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Keiji Nakamura Last modified date：2023.11.22

Lecturer / Pathobiology / Department of Basic Medicine / Faculty of Medical Sciences


Papers
1. Yukari Tanaka, Riu Yamashita, Junko Kawashima, Hiroshi Mori, Ken Kurokawa, Shinji Fukuda, Yasuhiro Gotoh, Keiji Nakamura, Tetsuya Hayashi, Yoshiyuki Kasahara, Yukuto Sato, Shin Fukudo, Omics profiles of fecal and oral microbiota change in irritable bowel syndrome patients with diarrhea and symptom exacerbation., Journal of gastroenterology, 10.1007/s00535-022-01888-2, 57, 10, 748-760, 2022.10, BACKGROUND: Irritable bowel syndrome (IBS) is a disorder of gut-brain interaction, including dysregulation of the hypothalamic-pituitary-adrenal axis with salivary cortisol changes. However, the role of gastrointestinal microbiota during IBS symptom exacerbation remains unclear. We tested the hypothesis that the microbial species, gene transcripts, and chemical composition of fecal and oral samples are altered during the exacerbation of IBS symptoms. METHODS: Fecal, salivary, and dental plaque samples were collected at baseline from 43 men with IBS with diarrhea (IBS-D) and 40 healthy control (HC) men. Samples in the IBS-D patients were also collected during symptom exacerbation. The composition of the fecal microbiota was determined by analyzing the 16S rRNA gene, RNA-based metatranscriptome, and metabolites in samples from HC and IBS patients with and without symptom exacerbation. Oral samples were also analyzed using omics approaches. RESULTS: The fecal microbiota during IBS symptom exacerbation exhibited significant differences in the phylogenic pattern and short-chain fatty acid compared with fecal samples during defecation when symptoms were not exacerbated. Although there were no significant differences in the phylogenic pattern of fecal microbiota abundance between HCs and IBS-D patients, significant differences were detected in the expression patterns of bacterial transcriptomes related to butyrate production and neuroendocrine hormones, including tryptophan-serotonin-melatonin synthesis and glutamine/GABA. The composition of plaque microbiota was different between HC and IBS-D patients during normal defecation. CONCLUSIONS: Our findings suggest that colonic host-microbial interactions are altered in IBS-D patients during exacerbation of symptoms. There were no overlaps between feces and oral microbiomes..
2. Keiji Nakamura, Kazuko Seto, Kenichi Lee, Tadasuke Ooka, Yasuhiro Gotoh, Itsuki Taniguchi, Yoshitoshi Ogura, Jacques Georges Mainil, Denis Piérard, Tetsuya Harada, Yoshiki Etoh, Saori Ueda, Mitsuhiro Hamasaki, Junko Isobe, Keiko Kimata, Hiroshi Narimatsu, Jun Yatsuyanagi, Makoto Ohnishi, Sunao Iyoda, Tetsuya Hayashi, Global population structure, genomic diversity and carbohydrate fermentation characteristics of clonal complex 119 (CC119), an understudied Shiga toxin-producing E. coli (STEC) lineage including O165:H25 and O172:H25, Microbial Genomics, 10.1099/mgen.0.000959, 9, 3, 2023.03,

Among Shiga toxin (Stx)-producing

Escherichia coli

 (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified.

