研究者データベース
| 山口 博之(ヤマグチ ヒロユキ) |
| 保健科学研究院 保健科学部門 病態解析学分野 |
| 教授 |
Last Updated :2024/05/09
研究活動情報
論文
- Ayano Konno, Torahiko Okubo, Yoshiaki Enoeda, Tomoko Uno, Toyotaka Sato, Shin-Ichi Yokota, Rika Yano, Hiroyuki YamaguchiPloS one 18 9 e0291765 2023年09月 [査読有り]
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. - Saicheng Zhang, Yuki Funahashi, Satoho Tanaka, Torahiko Okubo, Jeewan Thapa, Shinji Nakamura, Hideaki Higashi, Hiroyuki YamaguchiMicrobes and Infection 25 5 105097 - 105097 2023年06月 [査読有り]
- Ryoya Tsujikawa, Jeewan Thapa, Torahiko Okubo, Shinji Nakamura, Saicheng Zhang, Yoshikazu Furuta, Hideaki Higashi, Hiroyuki YamaguchiCurrent Microbiology 79 9 2022年09月 [査読有り]
- Jeewan Thapa, Gen Yoshiiri, Koki Ito, Torahiko Okubo, Shinji Nakamura, Yoshikazu Furuta, Hideaki Higashi, Hiroyuki YamaguchiFrontiers in Cellular and Infection Microbiology 12 2022年05月26日 [査読有り]
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 (Mtd) 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 Mtd 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. - Airi Kawashiro, Torahiko Okubo, Shinji Nakamura, Jeewan Thapa, Masaki Miyake, Hiroyuki YamaguchiMicrobiology (Reading, England) 167 8 2021年08月 [査読有り]
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. - 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 YamaguchiResearch in Microbiology 103864 - 103864 2021年07月 [査読有り]
- Miho Okude, Junji Matsuo, Tomohiro Yamazaki, Kentaro Saito, Yoshokazu Furuta, Shinji Nakamura, Jeewan Thapa, Torahiko Okubo, Hideaki Higashi, Hiroyuki YamaguchiMicrobiology and Immunology 65 3 115 - 124 2020年12月27日 [査読有り]
- Jeewan Thapa, Kent Hashimoto, Saori Sugawara, Ryoya Tsujikawa, Torahiko Okubo, Shinji Nakamura, Hiroyuki YamaguchiMicrobes and infection 22 9 441 - 450 2020年05月19日 [査読有り][通常論文]
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. - Junya Yamagishi, Kyoko Hayashida, Junji Matsuo, Torahiko Okubo, Makoto Kuroda, Hiroki Nagai, Tsuyoshi Sekizuka, Hiroyuki Yamaguchi, Chihiro SugimotoJournal of human genetics 65 1 41 - 48 2020年01月 [査読有り][通常論文]
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 strainsBMC Infectious Diseases 20 53 2020年01月 [査読有り][通常論文]
- Okubo T, Hasegawa T, Fukuda A, Thapa J, Usui M, Tamura Y, Yamaguchi HInternational journal of antimicrobial agents 54 6 831 - 833 2019年08月 [査読有り][通常論文]
- Rika Yano, Torahiko Okubo, Tomoko Shimoda, Junji Matsuo, Hiroyuki YamaguchiBMC medical education 19 1 53 - 53 2019年02月11日 [査読有り][通常論文]
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. - Kudo H, Usui M, Nagafuji W, Oka K, Takahashi M, Yamaguchi H, Tamura YThe Journal of antibiotics 72 2 79 - 85 2019年02月 [査読有り][通常論文]
- Tomoko Shimoda, Torahiko Okubo, Yoshiki Enoeda, Rika Yano, Shinji Nakamura, Jeewan Thapa, Hiroyuki YamaguchiPloS one 14 12 e0226952 2019年 [査読有り][通常論文]
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 hypoxiaMatsuo J, Sakai K, Okubo T, Yamaguchi HAPMIS in press 2018年12月 [査読有り][通常論文]
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。 - Matsuo J, Haga S, Hashimoto K, Okubo T, Ozawa T, Ozaki M, Yamaguchi HCanadian journal of microbiology 65 2 135 - 143 2018年10月 [査読有り][通常論文]
カスパーゼ-3基質配列挿入化学発光(ルシフェラーゼ)プローブを発現する細胞株を確立し、それを利用したアポトーシス検知系を用いて、クラミジア(性器クラミジア)が感染後期にカスパーゼ依存的にアポトーシスを誘導することを明らかにしました。 - Matsushita M, Okubo T, Hasegawa T, Matsuo J, Watanabe T, Iwasaki S, Fukumoto T, Hayasaka K, Akizawa K, Shimizu C, Yamaguchi HMicrobiology and immunology 62 11 720 - 728 2018年10月 [査読有り][通常論文]
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. - Sakai K, Matsuo J, Watanabe T, Okubo T, Nakamura S, Yamaguchi HJournal of microbiological methods 153 84 - 91 2018年09月 [査読有り][通常論文]
MIC-101ガス置換型チャンバー(Billups-Rothenberg製)を用いた培養系を確立し、低酸素での網羅的遺伝子発現解析を含めクラミジアの感染実験を再現性良く実施するための、最適条件を決定した。低酸素(2%)では、通常酸素条件下で使用する細胞濃度では、栄養分の枯渇が急速に起こり、それに伴い細胞内でのクラミジアの増殖も抑制される。またDMEMよりRPMI培養液の方が、その枯渇するスピードが緩やかであった。またそれら条件で精査した結果、網羅的遺伝子発現解析にてクラミジアの感染が線維化促進因子CTGFの発現を促進することを見つけた。 - Taki K, Watanabe T, Matsuo J, Sakai K, Okubo T, Matsushita M, Abe K, Minami K, Yamaguchi HJournal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy 24 10 815 - 821 2018年08月 [査読有り][通常論文]
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. - Torahiko Okubo, Mizue Matsushita, Shinji Nakamura, Junji Matsuo, Hiroki Nagai, Hiroyuki YamaguchiEnvironmental Microbiology Reports 10 3 344 - 354 2018年06月01日 [査読有り][通常論文]
Soil-borne amoeba Acanthamoeba S13WT has an endosymbiotic relationship with an environmental Neochlamydia bacterial strain. However, regardless of extensive experiments in liquid media, the biological advantage of the symbiosis remained elusive. We therefore explored the role of the endosymbiont in predator-prey interactions on solid media. A mixed culture of the symbiotic or aposymbiotic amoebae and GFP-expressing Escherichia coli or Salmonella Enteritidis was spotted onto the centre of a LB or B-CYE agar plate preinoculated with a ring of mCherry-expressing Legionella pneumophila (Legionella ‘wall’). The spread of the amoebae on the plate was assessed using a fluorescence imaging system or scanning electron microscopy. As a result, in contrast to the aposymbiotic amoebae, the symbiotic amoebae backpacked these GFP-expressing bacteria and formed flower-like fluorescence patterns in an anticlockwise direction. Other bacteria (Pseudomonas aeruginosa and Stenotrophomonas maltophilia), but not Staphylococcus aureus, were also backpacked by the symbiotic amoebae on LB agar, although lacked the movement to anticlockwise direction. Furthermore, in contrast to the aposymbiotic amoebae, the symbiotic amoebae backpacking the E. coli broke through the Legionella ‘wall’ on B-CYE agar plates. Thus, we concluded that Acanthamoeba S13WT required the Neochlamydia endosymbiont to backpack human pathogenic bacteria and resist Legionella infection on solid agar. - Chinatsu Maita, Mizue Matsushita, Masahiro Miyoshi, Torahiko Okubo, Shinji Nakamura, Junji Matsuo, Masaharu Takemura, Masaki Miyake, Hiroki Nagai, Hiroyuki YamaguchiMicrobes and Infection 20 4 236 - 244 2018年04月01日 [査読有り][通常論文]
Acanthamoeba isolated from environmental soil harbors the obligate intracellular symbiont Neochlamydia, which has a critical role in host amoebal defense against Legionella pneumophila infection. Here, by using morphological analysis with confocal laser scanning fluorescence microscopy and transmission electron microscopy, proteome analyses with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and liquid chromatography–mass spectrometry (LC/MS), and transcriptome analysis with DNA microarray, we explored the mechanism by which the Neochlamydia affected this defense. We observed that when rare uptake did occur, the symbiotic amoebae allowed Legionella to grow normally. However, the symbiotic amoebae had severely reduced uptake of Legionella when compared with the aposymbiotic amoebae. Also, in contrast to amoebae carrying the endosymbiont, the actin cytoskeleton was significantly disrupted by Legionella infection in aposymbiotic amoebae. Furthermore, despite Legionella exposure, there was little change in Neochlamydia gene expression. Taken together, we concluded that the endosymbiont, Neochlamydia prevents Legionella entry to the host amoeba, resulting in the host defense against Legionella infection. - Matsuo J, Nakamura S, Okubo T, Fukui M, Yamaguchi HParasitology research 117 3 937 - 941 2018年03月 [査読有り][通常論文]
A free-living amoeba, Naegleria is ubiquitously distributed in various natural environments. Since some Naegleria spp. are exclusively distributed in the Arctic and sub-Antarctic regions, we hypothesized that the amoeba may be useful to determine long-term survival of Naegleria in laboratory conditions at 4 A degrees C. The main objective of the study is to determine that a species of an environmental amoebal isolated can live at low temperatures after a long time. Here, we therefore show long-term survival of an amoeba, Naegleria polaris isolated from a sediment sample, which was collected from Antarctica 10 years ago, and since stored at 4 A degrees C. The sample was put on non-nutrient agar plates with heat-killed Escherichia coli, and then the plate was incubated at 4, 15, or 30 A degrees C. Motile amoebae were seen only when the plate was incubated at 15 A degrees C. The sequencing of ribosomal DNA including internal transcribed spacers (ITS) 1, 5.8S rDNA, and ITS2 region revealed the amoebae to be N. polaris, which is exclusively distributed in the Arctic and sub-Antarctic regions. Scanning electron microscopic observation showed that no typical sucker-like structure was seen on the surface of N. polaris, but the cysts were similar to those of Naegleria fowleri. Thus, our result shows, for the first time, that N. polaris can survive after 10 years of storage at 4 A degrees C. This finding may help us understand the still undescribed effects of environmental samples on viability of amoebae. - Kazuya Yamakawa, Junji Matsuo, Torahiko Okubo, Shinji Nakamura, Hiroyuki YamaguchiJournal of Infection and Chemotherapy 24 2 83 - 87 2018年02月01日 [査読有り][通常論文]
Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. Capsaicin, a component of chili pepper, which can stimulate actin remodeling via capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) and anti-inflammatory effects via PPARγ (peroxisome proliferator-activated receptor-γ) and LXRα (liver X receptor α), is a potential candidate to control chlamydial growth in host cells. We examined whether capsaicin could inhibit C. trachomatis growth in immortal human epithelial HeLa cells. Inclusion forming unit and quantitative PCR assays showed that capsaicin significantly inhibited bacterial growth in cells in a dose-dependent manner, even in the presence of cycloheximide, a eukaryotic protein synthesis inhibitor. Confocal microscopic and transmission electron microscopic observations revealed an obvious decrease in bacterial numbers to inclusions bodies formed in the cells. Although capsaicin can stimulate the apoptosis of cells, no increase in cleaved PARP (poly (ADP-ribose) polymerase), an apoptotic indicator, was observed at a working concentration. All of the drugs tested (capsazepine, a TRPV1 antagonist 5CPPSS-50, an LXRα inhibitor and T0070907, a PPARγ inhibitor) had no effect on chlamydial inhibition in the presence of capsaicin. In addition, we also confirmed that capsaicin inhibited Chlamydia pneumoniae growth, indicating a phenomena not specific to C. trachomatis. Thus, we conclude that capsaicin can block chlamydial growth without the requirement of host cell protein synthesis, but by another, yet to be defined, mechanism. - Watanabe T, Yamazaki S, Maita C, Matushita M, Matsuo J, Okubo T, Yamaguchi HEvolutionary bioinformatics online 14 1176934318788337 - 1176934318788337 2018年 [査読有り][通常論文]
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. - Yamaguchi Y, Okubo T, Matsushita M, Wataji M, Iwasaki S, Hayasaka K, Akizawa K, Matsuo J, Shimizu C, Yamaguchi HPeerJ 6 e5755 2018年 [査読有り][通常論文]
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. - Torahiko Okubo, Takako Osaki, Eriko Nozaki, Akira Uemura, Kouhei Sakai, Mizue Matushita, Junji Matsuo, Shinji Nakamura, Shigeru Kamiya, Hiroyuki YamaguchiPLOS ONE 12 9 e0184980 2017年09月 [査読有り][通常論文]
Although human occupancy is a source of airborne bacteria, the role of walkers on bacterial communities in built environments is poorly understood. Therefore, we visualized the impact of walker occupancy combined with other factors (temperature, humidity, atmospheric pressure, dust particles) on airborne bacterial features in the Sapporo underground pedestrian space in Sapporo, Japan. Air samples (n = 18; 4,800L/each sample) were collected at 8: 00 h to 20: 00 h on 3 days (regular sampling) and at early morning/late night (5: 50 h to 7: 50 h/22: 15 h to 24: 45 h) on a day (baseline sampling), and the number of CFUs (colony forming units) OTUs (operational taxonomic units) and other factors were determined. The results revealed that temperature, humidity, and atmospheric pressure changed with weather. The number of walkers increased greatly in the morning and evening on each regular sampling day, although total walker numbers did not differ significantly among regular sampling days. A slight increase in small dust particles (0.3-0.5 mu m) was observed on the days with higher temperature regardless of regular or baseline sampling. At the period on regular sampling, CFU levels varied irregularly among days, and the OTUs of 22-phylum types were observed, with the majority being from Firmicutes or Proteobacteria (gamma-), including Staphylococcus sp. derived from human individuals. The data obtained from regular samplings reveled that although no direct interaction of walker occupancy and airborne CFU and OTU features was observed upon Pearson's correlation analysis, cluster analysis indicated an obvious lineage consisting of walker occupancy, CFU numbers, OTU types, small dust particles, and seasonal factors (including temperature and humidity). Meanwhile, at the period on baseline sampling both walker and CFU numbers were similarly minimal. Taken together, the results revealed a positive correlation of walker occupancy with airborne bacteria that increased with increases in temperature and humidity in the presence of airborne small particles. Moreover, the results indicated that small dust particles at high temperature and humidity may be a crucial factor responsible for stabilizing the bacteria released from walkers in built environments. The findings presented herein advance our knowledge and understanding of the relationship between humans and bacterial communities in built environments, and will help improve public health in urban communities. - Rika Yano, Tomoko Shimoda, Reina Watanabe, Yasutoshi Kuroki, Torahiko Okubo, Shinji Nakamura, Junji Matsuo, Sadako Yoshimura, Hiroyuki YamaguchiJOURNAL OF INFECTION AND CHEMOTHERAPY 23 7 439 - 445 2017年07月 [査読有り][通常論文]
Previous works have demonstrated considerable variability in hospital cleanliness in Japan, suggesting that contamination is driven by factors that are currently poorly controlled. We undertook 16S rRNA sequence analysis to study population structures of hospital environmental microbiomes to see which factor(s) impacted contamination. One hundred forty-four samples were collected from surfaces of three hospitals with distinct sizes ("A": > 500 beds, "B": 100-500 beds, "C": < 100 beds). Sample locations of two ward types (Surgical and Internal) included patient room bed table (multiple) (4BT), patient overbed table (multiple) (4OT), patient room sink (multiple) (4S), patient room bed table (single) (SBT), patient overbed table (single) (SOT), patient room sink (single) (SS), nurse desk (ND), and nurse wagon (NW). Total DNA was extracted from each sample, and the 50 samples that yielded sufficient DNA were used for further 16S rRNA sequencing of hospital microbiome populations with cluster analysis. The number of assigned bacterial OTU populations was significantly decreased in hospital "C" compared to the other hospitals. Cluster analysis of sampling locations revealed that the population structure in almost all locations of hospital "C" and some locations in the other hospitals was very similar and unusually skewed with a family, Enterobacteriaceae. Interestingly, locations included patient area (4OT, 4BT, SBT) and nurse area (ND), with a device (NW) bridging the two and a place (4S and SS) shared between patients or visitors. We demonstrated diversity changes of hospital environmental microbiomes with a skewed population, presumably by medical staff pushing NWs or sinks shared by patients or visitors. (C) 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. - Torahiko Okubo, Mizue Matushita, Yukiko Ohara, Junji Matsuo, Satoshi Oguri, Tatsuya Fukumoto, Kasumi Hayasaka, Kouzi Akizawa, Hitoshi Shibuya, Chikara Shimizu, Hiroyuki YamaguchiINTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS 49 3 387 - 388 2017年03月 [査読有り][通常論文]
- Tatsuya Fukumoto, Junji Matsuo, Torahiko Okubo, Shinji Nakamura, Kentaro Miyamoto, Kentaro Oka, Motomichi Takahashi, Kouji Akizawa, Hitoshi Shibuya, Chikara Shimizu, Hiroyuki YamaguchiBMC MICROBIOLOGY 16 1 292 2016年12月 [査読有り][通常論文]
Background: Environmental chlamydiae belonging to the Parachlamydiaceae are obligate intracellular bacteria that infect Acanthamoeba, a free-living amoeba, and are a risk for hospital-acquired pneumonia. However, whether amoebae harboring environmental chlamydiae actually survive in hospital environments is unknown. We therefore isolated living amoebae with symbiotic chlamydiae from hospital environments. Results: One hundred smear samples were collected from Hokkaido University Hospital, Sapporo, Japan; 50 in winter (February to March, 2012) and 50 in summer (August, 2012), and used for the study. Acanthamoebae were isolated from the smear samples, and endosymbiotic chlamydial traits were assessed by infectivity, cytokine induction, and draft genomic analysis. From these, 23 amoebae were enriched on agar plates spread with heatkilled Escherichia coli. Amoeba prevalence was greater in the summer-collected samples (15/30, 50%) than those of the winter season (8/30, 26.7%), possibly indicating a seasonal variation (p = 0.096). Morphological assessment of cysts revealed 21 amoebae (21/23, 91%) to be Acanthamoeba, and cultures in PYG medium were established for 11 of these amoebae. Three amoebae contained environmental chlamydiae; however, only one amoeba (Acanthamoeba T4) with an environmental chlamydia (Protochlamydia W-9) was shown the infectious ability to Acanthamoeba C3 (reference amoebae). While Protochlamydia W-9 could infect C3 amoeba, it failed to replicate in immortal human epithelial, although exposure of HEp-2 cells to living bacteria induced the proinflammatory cytokine, IL-8. Comparative genome analysis with KEGG revealed similar genomic features compared with other Protochlamydia genomes (UWE25 and R18), except for a lack of genes encoding the type IV secretion system. Interestingly, resistance genes associated with several antibiotics and toxic compounds were identified. Conclusion: These findings are the first demonstration of the distribution in a hospital of a living Acanthamoeba carrying an endosymbiotic chlamydial pathogen. - Chinatsu Maita, Mizue Matushita, Torahiko Okubo, Junji Matsuo, Masaki Miyake, Hiroki Nagai, Hiroyuki YamaguchiGenome Announcements 4 4 2016年 [査読有り][通常論文]
