持つ分子をコードする共生細菌の遺伝子がアメーバ内で実際に発現していることをRT-PCRにて確認した。現在、Protochlamydia R18ドラフトゲノム情報を基にカスタマイズしたDNAマイクロアレイを用いてアメーバ内での共生細菌の遺伝子発現変化の網羅的な解析を進めている。
文部科学省, 挑戦的萌芽研究, 北海道大学, Principal investigator, Competitive research funding, 24659194

Molecular mechanism of pathogenic chlamydial infection to lymphocytes
Grants-in-Aid for Scientific Research(基盤研究(C))
2009 - 2011
Hiroyuki YAMAGUCHI, 松尾 淳司, Shigeru KAMIYA, Shinji NAKAMURA, Junji MATSUO
We assessed pathogenic chlamydial infection mechanism to lymphocytes when compared to epithelial cells. As a result, we found the unique chlamydial attachment process on lymphocytes, independent of heparin, which has a critical on bacterial general attachment to cells. It was also confirmed by using DNA microarray that pathogenic chlamydial survival in lymphocytes was controlled by granzyme K. Furthermore, we demonstrated that lymphocytes could provide a shelter for pathogenic chlamydiae to escape from IFNγ. Thus, pathogenic chlamydial infection mechanism to lymphocytes is unique when compared to epithelial cells, possibly connecting clinical significance.
Ministry of Education, Culture, Sports, Science and Technology, 基盤研究(C), 北海道大学, Principal investigator, Competitive research funding, 21590474

Clinical studies, immunological analysis, and antigen structure analysis about Alloiococcus otitidis
Grants-in-Aid for Scientific Research
2009 - 2010
HARIMAYA Atsushi, FUJII Nobuhiro, YOKOTA Shin-Ichi, IIDA Masahiro, HIMI Tetsuo, MONTEIRO Mario A., ALAR Sharif, YAMAGUCHI Hiroyuki, YLIKOSKI Jukka, MATTILA Petri, TARKKANEN Jussi
Optimal growth condition of Alloiococcus otitidis was analyzed and the frequency of this organism was investigated. In addition, host immune response against Alloiococcus otitidis was investigated and the antigen structure of the organism was analyzed.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), 21791627