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3. Tatsuya Miyata, Itsuki Taniguchi, Keiji Nakamura, Yasuhiro Gotoh, Dai Yoshimura, Takehiko Itoh, Shinichiro Hirai, Eiji Yokoyama, Makoto Ohnishi, Sunao Iyoda, Yoshitoshi Ogura, Tetsuya Hayashi, Alteration of a Shiga toxin-encoding phage associated with a change in toxin production level and disease severity in Escherichia coli., Microbial genomics, 10.1099/mgen.0.000935, 9, 2, 2023.02, Among the nine clades of Shiga toxin (Stx)-producing Escherichia coli O157:H7, clade 8 is thought to be highly pathogenic, as it causes severe disease more often than other clades. Two subclades have been proposed, but there are conflicting reports on intersubclade differences in Stx2 levels, although Stx2 production is a risk factor for severe disease development. The global population structure of clade 8 has also yet to be fully elucidated. Here, we present genome analyses of a global clade 8 strain set (n=510), including 147 Japanese strains sequenced in this study. The complete genome sequences of 18 of the 147 strains were determined to perform detailed clade-wide genome analyses together with 17 publicly available closed genomes. Intraclade variations in Stx2 production level and disease severity were also re-evaluated within the phylogenetic context. Based on phylogenomic analysis, clade 8 was divided into four lineages corresponding to the previously proposed SNP genotypes (SGs): SG8_30, SG8_31A, SG8_31B and SG8_32. SG8_30 and the common ancestor of the other SGs were first separated, with SG8_31A and SG8_31B emerging from the latter and SG8_32 emerging from SG8_31B. Comparison of 35 closed genomes revealed the overall structure of chromosomes and pO157 virulence plasmids and the prophage contents to be well conserved. However, Stx2a phages exhibit notable genomic diversity, even though all are integrated into the argW locus, indicating that subtype changes in Stx2a phage occurred from the γ subtype to its variant (γ_v1) in SG8_31A and from γ to δ in SG8_31B and SG8_32 via replacement of parts or almost entire phage genomes, respectively. We further show that SG8_30 strains (all carrying γ Stx2a phages) produce significantly higher levels of Stx2 and cause severe disease more frequently than SG8_32 strains (all carrying δ Stx2a phages). Clear conclusions on SG8_31A and SG8_31B cannot be made due to the small number of strains available, but as SG8_31A (carrying γ_v1 Stx2a phages) contains strains that produce much more Stx2 than SG8_30 strains, attention should also be paid to this SG..
4. 新居 佑規, 中村 佳司, 鈴木 孝一朗, 安倍 裕順, 新澤 直明, 堀口 安彦, 百日咳毒素の感染における役割の分析(Analysis of roles of pertussis toxin in infection), 日本細菌学雑誌, 71, 1, 74-74, 2016.02.
5. 鈴木 孝一朗, 新澤 直明, 中村 佳司, 堀口 安彦, Bordetella pertussis感染症に対する新規の抗原の探索(Seeking novel antigens against Bordetella pertussis infection), 日本細菌学雑誌, 71, 1, 99-99, 2016.02.
6. Keiji Nakamura, Kazuko Seto, Junko Isobe, Itsuki Taniguchi, Yasuhiro Gotoh, Tetsuya Hayashi, Insertion Sequence (IS)-Excision Enhancer (IEE)-Mediated IS Excision from the lacZ Gene Restores the Lactose Utilization Defect of Shiga Toxin-Producing Escherichia coli O121:H19 Strains and Is Responsible for Their Delayed Lactose Utilization Phenotype, Applied and Environmental Microbiology, 10.1128/aem.00760-22, 88, 16, e0076022, 2022.08, Insertion sequences (ISs) can modulate gene expression by gene inactivation or activation. While phenotypic changes due to IS insertion/transposition are frequently observed, gene reactivation by precise or simple IS excision rarely occurs..
7. Ruriko Nishida, Keiji Nakamura, Itsuki Taniguchi, Kazunori Murase, Tadasuke Ooka, Yoshitoshi Ogura, Yasuhiro Gotoh, Takehiko Itoh, Atsushi Toyoda, Jacques Georges Mainil, Denis Piérard, Kazuko Seto, Tetsuya Harada, Junko Isobe, Keiko Kimata, Yoshiki Etoh, Mitsuhiro Hamasaki, Hiroshi Narimatsu, Jun Yatsuyanagi, Mitsuhiro Kameyama, Yuko Matsumoto, Yuhki Nagai, Jun Kawase, Eiji Yokoyama, Kazuhiko Ishikawa, Takayuki Shiomoto, Kenichi Lee, Dongchon Kang, Koichi Akashi, Makoto Ohnishi, Sunao Iyoda, Tetsuya Hayashi, The global population structure and evolutionary history of the acquisition of major virulence factor-encoding genetic elements in Shiga toxin-producing Escherichia coli O121:H19., Microbial genomics, 10.1099/mgen.0.000716, 7, 12, 2021.12, Shiga toxin (Stx)-producing Escherichia coli (STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC..