We report here the draft genome sequences of two Legionella pneumophila variant strains (JR32 and Lp01_666) originally derived from a Philadelphia-1 clinical isolate, domesticated in Japan, with distinct susceptibility to amoebae. Detailed genomic analysis will allow us to better understand Legionella adaptation and survival mechanisms in host cells. - Tomoko Shimoda, Rika Yano, Shinji Nakamura, Mitsutaka Yoshida, Junji Matsuo, Sadako Yoshimura, Hiroyuki YamaguchiBMC research notes 8 807 - 807 2015年12月21日 [査読有り][通常論文]
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. - Tomohiro Yamazaki, Junji Matsuo, Satoshi Takahashi, Shouta Kumagai, Tomoko Shimoda, Kiyotaka Abe, Kunihiro Minami, Hiroyuki YamaguchiJournal of Infection and Chemotherapy 21 12 842 - 848 2015年12月01日 [査読有り][通常論文]
Although sexually transmitted disease due to Chlamydia trachomatis occurs similarly in both men and women, the female urogenital tract differs from that of males anatomically and physiologically, possibly leading to specific polymorphisms of the bacterial surface molecules. In the present study, we therefore characterized polymorphic features in a high-definition phylogenetic marker, polymorphic outer membrane protein (Pmp) F of C. trachomatis strains isolated from male urogenital tracts in Japan (Category: Japan-males, n = 12), when compared with those isolated from female cervical ducts in Japan (Category: Japan-females, n = 11), female cervical ducts in the other country (Category: Ref-females, n = 12) or homosexual male rectums in the other country (Category: Ref-males, n = 7), by general bioinformatics analysis tool with MAFFT software. As a result, phylogenetic reconstruction of the PmpF amino acid sequences showing three distinct clusters revealed that the Japan-males were limited into cluster 1 and 2, although there were only four clusters even though including an outgroup. Meanwhile, the phylogenetic distance values of PmpF passenger domain without hinge region, but not its full-length sequence, showed that the Japan-males were more stable and displayed less diversity when compared with the other categories, supported by the sequence conservation features. Thus, PmpF passenger domain is a useful phylogenetic maker, and the phylogenic features indicate that C. trachomatis strains isolated from male urogenital tracts in Japan may be unique, suggesting an adaptation depending on selective pressure, such as the presence or absence of microbial flora, furthermore possibly connecting to sexual differentiation. - Matsuo J, Nakamura S, Takeda S, Ishida K, Yamazaki T, Yoshida M, Chiba H, Hui SP, Yamaguchi HInfection and immunity 83 7 2917 - 2925 2015年07月 [査読有り][通常論文]
The obligate intracellular bacterium Chlamydia pneumoniae is not only a causative agent of community-acquired pneumonia but is also associated with a more serious chronic disease, asthma, which might be exacerbated by air pollution containing carbon nanoparticles. Although a detailed mechanism of exacerbation remains unknown, the proinflammatory cytokine interleukin- 1 beta (IL-1 beta) is a critical player in the pathogenesis of asthma. C. pneumoniae induces IL-1 beta in macrophages via NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activation and Toll-like receptor 2/4 (TLR2/4) stimulation. Carbon nanoparticles, such as carbon nanotubes (CNTs), can also evoke the NLRP3 inflammasome to trigger IL-1 beta secretion from lipopolysaccharide-primed macrophages. This study assessed whether costimulation of C. pneumoniae with CNTs synergistically enhanced IL-1 beta secretion from macrophages, and determined the molecular mechanism involved. Enhanced IL-1 beta secretion from C. pneumoniae-infected macrophages by CNTs was dose and time dependent. Transmission electron microscopy revealed that C. pneumoniae and CNTs were engulfed concurrently by macrophages. Inhibitors of actin polymerization or caspase-1, a component of the inflammasome, significantly blocked IL-1 beta secretion. Gene silencing using small interfering RNA (siRNA) targeting the NLRP3 gene also abolished IL-1 beta secretion. Other inhibitors (K+ efflux inhibitor, cathepsin B inhibitor, and reactive oxygen species-generating inhibitor) also blocked IL-1 beta secretion. Taken together, these findings demonstrated that CNTs synergistically enhanced IL-1 beta secretion from C. pneumoniae-infected macrophages via the NLRP3 inflammasome and caspase-1 activation, providing novel insight into our understanding of how C. pneumoniae infection can exacerbate asthma. - 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 2015年02月
- Chikayo Yamane, Tomohiro Yamazaki, Shinji Nakamura, Junji Matsuo, Kasumi Ishida, Sumire Yamazaki, Satoshi Oguri, Natsumi Shouji, Yasuhiro Hayashi, Mitsutaka Yoshida, Yimin, Hiroyuki YamaguchiPLOS ONE 10 2 e0116486 2015年02月 [査読有り][通常論文]
Ancient chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. However, how pathogenic chlamydiae adapted to mammalian cells that provide a stable niche at approximately 37 degrees C, remains unknown, although environmental chlamydiae have evolved as endosymbionts of lower eukaryotes in harsh niches of relatively low temperatures. Hence, we assessed whether an environmental chlamydia, Parachlamydia Bn-9, could grow in human HEp-2 cells at a low culture temperature of 30 degrees C. The assessment of inclusion formation by quantitative RT-PCR revealed that the numbers of bacterial inclusion bodies and the transcription level of 16SrRNA significantly increased after culture at 30 degrees C compared to at 37 degrees C. Confocal microscopy showed that the bacteria were located close to HEp-2 nuclei and were actively replicative. Transmission electron microscopy also revealed replicating bacteria consisting of reticular bodies, but with a few elementary bodies. Cytochalasin D and rifampicin inhibited inclusion formation. Lactacystin slightly inhibited bacterial inclusion formation. KEGG analysis using a draft genome sequence of the bacteria revealed that it possesses metabolic pathways almost identical to those of pathogenic chlamydia. Interestingly, comparative genomic analysis with pathogenic chlamydia revealed that the Parachlamydia similarly possess the genes encoding Type III secretion system, but lacking genes encoding inclusion membrane proteins (IncA to G) required for inclusion maturation. Taken together, we conclude that ancient chlamydiae had the potential to grow in human cells, but overcoming the thermal gap was a critical event for chlamydial adaptation to human cells. - Hiroyuki Yamaguchi, Junji Matsuo, Tomohiro Yamazaki, Kasumi Ishida, Kenji YagitaGenome Announcements 3 1 2015年 [査読有り][通常論文]
Here, we report the draft genome sequence of high-temperature-adapted Protochlamydia sp. strain HS-T3, an environmental chlamydia. This bacterium is an amoebal endosymbiont, found in Acanthamoeba isolated from a hot spring in Japan. Strain HS-T3 readily grew in mammalian cells at 37�C, a characteristic not previously reported for environmental chlamydiae. - Tomohiro Yamazaki, Junji Matsuo, Momoka Kikuchi, Kentaro Miyamoto, Kentaro Oka, Motomichi Takahashi, Satoshi Takahashi, Torahiko Okubo, Hiroyuki YamaguchiGenome Announcements 3 5 2015年 [査読有り][通常論文]
We report the draft genome sequence of Chlamydia trachomatis strain 54, isolated from the urogenital tract of a male in Japan, with unique polymorphic membrane proteins. Detailed genomic analysis will aid our understanding of the selective pressures that lead to sexual differentiation in chlamydial adaptive evolution. - Satoshi Oguri, Tomoko Hanawa, Junji Matsuo, Kasumi Ishida, Tomohiro Yamazaki, Shinji Nakamura, Torahiko Okubo, Tatsuya Fukumoto, Kouzi Akizawa, Chikara Shimizu, Shigeru Kamiya, Hiroyuki YamaguchiJOURNAL OF GENERAL AND APPLIED MICROBIOLOGY 61 5 203 - 210 2015年 [査読有り][通常論文]
We have previously demonstrated conjugation of Escherichia coli into vacuoles of the protozoal ciliate (Tetrahymena thermophila). This indicated a possible role of ciliates in evoking bacterial quorum sensing, directly connecting bacterial survival via accumulation in the ciliate vacuoles. We therefore assessed if ciliates promoted bacterial autoinducer (AI)-2 accumulation with vacuole formation, which controls quorum sensing. E. coli AI-2 accumulation was significantly enhanced in the supernatants of a mixed culture of ciliates and bacteria, likely depending on ciliate density rather than bacterial concentration. As expected, AI-2 production was significantly correlated with vacuole formation. The experiment with E. coli luxS mutants showed that ciliates failed to enhance bacterial AI-2 accumulation, denying a nonspecific phenomenon. Fluorescence microscopy revealed accumulation of fragmented bacteria in ciliate vacuoles, and, more importantly, expulsion of the vacuoles containing disrupted bacteria into the culture supernatant. There was no increase in the expression of luxS (encoding AI-2) or ydgG (a transporter for controlling bacterial export of AI-2). We conclude that ciliates promote bacterial AI-2 accumulation in a mixed culture, via accumulation of disrupted bacteria in ciliate vacuoles followed by ex-pulsion of the vacuoles, independently of luxS or ydgG gene induction. This is believed to be the first demonstration of a relationship between E. coli AI-2 dynamics and ciliates. In the natural environment, ciliate biotopes may provide a survival advantage to bacteria inhabiting such biotopes, via evoking quorum sensing. - Kasumi Ishida, Junji Matsuo, Yoshimasa Yamamoto, Hiroyuki YamaguchiBMC MICROBIOLOGY 14 330 2014年12月 [査読有り][通常論文]
Background: Pathogenic chlamydiae are obligate intracellular pathogens and have adapted successfully to human cells, causing sexually transmitted diseases or pneumonia. Chlamydial outer protein N (CopN) is likely a critical effector protein secreted by the type III secretion system in chlamydiae, which manipulates host cells. However, the mechanisms of its action remain to be clarified. In this work, we aimed to identify previously unidentified CopN effector target in host cells. Results: We first performed a pull-down assay with recombinant glutathione S-transferase (GST) fusion CopN proteins (GST-CpCopN: Chlamydia pneumoniae TW183, GST-CtCopN: Chlamydia trachomatis D/UW-3/CX) as "bait" and soluble lysates obtained from human immortal epithelial HEp-2 cells as "prey", followed by SDS-PAGE with mass spectroscopy (MS). We found that a host cell protein specifically bound to GST-CpCopN, but not GST-CtCopN. MS revealed the host protein to be fructose bisphosphate aldolase A (aldolase A), which plays a key role in glycolytic metabolism. We also confirmed the role of aldolase A in chlamydia-infected HEp-2 cells by using two distinct experiments for gene knockdown with an siRNA specific to aldolase A transcripts, and for assessment of glycolytic enzyme gene expression levels. As a result, both the numbers of chlamydial inclusion-forming units and RpoD transcripts were increased in the chlamydia-infected aldolase A knockdown cells, as compared with the wild-type HEp-2 cells. Meanwhile, chlamydial infection tended to enhance expression of aldolase A. Conclusions: We discovered that one of the C. pneumoniae CopN targets is the glycolytic enzyme aldolase A. Sequestering aldolase A may be beneficial to bacterial growth in infected host cells. - Yamazaki T, Matsuo J, Nakamura S, Oguri S, Yamaguchi HJournal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy 20 7-8 460 - 464 2014年07月 [査読有り][通常論文]
Chlamydia trachomatis is an obligate intracellular bacterium that causes a sexually transmitted disease. Ureaplasma parvum is commensal in the human genital tract, with a minimal contribution to urogenital infection. We have recently found that U. parvum has a significant effect on the presence of C trachomatis in the genital tract of healthy women. We therefore assessed the effect of U. parvum co-incubation on C. trachomatis maturation from reticulate bodies (RBs) to elementary bodies (EBs) in HeLa cells in the absence or presence of interferon (IFN)-gamma, which is a critical host defense factor. IFN-gamma stimulation of viable U. parvum significantly prompted chlamydial growth with an increase in infectious particles, EBs, in HeLa cells. IFN-gamma treatment of killed U. parvum had a similar effect on C trachomatis maturation in HeLa cells. There was no change in expression of indoleamine 2,3-dioxygenase (IDO) in cultures of viable or killed U. parvum. We concluded that U. parvum co-incubation by IFN-gamma helped C trachomatis to mature from RBs to EBs in HeLa cells, independent of IDO expression. This suggests a novel survival strategy of C trachomatis against IFN-gamma exposure, prompting secondary infection of the genital mucosa, with possible clinical implications. (C) 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. - 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 YamaguchiPLOS ONE 9 4 e95166 2014年04月 [査読有り][通常論文]
Previous work has shown that the obligate intracellular amoebal endosymbiont Neochlamydia S13, an environmental chlamydia strain, has an amoebal infection rate of 100%, but does not cause amoebal lysis and lacks transferability to other host amoebae. The underlying mechanism for these observations remains unknown. In this study, we found that the host amoeba could completely evade Legionella infection. The draft genome sequence of Neochlamydia S13 revealed several defects in essential metabolic pathways, as well as unique molecules with leucine-rich repeats (LRRs) and ankyrin domains, responsible for protein-protein interaction. Neochlamydia S13 lacked an intact tricarboxylic acid cycle and had an incomplete respiratory chain. ADP/ATP translocases, ATP-binding cassette transporters, and secretion systems (types II and III) were well conserved, but no type IV secretion system was found. The number of outer membrane proteins (OmcB, PomS, 76-kDa protein, and OmpW) was limited. Interestingly, genes predicting unique proteins with LRRs (30 genes) or ankyrin domains (one gene) were identified. Furthermore, 33 transposases were found, possibly explaining the drastic genome modification. Taken together, the genomic features of Neochlamydia S13 explain the intimate interaction with the host amoeba to compensate for bacterial metabolic defects, and illuminate the role of the endosymbiont in the defense of the host amoebae against Legionella infection. - Reina Watanabe, Tomoko Shimoda, Rika Yano, Yasuhiro Hayashi, Shinji Nakamura, Junji Matsuo, Hiroyuki YamaguchiBMC research notes 7 121 - 121 2014年03月04日 [査読有り][通常論文]
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. - Aya Sampo, Junji Matsuo, Chikayo Yamane, Kenji Yagita, Shinji Nakamura, Natsumi Shouji, Yasuhiro Hayashi, Tomohiro Yamazaki, Mitsutaka Yoshida, Miho Kobayashi, Kasumi Ishida, Hiroyuki YamaguchiENVIRONMENTAL MICROBIOLOGY 16 2 486 - 497 2014年02月 [査読有り][通常論文]
- Ishida K, Kubo T, Saeki A, Yamane C, Matsuo J, Yimin, Nakamura S, Hayashi Y, Kunichika M, Yoshida M, Takahashi K, Hirai I, Yamamoto Y, Shibata K, Yamaguchi HMicrobes and infection / Institut Pasteur 15 3 192 - 200 3 2013年03月 [査読有り][通常論文]
Lymphocytes are a potential host cell for Chlamydophila pneumoniae, although why the bacteria must hide in lymphocytes remains unknown. Meanwhile, interferon (IFN)-gamma is a crucial factor for eliminating chlamydiae from infected cells through indoleamine 2,3-dioxygenase (IDO) expression, resulting in depletion of tryptophan. We therefore assessed if lymphocytes could work as a shelter for the bacteria to escape IFN-gamma. C. pneumoniae grew normally in human lymphoid Jurkat cells, even in the presence of IFN-gamma or under stimulation with phorbol myristate acetate plus ionomycin. Although Jurkat cells expressed IFN-gamma receptor CD119, their lack of IDO expression was confirmed by RT-PCR and western blotting. Also, C. pneumoniae survived in enriched human peripheral blood lymphocytes, even in the presence of IFN-gamma. Furthermore, C. pneumoniae in spleen cells obtained from IFN-gamma knockout mice with C57BL16 background was maintained in a similar way to wild-type mice, supporting a minimal role of]FN-gamma-related response for eliminating C. pneumoniae from lymphocytes. Thus, we concluded that IFN-gamma did not remove C. pneumoniae from lymphocytes, possibly providing a shelter for C. pneumoniae to escape from the innate immune response, which has direct clinical significance. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. - 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 YamaguchiPLOS ONE 8 2 e56005 2013年02月 [査読有り][通常論文]