Chlamydia pneumoniae persistent infection and its mechanism
Grants-in-Aid for Scientific Research(基盤研究(C))
2005 - 2006
Hiroyuki YAMAGUCHI, Yoshimasa YAMAMOTO, Sunao KAWANO
Current studies have revealed that the obligate intracellular bacterium Chlamydia (Chlamydophila) pneumoniae is associated not only with respiratory diseases, but also with chronic diseases such as atherosclerosis. The detection of the C. pneumoniae DNA or antigen in coronary atherosclerotic plaque and peripheral blood of the patients with cardiovascular diseases has strengthened the likelihood of its possible involvement in the pathogenesis of atherosclerosis. Moreover, several studies indicate that viable C. pneumoniae can be detected in atherosclerotic plaques from patients of advanced age with coronary heart disease by both culture and RT-PCR methods. Our recent studies also showed that viable C. pneumoniae are readily detectable in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors, indicating that the presence of viable bacteria in the blood stream might be a risk factor for the development of atherosclerosis. C. pneumoniae preferentially infects respiratory tract epithelial cells as well as macrophages. On the other hand, our previous studies revealed that lymphocyte is another host cell permitting C. pneumoniae infection with a persistent nature. Since lymphocytes are a major immune cell type besides macrophages in the development of atherosclerosis, interaction between lymphocytes and this pathogen may contribute to the pathogenesis of chronic inflammatory diseases associated with C. pneumoniae. We first attempted to assess a possible association between development of diabetes in non-obese diabetic (NOD) mice and dissemination of C. pneumoniae from lung to peripheral blood. By real-time reverse transcription-polymerase chain reaction (RT-PCR) with primers for C. pneumoniae 16S rRNA, following multiple intranasal inoculations,. bacteria in lung were detected in NOD mice with diabetes (38.5 %) as well as ICR mice (40 %), but prevalence of bacteria in NOD mice without diabetes (Pre-diabetic NOD mice and non-diabetic retired NOD mice) was very low (4.8 %).The bacteria were only detected in peripheral blood mononuclear cells (PBMCs) cultured withhydrocortisone of the NOD mice with diabetes (53.8 %). Results of immunostaining with fluorescein isothiocyanate-conjugated anti-chlamydia monoclonal antibody also showed the presence of bacterial antigens m the lungs and the PBMCs judged as positive by the RT-PCR. The results clearly showed that the development of diabetes in NOD mouse appears to be one of critical factors for promoting the dissemination of C. pneumoniae from lung to peripheral blood. Second, we examined a possible alteration of CD3 and CD25 expressions of human lymphocytes [Molt-4 cells and enriched lymphocytes from peripheral blood mononuclear cells (PBMCs)] by C. pneumoniae infection. The expression levels of both molecules of Molt-4 cells were significantly decreased by C. pneumoniae infection. C. pneumoniae clinical isolates also caused the alteration of both expressions of the infected cells. In contrast, C. trachomatis did not cause any alteration of both expressions of Molt-4 cells. Heat-killed bacteria and HEp-2 lysate as mock did not cause any alteration of CD3 expression of lymphocytes. Addition of either NS-398 (Cox-2 inhibitor) or AH-23848 (EP4 prostanoid receptorantagonist) tothe culture abolishedthe alteration of CD3 expression ofthe infected cells. The enhanced prostaglandin E_2 (PGE_2) productions in the culture supernatant of infected cells were also confirmed by competitiveELISA. C. pneumoniae infection of enriched lymphocytesfromPBMCs also causedanalteration of CD3 expression.Thus, C. pneumoniae infection of human lymphocytes induces an alteration of CD3 expression mediated by PGE_2 production. Since CD3 molecule has a major role in the signal transduction following antigen recognitions, such alteration may be involved in C.pneumoniae persistent infection of lymphocytes. Also, we foundthat 2-amino-phenoxazine 3-one (phenoxazine derivate: Phx-3)inhibits C. pneumoniae replication in human monocytic cells as well as epithelial cells, partially depending on the tryptophan-metabolic pathway of host cells.
Ministry of Education, Culture, Sports, Science and Technology, 基盤研究(C), 大阪大学->北海道大学, Principal investigator, Competitive research funding, 17590391

Chlamydia pneumoniae dissemination and atherosclerosis
Grants-in-Aid for Scientific Research(基盤研究(C))
2003 - 2004
Hiroyuki YAMAGUCHI, Shigeru KAMIYA, Takako OSAKI, Haruhiko TAGUCHI
Current studies have revealed that the obligate intracellular bacterium Chlamydia (Chlamydophila) pneumoniae is associated not only with respiratory diseases but also with atherosclerosis. In the present study, to understand the viability and growth potential of C.pneumoniae detected in blood of healthy persons, we first attempted to detect C.pneumoniae transcript in peripheral blood mononuclear cells (PBMCs) obtained from 70 healthy donors by real-time RT-PCR method. The presence of C.pneumoniae transcript in PBMCs from blood of healthy human donors was assessed by real-time RT-PCR-using primers for C.pneumoniae 16S rRNA. Out of 70 donors (18.5 %) showed the presence of bacterial transcript in cultured PBMCs. Prevalence of bacterial detection and bacterial numbers were significantly increased in PBMNC cultures incubated with cycloheximide. These results showed that viable C.pneumoniae might be present in healthy human PBMCs. Although several studies reported C.pneumoniae systemic dissemination from lung in animal model by DNA-based qualitative methods, the lack of a suitable animal model for permitting viable C.pneumonia dissemination from lung to blood has impeded the understanding of how viable C.pneumoniae can reach blood vessels and trigger the development of atherosclerosis. Therefore, we next studied the susceptibility of non-obese diabetic (NOD) mice to dissemination of viable C.pneumoniae from lung to blood, as detected by real-time RT-PCR. Following multiple intranasal inoculations, bacteria in lung were detected in NOD mice with diabetes (38.5%, n=13) as well as ICR mice (40%, n=10), but prevalence of bacteria in NOD mice without diabetes [Pre-diabetic NOD mice (n=8) and non-diabetic retired NOD mice (n=13)] was very low (5.2%, n=21). The bacteria were only detected in cultured PBMCs (53.8 %) of the NOD mice with diabetes. The prevalence of bacterial detection appeared to be increased in PBMC cultures incubated with hydrocortisone. Cultivation of C.pneumoniae from cultured PBMCs from all NOD mice was unsuccessful. Results of immunostaining with fluorescein isothiocyanate-conjugated anti-Chlamydia monoclonal antibody also showed the presence of bacterial antigens in the lungs and the PBMCs judged as positive by the RT-PCR. Thus, diabetic NOD mouse is sensitive to C.pneumoniae infection in which a possible dissemination of viable bacteria from lung to blood, and the animal model established in this study may be useful for understanding dissemination of C.pneumoniae infection.
Ministry of Education, Culture, Sports, Science and Technology, 基盤研究(C), 杏林大学->大阪大学, Principal investigator, Competitive research funding, 15590399