8. Yasuhiro Gotoh, Yuya Atsuta, Takako Taniguchi, Ruriko Nishida, Keiji Nakamura, Yoshitoshi Ogura, Naoaki Misawa, Tetsuya Hayashi, Helicobacter cinaedi is a human-adapted lineage in the Helicobacter cinaedi/canicola/'magdeburgensis' complex., Microbial genomics, 10.1099/mgen.0.000830, 8, 5, 2022.05, Helicobacter cinaedi is an enterohepatic Helicobacter that causes bacteremia and other diseases in humans. While H. cinaedi-like strains are isolated from animals, including dog isolates belonging to a recently proposed H. canicola, little is known about the genetic differences between H. cinaedi and these animal isolates. Here, we sequenced 43 H. cinaedi- or H. canicola-like strains isolated from humans, hamsters, rats and dogs and collected 81 genome sequences of H. cinaedi, H. canicola and other enterohepatic Helicobacter strains from public databases. Genomic comparison of these strains identified four distinct clades (clades I-IV) in H. cinaedi/canicola/'magderbugensis' (HCCM) complex. Among these, clade I corresponds to H. cinaedi sensu stricto and represents a human-adapted lineage in the complex. We identified several genomic features unique to clade I. They include the accumulation of antimicrobial resistance-related mutations that reflects the human association of clade I and the larger genome size and the presence of a CRISPR-Cas system and multiple toxin-antitoxin and restriction-modification systems, both of which indicate the contribution of horizontal gene transfer to the evolution of clade I. In addition, nearly all clade I strains but only a few strains belonging to one minor clade contained a highly variable genomic region encoding a type VI secretion system (T6SS), which could play important roles in gut colonization by killing competitors or inhibiting their growth. We also developed a method to systematically search for H. cinaedi sequences in large metagenome data sets based on the results of genome comparison. Using this method, we successfully identified multiple HCCM complex-containing human faecal metagenome samples and obtained the sequence information covering almost the entire genome of each strain. Importantly, all were clade I strains, supporting our conclusion that H. cinaedi sensu stricto is a human-adapted lineage in the HCCM complex..
9. Tadasuke Ooka, Kazuko Seto, Yoshitoshi Ogura, Keiji Nakamura, Atsushi Iguchi, Yasuhiro Gotoh, Mikiko Honda, Yoshiki Etoh, Tetsuya Ikeda, Wakana Sugitani, Takayuki Konno, Kimiko Kawano, Naoko Imuta, Kiyotaka Yoshiie, Yukiko Hara-Kudo, Koichi Murakami, Tetsuya Hayashi, Junichiro Nishi, O-antigen biosynthesis gene clusters of Escherichia albertii: their diversity and similarity to Escherichia coli gene clusters and the development of an O-genotyping method, Microbial Genomics, 10.1099/mgen.0.000314, 5, 11, 2019.11, Escherichia albertii is a recently recognized human enteropathogen that is closely related to Escherichia coli. In many Gram-negative bacteria, including E. coli, O-antigen variation has long been used for the serotyping of strains. In E. albertii, while eight O-serotypes unique to this species have been identified, some strains have been shown to exhibit genetic or serological similarity to known E. coli/Shigella O-serotypes. However, the diversity of O-serotypes and O-antigen biosynthesis gene clusters (O-AGCs) of E. albertii remains to be systematically investigated. Here, we analysed the O-AGCs of 65 E. albertii strains and identified 40 E. albertii O-genotypes (EAOgs) (named EAOg1-EAOg40). Analyses of the 40 EAOgs revealed that as many as 20 EAOgs exhibited significant genetic and serological similarity to the O-AGCs of known E. coli/Shigella O-serotypes, and provided evidence for the inter-species horizontal gene transfer of O-AGCs between E. albertii and E. coli. Based on the sequence variation in the wzx gene among the 40 EAOgs, we developed a multiplex PCR-based O-genotyping system for E. albertii (EAO-genotyping PCR) and verified its usefulness by genotyping 278 E. albertii strains from various sources. Although 225 (80.9 %) of the 278 strains could be genotyped, 51 were not assigned to any of the 40 EAOgs, indicating that further analyses are required to better understand the diversity of O-AGCs in E. albertii and improve the EAO-genotyping PCR method. A phylogenetic view of E. albertii strains sequenced so far is also presented with the distribution of the 40 EAOgs, which provided multiple examples for the intra-species horizontal transfer of O-AGCs in E. albertii..
10. A. Habets, F. Crombé, K. Nakamura, V. Guérin, K. De Rauw, D. Piérard, M. Saulmont, T. Hayashi, J.G. Mainil, D. Thiry, Genetic characterization of Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 from diarrhoeic and septicaemic calves and relatedness to human Shigatoxigenic E. coli O80:H2, Journal of Applied Microbiology, 10.1111/jam.14759, 2020.07.