Obligate amoebal endosymbiotic bacterium Protochlamydia with ancestral pathogenic chlamydial features evolved to survive within protist hosts, such as Acanthamoba, 0.7-1.4 billion years ago, but not within vertebrates including humans. This observation raises the possibility that interactions between Protochlamydia and human cells may result in a novel cytopathic effect, leading to new insights into host-parasite relationships. Previously, we reported that Protochlamydia induces apoptosis of the immortalized human cell line, HEp-2. In this study, we attempted to elucidate the molecular mechanism underlying this apoptosis. We first confirmed that, upon stimulation with the bacteria, poly (ADP-ribose) polymerase (PARP) was cleaved at an early stage in HEp-2 cells, which was dependent on the amount of bacteria. A pan-caspase inhibitor and both caspase-3 and -9 inhibitors similarly inhibited the apoptosis of HEp-2 cells. A decrease of the mitochondrial membrane potential was also confirmed. Furthermore, lactacystin, an inhibitor of chlamydial protease-like activity factor (CPAF), blocked the apoptosis. Cytochalasin D also inhibited the apoptosis, which was dependent on the drug concentration, indicating that bacterial entry into cells was required to induce apoptosis. Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis. We also confirmed that the Protochlamydia used in this study possessed a homologue of the cpaf gene and that two critical residues, histidine-101 and serine-499 of C. trachomatis CPAF in the active center, were conserved. Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis. More interestingly, because C. trachomatis infection can block the apoptosis, our finding implies unique features of CPAF between pathogenic and primitive chlamydiae. - Tatsuya Fukumoto, Junji Matsuo, Yasuhiro Hayashi, Satoshi Oguri, Shinji Nakamura, Yoshihiko Mizutani, Takashi Yao, Kouzi Akizawa, Haruki Suzuki, Chikara Shimizu, Kazuhiko Matsuno, Hiroyuki YamaguchiJournal of Clinical Microbiology 51 1 385 2013年01月 [査読有り][通常論文]
- Miho Okude, Junji Matsuo, Shinji Nakamura, Kouhei Kawaguchi, Yasuhiro Hayashi, Haruna Sakai, Mitsutaka Yoshida, Kaori Takahashi, Hiroyuki YamaguchiMICROBES AND ENVIRONMENTS 27 4 423 - 429 2012年12月 [査読有り][通常論文]
Symbiosis between living beings is an important driver of evolutionary novelty and ecological diversity; however, understanding the mechanisms underlying obligate mutualism remains a significant challenge. Regarding this, we have previously isolated two different Acanthamoeba strains harboring endosymbiotic bacteria, Protochlamydia (R18 symbiotic amoebae: R18WT) or Neochlamydia (S13 symbiotic amoebae; S13WT). In this study, we treated the symbiotic amoebae R18WT and S13WT with doxycycline (DOX) and rifampicin (RFP), respectively, to establish the aposymbiotic amoebae R18DOX and S13RFP, respectively. Subsequently, we compared the growth speed, motility, phagocytosis, pinocytosis, and morphology of the symbiotic and aposymbiotic amoebae. The growth speed of R18DOX was decreased, although that of S13RFP was increased. A marked change in motility was observed only for R18DOX amoebae. There was no difference in phagocytic and pinocytic activities between the symbiotic and aposymbiotic amoebae. Meanwhile, we observed a significant change in the phalloidin staining pattern and morphological changes in R18DOX (but not S13RFP) aposymbiotic amoebae, indicating a change in actin accumulation upon removal of the Protochlamydia. Infection of C3 (a reference strain) or S13RFP amoebae with Protochlamydia had a harmful effect on the host amoebae, but R18DOX amoebae re-infected with Protochlamydia showed recovery in both growth speed and motility. Taken together, we conclude that endosymbiont environmental chlamydiae alter the growth speed and/or motility of their host Acanthamoeba, possibly implying an close mutual relationship between amoebae and environmental chlamydiae. - Fuhito Hojo, Daisuke Sato, Junji Matsuo, Masaki Miyake, Shinji Nakamura, Miyuki Kunichika, Yasuhiro Hayashi, Mitsutaka Yoshida, Kaori Takahashi, Hiromu Takemura, Shigeru Kamiya, Hiroyuki YamaguchiAPPLIED AND ENVIRONMENTAL MICROBIOLOGY 78 15 5247 - 5257 2012年08月 [査読有り][通常論文]
When Tetrahymena ciliates are cultured with Legionella pneumophila, the ciliates expel bacteria packaged in free spherical pellets. Why the ciliates expel these pellets remains unclear. Hence, we determined the optimal conditions for pellet expulsion and assessed whether pellet expulsion contributes to the maintenance of growth and the survival of ciliates. When incubated with environmental L. pneumophila, the ciliates expelled the pellets maximally at 2 days after infection. Heat-killed bacteria failed to produce pellets from ciliates, and there was no obvious difference in pellet production among the ciliates or bacterial strains. Morphological studies assessing lipid accumulation showed that pellets contained tightly packed bacteria with rapid lipid accumulation and were composed of the layers of membranes; bacterial culturability in the pellets rapidly decreased, in contrast to what was seen in ciliate-free culture, although the bacteria maintained membrane integrity in the pellets. Furthermore, ciliates newly cultured with pellets were maintained and grew vigorously compared with those without pellets. In contrast, a human L. pneumophila isolate killed ciliates 7 days postinfection in a Dot/Icm-dependent manner, and pellets harboring this strain did not support ciliate growth. Also, pellets harboring the human isolate were resuscitated by coculturing with amoebae, depending on Dot/Icm expression. Thus, while ciliates expel pellet-packaged environmental L. pneumophila for stockpiling food, the pellets packaging the human isolate are harmful to ciliate survival, which may be of clinical significance. - Takeru Kubo, Kasumi Ishida, Junji Matsuo, Shinji Nakamura, Yasuhiro Hayashi, Haruna Sakai, Mitsutaka Yoshida, Kaori Takahashi, Itaru Hirai, Yoshimasa Yamamoto, Hiroyuki YamaguchiMICROBIAL PATHOGENESIS 53 1 1 - 11 2012年07月 [査読有り][通常論文]
Chlamydia trachomatis L2 invasively attacks lymphatic and subepithelial tissues of the genital tract during the formation of primary lesions. This subsequently results in lymphadenopathy, and suggests a greater propensity for systemic dissemination. However, whether lymphocytes are a potential vehicle cell for the dissemination of this infection remains unknown. We therefore assessed the growth properties of C trachomatis L2 in lymphoid Jurkat cells compared with those observed in epithelial HeLa cells. Both cells supported the growth of C trachomatis with a similar increase in infective progenies. Enriched human-blood lymphocytes also supported the C trachomatis growth as well as Jurkat cells. Bacteria infecting the Jurkat cells were more susceptible to antibiotics (doxycycline, azithromycin, ofloxacin) than those in HeLa cells. Of the sphingomyelin biosynthesis inhibitors tested, both myriocin and fumonisin B1 significantly inhibited bacterial growth in both cells types. A Jurkat cell mutant that impaired bacterial growth was established using ethylmethanesulfonate treatment. DNA microarray analysis with real-time reverse transcription-polymerase chain reaction revealed that the mutant cells over-expressed granzyme K gene. Immunofluorescence staining also indicated that granzyme K irregularly over-expressed among the mutant cells as compared with that of the wild cells, suggesting a possible mechanism refractory to C. trachomatis infection. Thus, we concluded that C. trachomatis 12 could infect Jurkat cells with lymphoid properties, providing a new tool for studying C. trachomatis dissemination to tissues via lymphocyte movement. (C) 2012 Elsevier Ltd. All rights reserved. - Yasuhiro Hayashi, Yimin, Junji Matsuo, Shinji Nakamura, Miyuki Kunichika, Mitsutaka Yoshida, Kaori Takahashi, Hiroyuki YamaguchiMICROBIOLOGY-SGM 158 1607 - 1614 2012年06月 [査読有り][通常論文]
Parachlamydia acanthamoebae is an obligate intracellular bacterium that infects free-living amoebae (Acanthamoeba), and is a potential human pathogen associated with hospital-acquired pneumonia. The attachment mechanism of this bacteria to host cells is crucial in bacterial pathogenesis, yet remains undetermined. Hence, we obtained monoclonal antibodies (mAbs) specific to either P. acanthamoebae or amoebae in an attempt to elucidate the attachment mechanism involved. Hybridomas of 954 clones were assessed, and we found that four mAbs (mAb38, mAb300, mAb311, mAb562) that were reactive to the amoebae significantly inhibited bacterial attachment. All mAbs recognized amoebal released molecules, and mAb311 also recognized the amoebal surface. mAbs reacted with the bacteria not only within amoebae, but also when they were released from amoebae (except mAb311). Furthermore, a serine protease inhibitor had an inhibitory effect on the bacterial attachment to amoebae, although none of the mAbs had any synergistic effect on the inhibition of attachment by the protease inhibitor. Taken together, we conclude that concurrent P. acanthoamebae attachment to amoebae is required for several amoebal released molecules and serine protease activity, implying the existence of a complicated host parasite relationship. - Tomohiro Yamazaki, Megumi Matsumoto, Junji Matsuo, Kiyotaka Abe, Kunihiro Minami, Hiroyuki YamaguchiBMC INFECTIOUS DISEASES 12 82 2012年04月 [査読有り][通常論文]
Background: Although Chlamydia trachomatis is the most commonly reported pathogen that causes urogenital infection such as urethritis or cervicitis, Ureaplasma parvum and Ureaplasma urealyticum, which are commensals in the genital tract, have also now been recognized as contributors to urogenital infection. However, whether the presence of either U. parvum or U. urealyticum is related to that of C. trachomatis in the urogenital tract remains unknown. We therefore attempted to estimate by PCR the prevalence of C. trachomatis, U. parvum and U. urealyticum in endocervical samples obtained from healthy women attending their first prenatal visit in Sapporo, Japan. Methods: The samples were taken from 303 apparently healthy women, and the extracted DNAs (n = 280) were used for PCR detection targeting C. trachomatis, U. parvum and U. urealyticum. Statistical analysis of the data was performed by Fisher's exact test. Results: PCR detection revealed that the prevalence of C. trachomatis, U. parvum and U. urealyticum was 14.3% (40/280), 41.7% (117/280) and 8.9% (25/280), respectively. C. trachomatis ompA genotype D was most frequently identified. Surprisingly, either C. trachomatis or Ureaplasma spp. was detected in almost half of the healthy women. Mixed infection of C. trachomatis with either U. parvum or U. urealyticum was also observed in 9.2% (26/280) of the women. There was a significant association between C. trachomatis and either U. parvum (p = 0.023) or Ureaplasma total (p = 0.013), but not U. urealyticum (p = 0.275). Conclusion: This study demonstrated that the presence of Ureaplasma had a significant effect on the presence of C. trachomatis in the genital tract of healthy women, suggesting that mixed infection is an important factor in bacterial pathogenesis in the genital tract. - Ishida K, Yamazaki T, Motohashi K, Kobayashi M, Matsuo J, Osaki T, Hanawa T, Kamiya S, Yamamoto Y, Yamaguchi HJournal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy 18 1 22 - 29 1 2012年02月 [査読有り][通常論文]
We have previously demonstrated that the steroid receptor antagonist mifepristone (RU486) causes growth inhibition of Chlamydophila pneumoniae by binding to and subsequently destroying the bacteria during their normal developmental cycle in epithelial HEp-2 cells. In the present study, we assessed the efficacy of treatment with RU486 against persistent C. pneumoniae infection in interferon (IFN)gamma-treated HEp-2 cells. Assessment of bacterial growth modification, the number of infectious progenies, the formation of inclusions, and the expressions of the C. pneumoniae genes 16S rRNA and hsp60 were investigated in cells with or without IFN gamma stimulation in the presence of RU486, using an inclusion-forming unit (IFU) assay, fluorescence microscopic analysis, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Our results indicated that RU486 treatment produced growth inhibition and an absence of C. pneumoniae gene expression in normal HEp-2 cells and that this treatment failed to inhibit C. pneumoniae growth in HEp-2 cells stimulated with IFN gamma. These results indicate that treatment with RU486 had a limited effect on C. pneumoniae growth only during the active developmental stage of the bacteria, suggesting that the bacterial target molecule of RU486 is not expressed sufficiently during persistent infection in which there is an aberrant developmental cycle. Thus, our findings provide valuable insight into the complicated chlamydial biological processes involved in the recurrent cycling between normal and persistent infections. - Atsushi Ito, Junji Matsuo, Shinji Nakamura, Asahi Yoshida, Miho Okude, Yasuhiro Hayashi, Haruna Sakai, Mitsutaka Yoshida, Kaori Takahashi, Hiroyuki YamaguchiPLOS ONE 7 1 e30270 2012年01月 [査読有り][通常論文]
Protochlamydia, an environmental chlamydia and obligate amoebal endosymbiotic bacterium, evolved to survive within protist hosts, such as Acanthamobae, 700 million years ago. However, these bacteria do not live in vertebrates, including humans. This raises the possibility that interactions between Protochlamydia and human cells could induce a novel cytopathic effect, leading to new insights into host-parasite relationships. Therefore, we studied the effect of Protochlamydia on the survival of human immortal cell line, HEp-2 cells and primary peripheral blood mononuclear cells (PBMC). Using mainly 4',6-diamidino-2-phenylindole staining, fluorescent in situ hybridization, transmission electron microscopy, and also TUNEL and Transwell assays, we demonstrated that the Protochlamydia induced apoptosis in HEp-2 cells. The attachment of viable bacterial cells, but not an increase of bacterial infectious progenies within the cells, was required for the apoptosis. Other chlamydiae [Parachlamydia acanthamoebae and Chlamydia trachomatis (serovars D and L2)] did not induce the same phenomena, indicating that the observed apoptosis may be specific to the Protochlamydia. Furthermore, the bacteria had no effect on the survival of primary PBMCs collected from five volunteers, regardless of activation. We concluded that Protochlamydia induces apoptosis in human-immortal HEp-2 cells and that this endosymbiont could potentially be used as a biological tool for the elucidation of novel host-parasite relationships. - 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 YamaguchiMICROBIAL PATHOGENESIS 51 3 209 - 216 2011年09月 [査読有り][通常論文]
This study investigated the proteoglycan (PG)-dependent mechanism of Chlamydophila pneumoniae attachment to lymphocytic cells. Lymphoid Jurkat cells and epithelial HEp-2 cells were statically infected with C. pneumoniae (TW183). Transmission electron microscopy and assessment of inclusion-forming units indicated that the bacteria grew normally in Jurkat cells and were capable of producing secondary infection; however, they grew at a slower rate than in HEp-2 cells. RT-PCR analysis indicated that HEp-2 cells strongly expressed PG-core protein encoding genes, thereby sustaining glycosaminoglycans (GAGs), such as heparin, on the cellular surface. Similar gene expression levels were not observed in Jurkat cells, with the exception of glypican-1. Immunofluorescence analysis also supported strong heparin expression in HEp-2 cells and minimal expression in Jurkat cells, although heparan sulfate pretreatment significantly inhibited bacterial attachment to both cell types. Immunofluorescent co-staining with antibodies against chlamydial LPS and heparin did not identify bacterial and heparin co-localization on Jurkat cells. We also confirmed that when C. pneumoniae was statically infected to human CD4(+) peripheral blood lymphocytes known not expressing detectable level of heparin, the bacteria attached to and formed inclusion bodies in the cells. Thus, the attachment mechanism of C pneumoniae to Jurkat cells with low PG expression is unique when compared with HEp-2 cells and potentially independent of GAGs such as heparin. (C) 2011 Elsevier Ltd. All rights reserved. - Junji Matsuo, Atsushi Harimaya, Tatsuya Fukumoto, Shinji Nakamura, Mitsutaka Yoshida, Kaori Takahashi, Masahiro Iida, Nobuhiro Fujii, Hiroyuki YamaguchiJOURNAL OF INFECTION AND CHEMOTHERAPY 17 4 478 - 482 2011年08月 [査読有り][通常論文]
The survival of Alloiococcus otitidis (NCFB2890) with different nutritional supplements, including brain-heart infusion broth (BHI), phosphate-buffered saline (PBS), distilled water (DW), and middle ear effusion (MEE), as well as various atmospheres (aerobic, microaerobic, anaerobic), was compared using cultures, LIVE/DEAD staining, and transmission electron microscopy. The bacterial morphological traits and viability were maintained in BHI and MEE under aerobic conditions but were rapidly lost in PBS and DW. In contrast, anaerobic conditions did not support viability at all. Thus, the bacteria critically required an aerobic atmosphere for its survival as well as the appropriate nutrients, implying that culture of this pathogen from clinical specimens would become more difficult through oxygen depletion depending on a slight change in the middle ear atmosphere. - Ikuko Komiya, Yoshihiro Arimura, Kimimasa Nakabayashi, Akira Yamada, Takako Osaki, Hiroyuki Yamaguchi, Shigeru KamiyaMICROBIOLOGY AND IMMUNOLOGY 55 8 531 - 538 2011年08月 [査読有り][通常論文]