Pathogenic role of heat shock protein of Helicobacter pylori and host immune response to the heat shock protein
Grants-in-Aid for Scientific Research
2000 - 2001
KAMIYA Shigeru, YAMAGUCHI Hiroyuki, TAGUCHI Haruhiko
By using purified heat shock protein 60 (HSP60) of Helicobacter pylori and fusion protein of HSP60 with beta-galactosidase, it was shown that H. pylori HSP60 stimulated a secretion of interleukin-8 (IL-8) which is closely related with the induction of gastric mucosal inflammation from gastric epithelial cells. In addition, purified HSP60 induced apoptosis of gastric epithelial cells. These results indicate that HSP60 of H. pylori might play an important role in pathogenesis following H. pylori infection.
An epitope reactable with monoclonal antibody (Mab) to HSP60 was detected on the surface of gastric epithelial cells. Immune response to H. pylori HSP60 was stronger in the sera of the patients with H. pylori infection than in those without H. pylori infection. Not only fusion protein of HSP60 with beta-galactosidase but also oligopeptide pH9 reactable with the Mab showed a protective effect on colonization of H. pylori in mice. These results suggest that HSP60 of H. pylori might be a good candidate as a component of vaccine
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Kyorin University, 12670265

細菌病原因子としての分子シャペロン-DnaK およびGroEL による病原細菌表層構造構築の分子機構-
科学研究費助成事業
1999 - 1999
山本 友子, 花輪 智子, 山口 博之, 神谷 茂
(1)Listeria monocytogenes Dnak シャペロンの細胞表層構築機構における役割: L. Monocytogenes の分子シャペロンDnaK 変異株では、分子サイズ30kDa の細胞壁主要成分が欠失していた。この蛋白質のN末端アミノ酸配列ならびにそれに基づいた遺伝子のクローニングと決定された塩基配列から、この蛋白質はべん毛の主成分フラジェリンであることを明らかにした。さらに、フラジェリン遺伝子の発現を解析し、DnaKはフラジェリン遺伝子の転写を制御していることを明らかにした。又この変異株は、マクロファージによる貧食効率が顕著に低下していたが、DnaKはフラジェリンの発現を制御することにより貧食に関わっていると考えられる。
(2) Helicobacter pylori の表層発現型GroEL の役割: H. Pylori のGroEL に対するモノクローナル抗体を作製し、これらを用いてGroEL が細菌表層に発現していることを明らかにした。さらにこの表層局在GroELが、胃上皮細胞の付着に関与するとともに、炎症性サイトカインIL8産生誘導を引き起こすこと、又、SPFマウスに胃上皮に炎症をひきおこすことを明らかにした。本研究結果は、表層局在GroELが本菌の病原因子のひとつである可能性が示唆するものである。
日本学術振興会, 特定領域研究(A), 杏林大学, 11153225