11. Kai Ishimaru, Mari Sasaki, Hiroshi Narimatsu, Yoko Arimizu, Yasuhiro Gotoh, Keiji Nakamura, Tetsuya Hayashi, Yoshitoshi Ogura, Escherichia coli O8:H8 Carrying a Novel Variant of the Heat-Labile Enterotoxin LT2 Gene Caused Outbreaks of Diarrhea, Open Forum Infectious Diseases, 10.1093/ofid/ofaa021, 7, 1, ofaa021, 2020.01, Abstract
No outbreaks caused by Escherichia coli–producing heat-labile enterotoxin LT2 have been reported to date. Here, we revealed that the E. coli O8:H8 strains isolated from patients in 2 independent diarrhea outbreaks were negative for any known virulence determinants in routine microbiological tests, were very closely related, and carried a prophage-encoded gene for a novel LT2 variant (LT2d) and the genes for colonization factor antigen III. We also showed that LT2d has a cytotonic activity similar to LT1. These data indicate the importance of E. coli strains producing LT2d as a human pathogen..
12. Keiji Nakamura, Kazunori Murase, Mitsuhiko P. Sato, Atsushi Toyoda, Takehiko Itoh, Jacques Georges Mainil, Denis Piérard, Shuji Yoshino, Keiko Kimata, Junko Isobe, Kazuko Seto, Yoshiki Etoh, Hiroshi Narimatsu, Shioko Saito, Jun Yatsuyanagi, Kenichi Lee, Sunao Iyoda, Makoto Ohnishi, Tadasuke Ooka, Yasuhiro Gotoh, Yoshitoshi Ogura, Tetsuya Hayashi, Differential dynamics and impacts of prophages and plasmids on the pangenome and virulence factor repertoires of Shiga toxin-producing Escherichia coli O145:H28, Microbial Genomics, 10.1099/mgen.0.000323, 6, 1, 2020.01.
13. Shihono Teruya, Yukihiro Hiramatsu, Keiji Nakamura, Aya Fukui-Miyazaki, Kentaro Tsukamoto, Noriko Shinoda, Daisuke Motooka, Shota Nakamura, Keisuke Ishigaki, Naoaki Shinzawa, Takashi Nishida, Fuminori Sugihara, Yusuke Maeda, Yasuhiko Horiguchi, Bordetella Dermonecrotic Toxin Is a Neurotropic Virulence Factor That Uses CaV3.1 as the Cell Surface Receptor, mBio, 10.1128/mbio.03146-19, 11, 2, 2020.03, ABSTRACT
Dermonecrotic toxin (DNT) is one of the representative toxins produced by Bordetella pertussis, but its role in pertussis, B. pertussis infection, remains unknown. In this study, we identified the T-type voltage-gated Ca2+ channel CaV3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As CaV3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of B. pertussis. The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed.


IMPORTANCE Bordetella pertussis, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca2+ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of B. pertussis infection..
14. Keiji Nakamura, Yoshitoshi Ogura, Yasuhiro Gotoh, Tetsuya Hayashi, Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli, PLOS Pathogens, 10.1371/journal.ppat.1009073, 17, 4, e1009073-e1009073, 2021.04, Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species..
15. Mitsuhiko P Sato, Yoshitoshi Ogura, Keiji Nakamura, Ruriko Nishida, Yasuhiro Gotoh, Masahiro Hayashi, Junzo Hisatsune, Motoyuki Sugai, Itoh Takehiko, Tetsuya Hayashi, Comparison of the sequencing bias of currently available library preparation kits for Illumina sequencing of bacterial genomes and metagenomes, DNA Research, 10.1093/dnares/dsz017, 26, 5, 391-398, 2019.10, Abstract
In bacterial genome and metagenome sequencing, Illumina sequencers are most frequently used due to their high throughput capacity, and multiple library preparation kits have been developed for Illumina platforms. Here, we systematically analysed and compared the sequencing bias generated by currently available library preparation kits for Illumina sequencing. Our analyses revealed that a strong sequencing bias is introduced in low-GC regions by the Nextera XT kit. The level of bias introduced is dependent on the level of GC content; stronger bias is generated as the GC content decreases. Other analysed kits did not introduce this strong sequencing bias. The GC content-associated sequencing bias introduced by Nextera XT was more remarkable in metagenome sequencing of a mock bacterial community and seriously affected estimation of the relative abundance of low-GC species. The results of our analyses highlight the importance of selecting proper library preparation kits according to the purposes and targets of sequencing, particularly in metagenome sequencing, where a wide range of microbial species with various degrees of GC content is present. Our data also indicate that special attention should be paid to which library preparation kit was used when analysing and interpreting publicly available metagenomic data..