To determine serum antibody against human and bacterial heat shock protein (HSP) 60/70 in myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibody (ANCA) positive microscopic polyangiitis (MPA), 58 patients with MPO-ANCA positive MPA, 48 with RA (rheumatoid arthritis) and 40 with SLE (systemic lupus erythematosus) were studied. Serum antibodies against HSP (human HSP 70, human HSP 60, Mycobacterium HSP 70, and Escherichia coli HSP 60) were measured by sandwich ELISA. The frequency of anti-human HSP 60/70 antibody positive patients was significantly greater in MPO-ANCA positive MPA than SLE and healthy controls. Anti-human HSP 60/70 antibody titers in patients with MPO-ANCA positive MPA were significantly higher than those of healthy controls; anti-bacterial HSP 60/70 antibody titers were also higher. There was a significant correlation between titers of anti-human HSP 70 antibody and anti-Mycobacterium HSP 70 antibody. A correlation was also found between titers of anti-human HSP 70 antibody and anti-human HSP 60 antibody. Anti-human and bacterial HSP 60/70 antibody titers changed in parallel with disease activity in patients with antibody positive MPA. The anti-HSP antibody titer was also increased in patients with RA and SLE. These results suggest that an immunological background via anti-HSP 60/70 antibodies might be associated with pathogenesis in MPO-ANCA positive MPA. - Oguri S, Matsuo J, Hayashi Y, Nakamura S, Hanawa T, Fukumoto T, Mizutani Y, Yao T, Akizawa K, Suzuki H, Shimizu C, Matsuno K, Kamiya S, Yamaguchi HThe Journal of antimicrobial chemotherapy 66 3 527 - 530 3 2011年03月 [査読有り][通常論文]
The mechanism by which Escherichia coli acquires multidrug resistance genes from other bacteria in the natural environment or livestock is still unclear. The ability of ciliates to promote the transfer of genes encoding extended-spectrum beta-lactamases (ESBLs) between the CTX-M-27 donor and clinically isolated recipient E. coli strains was investigated. Equal amounts (similar to 10(9) cfu) of donor cefotaxime-resistant E. coli and recipient ciprofloxacin-resistant E. coli strains were mixed together in the presence or absence of 10(5) ciliates in Page's amoeba saline for 24 h, in the presence or absence of certain drugs (cytochalasin D, cycloheximide and latrunculin B). Gene transfer frequency in the presence of ciliates was estimated at similar to 10(-6); in the absence of ciliates it was similar to 10(-10). Protein synthesis (cycloheximide) or phagocytosis (cytochalasin D or latrunculin B) inhibitors significantly reduced the frequency of gene transfer. Ciliates promote the transfer of genes encoding ESBLs between E. coli strains, implying that the presence of ciliates may provide a significant impact on emerging multidrug-resistant bacteria. - Yasuhiro Hayashi, Shinji Nakamura, Junji Matsuo, Tatsuya Fukumoto, Mitsutaka Yoshida, Kaori Takahashi, Yoshihiko Mizutani, Takashi Yao, Hiroyuki YamaguchiMICROBIOLOGY AND IMMUNOLOGY 54 11 707 - 713 2010年11月 [査読有り][通常論文]
The obligate intracellular bacterium Parachlamydia acanthamoebae is a potential human pathogen, but the host range of the bacteria remains unknown. Hence, the growth of P. acanthamoebae Bn(9) in protozoa (Tetrahymena, Acanthamoeba, Dictyostelium) and mammalian cells (HEp-2, Vero, THP-1, PMA-stimulated THP-1, Jurkat) was assessed using an AIU assay which had been previously established by the current authors. P. acanthamoebae grew in Acanthamoeba but not in the other cell types. The growth was also confirmed using DAPI staining, FISH and TEM. These results indicate that the host range of P. acanthamoebae is limited. - 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 YamaguchiRESEARCH IN MICROBIOLOGY 161 8 711 - 719 2010年10月 [査読有り][通常論文]
The mechanism underlying bacterial conjugation through protozoa was Investigated. Kanamycin-resistant Escherichia colt SMI0 lambda(+) carrying pRT733 with TnphoA was used as donor bacteria and introduced by conjugation into ciprofloxacin-resistant E. colt clinical isolate recipient bacteria. Equal amounts of donor and recipient bacteria were Mixed together in the presence or absence of protozoa (ciliates, free-living amoebae, myxamoebae) in Page's amoeba saline for 24 h. Transconjugants were selected with Luria broth agar containing kanamycin and ciprofloxacin. The frequency of conjugation was estimated as the number of transconjugants for each recipient. Conjugation frequency in the presence of ciliates was estimated to be approximately 10(-6) but in the absence of ciliates, or in the presence of other protozoa, it was approximately 10(-8). Conjugation also occurred in culture of ciliates at least 2 h after incubation. Successful conjugation was confirmed by the polymerase chain reaction. Addition of cycloheximide or latrunculin B resulted in suppression of conjugation. Heat killing the ciliates or bacteria had no effect on conjugation frequency. Co-localization of green fluorescent protein-expressing E colt and PKH-67-vital-stained E. coli was observed in the same ciliate vesicles, suggesting that both donor and recipient bacteria had accumulated in the same vesicle. In this study, the conjugation frequency of bacteria was found to be significantly higher in vesicles purified from ciliates than those in culture suspension We conclude that ciliates rapidly enhance the conjugation of E. coli strains through bacterial accumulation in vesicles. (C) 2010 Elsevier Masson SAS. All rights reserved. - Tatsuya Fukumoto, Junji Matsuo, Masahiro Hayashi, Satoshi Oguri, Shinji Nakamura, Yoshihiko Mizutani, Takashi Yao, Kouzi Akizawa, Haruki Suzuki, Chikara Shimizu, Kazuhiko Matsuno, Hiroyuki YamaguchiJOURNAL OF CLINICAL MICROBIOLOGY 48 9 3360 - 3365 2010年09月 [査読有り][通常論文]
Parachlamydia acanthamoebae is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen in hospital-acquired pneumonia. We examined whether the presence of P. acanthamoebae is related to the presence of Acanthamoeba in an actual hospital environment and assessed the in vitro survival of P. acanthamoebae. Ninety smear samples were collected between November 2007 and March 2008 (trial 1, n = 52) and between October 2008 and February 2009 (trial 2, n = 38) from the floor (dry conditions, n = 56) and sink outlets (moist conditions, n = 34) of a hospital. The prevalences of P. acanthamoebae DNA in the first and second trials were 64.3% and 76%, respectively. The prevalences of Acanthamoeba DNA in the first and second trials were 48% and 63.1%, respectively. A statistical correlation between the prevalence of P. acanthamoebae and that of Acanthamoeba was found (trial 1, P = 0.011; trial 2, P = 0.022), and that correlation increased when samples from just the dry area (floor smear samples, P = 0.002) were analyzed but decreased when samples from a moist area were analyzed (P = 0.273). The in vitro experiment showed that, without Acanthamoeba, P. acanthamoebae could not survive in dry conditions for 3 days at 30 degrees C or 15 days at 15 degrees C. Thus, both organisms were coincidentally found in an actual hospital environment, with the presence of Acanthamoeba having a significant effect on the long-term survival of P. acanthamoebae, suggesting that this potential human pathogen could spread through a hospital environment via Acanthamoeba. - Junji Matsuo, Kouhei Kawaguchi, Shinji Nakamura, Yasuhiro Hayashi, Mitsutaka Yoshida, Kaori Takahashi, Yoshihiko Mizutani, Takashi Yao, Hiroyuki YamaguchiENVIRONMENTAL MICROBIOLOGY REPORTS 2 4 524 - 533 2010年08月 [査読有り][通常論文]
P>Obligate intracellular bacteria are commonly found as endosymbionts of acanthamoebae; however, their survival in and ability to transfer to amoebae are currently uncharacterized. In this study, six bacterial endosymbionts, found in five environmental Acanthamoeba isolates (S13, R18, S23, S31, S40) from different locations of Sapporo city, Japan, were characterized. Phylogenetic analysis revealed that three bacterial endosymbionts (eS23, eS31, eS40a) belonged to alpha- and beta-Proteobacteria phyla and the remaining endosymbionts (eS13, eR18, eS40b) belonged to the order Chlamydiales. The Acanthamoeba isolate (S40) contained two phylogenetically different bacterial endosymbionts (eS40a, eS40b). Fluorescent in situ hybridization analysis showed that all bacterial endosymbionts were diffusely localized within amoebae. Transmission electron microscopy also showed that the endosymbionts were rod-shaped (eS23, eS31, eS40a) or sphere- or crescent-shaped (eS13, eR18, eS40b). No successful culture of these bacteria was achieved using conventional culture methods, but the viability of endosymbionts was confirmed by live/dead staining and RT-PCR methods. However, endosymbionts (except eR18) derived from original host cells lost the ability to be transferred to another Acanthamoebae strains [ATCC strain (C3), environmental strains (S14, R23, S24)]. Thus, our data demonstrate that phylogenetically diverse bacterial endosymbionts found in amoebae maintain a stable interaction with amoebae, but the transferability is limited. - Shinji Nakamura, Junji Matsuo, Yasuhiro Hayashi, Kouhei Kawaguchi, Mitsutaka Yoshida, Kaori Takahashi, Yoshihiko Mizutani, Takashi Yao, Hiroyuki YamaguchiENVIRONMENTAL MICROBIOLOGY REPORTS 2 4 611 - 618 2010年08月 [査読有り][通常論文]
P>Obligate intracellular bacteria are commonly seen as endosymbionts of acanthamoebae. However, whether endosymbionts can survive amoebal encystations remains a significant challenge in cellular biology. The survival of the endosymbiotic bacteria Protochlamydia belonging to environmental chlamydiae found in an amoebal isolate that we have previously reported (Environmental Microbiology Reports, DOI: 10.1111/j.1758-2229.2009.00094.x, 2009) following encystation was therefore assessed. The bacteria were observed in cysts and trophozoites reverted from cysts by analysis with transmission electron microscope, and the bacterial 16S rRNA transcripts were detected in amoeba cultures following encystations by reverse transcription polymerase chain reaction method. Furthermore, the bacterial growth was also confirmed, by fluorescent in situ hybridization analysis and the AIU assay that we have previously established (Applied Environmental Microbiology, 74: 6397-6404, 2008), in trophozoites reverted from cysts stored at 4 degrees C for up to a month after encystation. Thus, these results demonstrated that Protochlamydia could survive in acanthamoebae following encystation. Our findings suggest that amoeba cysts might be further studied in order to understand their role in the environmental survival of endosymbionts. - Itaru Hirai, Maki Utsumi, Hiroyuki Yamaguchi, Yoshimasa YamamotoCANADIAN JOURNAL OF MICROBIOLOGY 56 4 289 - 294 2010年04月 [査読有り][通常論文]
Chlamydia pneumoniae (Chlamydophila pneumoniae) infects lymphocytes and modulates their immune functions; this is critical in the development of chronic inflammatory diseases associated with this pathogen. Therefore, to clarify this immune modulation due to C. pneumoniae infection, the effect of this infection on the proliferation of human peripheral blood lymphocytes was examined. Lymphocytes were proliferated by stimulation with Staphylococcus aureus enterotoxin B, and the cell number was increased up to 3 times the unstimulated lymphocyte number. Further, induction of CD25 expression was observed in 55.8% of lymphocytes. Infection with C. pneumoniae suppressed the proliferation of almost half the lymphocytes induced by stimulation with S. aureus enterotoxin B, and CD25 induction was inhibited in 64.7% of lymphocytes. Inhibition of CD25 expression was observed in both infected and uninfected lymphocytes in culture. However, the expression of VLA4 was not affected by C. pneumoniae infection. Furthermore, inhibition was observed only by infection with viable C. pneumoniae and not by the heat-killed bacteria. These results suggest that C. pneumoniae affects lymphocyte function by inhibiting proliferation and CD25 expression in response to immunological stimulation, possibly via humoral mediator(s). - Stability of Chlamydophila pneumoniae in a harsh environment without a requirement for acanthamoebaeJunji Matsuo, Miho Kobayashi, Shinji Nakamura, Yoshihiko Mizutani, Takashi Yao, Itaru Hirai, Yoshimasa Yamamoto, Hiroyuki YamaguchiMICROBIOLOGY AND IMMUNOLOGY 54 2 63 - 73 2010年02月 [査読有り][通常論文]
The effect of actual interactions between Chlamydophila pneumoniae and amoebae (Acanthamoeba) on the survival of C. pneumoniae was investigated. C. pneumoniae and amoebae were detected in 75 soil samples by IFU assay and PCR. Although C. pneumoniae could not be cultured, the DNA prevalence of C. pneumoniae and amoebae in natural soil was 20% and 92% (no correlation between the prevalence of DNA was observed). The viability of C. pneumoniae spiked in autoclaved soil was assessed by IFU assay and RT-PCR. Although the number of infective progeny decreased for three days, transcripts of C. pneumoniae were detected for up to 98 days independently of amoebae. The stability of C. pneumoniae in liquid medium was also assessed by IFU assay and transmission electron microscopy. The bacteria could survive at 15 degrees C for 14 days independently of amoebae. Bacteria cultured without amoebae were confirmed to have normal structures. Thus, the presence of amoebae has no effect on C. pneumoniae survival, and the bacteria can survive in the absence of host cells for an extended period of time. - K. Kawaguchi, J. Matsuo, T. Osaki, S. Kamiya, H. YamaguchiLETTERS IN APPLIED MICROBIOLOGY 48 4 465 - 471 2009年04月 [査読有り][通常論文]
We examined whether the presence of Helicobacter is related to that of Acanthamoeba in river and soil environments. The samples (river n = 51, soil n = 75) were collected in Sapporo City, Japan. PCR with primers for Helicobacter genus-specific and standard culture techniques were used to detect helicobacter. Prevalence of acanthamoeba was also evaluated by genus-specific PCR. The prevalence of Helicobacter genus-specific DNA in river water samples and in soil samples was 88% and 0%, respectively. No successful culture of helicobacter was achieved. The prevalence of Acanthamoeba genus-specific DNA in river samples and in soil samples was 61% and 96%, respectively. No statistical correlation between the prevalence of helicobacter and either that of acanthamoeba or water quality parameters (pH, turbidity and coliform group) except for temperature was found. We revealed the presence of helicobacter in river water and non-existence of helicobacter in soil. However, the distribution of helicobacter did not overlap with that of acanthamoeba in rivers. The role of acanthamoeba on the survival of helicobacter might be limited as the both are coincidentally present in the environment. - Hiroyuki Yamaguchi, Junji Matsuo, Shigehiro Sugimoto, Maki Utsumi, Yoshimasa YamamotoMICROBIAL PATHOGENESIS 45 4 290 - 296 2008年10月 [査読有り][通常論文]
Since lymphocytes are a major immune cell besides macrophages in the development of atherosclerosis, interaction between lymphocytes and Chlamydophila pneumoniae may contribute to the pathogenesis of chronic inflammatory diseases associated with C pneumoniae. In this regard, we examined a possible alteration of CD3 expression of human lymphocyte Molt-4 cells by C pneumoniae infection. The expression levels of CD3 molecules of lymphocyte Molt-4 cells were significantly decreased by C. pneumoniae infection. In contrast, heat-killed C. pneumoniae as well as mock (cell lysates) did not cause any alteration of CD3 expression of the cells. Treatment of the infected cells with NS-398 (cyclooxyganase-2 inhibitor) or AH-23848 (EP(4) prostanoid receptor antagonist) abolished the inhibition of CD3 expression. The enhanced prostaglandin E(2) (PGE(2)) productions in the culture supernatants of infected cells were confirmed by competitive enzyme-immunosorbent assay (ELISA). C pneumoniae infection of enriched lymphocytes from human peripheral blood mononuclear cells also induced a decrease of CD3 expression. Thus, C pneumoniae infection of lymphocytes induces a decrease of CD3 expression mediated by possibly PGE(2) production. (C) 2008 Elsevier Ltd. All rights reserved. - Junji Matsuo, Yasuhiro Hayashi, Shinji Nakamura, Marie Sato, Yoshihiko Mizutani, Masahiro Asaka, Hiroyuki YamaguchiAPPLIED AND ENVIRONMENTAL MICROBIOLOGY 74 20 6397 - 6404 2008年10月 [査読有り][通常論文]
Parachlamydia acanthamoebae, belonging to the order Chlamydiales, is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen. However, no method exists to accurately quantify viable bacterial numbers. We present a novel quantification method for P. acanthamoebae based on coculture with amoebae. P. acanthamoebae was cultured either with Acanthamoeba spp. or with mammalian epithelial HEp-2 or Vero cells. The infection rate of P. acanthamoebae (amoeba-infectious dose [AID]) was determined by DAPI (4', 6-diamidino-2-phenylindole) staining and was confirmed by fluorescent in situ hybridization. AIDs were plotted as logistic sigmoid dilution curves, and P. acanthamoebae numbers, defined as amoeba-infectious units (AIU), were calculated. During culture, amoeba numbers and viabilities did not change, and amoebae did not change from trophozoites to cysts. Eight amoeba strains showed similar levels of P. acanthamoebae growth, and bacterial numbers reached ca. 1,000-fold (10(9) AIU preculture) after 4 days. In contrast, no increase was observed for P. acanthamoebae in either mammalian cell line. However, aberrant structures in epithelial cells, implying possible persistent infection, were seen by transmission electron microscopy. Thus, our method could monitor numbers of P. acanthamoebae bacteria in host cells and may be useful for understanding chlamydiae present in the natural environment as human pathogens. - Hiroyuki Yamaguchi, Shigerul Kamiya, Tomonori Uruma, Takako Osaki, Haruhiko Taguchi, Tomoko Hanawa, Minoru Fukuda, Hayato Kawakami, Hajime Goto, Herman Friedman, Yoshimasa YamamotoANTIMICROBIAL AGENTS AND CHEMOTHERAPY 52 6 1991 - 1998 2008年06月 [査読有り][通常論文]