無菌マウスを用いた腸管出血性大腸菌感染症・病態解析モデルの開発
科学研究費助成事業
1997 - 1998
大崎 敬子, 山口 博之
感染モデルの作成:無菌マウス(IQI/jic,8w,メス)に腸管出血性大腸菌(EHEC)O157:H7志賀様毒素SLT-I,SLT-II陽性)を経口投与することにより、感染モデルを作成した(昨年度報告)。感染マウスの病理所見では腸管において出血・炎症像が観察され,腎臓では尿細管の壊死と軽度ながらもボーマン嚢の炎症像,小脳ではプルキンエ細胞の脱落と不均一な配列,さらに大脳では神経細胞の壊死が観察された。これらの症状はヒトのEHEC感染症にみられる症状と同様のもので、感染モデルとしての有用性を示すものと考えられた。
プロバイオテクスを用いた予防および治療効果の検討:EHEC感染の予防として防としてC.butyricum588株の前投与は有用であることを昨年度報告した。今年度はEHEC感染の後にC.butyricum588株を用いて治療効果があるかどうかを検討した。EHECを経口感染(10^8cfu/mouse)後2日目にC.butyricum588株約10^9を経口投与したところ単独感染ではマウスの生残が0%となる感染8日目においても50%が生残し,12日目には37.5%の生残であった。また糞便内EHEC菌数の消長においても抑制効果が著明で感染6日目に単独群において10^<9.25>cfu/gfeces,投与群では10^<8.30>cfu/gfecesであった。
能動および受動免疫の効果:能動免疫として不活化EHEC菌体と不活化SLT-IおよびSLT-IIを経口免疫した後に,EHECを感染させた群では,対照群と同様にすべてのマウスが感染5日までに死亡した。受動免疫は家兎抗SLT-I,SLT-II血清を、EHEC感染と同日より3回投与する群と感染2日前より6回投与する群を作成した。3回免疫群では感染10日後に50%生残,6回免疫群では同じく10日後に100%の生残率であった。以上の結果,受動免疫によってEHEC感染マウスの死亡率を低下させる効果を示すことができた。
日本学術振興会, 萌芽的研究, 杏林大学, 09877060

Study on pathogenecity of Helicobacter pylori in gastroduodenum using germ-free mice.
Grants-in-Aid for Scientific Research
1997 - 1998
KAMIYA Shigeru, OSAKI Takako, YAMAGUCHI Hiroyuki, TAGUCHI Haruhiko
Germ-free (GF) mouse was orally infected with Helicobacter pylon TK1402 strain (10^<8-9> cfu/ 0.5 ml, three times). Persistent infection of H.pylon in gastric mucosa of the mouse was demonstrated from 1-9 weeks after the infection, and infiltration of inflammatory cells and partial atrophy of gastric mucosa was detected. These results indicate that GF mouse is a useful model for persistent H.pylon infection.
Heat shock protein 60 (HSP6O) of H.pylon was shown to be associated with adhesion of H.pylon to gastric epithelial cells. Pretreatment of H.pylon with anti-HSP6O monoclonal antibody resulted in a significant decrease of the number of h. pylon colonized in gastric mucosa of CF mouse.
Administration of HSP6O into gastric mucosa of GF mouse induced no significant gastric damage. In contrast, oral administration of HSP6O followed by challenge of H.pylon induced severe inflammatory change in the gastric mucosa. Interestingly, the number of H.pylon colonized was significantly lower by adminstration of HSP6O than that of control. Administration of imidazole derivatives or probiotics (Lactobacillus salivanius, Clostnidium butynicum) cured the persistent infection with H.pylon in CF mouse. These results indicate that GF mouse is also a useful model for evaluation of bactericidal drug or bacteriotherapy for H.pylon infection.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), KYORIN UNIVERSITY, 09670297