16. BspR/BtrA, an Anti-σ factor, regulates the ability of Bordetella bronchiseptica to cause cough in rats
Bordetella pertussis, B. parapertussis, and B. bronchiseptica cause respiratory infections, many of which are characterized by coughing of the infected hosts. The pathogenesis of the coughing remains to be analyzed, mainly because there were no convenient infection models of small animals that replicate coughing after Bordetella infection. Here, we present a coughing model of rats infected with B. bronchiseptica. Rats, which are one of natural hosts of B. bronchiseptica, were readily infected with the organisms and showed frequent coughing. B. pertussis also caused coughing in rats, which is consistent with previous reports, but the cough response was less apparent than the B. bronchiseptica-induced cough. By using the rat model, we demonstrated that adenylate cyclase toxin, dermonecrotic toxin, and the type III secretion system are not involved in cough production, but BspR/BtrA (different names for the same protein), an anti-σ factor, regulates the production of unknown factor(s) to cause coughing. Rat coughing was observed by inoculation of not only the living bacteria but also the bacterial lysates. Infection with bspR (btrA)-deficient strains caused significantly less frequent coughing than the wild type; however, intranasal inoculation of the lysates from a bspR (btrA)-deficient strain caused coughing similarly to the wild type, suggesting that BspR/BtrA regulates the production of the cough factor(s) only when the bacteria colonize host bodies. Moreover, the cough factor(s) was found to be heat labile and produced by B. bronchiseptica in the Bvg phase. We consider that our rat model provides insight into the pathogenesis of cough induced by the Bordetella infection. +.
17. Genomic Characterization of β-Glucuronidase-Positive Escherichia coli O157:H7 Producing Stx2a.
Among Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a β-glucuronidase-positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (β-glucuronidase-negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones..
18. Shu Kuan Wong, Susumu Yoshizawa, Yu Nakajima, Marie Johanna Cuadra, Yuichi Nogi, Keiji Nakamura, Hideto Takami, Yoshitoshi Ogura, Tetsuya Hayashi, Hiroshi Xavier Chiura, Koji Hamasaki, Amylibacter kogurei sp. Nov., a novel marine alphaproteobacterium isolated from the coastal sea surface microlayer of a marine inlet, International Journal of Systematic and Evolutionary Microbiology, 10.1099/ijsem.0.002911, 68, 9, 2872-2877, 2018.09, A novel Gram-negative bacterium, designated 4G11T, was isolated from the sea surface microlayer of a marine inlet. On the basis of 16S rRNA gene sequence analysis, the strain showed the closest similarity to Amylibacter ulvae KCTC 32465T (99.0 %). However, DNA-DNA hybridization values showed low DNA relatedness between strain 4G11Tand its close phylogenetic neighbours, Amylibacter marinus NBRC 110140T (8.0±0.4 %) and Amylibacter ulvae KCTC 32465T(52.9±0.9 %). Strain 4G11T had C18: 1, C16: 0and C18: 2as the major fatty acids. The only isoprenoid quinone detected for strain 4G11T was ubiquinone-10. The major polar lipids were phosphatidylglycerol, phosphatidylcholine, one unidentified polar lipid, one unidentified phospholipid and one unidentified aminolipid. The DNA G+C content of strain 4G11T was 50.0 mol%. Based on phenotypic and chemotaxonomic characteristics and analysis of the 16S rRNA gene sequence, the novel strain should be assigned to a novel species, for which the name Amylibacter kogurei sp. nov. is proposed. The type strain of Amylibacter kogurei is 4G11T(KY463497=KCTC 52506T=NBRC 112428T)..