Since steroids are powerful anti-inflammatory agents and increase susceptibility to a variety of infections, including Chlamydia (Chlamydophila) pneumoniae respiratory tract infections, the effect of the steroid receptor antagonist RU486 (mifepristone) on C pneumoniae growth in epithelial HEp-2 cells was examined. Treatment of HEp-2 cells with RU486 significantly inhibited the growth of C pneumoniae in a dose-dependent manner. Electron microscopic studies also revealed that the treatment of infected cells with RU486 resulted in a marked destruction of infecting organisms. The addition of the host cell protein synthesis inhibitor cycloheximide to the infected cells did not alter the inhibition of C pneumoniae growth by RU486. Pretreatment of C pneumoniae organisms with RU486 before addition to culture also did not result in any modulation of bacterial growth in the cells. However, the binding of RU486 to C. pneumoniae organisms in cells at 24 h after infection was demonstrated by immune electron microscopy with anti-RU486 antibody. Incubation of cells with anti-RU486 antibody completely diminished the inhibition of C pneumoniae growth by RU486. These results indicate that RU486 may directly bind to the bacteria within cells and cause the destruction of C pneumoniae. This novel mode of regulation of C. pneumoniae growth in cells by RU486 might provide a new approach to understanding complicated aspects of C pneumoniae infection. - Takako Osaki, Tomoko Hanawa, Taki Manzoku, Minoru Fukuda, Hayato Kawakami, Hidekazu Suzuki, Hiroyuki Yamaguchi, Xu Yan, Haruhiko Taguchi, Satoshi Kurata, Shigeru KamiyaJOURNAL OF MEDICAL MICROBIOLOGY 55 11 1477 - 1485 2006年11月 [査読有り][通常論文]
Helicobacter pylori is associated with gastric disorders in humans and some experimental animals, and possesses the luxS/type 2 autoinducer (AI-2) system. The effects of a specific luxS mutation on the characteristics of H. pylori were examined. On 0(.)3% agar medium, motility of H. pylori HPKY08 (luxS: : cat) was significantly lower than that of wild-type H. pylori TK1402. The luxS-complemented strain HPKY21 exhibited motility comparable to that of H. pylori TK1402. It was shown that the luxS/AI-2 system plays an important role in H. pylori motility. The luxS mutant exhibited a reduced infection rate relative to the wild-type parent strain TK1402 in a Mongolian gerbil model. At 3 months after oral inoculation, lower numbers of H. pylori were detected by semi-quantitative real-time reverse transcription PCR (qRT-PCR) in luxS(-) mutant-infected gerbils than in TK1402-infected gerbils. Gastric inflammation and increased antibody titre for H. pylori were observed in TK1402-infected gerbils only. - H Yamaguchi, Oshio, I, T Osaki, S Kurata, Y Yamamoto, S KamiyaINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY 87 2 121 - 129 2006年04月 [査読有り][通常論文]
We examined a possible association between development of diabetes in non-obese diabetic (NOD) mice and dissemination of Chlamydia (Chlamydophila) 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, we detected bacteria in lung in NOD mice with diabetes (38.5%) as well as Institute of Cancer Research, USA (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 with hydrocortisone of the NOD mice with diabetes (53.8%). Results of immunostaining with fluorescein isothiocyanate-conjugated antichlamydia monoclonal antibody also showed the presence of bacterial antigens in the lungs and the PBMCs judged as positive by the RT-PCR. However, C. pneumoniae from cultured PBMCs of all NOD mice was undetected by cultivation method with inclusion-forming units assay. In addition, no influence of C. pneumoniae intranasal inoculation on development of diabetes in NOD mice was confirmed. Thus, 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. - T Uruma, H Yamaguchi, M Fukuda, H Kawakami, H Goto, T Kishimoto, Y Yamamoto, A Tomoda, S KamiyaJOURNAL OF MEDICAL MICROBIOLOGY 54 12 1143 - 1149 2005年12月 [査読有り][通常論文]
In this study the effects of 2-amino-phenoxazine-3-one (phenoxazine derivate, Phx-3) on Chlamydia (Chlamydophila) pneumoniae growth in human monocytic THP-1 cells as well as human epithelial HEp-2 cells were examined. Cells were infected with bacteria at an m.o.i. of 10 by centrifugation. After washing to remove any remaining bacteria, the cells were incubated with or without Phx-3 in the presence or absence of tryptophan for 72 h. The bacteria in cells were assessed by staining of chlamydial inclusions with FITC-labelled anti-chlamydial antibody, electron microscopic analysis, real-time RT-PCR specific for C. pneumoniae 16S rRNA and propagation on HEp-2 cells. Treatment with Phx-3 significantly inhibited growth of C. pneumoniae in THP-1 and HEp-2 cells. A decrease in the number of bacterial 16S rRNA transcripts was also confirmed in both cell lines by real-time RT-PCR. Electron microscopic studies revealed that treatment with Phx-3 induces bacterial destruction in most of the inclusion bodies in these cells. Addition of tryptophan to the culture slightly blocked the growth inhibition of C. pneumoniae by Phx-3. The reagents did not show any cytotoxicity to the cells at the concentrations used. The results suggest that 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. Thus, Phx-3 might be a useful compound for controlling C. pneumoniae growth in cells and may be an alternative conventional therapy. - R Takano, H Yamaguchi, S Sugimoto, S Nakamura, H Friedman, Y YamamotoCURRENT MICROBIOLOGY 50 3 160 - 166 2005年03月 [査読有り][通常論文]
Chlamydia pneumoniae infection of lymphocytes in blood has been documented, and it is apparent that control of this pathogen in lymphocytes as well as immune functions of the infected lymphocytes may be critical in the development of chronic inflammatory diseases associated with infection by this bacterium. Since immune function of lymphocytes infected with C. pneunioniae has not been well studied, the cytokine response of lymphocytes infected with this pathogen was analyzed using an in vitro infection model of the Molt-4 human lymphoid cell line. C. pneunioniae infection of the cells showed a persistent infection without any vigorous growth of the bacteria. Analysis of the cytokine response of the cells persistently infected with C. pneumoniae showed minimum induction of inflammatory cytokine TNF-alpha message, determined by real-time reverse transcription (RT)-PCR in the lymphocytes, even though the infection of THP-1 monocylic cells showed a marked induction of this cytokine messages. BIC (a lymphocyte activation marker gene) as well as IFN-gamma messages were also minimally induced by the infection in Molt-4 lymphocytes. In contrast, constitutive expression of interleukin 8 (IL-8) messages of Molt-4 cells was suppressed by the infection. Thus, these results suggest that lymphocytes persistently infected with C. pneumoniae. tie may have attenuated cytokine responses. - S Nakagawa, T Osaki, Y Fujioka, H Yamaguchi, S KamiyaCLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY 12 2 347 - 353 2005年02月 [査読有り][通常論文]
The effects of long-term infection with Helicobacter pylori on the gastric mucosa of Mongolian gerbils were examined. Colonization by H. pylori was evaluated by both microaerobic cultivation and real-time reverse transcriptase PCR (RT-PCR). Persistent infection with H. pylori in gastric mucosa was detected by real-time RT-PCR during 6 months after infection, but no H. pylori was isolated 4 months after infection by cultivation. Infiltration with neutrophils and mononuclear cells was observed from 2 months after infection. Both intestinal metaplasia and gastric atrophy were also detected from 2 months after infection. The results by enzyme-linked immunosorbent assay indicated that antibody titers against whole H. pylori antigens, H. pylori heat shock protein 60 (HSP60), and Escherichia coli GroEL were significantly higher in the infected gerbils than in noninfected gerbils. After long-term infection with H. pylori for 18 months, marked atrophy of gastric mucosa and multiple cysts in the submucosa were observed in the glandular stomach of the infected gerbils. In addition, squamous cell papilloma with hyperkeratosis was observed in cardia of all the infected gerbils. These results indicate that evaluation of bacterial colonization during long-term infection can be done by real-time RT-PCR and that mucosal damage might be induced by host immune response against whole H. pylori antigen. - Atsushi Toyoda, Takako Osaki, Hiroyuki Yamaguchi, Tomoko Hanawa, Haruhiko Taguchi, Makoto Hasegawa, Shigeru KamiyaJournal of Infection and Chemotherapy 11 3 129 - 135 2005年 [査読有り][通常論文]
The effect of Helicobacter pylori on DNA synthesis of human cultured cells was examined. Viable H. pylori strains were able to stimulate DNA synthesis by gastric cancer MKN45 cells (eight of eight strains) and laryngeal cancer HEp-2 cells (seven of eight strains) but not by lung cancer A549 cells or fetal intestine Int407 cells. In contrast, neither heat-killed H. pylori nor culture supernatants of H. pylori other than SS-1 strain affected DNA synthesis of MKN45 and HEp-2 cells. In contrast, water extracts of H. pylori strains inhibited DNA synthesis. Soluble outer membrane protein (OMP) of the SS-1 strain stimulated DNA synthesis of MKN45 cells but produced no alteration in cell cycle or apoptosis induction of MKN45 cells. To identify those proteins in the soluble OMP that were capable of stimulating DNA synthesis, the OMP was fractionated by fast performance liquid chromatography with a Mono Q column. The results suggest that two proteins with molecular weights of 18 and 45kDa, respectively, are associated with stimulation of DNA synthesis by MKN45 cells. © Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2005. - M Takahashi, H Taguchi, H Yamaguchi, T Osaki, A Komatsu, S KamiyaFEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY 41 3 219 - 226 2004年07月 [査読有り][通常論文]
Enterohemorrhagic Escherichia coli (EHEC) 0 157:117 has been considered as an agent responsible for outbreak of hemorrhagic colitis and the hemolytic uremic syndrome. We examined the effect of the probiotic agent Clostridium butyricum MIYAIRI strain 588 on EHEC O157:147 infections in vitro and in vivo using gnotobiotic mice. The growth of EHEC 01 57:H7 and the production of Shiga-like toxins in broth cultures were inhibited by co-incubation with C. butyricum. The antibacterial effects of butyric and lactic acid were demonstrated in a dose-dependent manner. In addition, the inhibitory effect of butyric acid on the viability of EHEC was demonstrated not only at low pH, but also at neutral pH adjusted to 7.0. Flowcytometric analysis showed that pre-incubation of Caco-2 cells with C. butyricum and E. coli K12 inhibited the adhesion of EHEC O157:H7. However, the effect of C butyricum on adhesion of EHEC to Caco-2 cells was more inhibitory than that of E. coli K12. Gnotobiotic mice mono-associated with EHEC O157:H7 died within 4-7 days after the infection. On the other hand, all gnotobiotic mice prophylactically pre-treated with C. butyricum survived exposure to EHEC O157:H7 and of the gnotobiotic mice therapeutically post-treated with C. butyricum, 50% survived. Both counts of EHEC O157:147 and the amounts of shiga-like toxins (Stx1 and Stx2) in fecal contents of gnotobiotic mice di-associated with EHEC O157:H7 and C. butyricum were less than those of gnotobiotic mice mono-associated with EHEC O157:H7. These results indicated that the probiotic bacterium C butyricum MIYAIRI strain 588 has preventive and therapeutic effects on EHEC O157:1-17 infection in gnotobiotic mice. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. - H Yamaguchi, M Yamada, T Uruma, M Kanamori, H Goto, Y Yamamoto, S KamiyaTRANSFUSION 44 7 1072 - 1078 2004年07月 [査読有り][通常論文]
BACKGROUND: Demonstration of viable Chlamydia (Chlamydophila) pneumoniae in peripheral blood mononuclear cells (PBMNCs) is essential to understand the involvement of C. pneumoniae in atherosclerosis. Nevertheless, the prevalence of viable C. pneumoniae in the blood of healthy donors has not yet been studied. STUDY DESIGN AND METHODS: The presence of C. pneumoniae transcript in PBMNCs from blood of healthy human donors was assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR) with primers for C. pneumoniae 16S rRNA, which is more sensitive than genomic-DNA-based analysis, and by the use of staining with fluorescein isothiocyanate-conjugated chlamydia monoclonal antibody (MoAb). RESULTS: Thirteen of 70 donors (18.5%) showed the presence of bacterial transcript in cultured PBMNCs. The prevalence of bacterial detection and bacterial numbers was significantly increased in PBMNC cultures incubated with cycloheximide. Immunostaining of PBMNCs with antichlamydial MoAb also revealed the presence of bacterial antigen in the PBMNCs judged as positive. Nevertheless, cultivation of C. pneumoniae from all PCR-positive donors was unsuccessful. There was no significant correlation between the presence of chlamydia and either sex or current smoking habits. A possible age variation, however, in the presence of chlamydia in blood of healthy donors was suggested by the results obtained. CONCLUSION: The bacterial transcripts in PBMNCs obtained from healthy donors were detected by the RTPCR method. Viable C. pneumoniae may be present in healthy human PBMNCs. - K Matsunaga, H Yamaguchi, TW Klein, H Friedman, Y YamamotoINFECTION AND IMMUNITY 71 11 6672 - 6675 2003年11月 [査読有り][通常論文]
A possible involvement of the mitogen-activated protein (MAP) kinase cascade in the inhibition of macrophage interleukin-12 (IL-12) production by Legionella pneumophila infection was examined. The results of MAP kinase inhibition by p42/44 and p38 MAP kinase inhibitors and of p42/44 MAP kinase activity assays indicate that L. pneumophila infection of macrophages causes a selective inhibition of lipopolysaccharide-induced IL-12 production by activating the p42/44 MAP kinase cascade. In addition, it was also revealed that the p38 MAP kinase may be important for the production of IL-12 but not for the inhibition caused by L. pneumophila infection. - H Yamaguchi, T Osaki, H Taguchi, N Sato, A Toyoda, M Takahashi, M Kai, N Nakata, A Komatsu, Y Atomi, S KamiyaCLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY 10 5 808 - 812 2003年09月 [査読有り][通常論文]
In order to assess the efficacy of oral Helicobacter pylori heat shock protein 60 (HSP60) as a vaccine, protection against H. pylori infection in specific-pathogen-free (SPF) C57BL/6 and germfree (GF) IQI mice was examined. Prophylactic oral vaccination of these two strains of mice with either H. pylori HSP60 or Escherichia coli GroEL inhibited H. pylori colonization by 90 to 95% at 3 weeks postinfection (p.i.). However, these mice were only partially protected because bacterial loads increased in all animals at 10 weeks p.i. Anti-H. pylori HSP60 immunoglobulin G was detected in serum at 3 weeks p.i. in mice vaccinated with either H. pylori HSP60 or GroEL. Significant increases in the gastritis scores were observed only in SPF mice immunized with H. pylori HSP60. These results indicate that oral vaccination with H. pylori HSP60 has partial protective effects on subsequent H. pylori infection but also induces postimmunization gastritis. However, GF mice immunized with H. pylori HSP60 did not suffer from severe gastritis. Therefore, the presence of bacterial flora appears to contribute to the induction of postimmunization gastritis. - H Yamaguchi, H Friedman, M Yamamoto, K Yasuda, Y YamamotoANTIMICROBIAL AGENTS AND CHEMOTHERAPY 47 6 1972 - 1975 2003年06月 [査読有り][通常論文]
Chlamydia pneumoniae infection of lymphocytes in blood has been well documented, and it is apparent that control of this pathogen in these cells may be critical in the development of chronic inflammatory diseases associated with infection by this bacterium. The activity of antibiotics against C. pneumoniae in lymphocytes was assessed in this study by utilizing an in vitro infection model with lymphoid cells. The results obtained indicated that although all of the antibiotics tested showed remarkable activity against bacterial growth in epithelial cells, C. pneumoniae in lymphocytes was less susceptible to antibiotics than was bacterial growth in epithelial cells, which are widely used for the evaluation of anti-C. pneumoniae antibiotics. - H Yamaguchi, H Friedman, Y YamamotoINFECTION AND IMMUNITY 71 6 3645 - 3647 2003年06月 [査読有り][通常論文]