ヘリコバクター・ピロリ熱ショック蛋白の胃・十二指腸慢性炎症反応における役割
科学研究費補助金(奨励研究(A))
1997 - 1998
山口 博之
1. H.pyloriHSP60の発現と病原因子との関連性(1) H.pyloriHSP60の発現と病原因子との関連性:本菌のHSP60の発現、ウレアーゼ活性、空胞化毒素産生性およびヒト胃癌細胞株への付着率との間の相関性を検討した結果、本菌HSP60の発現と付着率との間に相関性が認められることを明らかにした(J.Gastroenterol.,1998,33:6-9)。(2) H.pylonHSP60の局在:本菌HSP60に対するH20モノクローナル抗体(mAb)によりH.pyloriの免疫電顕を行った。その結果、H20mAbは菌体内部のみならず菌体表層とも反応することから、本菌HSP60が菌体表層にも存在することを明らかにした(Microbiol.lmmunol.,1997,41:909-916)。(3) 本菌HSP60に対するH20モノクローナル抗体(mAb)によるH.pyloriのヒト胃癌由来細胞MKN4S株への付着阻止効果の検討:H20mAbでH.pylonを前処理することにより本菌のMKN45細胞への付着は阻止された。この結果は本菌のHSP60がヒト胃細胞への付着に直接関与していることを明らかにした(J.Med.Microbiol.,1997,46:825-831)。2. H.pyloriHSP60(1) H.pyloriHSP60を認識するmAbのエピトープの決定とそれに対するヒト免疫応答:以前確立したH.pyloriHSP60を認識するmAbのエピトープを決定し、その領域がヒトから種々の細菌HSP60に至まで広く保存されていることを明らかにした。またこの領域に対する免疫応答が感染防御に関与している可能性を示す結果も得ている。(2) H.pyloriHSP60のヒト胃癌由来細胞からのIL-8誘導能:アフィニティー精製したH.pyloriHSP60によりヒト胃癌由来細胞を刺激した結果、IL-8の誘導が確認された(J.Gastroenterol.,inpress)。またリコンビナント(r)H.pyloriHSP60の刺激においても同様な結果が得られた。これらの結果より本菌HSP60はIL-8の誘導に関与することが明らかになった。(3)rH.pyloriHSP60投与マウスにおける胃内病理学的変化の検討:rH.pyloriHSP60をコレラ毒素とともにC57BL/6マウスに経口投与したが胃内病理学的変化は認められなかった.しかしながらrH.pyloriHSP60とコレラ毒素を経口免疫した同系マウスにH.pyloriを感染させたところ顕著なびらん形成が確認された.
文部科学省, 奨励研究(A), 杏林大学, Principal investigator, Competitive research funding, 09770191

Role of Stress Protein on Bacterial Infection
Grants-in-Aid for Scientific Research
1996 - 1998
YAMAMOTO Tomoko, YAMAGUCHI Hiroyuki, TAGUCHI Haruhiko, HANAWA Tomoko
All organisms respond to unfavorable conditions such as stressful environment by the rapid and transient acceleration in the synthesis of a group of proteins called the stress proteins. For pathogenic bacteria, the intracellular environment of macrophages is one of the most hostile environments. Therefore, the bacteria in the phagocytes may respond to the hostile stimuli by the expression of the stress proteins, and those pro. teiris may put the intracellular bacteria at advantages in the survival in the phagocytes. To elucidate the role of bacterial stress protein on the survival in the macrophages and expression of virulence, we studied the stress response and the role of stress proteins of Yersinia enterocolitica and Listeria monocytogenes within macrophages after phagocytosis. The results are as follows ; (1) The Y.enterocolitica gsrLAMBDA was identified as essential for protecting cells under both extracellular environmental stress and intracellular stress in macrophages due to phagocytosis. The gsrLAMBDA encodes a basic 49.5 kDa protein which is a periplasmic protease. (2) The GsrA stress protein was induced by macrophage phagocytosis. (3) L.monocylogenes could grow in macrophages without the induction of stress proteins. (4) The dnaK gene was cloned and subjected to the molecular analysis to understand the role of stress protein DnaK for intracellular survival of L.monocylogenes . (5) The DnaK does not largely contribute to the survival of L.inonocytogenes in macrophage cells but is involved in the step of the phagocytosis. From these results, it is speculated that common mechanisms with the stress proteins would contribute to the expression of the bacterial pathogen esis besides the species-specific mechanisms.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Kyorin University, 08670317