19. Aya Fukui-Miyazaki, Hirono Toshima, Yukihiro Hiramatsu, Keisuke Okada, Keiji Nakamura, Keisuke Ishigaki, Naoaki Shinzawa, Hiroyuki Abe, Yasuhiko Horiguchi, The eukaryotic host factor 14-3-3 inactivates adenylate cyclase toxins of Bordetella bronchiseptica and B. Parapertussis, but not B. pertussis, mBio, 10.1128/mBio.00628-18, 9, 4, 2018.07, Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis share highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles in Bordetella infections. We herein demonstrate that CyaAs function as virulence factors differently between B. bronchiseptica/B. parapertussis and B. pertussis. B. bronchiseptica CyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly to B. pertussis CyaA. The hemolytic activity of B. bronchiseptica CyaA on sheep erythrocytes was also preserved. However, in nucleated target cells, B. bronchiseptica CyaA was phosphorylated at Ser 375 , which constitutes a motif (RSXpSXP [pS is phos-phoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects of B. bronchiseptica CyaA based on its enzyme activity were markedly attenuated. B. parapertussis CyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly to B. bronchiseptica CyaA; however, B. pertussis CyaA has Phe 375 instead of Ser, and thus, was not affected by 14-3-3. In addition, B. pertussis CyaA impaired the barrier function of epithelial cells, whereas B. bronchiseptica CyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity between B. bronchiseptica/B. parapertussis and B. pertussis. IMPORTANCE Bordetella pertussis, B. bronchiseptica, and B. parapertussis are bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by each Bordetella species. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor of Bordetella. The toxins of B. bronchiseptica and presumably B. parapertussis were inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas the B. pertussis toxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences in Bordetella are attributed to the different activities of adenylate cyclase toxins..
20. Masumi Hasegawa, Yu Nakajima, Shu Kuan Wong, Keiji Nakamura, Yoshitoshi Ogura, Tetsuya Hayashi, Kazuhiro Kogure, Susumu Yoshizawa, Draft genome sequence of Saccharospirillum sp. strain MSK14-1, isolated from surface seawater collected at Aburatsubo Inlet in Japan, Genome Announcements, 10.1128/genomeA.00469-18, 6, 22, 2018.05, Here, we report the draft genome sequence of Saccharospirillum sp. strain MSK14-1, isolated from surface seawater collected at Aburatsubo Inlet in Japan. The genome sequence of strain MSK14-1 should contribute to our understanding of the characteristics of the genus Saccharospirillum..
21. Tomoko Kohda, Keiji Nakamura, Koji Hosomi, Yasushi Torii, Shunji Kozaki, Masafumi Mukamoto, Response to “Standardized methods must be used to compare the properties of botulinum toxin serotypes”, MICROBIOLOGY and IMMUNOLOGY, 10.1111/1348-0421.12552, 61, 12, 2017.12.
22. Kohda Tomoko, Nakamura Keiji, Hosomi Koji, Torii Yasushi, Kozaki Shunji, Mukamoto Masafumi, Characterization of the functional activity of botulinum neurotoxin subtype B6, Microbiology and immunology, 10.1111/1348-0421.12540, 61, 11, 482-489, 2017.11.
23. Yohei Kumagai, Susumu Yoshizawa, Keiji Nakamura, Yoshitoshi Ogura, Tetsuya Hayashi, Kazuhiro Kogure, Complete and draft genome sequences of eight oceanic Pseudomonas aeruginosa strains, Genome Announcements, 10.1128/genomeA.01255-17, 5, 44, 2017.11, Pseudomonas aeruginosa is one of the most common model bacterial species, and genomes of hundreds of strains of this species have been sequenced to date. However, currently there is only one available genome of an oceanic isolate. Here, we report two complete and six draft genome sequences of P. aeruginosa isolates from the open ocean..
24. Koichiro Suzuki, Naoaki Shinzawa, Keisuke Ishigaki, Keiji Nakamura, Hiroyuki Abe, Aya Fukui-Miyazaki, Kazuyoshi Ikuta, Yasuhiko Horiguchi, Protective effects of in vivo-expressed autotransporters against Bordetella pertussis infection, MICROBIOLOGY and IMMUNOLOGY, 10.1111/1348-0421.12504, 61, 9, 371-379, 2017.09, Bordetella pertussis causes whooping cough, a severe and prolonged respiratory disease that results inhas high morbidity and mortality rates, particularly in developing countries. The number incidence of whooping cough cases is increasing in many countries despite high vaccine coverage. Causes for the re-emergence of the disease include the limited duration of protection conferred by the acellular pertussis vaccines (aP)s and pathogenic adaptations that involve antigenic divergence from vaccine strains. Therefore, current vaccines therefore need to be improved. In the present study, we focused on five autotransporters: namely SphB1, BatB, SphB2, Phg, and Vag8, which were previously found to be expressed by B. bronchiseptica during the course of infection in rats and examined their protective efficiencies as vaccine antigens. The passenger domains of these proteins were produced in recombinant forms and used as antigens. An intranasal murine challenge assay showed that immunization with a mixture of SphB1 and Vag8 (SV) significantly reduced bacterial load in the lower respiratory tract and a combination of aP and SV acts synergistically in effects of conferring protection against B. pertussis infection, implying that these antigens have potential as components to for improvinge th the currently available acellular pertussis vaccine..