Nicotinic acetylcholine receptors (nAChRs) play an essential role in neurotransmission. Recent studies have indicated that nAChRs may be involved in the regulation of some bacterial infections through immunological mechanisms in macrophages. However, the regulation of infection with Chlamydia pneumoniae, which is a ubiquitous pneumonia-causing bacterium, by an nAChR-mediated mechanism is still unclear. In the present study, it was found that stimulation of nAChRs with ligands such as nicotine and acetylcholine altered the growth of C. pneumoniae in epithelial HEp-2 cells. Thus, the results revealed a possible pathophysiological role of nAChRs in the regulation of intracellular bacterial infection. - S Haranaga, H Yamaguchi, H Ikejima, H Friedman, Y YamamotoJOURNAL OF INFECTIOUS DISEASES 187 7 1107 - 1115 2003年04月 [査読有り][通常論文]
Chlamydia ( Chlamydophila) pneumoniae is a common respiratory pathogen, and it seems likely that alveolar macrophages may have an important role in infection with this bacterium. In the present study, we examined the usefulness of a continuous cell line of murine alveolar macrophages, designated "MH-S," as an in vitro C. pneumoniae infection model. Infection of MH-S cells with C. pneumoniae resulted in the development of typical inclusion bodies in the cells, similar to that seen in primary alveolar macrophages. However, we noted that, although the number of bacteria in the cultures increased during the infection, there was a restricted production of infective elementary bodies. The analysis of bacterial messenger RNA in the cultures showed that the message levels for the omcB gene were present only at a moderate level, but the levels of hsp60 messages increased markedly during infection. Neutralization of tumor necrosis factor (TNF)-alpha induced by inoculation with antibody significantly enhanced the infection, but omcB message levels were still inhibited. These results indicate that the growth of C. pneumoniae in alveolar macrophages may be restricted. Endogenous TNF-alpha may be one of the factors responsible for such restriction, but other factors also may be involved. - Shigeru Kamiya, Takako Osaki, Haruhiko Taguchi, Hiroyuki YamaguchiKeio Journal of Medicine 51 2 24 - 25 2002年12月 [査読有り][通常論文]
The reactive epitope of heat shock protein 60 (HSP60) of Helicobacter pylori to its monoclonal antibody (H9) was determined, and its synthesized peptide designated pH9 was used for ELISA. The patients with H. pylori infection had significantly lower titers of pH9 antibody than did uninfected patients. In C57BL/6 mice immunized intraperitoneally with the pH9 peptide with Freund's complete adjuvant (FCA), the number of H. pylori organisms colonizing the stomach was significantly lower than that in mice immunized with FCA only. These results suggest that HSP60 of H. pylori is effective in protection against H. pylori infection and might be a good candidate as a vaccine component. - H Yamaguchi, S Haranaga, H Friedman, JA Moor, KE Muffly, Y YamamotoFEMS MICROBIOLOGY LETTERS 216 2 229 - 234 2002年11月 [査読有り][通常論文]
Since current studies indicate possible infection of human lymphocytes with Chlamydia (Chlamydophila) pneumoniae, establishment of an in vitro C. pneumoniae infection model using lymphocyte cell lines was demonstrated. Human lymphoid cell lines (Molt 4 [T-cell] and P3HR1 [B-cell]) were utilized for this purpose besides human monocyte cell line (THP-1) and human epithelial cell line (HEp-2), as a reference of monocyte/macrophage cells and a positive control for support of C. pneumoniae growth, respectively. Both lymphoid cells (Molt 4 and P3HR1) supported the growth of C. pneumoniae as demonstrated by Chlamydia inclusion formation, detection of increased infective progenies and increased bacterial antigen levels. Similar data were obtained using monocyte THP-1 cells. However, the bacterial growth in these cells was less than that in HEp-2 cells. The electron microscopic study showed typical inclusions with many Chlamydia elementary bodies in lymphoid cells tested, similar to that seen in HEp-2 cells. These results indicate that C. pneumoniae can infect cells with lymphocyte properties and this infection model with lymphoid cell line cells could be valuable to study details of lymphocyte C. pneumoniae interaction. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. - H Yamaguchi, S Haranaga, R Widen, H Friedman, Y YamamotoINFECTION AND IMMUNITY 70 5 2392 - 2398 2002年05月 [査読有り][通常論文]
Migration and differentiation of monocytes to the intima of blood vessels may be a crucial first step in the development of atherosclerosis associated with Chlamydia (Chlamydophila) pneumoniae. However, the involvement of C. pneumoniae infection in such steps is not clear. In the present study, therefore, the differentiation-inducing activity, of C. pneumoniae to monocytes was examined. Human THP-1 monocytic cell line cells were infected with C. pneumoniae, and the differentiation of monocytes to macrophages was assessed by cell morphology, phagocytic activity, and expression of a cell surface adhesion molecule. The monocytic cells infected with viable bacteria markedly differentiated into macrophages associated with diffused cell morphology, increased uptake of polystyrene beads and increased ICAM-1 (intercellular adhesion molecule 1) expression on the cell surfaces. Heat-killed bacteria did not induce any morphological changes or increase of phagocytosis, but they did induce an increase of cell surface ICAM-I expressions in THP-1 monocytic cells. The antibiotic minocycline treatment of infected cells resulted in marked inhibition of the cell differentiation as well as C. pneumonia e growth in the cells, but not ICAM-I expression. In addition, the experiments with human peripheral blood monocytes infected with C. pneumoniae also showed the differentiation of macrophages assessed by morphological change and phagocytic activity. These results indicate that C. pneumoniae infection may directly induce the differentiation of monocytes to macrophages. However, antigenic stimulation of monocytes with bacteria may not be sufficient for a full macrophage differentiation. - T Osaki, H Yamaguchi, H Taguchi, M Fukada, H Kawakami, H Hirano, S KamiyaJOURNAL OF MEDICAL MICROBIOLOGY 51 4 295 - 299 2002年04月 [査読有り][通常論文]
To determine the pathological significance of the coccoid form of Helicobacter pylori, its adhesion to and induction of secretion of interleukin-8 (IL-8) by gastric epithelial (MKN45) cells were studied. By flow cytometry, the adhesion of the coccoid form to MKN45 cells was significantly lower than that of the helical form. The monoclonal antibody A20 recognising lipopolysaccharide of H. pylori inhibited the adhesion of the coccoid form to MKN45 cells as much as that of the helical form. There was significantly lower induction of IL-8 secretion by MKN45 cells exposed to the coccoid form (two of four strains) as compared with the helical form. - H Taguchi, M Takahashi, H Yamaguchi, T Osaki, A Komatsu, Y Fujioka, S KamiyaJOURNAL OF MEDICAL MICROBIOLOGY 51 4 336 - 343 2002年04月 [査読有り][通常論文]
A mouse enterohaemorrhagic Escherichia coli (EHEC) infection model was developed with a combination of germ-free (GF) mice and hyper-toxigenic EHEC (HT-EHEC) O157:H7 strain 6. The HT-EHEC strain 6 produced both Shiga-like toxin (SLT)-1 1.0 mug/ml and SLT-2 8.2 mug/ml in vitro. Eight-week-old germ-free mice were inoculated intragastrically with 5.0 x 10(7) cfu of HT-EHEC strain 6. All GF mice challenged with the HT-EHEC developed ruffled fur and convulsion of limbs or hindleg weakness within 3 days after the challenge, culminating in death within 5 days. The HT-EHEC colonised well at a level of 10(8)-10(9) cfu/g of faeces 5 days after the challenge. Both SLT-1 and SLT-2 were demonstrated in the faeces of the mice for 5 days after challenge. Histological examination of the colons of the infected mice showed congestion of the lamina propria mucosa, mild inflammatory cell infiltration and goblet cell depletion. In proximal tubules of the renal cortex, epithelial swelling with scattered necrotic cells was recognised. Endothelial swelling and mononuclear cell infiltration were also observed in the glomeruli. The brain showed acute neuronal necrosis in the cerebrum and slight loss of Purkinje cells in the cerebellum. Passive immunisation with anti-SLT antisera prolonged the life of the mice without any neural symptoms. Microscopically, all tissue specimens from the passively immunised mice were not remarkable. These results indicate that the infection of GF mice with HT-EHEC is a useful animal model to study the pathogenicity of SLT-producing E. coli and the toxins. - S Haranaga, H Ikejima, Yamaguchi, I, H Friedman, Y YamamotoCLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY 9 2 313 - 319 2002年03月 [査読有り][通常論文]
Chlamydia pneumoniae is an obligate intracellular bacterium and has a unique development cycle consisting of an elementary body (EB) and reticular body (RB). EBs survive in extracellular environments as well as infect susceptible host cells. However, EBs display no measurable metabolic activity. In contrast, RBs are metabolically active and can replicate in a host cell but are noninfectious. Therefore, analysis of C. pneumoniae growth in infected cells by conventional bacterial culture may not permit sufficient information about growth of the bacteria in cells. In this study, therefore, we examined the usefulness of the reverse transcription (RT)-PCR method for analysis of bacterial transcripts to evaluate C. pneumoniae growth in HEp-2 cells because the levels of bacterial gene transcripts are known to show the metabolic activity of bacteria. The transcripts for the C. pneumoniae hsp60 gene and 16S rRNA in the cells were easily detected just after infection, followed by a marked increase. In contrast, pyk and omcB transcripts slowly increased after a latent period. The hydrocortisone treatment of C. pneumoniae-infected cells induced an increase of all bacterial transcripts tested compared with the control group. The treatment of the infected cells with the antibiotic minocycline showed a selective inhibition of bacterial gene transcripts, even though the complete inhibition of EB production determined by the bacterial culture assay was evident. These results indicate that the determination of bacterial gene transcripts by RT-PCR might be a powerful method to analyze in detail growth of C. pneumoniae in host cells, particularly altered bacterial growth caused by agents such as antimicrobials. - S Haranaga, H Yamaguchi, H Friedman, SI Izumi, Y YamamotoINFECTION AND IMMUNITY 69 12 7753 - 7759 2001年12月 [査読有り][通常論文]
The obligate intracellular pathogen Chlamydia (Chlamydophila) pneumoniae is known to be associated with some chronic inflammatory diseases, such as atherosclerosis. Interaction between C. pneumoniae and immune cells is important in the development of such diseases. However, susceptibility of immune cells, particularly lymphocytes, to C. pneumoniae infection has not been reported, even though lymphocytes play a pivotal role in the development of the diseases caused by this bacterium. In this regard, we examined the susceptibility of lymphocytes to C. pneumoniae infection in vitro. The results demonstrated that human peripheral blood lymphocytes as well as mouse spleen lymphocytes could be infected with C pneumoniae. Furthermore, purified T lymphocytes as well as established T-lymphocyte cell line cells showed an obvious susceptibility to C pneumoniae infection, indicating that T cells could be one of the host cells for this bacterial infection. These findings reveal a new infection site for C. pneumoniae, i.e., lymphocytes. - S Haranaga, H Yamaguchi, GF Leparc, H Friedman, Y YamamotoTRANSFUSION 41 9 1114 - 1119 2001年09月 [査読有り][通常論文]
BACKGROUND: Because it has been increasingly recognized that Chlamydia pneumoniae may be linked to some chronic inflammatory diseases, including atherosclerosis, detection of this pathogen in blood from patients may be valuable in the diagnosis of such diseases. However, the prevalence of chlamydia in the blood of healthy donors has not yet been extensively studied. STUDY DESIGN AND METHODS: The presence of C. pneumoniae in PBMNCs obtained from healthy persons who donated blood for blood transfusion was assessed by a PCR that was specific for the C. pneumoniae 16S rRNA gene and by the use of staining with FITC-conjugated chlamydia MoAb. RESULTS: Twenty-one (8.9%) of 237 blood samples tested showed the presence of C. pneumoniae DNA and antigen in the PBMNCs. There was no significant difference in the presence of chlamydia in blood according to sex or to age between 20 and 59 years of age. However, a possible seasonal variation in the presence of chlamydia in blood from healthy donors was suggested by the results obtained. CONCLUSION: A significant percentage of healthy donors carry C. pneumoniae, which may be a risk factor for some chronic diseases. - H Yamaguchi, T Osaki, N Kurihara, M Kitajima, M Kai, M Takahashi, H Taguchi, S KamiyaJOURNAL OF MEDICAL MICROBIOLOGY 48 10 927 - 933 1999年10月 [査読有り][通常論文]
Escherichia coli cells expressing fusion proteins consisting of P-galactosidase and bacterial heat-shock protein (HSP) 60 of E. coli, Yersinia enterocolitica or Helicobacter pylori were constructed, and designated as HY1, HY2 or HY3, respectively, Fusion proteins prepared from HY2 and HY3 induced secretion of interleukin-8 (IL-8) from human gastric epithelial KATO III cell cultures. On the other hand, the parent strain (E. coli pop2136), PEX (pop2136 transformed by vector) and fusion protein prepared from HY1 did not induce IL-8 secretion from KATO III cells, Other human gastric (MKN45) and non-gastric cell lines (Int 407 and A549) did not secrete IL-8 following treatment with these proteins. These results indicate that H. pylori HSP60 induces IL-8 secretion from human gastric cells and the levels of IL-8 differ among the various gastric cell lines, suggesting that HSP60 might be an important virulence factor associated with chronic gastric inflammation following H. pylori infection in man. - H Yamaguchi, T Osaki, M Takahashi, H Taguchi, S KamiyaFEMS MICROBIOLOGY LETTERS 175 1 107 - 111 1999年06月 [査読有り][通常論文]
To evaluate the viability of Helicobacter pylori cultured under anaerobic conditions, H. pylori strain TK1029 was grown on blood agar in a microaerophilic environment at 37 degrees C for 4 days, and subsequently cultured under anaerobic conditions for 1 to 35 days. Colony formation by bacteria on blood agar plates cultured under anaerobic conditions was observed only for up to 4 days of microaerophilic incubation. By Gram staining, the morphological form of the bacteria was shown to be predominantly coccoid. However, bacteria cultured under anaerobic conditions for 15 to 35 days formed colonies on blood agar after pre-incubation of bacteria with PBS, but not without pre-incubation, These results suggest that H. pylori survives long-term culture under anaerobic conditions and that both pre-incubation in non-nutrient solution and high density of bacterial concentration might be important for recovery of H. pylori cultured for a prolonged time under anaerobic conditions. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. - H Yamaguchi, T Osaki, H Taguchi, T Hanawa, T Yamamoto, S KamiyaJOURNAL OF GASTROENTEROLOGY 33 Suppl 10 6 - 9 1998年11月 [査読有り][通常論文]
The relationship between the expression of heat shock protein 60 (HSP60) of Helicobacter pylori on the bacterial surface and its pathogenic factors of urease activity, production of vacuolating toxin, and adherence to human gastric carcinoma cells was examined. There was no correlation between urease activity, production of vacuolating toxin, and the expression of HSP60. However, there was a significant correlation between the adherence activity to human gastric carcinoma MKN45 cells and the expression of HSP60 (correlation coefficient; r = 0.68). In an inhibition assay with anti-HSP60 antibody, we examined the adhesion of H. pylori to MKN45 cells. The adherence of 3 of 13 H. pylori strains was inhibited by anti-HSP60 antibody. The results suggest that H. pylori HSP60 may be associated with the adhesion of H. pylori to human gastric cells. - H Yamaguchi, T Osaki, H Taguchi, T Hanawa, T Yamamoto, S KamiyaJOURNAL OF MEDICAL MICROBIOLOGY 46 10 819 - 824 1997年10月 [査読有り][通常論文]
Two monoclonal antibodies (MAbs), designated as H9 (IgG2a) and H20 (IgM), directed against heat-shock protein 60 (HSP60) of Helicobacter pylori strain TK1029 were established, Affinity-purified antigens cross-reacted in immunoblots with MAb H9 and MAb H20 respectively, These antigens also reacted with the 3C8 MAb previously established in this laboratory, which recognised Yersinia enterocolitica HSP60, By aminoacid sequence analysis, the N-terminal amino-acid sequence of the protein recognised by both H9 and H20 MAbs was confirmed as the amino-acid sequence of H, pylori HSP60 reported previously, Both MAbs reacted with nine strains of H, pylori in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis, In addition, MAb H9 reacted with extracts of other bacteria including H, mustelae, Pseudomonas aeruginosa, Vibrio cholerae, Serratia marcescens, Proteus mirabilis, Escherichia coli and Shigella sonnei, In contrast, MAb H20 reacted only with strains H, pylori. These results suggest that both the species-specific epitope recognised by MAb H20 and the common epitope recognised by MAb H9 exist on HSP60 of the bacterial cell, Both MAbs also reacted with the 60-kDa protein in the lysate of human gastric carcinoma (MKN45) cells, It was shown by immunohistochemical staining that gastric epithelial cells of four out of six biopsy specimens examined stained positively with MAb H20, These results suggest that there is a common epitope in H, pylori HSP60 and human gastric epithelial cells. - H Yamaguchi, T Osaki, N Kurihara, H Taguchi, T Hanawa, T Yamamoto, S KamiyaJOURNAL OF MEDICAL MICROBIOLOGY 46 10 825 - 831 1997年10月 [査読有り][通常論文]