Analysis for the mechanism of gastric mucosal damage by Helicobacter pylori infection
Grants-in-Aid for Scientific Research
1995 - 1996
KAMIYA Shigeru, OSAKI Takako, YAMAGUCHI Hiroyuki
Among various virulence factors of Helicobacter pylori, adhesion factor, vacuolating toxin (VT), cytokines (particularly, interleukin-8 (IL-8)), heat shock protein (HSP) and active oxygen were focused to analyze the mechanism of gastric mucasal damage by H.pylori infection.
According to flowcytometric (FCM) analysis, it was shown that H.pylori strains adhere more effectively to gastric epithelial cells than intestinal cells. In addition, H.pylori has hemagglutinating (HA) activity with erythrocytes of guinea pig, rabbit, human, sheep, chicken, horse and bovine. It was also demonstrated that HA activity of H.pylori strains may correlate with adhesion activity to gastric cells. About half of H.pylori strains produce VT.Culture supernates of VT-producing H.pylori strains inhibited secretion of gastric acid by guinea pig parietal cells, but that of VT-non-producing strains not. By analysis of 2nd messengers (Ca^<2+> and cyclic AMP) in signal transduction for secretion of gastric acid, it was suggested that VT may directly inhibit proton pump H^+-K^+- ATPase. It was shown that H.pylori induces secretion of IL-8 by gastric epithelial cells following its adhesion to the cells. Similarly, it was demonstrated that co-incubation of human peripheral neutrophils with H.pylori resulted in a generation of active oxygen (superoxide). However, neither IL-8 nor active oxygen was induced by culture supernates of H.pylori strains. HSP60 (molecular weight ; 60kDa) was equally detected in 10 H.pylori strains examined in immunoblot analysis. On the other hand, HSP60 on the surface of H.pylori was differently expressed among H.pylori strains in FCM analysis. In addition, the expression rate of HSP60 on the surface correlated with adhesion activity of H.pylori strains. The epitope reactable with monoclonal antibody against H.pylori HSP60 was detected on the surface of gastric epithelial cells, suggesting that HSP60 might be a trigger for chronic inflammation or mucosal damage in gastric tissue.
These results indicate that adhesion factor, VT,cytokines, active oxygen and HSP60 are all closely associated with the occurrence of gastric mucosal damages, and the mechanism for the mucosal damage secms to be multifactorial.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Kyorin University, 07670322