25. Yu Nakajima, Susumu Yoshizawa, Keiji Nakamura, Yoshitoshi Ogura, Tetsuya Hayashi, Kazuhiro Kogure, Draft genome sequences of Tersicoccus phoenicis DSM 30849T, isolated from a cleanroom for spacecraft assembly, and Tersicoccus sp. strain Bi-70, isolated from a freshwater lake, Genome Announcements, 10.1128/genomeA.00079-17, 5, 13, 2017.01, Here, we report the draft genome sequences of Tersicoccus phoenicis DSM 30849T, isolated from a spacecraft assembly cleanroom at the National Aeronautics and Space Administration (NASA), and Tersicoccus sp. strain Bi-70, isolated from Lake Biwa, the largest lake in Japan. These genome sequences facilitate our understanding of the adaptation of these closely related strains to different habitats..
26. Sayaka Nishikawa, Naoaki Shinzawa, Keiji Nakamura, Keisuke Ishigaki, Hiroyuki Abe, Yasuhiko Horiguchi, The bvg-repressed gene brtA, encoding biofilm-associated surface adhesin, is expressed during host infection by Bordetella bronchiseptica, MICROBIOLOGY and IMMUNOLOGY, 10.1111/1348-0421.12356, 60, 2, 93-105, 2016.02, Bordetella species display phase modulation between Bvg+ and Bvg- phases. Because expression of known virulence factors is up-regulated in the Bvg+ phase, bacteria in this phase are considered competent for infection. However, the Bvg- phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg- phase (bvg-repressed factors) are expressed in the course of Bordetella infection. In the present study, the gene brtA (Bordetella RTX-family Adhesin), which is a typical bvg-repressed gene but is expressed in B. bronchiseptica infecting hosts, was characterized. BrtA is composed of repeated pairs of the VCBS unit and dystroglycan-type cadherin-like unit, the von Willebrand Factor A domain, RTX motif and type I secretion target signal. It is herein demonstrated that BrtA is secreted by the type I secretion system and is essential for Ca2+-dependent bacteria-to-substrate adherence, followed by biofilm formation. Although the contribution of BrtA to bacterial colonization of the rat trachea currently remains unclear, this is the first study to present concrete evidence for the expression of a bvg-repressed gene during infection, which may provide a novel aspect for analyses of Bordetella pathogenesis..
27. Hiroyuki Abe, Shigeki Kamitani, Aya Fukui-Miyazaki, Naoaki Shinzawa, Keiji Nakamura, Yasuhiko Horiguchi, Detection of genes expressed in Bordetella bronchiseptica colonizing rat trachea by in vivo expressed-tag immunoprecipitation method, MICROBIOLOGY and IMMUNOLOGY, 10.1111/1348-0421.12247, 59, 5, 249-261, 2015.05, Analyses of bacterial genes expressed in response to the host environment provide clues to understanding the host-pathogen interactions that lead to the establishment of infection. In this study, a novel method named In Vivo Expressed-Tag ImmunoPrecipitation (IVET-PI) was developed for detecting genes expressed in bacteria that are recovered in a small numbers from host tissues. IVET-IP was designed to overcome some drawbacks of previous similar methods. We applied IVET-IP to Bordetella bronchiseptica colonizing rat trachea and identified 173 genes that were expressed in the bacteria over the entire course of an infection. These gene products included two transcriptional factors that are involved in the expression of filamentous hemagglutinin, adenylate cyclase toxin, and major virulence factors for the bordetellae. We consider that this method might provide novel insight into the course of Bordetella infection..