A previous study reported a relationship between the expression of heat-shock protein 60 (HSP60) by Helicobacter pylori and its adhesion to human gastric carcinoma (MKN45) cells. To examine whether the HSP60 homologue of H. pylori is associated with the adhesion of H, pylori to human gastric epithelial cells, an inhibition assay of adhesion of H, pylori to MKN45 cells was performed by flow cytometric analysis with monoclonal antibody (MAb) designated as H20 recognising HSP60 of H, pylori. The rate of adhesion of H, pylori pretreated with MAbH20 to MKN45 cells was lower than that of untreated H. pylori, Primary human gastric epithelial cells from a patient with gastric cancer were also prepared for comparison in the inhibition assay with MAbH20. H. pylori adhered to the primary human gastric epithelial cells, and this adhesion was significantly inhibited by MAbH20, These results suggest that the H, pylori HSP60 homologue recognised by MAbH20 might be associated with the adhesion of H, pylori to primary human gastric epithelial cells as well as to cultured gastric cancer cells. - T Osaki, H Yamaguchi, H Taguchi, J Kumada, S Ogata, S KamiyaJOURNAL OF MEDICAL MICROBIOLOGY 46 2 117 - 121 1997年02月 [査読有り][通常論文]
The adhesion of Helicobacter pylori to gastric carcinoma cells (MKN45, KatoIII and MKN28) and Intestine-407 cells was tested by flow cytometric analysis. The mean adhesion rates of H. pylori strains to MKN45, KatoIII and Intestine-407 cells were 90.5, 42.7 and 15.1%, respectively, There was no statistical correlation between the adhesion rates to MKN45 cells and haemagglutination (HA) activity of H. pylori strains, although H. pylori strains with high HA activity with human type O erythrocytes tended to adhere effectively to MKN45 cells. No correlation between adhesion and production of vacuolating toxin was observed. - H Yamaguchi, T Osaki, H Taguchi, T Hanawa, T Yamamoto, S KamiyaJOURNAL OF GASTROENTEROLOGY 31 Suppl 9 12 - 15 1996年11月 [査読有り][通常論文]
Induction of heal shock proteins (HSPs) was analyzed in Helicobacter pylori strains. With hear shock at 42 degrees C, a synthesized 60 kDa-HSP (HSP60) was detected on autoradiography. The expression of HSP60 on the cell surface of H. pylori was examined by flow cytometric analysis. All strains used in this study expressed HSP60 on the cell surface. although the intensity differed among the strains, depending on culture conditions. The reactivity of a monoclonal antibody (mAb), 3C8, directed against bacterial HSP60, with HSP60 derived from ten strains of H. pylori and with human gastric carcinoma cell HSP60 was examined by immunoblot analysis. An epitope that reacted with the mAb was detected in the HSP60 of H. pylori and on the surface of human gastric carcinoma cells. - H Yamaguchi, H Taguchi, S KamiyaJAPANESE JOURNAL OF MEDICAL SCIENCE & BIOLOGY 49 5-6 262 - 263 1996年10月 [査読有り][通常論文]
- H Yamaguchi, T Osaki, H Taguchi, T Hanawa, T Yamamoto, S KamiyaJOURNAL OF MEDICAL MICROBIOLOGY 45 4 270 - 277 1996年10月 [査読有り][通常論文]
The expression of a 60-kDa heat shock protein (HSP60) on the cell surface of Helicobacter pylori was analysed by flow cytometry with polyclonal antibody directed to HSP60. All 13 strains of H. pylori examined expressed HSP60 on the cell surface, although the intensity of expression was different among the strains and depended on culture conditions. There was a correlation between the intensity of HSP60 expressed on the cell surface and the rate of adherence to human gastric carcinoma cells (MKN45) by H. pylori, but not with urease activity and production of vacuolating toxin. By flow cytometric analysis with monoclonal antibody (MAb) 3C8 against HSP60, the reactive epitope in the HSP60 of H. pylori was detected, on the surface of MKN45 cells. Furthermore, it was shown that gastric epithelial cells were positively stained with MAb 3C8 in one of two biopsy specimens examined. These results suggest that there is a common epitope showing homology between H. pylori HSP60 and human gastric epithelial cells. - H Yamaguchi, H Miura, K Ohsumi, T Osaki, H Taguchi, T Yamamoto, T Hanawa, S Ogata, S KamiyaMICROBIOLOGY AND IMMUNOLOGY 40 1 77 - 80 1996年 [査読有り][通常論文]
To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gin Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3. - H YAMAGUCHI, T YAMAMOTO, Y KONOEDA, H TAGUCHI, S OGATAAPMIS 100 11 957 - 962 1992年11月 [査読有り][通常論文]
Mouse monoclonal antibodies (MAbs) showing distinct reactivity against the 60-kilodalton (kDa) antigen heat shock protein of Yersinia enterocolitica, designated cross-reacting protein antigen (CRPA), have previously been established. The reactivities of these MAbs (5C3 and 3C8) against mouse and human host cells were studied by Western blotting and flow cytometric analysis. The results indicated that epitopes on the bacterial 60-kDa heat shock protein are present on various molecules in mouse spleen cells and human B cells. An epitope recognized by MAb 5C3 was expressed on the mouse and human host cell surface, and an epitope recognized by MAb 3C8 was also expressed on the human host cell surface. - 山口 博之, 田口 晴彦, 石山 業弘, 金森 政人, 緒方 幸雄日本細菌学雑誌 41 4 701 - 707 JAPANESE SOCIETY FOR BACTERIOLOGY 1986年Yersinia enterocoliticaとVibrio choleraeに存在する共通たんぱく質抗原cross-reacting protein antigen (CRPA)が他の菌属種にも存在するか否かShigella sonnei, Proteus mirabilis, Salmonella enteritidis, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Pseudomonas aeruginosa, Neisseria gonorrhoeaeおよびStaphylococcus aureusについて免疫電気泳動法により調べた。その結果,N. gonorrhoeaeとS. aureusを除くグラム陰性9菌種にCRPAの存在が認められた。これら9菌種についてimmunoblotting法を行つた結果,各CRPAには主として60キロダルトンのたんぱく質が共通に存在しているとの示唆が得られた。確認のためにさらにV. choleraeおよびY. enterocoliticaよりCRPAを特に部分精製し,immunoblottingを行つた結果,60キロダルトンたんぱく質がそれぞれのCRPAの主要構成たんぱく質と判明し,またV. cholerae外膜たんぱく質とV. choleraeおよびY. enterocoliticaのCRPAとの異同について検討した結果,いずれのCRPAもV. cholerae外膜標品中には認められない成績を得た。
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- 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 [通常講演]Jeewan Thapa, Takanori Watanabe, Keisuke Taki, Junji Matsuo, Torahiko Okubo, Hiroyuki Yamaguchi第92回日本細菌学会総会 2019年04月 ポスター発表
- 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 [通常講演]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 2019年04月 ポスター発表
- 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 [通常講演]大久保寅彦, 松尾淳司, 山口博之第92回日本細菌学会総会 2019年04月 シンポジウム・ワークショップパネル（指名）
- 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 [通常講演]鷲見優斗, 大久保寅彦, 山口博之第92回日本細菌学会総会 2019年04月 ポスター発表
- 低酸素環境はChlamydia trachomatis L2の細胞内増殖を促進する [通常講演]橋本拳人, 大久保寅彦, 山口博之第92回日本細菌学会総会 2019年04月 ポスター発表
- 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 [通常講演]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) 2018年08月 口頭発表（一般）
- Subtle change of host-cell density causes fatal error on monitoring the intracellular growth of Chlamydia trachomatis in a low-oxygen environment [通常講演]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 2018年06月 口頭発表（一般）
- Ciliates promote the interactive transfer of plasmid encoding blaNDM-5 between human pathogenic Escherichia coli and environmental Aeromonas caviae [通常講演]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 2018年06月 口頭発表（一般）
- Cyclic Luciferase Probe Reveals Caspase-3 Activation in Chlamydia-Infected Cells At Late Times During Infection [通常講演]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) 2018年06月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 [通常講演]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) 2018年06月 シンポジウム・ワークショップパネル（公募）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。
- 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 [通常講演]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) 2018年03月 口頭発表（一般）
- 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 [通常講演]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) 2018年03月 口頭発表（一般）
- 低酸素環境においてChlamydia trachomatisの感染動態を修飾する要因 [通常講演]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) 2018年03月 口頭発表（一般）
- 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 [通常講演]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) 2018年03月 口頭発表（一般）
- 鬩ぎ合う原生生物と細菌から学ぶ [通常講演]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 2017年12月 公開講演，セミナー，チュートリアル，講習，講義等
- Interaction between protozoa and bacteria evokes a novel paradigm on understanding unseen life [通常講演]Hiroyuki YamaguchiTMU Medical Laboratory Forum (台北医科大学検査医学フォーラム) 招待講演 2017年12月 口頭発表（招待・特別）
その他活動・業績
- 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年
- 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 2016年02月 [査読無し][通常論文]
- 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年
- SURVIVAL STRATEGY OF HELICOBACTER PYLORI IN ENVIRONMENTS BY CO-EXISTENCE IN ACANTHAMOEBA CASTELLANIIF. Hojo, T. Osaki, H. Yonezawa, S. Kurata, T. Hanawa, H. Yamaguchi, S. Kamiya HELICOBACTER 19 103 -103 2014年09月 [査読無し][通常論文]
- YAMAGUCHI Hiroyuki, MATSUO Junji, SEKIZUKA Tsuyoshi, KURODA Makoto, NAGAI Hiroki, SUGIMOTO Chihiro 日本細菌学雑誌 68 (1) 186 2013年02月25日 [査読無し][通常論文]
- 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 2012年03月 [査読無し][通常論文]
- 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年 [査読無し][通常論文]
- 山口 博之 無菌生物 = Japanese journal of germfree life and gnotobiology 41 (1) 54 -58 2011年09月01日
- 神谷 茂, 田口 晴彦, 山口 博之, CHAKAYA James M, BII Christine C 日本マイコプラズマ学会雑誌 = Japanese Journal of Mycoplasmology 37 75 -77 2011年03月31日
- 【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 2010年11月 [査読無し][通常論文]
- 松尾 淳司, 山口 博之 日本細菌学雑誌 62 (1) 79 -79 2007年02月25日
- 松尾 淳司, 山口 博之 日本細菌学雑誌 62 (1) 58 -58 2007年02月25日
- 島田 太一, 大崎 敬子, 山口 博之 Food function 1 (1) 40 -47 2005年03月
- 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年
- 山口 博之, 大崎 敬子, 田口 晴彦, 神谷 茂 無菌生物 = Japanese journal of germfree life and gnotobiology 33 (2) 84 -86 2003年12月01日
- 田口 晴彦, 関根 秀明, 早川 雅之, 大崎 敬子, 花輪 智子, 山口 博之, 神谷 茂 無菌生物 = Japanese journal of germfree life and gnotobiology 33 (2) 87 -90 2003年12月01日
- 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 2003年03月30日
- 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 2003年03月30日
- Y Yamamoto, H Ikejima, H Yamaguchi, G Leparc, H Friedman CLINICAL IMMUNOLOGY 103 (3) S5 -S5 2002年06月 [査読無し][通常論文]
- H Yamaguchi, H Ikejima, GF Leparc, SE Phillips, CA Weideman, H Friedman, Y Yamamoto FASEB JOURNAL 16 (4) A218 -A218 2002年03月 [査読無し][通常論文]
- 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 2002年02月 [査読無し][通常論文]
- 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年
- H Yamaguchi, A Toyoda, M Takahashi, H Taguchi, S Kamiya GUT 47 A60 -A61 2000年10月 [査読無し][通常論文]
- M Takahashi, H Taguchi, H Yamaguchi, T Osaki, S Kamiya JOURNAL OF MEDICAL MICROBIOLOGY 49 (7) 635 -642 2000年07月 [査読無し][通常論文]
The interaction between Clostridium butyricum and Helicobacter pylori was examined in vitro and in vivo. The culture supernate of C, butyricum MIYAIRI 588 inhibited the growth of H. pylori even when its pH was adjusted to 7.4. The bactericidal effect of butyric acid on H. pylori was stronger than that of lactic, acetic or hydrochloric acids. Flow cytometric analysis showed that pre-incubation of gastric epithelial (MKN45) cells with H, pylori and C, butyricum inhibited the adhesion of H, pylori to the cells. Persistent infection with H, pylori in the gastric mucosa of germ-free mice was observed for 5 weeks. Cure of persistent infection with H, pylori in the gnotobiotic mice was demonstrated following infection with C, butyricum, The probiotic agent, C, butyricum MIYAIRI 588 may have some beneficial effects on H, pylori infection. - H Yamaguchi, T Osaki, M Kai, H Taguchi, S Kamiya INFECTION AND IMMUNITY 68 (6) 3448 -3454 2000年06月 [査読無し][通常論文]
We previously established a monoclonal antibody (MAb), designated H9, which reacts with the heat shock protein 60 (HSP60) homologue of Helicobacter pylori as well as with other bacterial and human HSP60s. To determine the importance of a cross-reactive epitope on H. pylori HSP60 in H. pylori immunopathogenesis, me performed (i) mapping of an epitope on H. pylori HSP60 recognized by the H9 MAb, (ii) analysis of immunoglobulin G responses of patients with or without H. pylori infection to its epitope region, and (iii) studies of the protective effect of immunization with its epitope region on H. pylori infection in mice. The epitope recognized by the H9 MAb was mapped to the sequence of amino acids 189 to 203 (VEGMQFDRGYLSPYF) on the H. pylori HSP60 molecule. It was confirmed that the synthesized peptide designated pH9 was recognized by the H9 MAb. Enzyme-linked immunosorbent assay analysis showed that patients with H. pylori infection (n = 319) had significantly lower titers of pH9 antibody than did uninfected patients (n = 200) (P < 0.001), but this was not the case with purified H. pylori HSP60 recombinant Escherichia coli GroEL, or recombinant human HSP60. In C57BL/6 mice immunized with the pH9 peptide with Freund's complete adjuvant (PCA), the number of H. pylori organisms colonizing the stomach was significantly lower than that in mice immunized with pCont plus FCA (P < 0.0001) or FCA only (P < 0.005). The results suggest that the immune response to the cross-reactive epitope (pH9 region) on H, pylori HSP60 is unique and might be associated with protection against H. pylori infection. - 田口 晴彦, 高橋 志達, 山口 博之, 大崎 敬子, 神谷 茂 無菌生物 = Japanese journal of germfree life and gnotobiology 29 (2) 93 -95 1999年12月01日
- H Yamaguchi, T Osaki, N Kurihara, H Taguchi, S Kamiya JOURNAL OF GASTROENTEROLOGY 34 (Supplement 11) 1 -5 1999年12月 [査読無し][通常論文]
The monoclonal antibody (mAb) designated H20, which recognizes heat shock protein 60 (HSP60) of Helicobacter pylori, was previously established; and the epitope recognized by the mAb was shown to be species-specific. Using immunohistochemical staining of six gastric biopsy specimens with the H20 mAb, gastric epithelial cells of four biopsy samples stained positively. Flow cytometric analysis showed that H20 mAb reacted with primary human gastric epithelial (PHGE) cells, though the reactivities of the mAbs were different among the PHGE cells prepared. These results indicate that the species-specific epitope recognized by H20 mAb exists on human gastric cells. In addition, affinity-purified HSP60 from H. pylori by H20 mAb induced interleukin-8 (IL-8) secretion from PHGE cells (in one of four cases). These results indicate that H. pylori HSP60 induces IL-8 secretion from human gastric cells, and the levels of IL-8 differ among the various prepared PHGE cells. - H Yamaguchi, T Osaki, M Takahashi, H Taguchi, S Kamiya GUT 45 (Supple111) A36 -A36 1999年09月 [査読無し][通常論文]
- 高橋 俊雄, 田口 晴彦, 山口 博之, 大崎 敬子, 佐藤 進, 亀井 優徳, 橋爪 秀一, 神谷 茂 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 1999年07月20日
- H Yamaguchi, T Osaki, H Taguchi, S Kamiya FEMS MICROBIOLOGY LETTERS 168 (2) 277 -282 1998年11月 [査読無し][通常論文]
We have succeeded in producing monoclonal antibodies which neutralize the vacuolation of rabbit kidney cells by a cytotoxin produced by Helicobacter pylori. Vacuolating activity of several H. pylori strains correlated with ELISA values using these monoclonal antibodies and culture supernatants of the strains. These results indicate that the molecules recognized by the monoclonal antibodies might be the vacuolating toxin produced by H. pylori. In addition, the sera from patients with gastritis and gastric cancer reacted strongly with the antigens captured by the monoclonal antibodies from the supernatant containing H. pylori vacuolating toxin. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. - 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 1998年11月 [査読無し][通常論文]
The pathogenesis of chronic gastritis, gastric ulcer, duodenal ulcer, gastric cancer, and remnant gastritis is considered to be related to infection by Helicobacter pylori. The purpose of this study was to investigate the characteristics of H. pylori strains isolated from patients with various gastric diseases. Fifty strains of H. pylori were clinically isolated at Keio University Hospital and were assessed for (i) vacuolating cytotoxin (VT) production, by cytotoxic assay using rabbit kidney (RK) 13 cells; (ii) the expression of cytotoxin associated gene (cagA), by polymerase chain reaction; and (iii) motility on a semi-solid plate. Positivity for VT production by H. pylori strains isolated from patients with chronic gastritis, gastric ulcer, duodenal ulcer, gastric cancer, and remnant gastritis, was 100%, 89%, 92%, 100%, and 60%, respectively. There was no significant difference in the incidence of positivity for cagA among the strains isolated from different gastric diseases. Swarming distances, which indicate motility of PI. pylori strains, were 12.7, 13.2, 12.8, 16.6 and 9.2mm in strains from chronic gastritis, gastric ulcer, duodenal ulcer, gastric cancer, and remnant gastritis, respectively, with H. pylori isolated from remnant gastritis showing significantly less motility than H. pylori isolates from other diseases. There were no significant differences in VT production and cagA positivity among the H. pylori strains. - H Yamaguchi, T Osaki, N Kurihara, H Taguchi, S Kamiya GUT 43 A33 -A34 1998年09月 [査読無し][通常論文]
- 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 1998年06月 [査読無し][通常論文]