Helicobacter pylori熱ショック蛋白の精製,製造解析と発現機構
科学研究費補助金(奨励研究(A))
1995 - 1995
山口 博之
1.H. pylori HSP60の精製と構造解析(1)H. Pylori HSP60を確認するマウスモノクローナル抗体(mAb)の確立:H. Pylori HSP60の精製を目的として本菌HSP60 を認識するmAbの作製を試みた。BALB/cマウスへの投与抗原にはSDS-PAGEのゲルより回収したH. Pylori 60kDa蛋白抗原を用いた。またこの抗原を固相化したELISAによりスクリーニングを行った。その結果、2つのmAb(H9およびH20)を確立した。H9およびH20mAbのクラスおよびサブクラスはそれぞれlgG2aおよびlgMであった。どちらのmAbも熱ショック応答により大量に誘導された本菌60kDa抗原を認識したことより,H. Pylori HSP60を認識していると考えられた。(2)H. Pylori HSP60を抗原構造:ELISAおよびイムノブロット法においてH9およびH20mAbはH. Pyloriと反応したが,FACSにおいてはH20mAbのみが反応した。これらの結果より,どちらのmAbも未変性および変性したどちらの状態のHSP60も認識した。またH20mAbの認識するエピトープは本菌菌体表層に存在しているが,H9mAbの認識するエピトープは菌体内に存在していると考えられた。2.H. Pylori HSP60とヒト胃粘膜組織中HSP60とエピトープの相同性についての検討(1)H. Pylori HSP60を認識する3C8mAbの認識するエピトープのアミノ酸配列:既に確立していたYersinia enterocolitica HSP60を認識するmAb,3C8がH. Pylori HSP60とヒト胃癌由来細胞株と反応することを明らかにした(J.Gastroenterol. 1996 inpress)。また本3C8mAbの認識するエピトープのアミノ酸配列にLue Gly Valの3つのアミノ酸が関与することを明らかにした(Microbiol. lmmunol. 40(1) 77-80 1996)。(2)H. Pylori HSP60を認識するH9およびH20mAbの反応性:どちらのmAbもH. Pylori HSP60およびヒト胃癌由来細胞株MKN45と反応性を示した。しかしながらH9mAbはEsherichia coli, Shigella sonnei, Salmonella enteritidis, Vibrio cholerae, Pseudomonas aeruginosaと反応したがH20mAbは反応しなかった。これらの結果より,H. Pylori HSP60とヒト胃細胞に特異的なエピトープの存在する可能性が示唆された。3.H. Pylori HSP60と病原因子との関連性H. Pylori HSP60の発現と病原因子との相関性:本菌のHSP60の発現,ウレアーゼ活性,空胞化毒素産生性およびヒト胃癌由来細胞株への付着率との間の相関性について検討した結果,本菌HSP60の発現と付着率との間に相関性が認められることを明らかにした(J.Med.Microbiol. 1996 inpress)。
文部科学省, 奨励研究(A), 杏林大学, Principal investigator, Competitive research funding, 07770205

Studies on the Bacterial Stress Proteins as Virulence Fctors
Grants-in-Aid for Scientific Research
1994 - 1995
YAMAMOTO Tomoko, HANAWA Tomoko, YAMAGUCHI Hiroyuki, TAGUCHI Haruhiko
Yersinia enterocolitica is a facultative intracellular pathogen which is able to resist the microbicidal mechanisms of macrophages and to grow within phagocytic cells. Some bacteria including Y.enterocolitical have been shown to respond to the hostile environment in macrophages by producing a set of stress proteins which are also induced by environmental stresses. To understand the role of stress proteins on the intracellular survival of bacteriawe identified and cloned a Y.enterocolitica gene, celled gsrA (global stress requirement). The gsrA gene was identified because its insertional inactivation by a transposon resulted in the inability of the organism to grow at an elevated temperature and to survive within macrophages after phagocytosis. The gsrA gene was sequenced and shown to encode for a basic, 49500 Da protein. The GsrA protein shows significant amino acid sequence homology to the HtrA stress protein which was originally indentified in Escherichia coli. Furthermore, the genetically defined Y.enterocolitica gsrA mutant was constructed and characterized. The insertional mutation of gsrA resulted in the inhibition of growth at temperatures above 39ﾟC and the grealty increased susceptibility to oxidative and osmotic stresses. The mutant additionally lost the ability to survive and replicate within macrophages. These results, taken together, indicate that the gsrA gene is an essential component of the protection mechanism employed by Y.enterocolitica, allowing it to respond against the intracellular stress found in macrophages as well as extracellular environmental stress.
Japan Society for the Promotion of Science, Grant-in-Aid for General Scientific Research (C), Kyorin University, 06670305