28. Keisuke Okada, Hiroyuki Abe, Fumio Ike, Yoshitoshi Ogura, Tetsuya Hayashi, Aya Fukui-Miyazaki, Keiji Nakamura, Naoaki Shinzawa, Yasuhiko Horiguchi, Polymorphisms influencing expression of dermonecrotic toxin in Bordetella bronchiseptica, PloS one, 10.1371/journal.pone.0116604, 10, 2, e0116604, 2015.02, Bordetella bronchiseptica is a pathogenic bacterium causing respiratory infections in a broad range of mammals. Recently, we determined the whole genome sequence of B. bronchiseptica S798 strain isolated from a pig infected with atrophic rhinitis and found four single-nucleotide polymorphisms (SNPs) at positions -129, -72, +22, and +38 in the region upstream of dnt encoding dermonecrotic toxin (DNT), when compared with a rabbit isolate, RB50. DNT is known to be involved in turbinate atrophy observed in atrophic rhinitis. Immunoblotting, quantitative real-time PCR, and β-galactosidase reporter assay revealed that these SNPs resulted in the increased promoter activity of dnt and conferred the increased ability to produce DNT on the bacteria. Similar or identical SNPs were also found in other pig isolates kept in our laboratory, all of which produce a larger amount of DNT than RB50. Our analysis revealed that substitution of at least two of the four bases, at positions -72 and +22, influenced the promoter activity for dnt. These results imply that these SNPs are involved in the pathogenicity of bordetellae specific to pig diseases..
29. Kentaro Tsukamoto, Hideyuki Arimitsu, Sadayuki Ochi, Keiji Nakamura, Yoshikazu Tanaka, Nipawan Nuemket, Koki Taniguchi, Shunji Kozaki, Takao Tsuji, P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins, MICROBIOLOGY and IMMUNOLOGY, 10.1111/j.1348-0421.2012.00490.x, 56, 10, 664-672, 2012.10, Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs..
30. Haiyang Zhao, Keiji Nakamura, Tomoko Kohda, Masafumi Mukamoto, Shunji Kozaki, Characterization of the monoclonal antibody response to botulinum neurotoxin type A in the complexed and uncomplexed forms, Japanese Journal of Infectious Diseases, 65, 2, 138-145, 2012.04, Clostridium botulinum produces large complex toxins, which include botulinum neurotoxin (BoNT) and auxiliary non-toxic proteins. We prepared monoclonal antibodies (mAbs) from mice that were immunized several times with BoNT/A after basal immunization with toxoid. We then examined the reactivities of these mAbs to BoNT and toxoid and showed that some mAbs reacted to only BoNT. This result indicates that the antigenicity of BoNT/A partially disappeared with formalin treatment. Some mAbs that specifically recognized either BoNT/A1 or BoNT/A2 were considered useful as detection antibodies specific for the BoNT/A subtype. Results of a neutralizing test with mAbs against either BoNT/A1 or BoNT/A2 showed that neutralizing antibody recognition sites were present in the light chain, heavy chain (N-terminal half), and heavy chain (C-terminal half) domains. Investigation of the different binding capabilities of the mAbs to BoNT and the complex toxin by immunoprecipitation suggested that the light chain of BoNT is exposed at the molecular surface of the complex toxin since there was no difference in the binding of light chain-specific mAb to BoNT and the complex toxin. The heavy chain is related to BoNT binding to non-toxic components, because the reactivity of the heavy chain to some mAbs was influenced by non-toxic components..
31. Nipawan Nuemket, Yoshikazu Tanaka, Kentaro Tsukamoto, Takao Tsuji, Keiji Nakamura, Shunji Kozaki, Min Yao, Isao Tanaka, Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin Insight into the ganglioside binding mechanism, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2011.06.173, 411, 2, 433-439, 2011.07, Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3′-sialyllactose at a resolution of 3.0 Å. In the structure, an electron density derived from the 3′-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites..
32. Nipawan Nuemket, Yoshikazu Tanaka, Kentaro Tsukamoto, Takao Tsuji, Keiji Nakamura, Shunji Kozaki, Min Yao, Isao Tanaka, Preliminary X-ray crystallographic study of the receptor-binding domain of the D/C mosaic neurotoxin from Clostridium botulinum, Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 10.1107/S1744309110012182, 66, 5, 608-610, 2010.04, Botulinum toxin (BoNT) from Clostridium botulinum OFD05, isolated from bovine botulism, is a D/C mosaic-type BoNT. BoNTs possess binding, translocation and catalytic domains. The BoNT/OFD05 binding domain exhibits significant sequence identity to BoNT/C, which requires a single ganglioside as a binding receptor on neuronal cells, while BoNT/A and BoNT/B require two receptors for specific binding. To determine the binding mechanism of BoNT/OFD05 and its ganglioside receptors on neuronal cells, recombinant BoNT/OFD05 receptor-binding domain has been expressed, purified and crystallized. Native and SeMet-derivative crystals showed X-ray diffraction to 2.8 and 3.1 Å resolution, respectively. The crystals belonged to space group P212121..


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