Monoclonal antibodies (MAbs) that inhibit adhesion of pylori to human gastric cancer (MKN45) cells were established to clarify the mechanism of adhesion of H. pylori. Of 53 hybridoma clones screened by the primary inhibition assay for adhesion, MAb A20 of IgM class was selected on the basis of both its reactivity to whole cells of H. pylori by ELISA and its inhibitory effect on adhesion of H. pylori. The H. adhesion of pylori strain TK1029 to MKN45 cells was inhibited by MAb A20, depending on the concentration of the MAb. The MAb recognised the surface antigen, lipopolysaccharide (LPS) of H. pylori, suggesting that LPS is associated with adhesion of H. pylori to human gastric epithelial cells. - 山口 博之, 大崎 敬子, 栗原 直人, 田口 晴彦, 神谷 茂 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 1998年05月20日
- 田口 晴彦, 山口 博之, 神谷 茂, 小林 宏行, 堤 裕幸, 千葉 峻三, 神谷 保彦 感染症学雑誌 72 (12) 29 -34 1998年 [査読無し][通常論文]
1997年2月より1998年2月の1年間, ケニア共和国ナイロビ市内キベラスラムにおいて, 5歳以下の急性呼吸器感染症の患者を対象に, 細菌・真菌・ウイルスの病原体検索を行った.入院患者総数226人中, 何らかの病原体が検索された病原体陽性患者数は128人 (56.6%) であった.病原体陽性患者の年齢構成は6ヵ月以下が33.3%, 7ヵ月以上1歳以下が28.5%であり, それらは全体の6割をしめた.分離・同定, 検索された病原体数は192検体であった.高頻度に検索された病原体はStreptococcus pneumoniae (60株, 31.3%) で, 次いでrespiratory syncytial virus (20検体, 10.4%), Candida albicans (19株, 9.9%), Moraxella (Branhamella) catarrhalis (15株, 7.8%), adenovirus (10検体, 5.2%), Klebsiella pneumoniae (9株, 4.7%) であった.分離されたS.pneumoniae, M.catarrhalis, K.pneumoniaeはいずれもアフリヵで汎用されている抗生物質に対して耐性を示した. - T Osaki, H Taguchi, H Yamaguchi, S Kamiya JOURNAL OF CLINICAL MICROBIOLOGY 36 (1) 321 -323 1998年01月 [査読無し][通常論文]
By an immunomagnetic-bead (IMB) separation technique, isolation of Helicobacter pylori from gastrointestinal and fecal samples of gnotobiotic mice infected with the microorganism was tried. The isolation rate of H. pylori from stomach samples after IMB separation was not higher than that of direct culture of the samples. After IMB separation of feces, H. pylori was detectable by PCR, although H. pylori was not culturable. - 山口 博之, 大崎 敬子, 田口 晴彦, 神谷 茂 Cytometry research : 日本サイトメトリー学会機関誌 7 (2) 51 -55 1997年12月12日
- H Yamaguchi, T Osaki, N Kurihara, S Kamiya GUT 41 A24 -A24 1997年09月 [査読無し][通常論文]
- 山口 博之, 田口 晴彦, 大崎 敬子, 花輪 智子, 山本 友子, 神谷 茂 無菌生物 = Japanese journal of germfree life and gnotobiology 27 (1) 68 -71 1997年06月
- 高橋 志達, 田口 晴彦, 山口 博之, 大崎 敬子, 神谷 茂 無菌生物 = Japanese journal of germfree life and gnotobiology 27 (1) 40 -43 1997年06月
- 田口 晴彦, 山口 博之, 大崎 敬子, 高橋 志達, 神谷 茂 無菌生物 = Japanese journal of germfree life and gnotobiology 27 (1) 65 -67 1997年06月
- 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年 [査読無し][通常論文]
The H20mAb recognizing the 60-kilodalton protein, which existed in the outer membrane and was induced by heat shock at 42 C, was established, The molecule recognized with the mAb was a heat-shock protein 60 (HSP60) of Helicobacter pylori, To understand the role of HSP60 on the cell surface of H. pylori, whether or not H20mAb affects the growth of H. pylori was investigated, When bacteria were cultured with H20mAb, growth was markedly inhibited after 24 hr, although an initial 5 hr-incubation with the mAb induced no significant inhibition of H. pylori growth, The 24- and 48 hr growth of the bacteria after washing to remove the mAb at 5 hr was also inhibited though the inhibitory effect was not strong, In electron microscopical analysis, the spots with high electron density in the cytoplasm of the bacteria treated with H20mAb were increased, depending on the length of incubation time from 5 to 24 hr. After 24 hr treatment with H20mAb, bacterial destruction was also observed, indicating bactericidal activity by H20mAb, These results suggest that the HSP60 on the cell surface of H. pylori might have an essential role in the growth of the bacteria. - 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年
- 神谷 茂, 山口 博之, 田口 晴彦, 甲斐 雅規, 塩沢 寛治, 緒方 幸雄 日本マイコプラズマ学会雑誌 = Japanese Journal of Mycoplasmology 21 162 -165 1995年10月01日
- H TAGUCHI, T OSAKI, H YAMAGUCHI, S KAMIYA MICROBIOLOGY AND IMMUNOLOGY 39 (11) 891 -894 1995年 [査読無し][通常論文]
A comparative study of indirect and direct flow cytometric analysis for adherence of Vibrio cholerae to Intestine 407 cells was performed. The direct flow cytometric analysis employed the lipophilic dye PKH-2. It was concluded that direct flow cytometry using the lipophilic dye PKH-2 is useful and convenient for analyzing bacterium-host cell interactions, since it does not require any specific antibody as the first antibody. - 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年 [査読無し][通常論文]
The enzyme-linked immunosorbent assay (ELISA) with bacterial heat-shock protein 60 (HSP60) purified from Yersinia enterocolitica (Ye) revealed that the antibodies directed against YeHSP60 existed in sera of patients with primary biliary cirrhosis (PBC). To characterize the epitope specificity of the antibodies in patients, the epitope mapping of HSP60 by means of the antibodies was performed. The results have suggested that the epitope recognized with anti-HSP60 antibodies in PBC relates to the amino acid sequence of YeHSP60 molecule as follows: DLGQA-KRVVINKDTTIIIDGVGDEAAIQGRLAQIRQQIEEATSDYDKEK. - H YAMAGUCHI, T YAMAMOTO, H TAGUCHI, S OGATA JOURNAL OF GENERAL MICROBIOLOGY 136 1091 -1097 1990年06月 [査読無し][通常論文]
共同研究・競争的資金等の研究課題
- 日本学術振興会：科学研究費助成事業研究期間 : 2022年04月 -2025年03月代表者 : 大久保 寅彦, 中村 眞二, 山口 博之
- 日本学術振興会：科学研究費助成事業 基盤研究(B)研究期間 : 2021年04月 -2025年03月代表者 : 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。
- 日本学術振興会：科学研究費助成事業研究期間 : 2020年04月 -2024年03月代表者 : 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：科学研究費助成事業 挑戦的研究(開拓)研究期間 : 2020年07月 -2024年03月代表者 : 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。
- 日本学術振興会：科学研究費助成事業研究期間 : 2019年04月 -2022年03月代表者 : 松尾 淳司, 山口 博之, 中村 眞二クラミジア感染における宿主細胞修飾を明らかにするために、クラミジア感染細胞のDNA損傷誘導ならびにその制御について検討した。クラミジア感染細胞のDNA損傷はクラミジア感染後期に検出された。そこでDNA損傷誘導メカニズムを明らかにするために、ROS産生に関わる遺伝子ノックダウン細胞で検討したものの明らかな違いは見いだせなかった。一方、損傷DNAの応答に関わる遺伝子群の発現解析をqPCRアレイで行ったところ、発現変動する遺伝子群が見いだされた。このように、クラミジア感染細胞において損傷DNAの誘導とその応答が発現制御されている可能性が示唆された。
- 第51回内藤記念科学奨励金研究助成研究期間 : 2019年10月 -2020年10月代表者 : 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。
- 日本学術振興会：科学研究費助成事業研究期間 : 2014年05月 -2019年03月代表者 : 小澤 岳昌, 尾崎 倫孝, 山口 博之, 芳賀 早苗生細胞内の分子の素過程をネットワークとして理解するために，生体分子を可視化および操作するための新たな分析方法を確立した．具体的には，１）小数生体分子の可視化・定量法，２）光による酵素活性制御法，３）Gタンパク質共役受容体活性を制御する光操作法を開発した．開発した方法は，基礎生命科学研究の基盤技術になるとともに，医学や農学や創薬分野等における革新的技術となる．
- 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 研究課題文部科学省(日本学術振興会)(挑戦的萌芽研究)：研究期間 : 2016年04月 -2019年03月代表者 : 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。
- 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日本学術振興会：科学研究補助金(基盤研究B)研究期間 : 2016年04月 -2019年03月代表者 : 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：科学研究費助成事業研究期間 : 2016年04月 -2018年03月代表者 : 大久保 寅彦, 山口 博之, 松尾 淳司, 中村 眞二, 松下 瑞江, 秋沢 宏次, 早坂 かすみ, 福元 達也, 岩崎 澄夫細菌はシグナル分子AI-2を介して周囲の細菌と情報交換を行なっている。本研究室では以前、原生生物と細菌を共培養するとAI-2濃度が上昇する現象を発見し、微生物間相互作用においてAI-2量の変動が起こることを明らかにした。一方、AI-2が原生生物に対してどのような影響を与えるかは知られていない。本研究ではAI-2添加下でアメーバおよび繊毛虫を培養し、非添加下と性状を比較した。その結果、固体（寒天培地）上ではアメーバの運動速度が上昇することが明らかとなり、アメーバはAI-2濃度を感知して自身の運動性を上昇させることが示された。
- 日本学術振興会：科学研究費助成事業研究期間 : 2015年04月 -2018年03月代表者 : 松尾 淳司, 中村 眞二, 山口 博之クラミジア感染における宿主炎症応答制御機構を明らかにするために、クラミジア感染を感知する新規レセプターの探索や、環境クラミジアを用いた保存性の高いクラミジア因子の探索を行ったところ、非TLRレセプターおよび易熱性のクラミジア分子が炎症誘導に関与することが示唆された。さらにこれらクラミジアによる炎症誘導が低酸素条件下で促進されることも明らかとなった。一方、炎症誘導に関連する宿主細胞のアポトーシス下流分子カスパーゼ3活性測定を行ったところ、感染後期で促進していた。またクラミジア感染においてmiRNAの発現変動が宿主炎症応答に関与する可能性が示唆された。
- 日本学術振興会：科学研究費助成事業研究期間 : 2011年04月 -2016年03月代表者 : 永井 宏樹, 山口 博之, 丸山 史人ヒトをはじめとする動物や植物などに代表される真核生物の誕生には、細菌が別の細胞内へはいりこんでミトコンドリアや葉緑体になるという、一次共生の成立が鍵となっています。しかしながら、細菌が真核細胞へ侵入するという現象自体は、細菌感染の現場で今日でも日常的に起こっています。本研究では、ヒト病原菌レジオネラ、アメーバ共生菌や病原性細胞内寄生菌とそれらの宿主である真核生物細胞との関わり方を解析することにより、生物が入れ子になって進化を駆動するというマトリョーシカ型進化の第一段階における進化原理の一端を明らかにすることができました。
- 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文部科学省(日本学術振興会)(挑戦的萌芽研究)：研究期間 : 2014年04月 -2016年03月代表者 : 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。
- 日本学術振興会：科学研究費助成事業研究期間 : 2013年04月 -2015年03月代表者 : 中村 眞二, 松尾 淳司, 石津 明洋, 山口 博之, 伊藤 晋クラミジア(Ct)は，約10%程度の感染がみられる性感染症のポプュラーな原因菌である．このCtを無治療でいると，一部の感染者に繊維化を伴う卵管閉塞など難治化することが知られているが，その理由は不明である．IL-1やPGE2などの炎症性メディエーターの動態を検索し，Ct感染症の難治化の原因を検討した結果，間質の浸潤細胞にIL-1・IL-1Ra・PGE2の陽性細胞を認めたが，Ct感染の有無による所見の差は無かった．Ct感染患者では，扁平及び腺上皮細胞にIL-1αとPGE2の強く発現されていた．この結果はPGE2が炎症を慢性化し難治化への誘因の１つとして重要な働きをしていること示唆している．
- 文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))研究期間 : 2012年 -2013年代表者 : 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：科学研究費補助金(挑戦的萌芽研究)研究期間 : 2012年 -2013年代表者 : 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：科学研究費補助金(基盤研究(C))研究期間 : 2009年 -2011年代表者 : 山口 博之, 松尾 淳司, 神谷 茂, 中村 眞二, 松尾 淳司申請者らは、病原性クラミジアのリンパ球細胞への感染様式と感染細胞に於ける細胞機能修飾機構について上皮細胞への感染様式と比較検討した。その結果、上皮細胞への付着にはヘパリングルカンが必要であるが、リンパ球細胞へ付着侵入には、この細胞外マトリックスを要求せず、未知の付着機構の存在が示唆された。また変異源物質EMSを用いて病原性クラミジア感染抵抗性リンパ球細胞株の樹立に成功し、グランザイムKの発現異常がこの抵抗性に寄与している可能性について報告した。さらにリンパ球細胞に感染した病原性クラミジアは、生体防御因子IFNγからエスケープできることを発見した。
- 日本学術振興会：科学研究費助成事業研究期間 : 2009年 -2010年代表者 : 播摩谷 敦, 藤井 暢弘, 横田 伸一, 飯田 政弘, 氷見 徹夫, 山口 博之中耳炎に関する新種細菌アロイオコッカスについての、発育至適条件や臨床症例における頻度、ならびに本細菌に対する宿主免疫応答の詳細について、新たな事実を発見した。また、本細菌の抗原を同定、さらには抗原構造の解析を遂行し、新たな知見を得た。
- 文部科学省：科学研究費補助金(基盤研究(C))研究期間 : 2005年 -2006年代表者 : 山口 博之, 山本 容正, 川野 淳偏性細胞内寄生性細菌である肺炎クラミジア[Chlamydia (Chlamydophila) pneumoniae]は10数年前より、ヒトからヒトへ伝播する呼吸器感染症の原因微生物として知られるようになった新興感染症の病原体である。本菌呼吸器感染症は、しばしば慢性化し、喘息や慢性気管支炎の原因としても注目されている。一方、近年になって、C.pneumoniae感染症と糖尿病、高血圧、肥満等の生活習慣病に併発する動脈硬化症との関連性が明らかになってきた。その詳細な機構は未だ明らかではないが、末梢血液単核球細胞(PBMC)や動脈硬化部位からC.pneumoniae遺伝子や生菌が検出されることより、本菌が呼吸器から何等かの細胞を介して動脈血管に移行し、動脈硬化病態形成に関与しているものと考えられる。C.pneumoniaeは肺ならびに気道上皮系細胞に感染するが、肺胞マクロファージ等の単球/マクロファージ系細胞においても同様に感染することが既に知られている。それ故に、呼吸器から動脈血管への媒介細胞として単球/マクロファージ系細胞が重要な役割を演じていると考えられている。最近になって、ヒト末梢血液中のCD3陽性リンパ球よりC.pneumoniae遺伝子が検出されるとともにC.pneumoniaeがリンパ球中においても単級/マクロファージ系細胞と同様に感染し、発育・増殖が可能であることが明らかになり、リンパ球も(C.pneumoniaeを肺から動脈血管に移行させうる媒介細胞として重要な役割を演じていると考えられる様になって来た。またリンパ球内に存在するC.pneumoniaeは、抗生物質やサイトカインにより活性化した細胞の影響を受け難く、容易に生体の排除機構からエスケープし潜伏・持続感染を成立しうる可能性がある。さらに動脈硬化症病態形成においてリンパ球等免疫担当細胞が重要な役割を演じているが、C.pneumoniaeのリンパ球への潜伏・持続感染により誘導される免疫応答の変化が動脈硬化病態形成機序に極めて大きな影響を及ぼす可能性もある。それ故に、リンパ球細胞内でのC.pneumoniaeの感染様式を明らかにすることは、本菌による慢性呼吸器疾患や動脈硬化症発症機序を明らかにする上で極めて有用であると考えられるが、未だその詳細は明らかになっていない。そこで申請者等は、本研究において(C.pneumoniae感染リンパ球の細胞レベルでの動態ならびに本菌のリンパ細胞内での潜伏・持続感染様式を明らかにすることを目的として本研究を実施した。まずリンパ球細胞球株やマウスPBMCにC.pneumoniaeをin vitroにて感染させ細胞内での菌の動態ならびに細胞機能修飾株やマウスPBMCにC.pneumoniaeをin vitroにて感染させ細胞内での菌の動態ならびに細胞機能修飾について検討を行いCD3ならびにCD25分子の発現が修飾されることを見いだした。C.pneumoniae感染免疫担当細胞からの除菌が極めて困難であることを分け分けは以前報告しているが、免疫担当細胞(単球系細胞株)に感染した(C.pneumoniaeを効果的に排除する薬剤を見つけ出すことに成功した。さらにC.pneumoniae感染により本菌が生菌レベルにて末梢血液に移行することをNODマウスにて確認し報告することができた。
- 文部科学省：科学研究費補助金(基盤研究(C))研究期間 : 2003年 -2004年代表者 : 山口 博之, 神谷 茂, 大崎 敬子, 田口 晴彦アテローム性動脈硬化症は脳、心臓、その他の重要な器官および四肢の動脈に起こる血管壁内層への脂質の蓄積を伴う動脈硬化の一種である。アテローム性動脈硬化症による心臓発作や脳卒中は米国、西洋諸国そして本邦における死亡の主要な原因であり、その病態機序解明および根本的治療法の開発は心臓発作および脳卒中の予防・治療の本質的なストラテジーである。1999年、Rossはアテローム性動脈硬化症の病態形成がリンパ球や単球/マクロファージを主体とする免疫担当細胞の関与する慢性炎症反応であるという説を初めて提示した。現在、この考えは広く認められ、偏性細胞内寄生性細菌Chlamydia(Chlamydophila)pneumoniaeがこの慢性炎症反応を惹起する要因であると考えられている。またC.pneumoniae遺伝子や抗原が動脈硬化プラークより高率に検出されることより、動脈硬化病態形成には本菌の硬化巣への感染そのものが極めて重要な要因になっている可能性も考えられる。しかしながら本来、経気道感染により肺炎を誘導する普遍的な日常感染菌がどのように動脈内に移行し動脈硬化病変形成に関与しているのか明らかにされておらずそのメカニズム解明は進んでいない。そこで本研究ではC.pneumoniaeの生体内伝播様式を解明することを目的として、健常人ならびに循環器障害を持つ患者末梢血液細胞PBMCからの生菌レベルでのC.pneumoniaeの検出を試みた。また肺に感染したC.pneumoniaeを生菌レベルにて末梢血液に移行しうる動物モデルの確立についても検討を検行った。その結果、ヒト末梢血液細胞PBMCからC.pneumoniaeが生菌レベルで検出された。本菌の検出レベルが健康人に比べ循環器障害を持つ入院患者にて有意に高かった事より、ヒト末梢血液細胞での本菌の存在は動脈硬化症発症において大きな危険因子になりうる可能性が示唆された。またC.pneumoniae感染NODマウスにおいて本菌の肺から末梢血液細胞への生菌レベルでの移行が確認された。C.pneumoniaeの肺から末梢血液細胞への移行機序ならびに末梢血液細胞内でのC.pneumoniaeの存在意義を解明するためには更なる検討が必要であるが、本研究により得られた結果は、本菌感染症の動脈硬化症病態形成機構を考える上で極めて有用な結果であると思われた。
- 日本学術振興会：科学研究費助成事業研究期間 : 2000年 -2001年代表者 : 神谷 茂, 山口 博之, 田口 晴彦H.pylorの主要なストレス蛋白であるHSP60が胃粘膜炎症の大きな役割を担うサイトカインの二種、IL-8の産生を誘導することが、精製HSP60およびHSP60融合蛋白を用いた実験により明らかにされた。加えて、精製HSP60が胃上皮細胞にアポトーシスを誘導することも示された。これらの研究結果は本菌HSP60が種々のストレスにより誘導される蛋白であるとともに、胃粘膜にストレスを与える病原因子としても機能する可能性を提示している。 また、H.pylori HSP60は宿主細胞性のHSP60と相同性を有するため、ヒト胃粘膜細胞上に本菌HSP60エピトープが高頻度で検出された。この頻度はH.phlori感染者と非感染者の間で有意な差は認められなかったが、高い反応性エピトープを有する感染者の長期にわたる臨床観察に注目したい。whole HSP60を抗原とした場合、H.pylori感染者の血清は非感染者に比べ、有意に強い反応性を有していた。しかしながら、部分エピトープpH9を抗原とした場合には、非感染者血清が感染者血清に比べ有意に強い反応性を示した。動物実験においても、pH9ペプチドが感染防御を誘導し得ることが示された。H.pylori感染予防にためのワクチンが現在開発されており、本菌HSP60の部分ペプチドも優れたワクチンコンポーネントになり得るものと考えられる。
- 日本学術振興会：科学研究費助成事業研究期間 : 1999年 -1999年代表者 : 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：科学研究費助成事業研究期間 : 1997年 -1998年代表者 : 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：科学研究費助成事業研究期間 : 1997年 -1998年代表者 : 神谷 茂, 大崎 敬子, 山口 博之, 田口 晴彦Helicobacter pylori TK1402株を無菌(GF)マウスに経口接種(10^8-10^9cfu/0.5ml,3日間連続投与)した結果,感染9週にわたるまで安定な本菌の持続感染が認められた。幽門部粘膜には著明な炎症細胞の浸潤と部分的な粘膜萎縮が検出され,GFマウスはH.pyloriの定着-持続感染のモデルになり得ることが示された。また感染マウス糞便より本菌の存在を免疫磁気ビーズ法とPCR法との併用により検出することができた。本法により感染マウスを斃死させることなく,定着H.pylori,を評価することが可能となった。 H.pyloriの熱ショック蛋白(HSP60)は本菌の胃上皮細胞への付着に関与することがin vitro実験で示された。また本菌を抗HSP60モノクローナル抗体で前処理することにより,無菌マウスへの胃内定着菌数が著減することか明らかにされた。本結果はin vivoでもHSP60が本菌の胃上皮細胞への付着に深く関与することが示された。HSP60のGFマウスへの経口投与は胃粘膜に著名な病変を引き起こさなかった。 しかし,HSP60経口投与後,H.pyloriの感染を行った結果,対照に比べ胃炎病変の増強が認められたが,定着菌数の著明な減少がみられた。 H.pylori定着マウスへのイミダゾール誘導体およびLactobacillus salivarius又はClostridium butyricumの投与により,H.pyloriの除菌が認められた。これらの結果はGFマウスが副作用(下痢,耐性菌の誘導等)のない抗菌剤(因子)の開発および評価に有用であることを示している。
- 文部科学省：科学研究費補助金(奨励研究(A))研究期間 : 1997年 -1998年代表者 : 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.
- 日本学術振興会：科学研究費助成事業研究期間 : 1996年 -1998年代表者 : 山本 友子, 山口 博之, 田口 晴彦, 花輪 智子細菌は,生体への感染に際して様々なストレスに曝される。従って,感染宿主内での細菌の生育および病原性発現には、ストレス蛋白質が重要な役割を果たす可能性を考えることができる。感染初期の生体防御機構の中心であるマクロファージ(MΦ)等の食細胞による殺菌作用は,細菌が感染時に遭遇する最も過酷なストレスであり、細胞内寄生細菌とってMΦでの殺菌機構からのエスケープと増殖能は、重要な病原因子と考えられている。このような観点から,異なるエスケープ機構を持つ2種の細胞内寄生細菌を用いて,MΦ内での細菌の増殖におけるストレス蛋白質の役割解明を目的として本研究を行い、次のような成果を得た。(1)Yersinia enterocoliticaは、MΦに貪食されると、殺菌物質の産生を抑制しながらファゴソーム内で増殖するが、その際誘発されたストレス蛋白質の一つであり、ペリプラスム極在型プロテアーゼGsrAが、その増殖に必須であることを明らかにした。(2)Listeria monocytogenesは、貪食されると極めて早い時期にファゴソームを脱出し、細胞質内で増殖する。このようなエスケープ機構を持つ細菌は、貪食後ストレス蛋白質の合成を誘発しないことを明らかにした。そこで、平常時においても産生されるストレス蛋白質DnaKの役割を検討した。(3)dnaK遺伝子のクローニングを行い、hrcA,grpE,dnaK,dnaJ,orf35,orf29の6つの遺伝子からなるオペロンの存在を明らかにした。(4)dnaK挿入変異株を分離し、MΦ内増殖能を検討した結果、シャペロン機能を持つストレス蛋白質DnaKは,本菌のMΦ内増殖には必須ではないが,貪食過程に関与することが明らがとなった。これらの研究を通じて,細菌の病原性発現に関わる菌種特異的な機構を超えた普遍的な機構の存在を明らかにすることができた。
- 日本学術振興会：科学研究費助成事業研究期間 : 1995年 -1996年代表者 : 神谷 茂, 大崎 敬子, 山口 博之Helicobacter pyloriの種々の病原因子のうち,付着因子,空胞化毒素(vacuolating toxin:VT),サイトカイン(特にインターロイキン8(IL-8),熱ショック蛋白(heat shock protein:HSP)および活性酸素に注目し,これらの因子の性状を解析することによりH.pylori感染による胃粘膜障害発現機構の解明を行った。 フローサイトメーター(FCM)による付着性の解析の結果,H.pyloriは胃由来上皮細胞株と強い親和性をもつことが明らかにされた。また,本菌はモルモット,ウサギ,ヒト,ヒツジ,ニワトリ,ウマ,ウシの赤血球を凝集する活性を有すると共に,これらの活性が菌の細胞への付着性と関連することも明らかにされた。 H.pyloriの約半数の菌株はVTを産生する。VT産生性菌株培養上清は,モルモット胃より調整した壁細胞の胃酸分泌能を抑制したが,VT非産生性菌株培養上清はこのような効果をもたなかった。細胞内シグナル伝達のセカンドメッセンジャーの解析より,VTは直接プロトンポンプを抑制していることが考えられた。 H.pyloriは胃上皮細胞および好中球と接触することにより,IL-8および活性酸素を生成させることが明らかにされた。またこれらの効果は菌株培養上清のみでは引き起こされなかった。 HSP60(分子量6万)はH.pylori菌体内には被験全菌株に同程度検出されたが,菌体表面での発現は菌株間により異なっていた。また,この発現率は菌株の胃上皮細胞への付着性と相関していた。胃上皮細胞表面に本菌HSP60と交叉反応性を示すエピトープの存在が確認され,HSP60が慢性炎症および粘膜障害のトリガーとなる可能性が示された。 以上,付着因子,VT,サイトカイン,活性酸素,HSP60等のいずれも胃粘膜障害に関与するものと考えられ,障害機構は現在のところmultifactorialであると想定される。
- 文部科学省：科学研究費補助金(奨励研究(A))研究期間 : 1995年 -1995年代表者 : 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)。
- 日本学術振興会：科学研究費助成事業研究期間 : 1994年 -1995年代表者 : 山本 友子, 花輪 智子, 山口 博之, 田口 晴彦Yersinia enterocoliticaは、マクロファージ等の食細胞による食菌作用に抵抗し、増殖することが可能な細胞寄生性を有している。本研究においてY.enterocoliticaのマクロファージ内生残と増殖には、高温や過酸化物等の環境ストレス下での生育に必須なストレスタンパク質のひとつであるGsrAが要求されることが明らかとなった。gsrA遺伝子の全塩基配列を決定し、それに基ずくアミノ酸配列のホモロジー検索を行ったところ、GsrAは大腸菌HtrAのホモログであることが明らかとなった。HtrAはペリプラズムに存在するプロテアーゼであるが、種々の環境ストレスおよびマクロファージ内ストレスにより生じた変性タンパク質を分解する機能が、Y.enterocoliticaの細胞外および細胞内ストレス下での生育に要求されるものと考えられる。 Y.enterocoliticaのマクロファージ内増殖能は、上皮細胞に侵襲する能力とあわせて病原性発現に必須の因子である。本菌固有の病原因子としては病原プラスミド性のYop蛋白質群が知られているが、それらの中で,YopHとYopDはマクロファージ抵抗性に関与すると報告されている。これらのY.enterocolitica特有の病原因子に加え、GsrAストレス蛋白質が病原性発現に至る複雑なネットワークに関与しているものと考えられる。 今後は、マクロファージ貧食で誘発される一群のストレスタンパク質の機能解明をはじめ、ストレスタンパク質の病原因子としての役割を明らかにしていきたい。
- 日本学術振興会：科学研究費助成事業研究期間 : 1993年 -1994年代表者 : 神谷 茂, 山口 博之, 白井 孝之Helicobacter pylori感染と胃十二指腸疾患発症との関連性を調べるために、胃十二指腸疾患患者より本菌の分離を試みた。胃潰瘍、十二指腸潰瘍、萎縮性胃炎患者からの本菌検出率は各々83、85、75%であり、急性表層性胃炎(25%)、粘膜下腫瘍(33%)患者のそれらに比べて高率であった。胃生検材料のウレアーゼテストの培養法に対する感度、特異性、陽性および陰性予知率は各々76、85、91、63%であり、本テストは迅速スクリーニングテストとして有用であることが判明した。定量培養の結果、1生検材料あたり5,000-1,000CFUのH.pyloriが存在するとウレアーゼテストが陽性となることが示唆された。 H.pylori の約半数の菌株は培養細胞の空胞化を引き起こす細胞障害毒素 cytotoxin(CT)を産生する。CTは70℃で易熱性な分子量2万以上の蛋白であることが明らかにされた。50%硫安塩析後、セファクリルS300ゲル濾過、フェニルスーパーロース疎水結合カラム、Qセファロース FFおよびモノQ陰イオン交換カラム等によりCTの高度精製を試みたが成功に到らなかった。H.pylori 33菌株のCT産生性を検討した。ウサギ腎(RK13)、ヒト羊膜上皮(FL)、サル腎(Vero)、ハムスター腎(BHK-21)、ヒト子宮癌(HeLa)細胞を指示細胞とした場合のCT検出率は各々73、61、27、27、21%であり、CT検出細胞としてRK-13細胞が優れていることが明らかとなった。 H.pylori 15菌株のMKN45、KATO III(いずれもヒト胃癌由来)およびInt-407(ヒト小腸上皮由来)細胞への付着性をフローサイトメーターにより解析した。被検15菌株のMKN45、KATO III、Int-407への平均付着率はそれぞれ76.6、42.7、15.1%であり、H.pyloriは、小腸由来細胞よりも胃上皮由来細胞に対して強い親和性を有し、特にMKN45細胞に対して強い付着性をもつことが示された。CT産生性と付着率の間に有意な相関は認められなかった。
- 日本学術振興会：科学研究費助成事業研究期間 : 1992年 -1993年代表者 : 山本 友子, 山口 博之, 田口 晴彦, 緒方 幸雄細菌の病原性発現におけるストレス蛋白質の役割を解明するために、Yersinia enterocoliticaのストレス蛋白質変異株を分離後、それらの病原性発現に関する性状を解析し、以下のような結果を得た。さらにマクロファージ貪食によりY.enterocoliticaのストレス応答が誘発されることを見いだした。 1.我々が開発したトランスポゾン挿入変異株を分離するためのsuicide plasmidを用いて、Y.enterocolitica 0:8より1800株のカナマイシントランスポゾン挿入変異株を得た。その中で温度感受性の性質を示す48株についてマクロファージ内増殖能を検討した。その結果1株のマクロファージ増殖能欠損変異株が得られた。このマクロファージ感受性変異株は、マウスに対する病原性が低下していた。この変異株は、過酸化水素などのoxidative stressに対しても感受性をしめした。現在、対応する遺伝子をクローニングし、遺伝子レベルでの解析を行っているが、本研究の成果は、細菌のストレス蛋白質が病原性の発現に重要な役割をもつだろうという我々の推論を強く支持するものであった。 2.マクロファージ貪食により、1時間以内にY.enterocoliticaの約20種類の蛋白質の新たな合成あるいは、その産生量が増加することを確認した。そのなかで6種類の蛋白質が熱ショックで誘発されるストレス蛋白質と2次元ゲル電気泳動上で一致した。誘発された蛋白質のうち、免疫学的反応性からhsp60およびhsp70が同定された。貪食後の主要なストレス蛋白質の合成の変化を経時的に検討した結果、誘発は1時間以内におこり、22時間後においても続くことが明らかとなった。誘発量の最も多い22,000ダルトンの塩基性蛋白質に注目し、遺伝子の解析を行っている。
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