Purification and characterization of cytotoxin produced by H.phlori, and mechanisms for the occurrence of gastroduodenal diseases by H.pylori infection.
Grants-in-Aid for Scientific Research
1993 - 1994
KAMIYA Shigeru, YAMAGUCHI Hiroyuki, SHIRAI Takayuki
To examine association of Helicobacter pylori infection with the occurrence of gastroduodenal diseases, isolation of H.phlori from patients with the diseases was carried out. Isolation rates of H.pylori from the patients with gastric ulcer, duodenal ulcer and atrophic gastritis were 83,85 and 75%, respectively. In contrast, those from the patients with acute superficial gastritis and submucosal tumour were 25 and 33%, respectively. Sensitivity, specificity, positive^- and negative^-predictive values of rapid urease test of biopsy specimens to cultivation method were 76,85,91 and 63%, respectively, indicating that the rapid urease test is an useful screening test. By quantitative culture, it was shown that 5,000 - 10,000 CFU of H.phlori were required for positive reaction in the urease test.
Nearly half strains of H.phlori produce cytotoxin (CT) which induces vacuolation in cultured cells. It was demonstrated that CT was heat-labile at 70ﾟC and proteinous toxin with molecular weight of more than 20kDa. After precipitation of CT with 50% ammonium sulphate, purification of CT was tried using Sephacryl S300, phenylsuperose, Q Sepharose FF and Mono Q columns. However, highly purified CT was not obtained by these procedures. Positive rates for detection of CT using RK-13 (rabbit kidney), FL (human amnion), Vero (monkey kidney), BHK-21 (hamster kidney) and HeLa (human uterine cancer) cells were 73,61,27,27 and 21%, respectively, indicating that RK-13 cell line is a good indicator.
Adherence activity of H.phlori strains to human gastric cancer (MKN45, KATO III) cells and human intestine (Int-407) cells was analyzed by flowcytometer. Average adherence rates of H.pylori straius to MKN 45, KATO III and Int-407 cells were 76.6,42.7 and 15.1%, respectively. These results suggest that H.pylori has an affinity to gastric cells, particularly MKN 45 cells, rather than instinal cells. No significant correlation between adherence activity of and CT production by H.pylori was observed.
Japan Society for the Promotion of Science, Grant-in-Aid for General Scientific Research (C), 05807022

Studies on the Bacterial Stress Proteins as Virulence Factors
Grants-in-Aid for Scientific Research
1992 - 1993
YAMAMOTO T., YAMAGUCHI H., TAGUCHI H., OGATA S
Yersinia enterrocolitica is a facultative intracellular pathogen capable of surviving within professional phagocytic cells. Since the internal environment is stressful for the phagocytosed bacteria, the bateria responds by induction of stress proteins which possibly contribute to intracellular survival. To identify the stress-responding genes important for intracellular survival, a total 1800 mutants of Y.enterrocolitica with independent Tn10-insertion were screened for the stress-sennsitive mutations that allow normal growth at 30ﾟC but either block or result in very poor bacterial growth at high temperature, 39ﾟC.The resulting 42 mutants with thermosensitive phenotype for vacterial growth were then screened for the mutation with a diminished capacity for intracellular survival using an in vitro asay for survival in macrophagges. A mutant named Gts 154 unable to survive within macrophages was less virulent than the wild-type parent in vivo. Theresult suggest the stress-responding gene at the stress envitonment in vitro involves in the survival and growth in the macrophage which is an imoportant aspect of virulence of Y.enterocolitica. We then studied a coordinate regulation in gene expression of Y.enterrocolitica which possibly contributes to bacterial survival within phagocytes. At least 16 proteins were selectively induced in response to phagocytosis. Several macrophage-induced proteins were also induced by heat shock and oxidative stresses in vitro. Of those, two major stress proteins, DnaK and CRPA, were immunologically identified. These results indicated that intracellular Y.enterocolitica exhibits a global stress respponse to the hostille environment of the macrophage.
Japan Society for the Promotion of Science, Grant-in-Aid for General Scientific Research (C), Kyorin University, 04670252