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Tetsuya Iida

Publications and source records attributed to Tetsuya Iida.

At least 19 recordsLinked to original sources
PubMed2026

Multidimensional prophage profiling of carbapenem-resistant Enterobacteriaceae in Thailand: a nationwide, multicentre, genomic study.

BACKGROUND: Prophages influence bacterial fitness, resistance, and evolution, yet their epidemiology remains poorly understood in carbapenem-resistant Enterobacteriaceae (CRE). In this nationwide study in Thailand, we aimed to describe prophage repertoires in clinical CRE isolates and to explore their potential relevance for molecular epidemiology. METHODS: We performed a nationwide, retrospective, genomic analysis of all CRE clinical isolates collected through our previous national surveillance study involving 11 hospitals in 11 provinces in Thailand between March 25, 2012, and Jul 21, 2017. Whole-genome sequencing data from 747 CRE isolates were analysed. Intact prophages were identified using PHAge Search Tool Enhanced Release (PHASTER) and clustered by nucleotide sequence similarity. Prophage profiles were compared across multilocus sequence types, carbapenemase genotypes, specimens, geography, and patient demographics (age and sex). FINDINGS: Of the included 747 CRE isolates, 170 (23%) were Escherichia coli and 577 (77%) were Klebsiella pneumoniae. 220 (29%) of 747 strains had been isolated from female patients and 264 (35%) from male patients; metadata on patient sex were missing for 263 (35%) isolates. The median patient age was 63 years (IQR 50-72). 71 (10%) of isolates were from blood, 283 (38%) from sputum, 284 (38%) from urine, and 109 (15%) from other specimens. 374 distinct prophage clusters were identified, with significantly more prophages per genome in K pneumoniae (mean 3&#xb7;01 [SD 1&#xb7;55]) than in E coli (1&#xb7;64 [1&#xb7;46]; p<0&#xb7;0001). Prophage repertoires largely mirrored bacterial multilocus sequence types. However, even within the highly clonal K pneumoniae sequence type 16 lineage, discrete prophage variation was identified, with common profiles observed in geographically dispersed patients. Respiratory K pneumoniae frequently carried a mosaic prophage with environmental signatures and a type VI secretion system, whereas blood-derived E coli harboured a prophage with immune-modulating genes. Distinct prophage clusters were observed across clinical specimens, age groups, carbapenemase genotype, and geographical region. Strains coharbouring blaNDM-1 plus blaOXA-232 (114 [15%] of 747) had the highest prophage loads. INTERPRETATION: The prophage content was shaped by the bacterial lineage, ecological niche, and temporal dynamics, providing an additional layer of epidemiological resolution beyond conventional genome typing. Integrating prophage profiling into molecular surveillance frameworks could help to identify transmission events, improve infectious source attribution, and enhance infection control strategies. FUNDING: Japan Agency for Medical Research and Development.

Female↗
PubMed2007

Polyclonality of Staphylococcus epidermidis residing on the healthy ocular surface.

Staphylococcus epidermidis is part of the normal bacterial flora on the ocular surface. The chromosomal DNA of bacterial isolates obtained from the conjunctival sac, upper and lower lid margins, and upper and lower Meibomian glands of healthy volunteers was subjected to SmaI digestion and PFGE to study the genetic diversity of the organisms. Multiple colonies were also examined of S. epidermidis derived from the conjunctival sac of the same subjects. Lastly, commensal bacteria were harvested from the ocular surfaces of four healthy subjects once a month for 6 months, and the genetic background of the S. epidermidis isolates was analysed. It was found that bacterial strains not only from different subjects but also from multiple ocular surface sites of the same subject exhibited different PFGE patterns. In five of 42 subjects multiple colonies of S. epidermidis were isolated from the conjunctival sac; three harboured multiple colonies with different PFGE patterns, and two manifested multiple colonies with identical PFGE patterns. S. epidermidis isolated from the conjunctival sac of the same subjects over a 6-month period exhibited varying PFGE patterns. The data demonstrate the polyclonality of S. epidermidis on the healthy ocular surface.

Conjunctiva↗
PubMed2006

Lateral transfer of the lux gene cluster.

The lux operon is an uncommon gene cluster. To find the pathway through which the operon has been transferred, we sequenced the operon and both flanking regions in four typical luminous species. In Vibrio cholerae NCIMB 41, a five-gene cluster, most genes of which were highly similar to orthologues present in Gram-positive bacteria, along with the lux operon, is inserted between VC1560 and VC1563, on chromosome 1. Because this entire five-gene cluster is present in Photorhabdus luminescens TT01, about 1.5 Mbp upstream of the operon, we deduced that the operon and the gene cluster were transferred from V. cholerae to an ancestor of Pr. luminescens. Because in both V. fischeri and Shewanella hanedai, luxR and luxI were found just upstream of the operon, we concluded that the operon was transferred from either species to the other. Because most of the genes flanking the operon were highly similar to orthologues present on chromosome 2 of vibrios, we speculated that the operon of most species is located on this chromosome. The undigested genomic DNAs of five luminous species were analysed by pulsed-field gel electrophoresis and Southern hybridization. In all the species except V. cholerae, the operons are located on chromosome 2.

Bacterial Proteins↗
PubMed2006

VP1686, a Vibrio type III secretion protein, induces toll-like receptor-independent apoptosis in macrophage through NF-kappaB inhibition.

Vibrio parahaemolyticus, causative agent of human gastrointestinal diseases, possesses several virulent machineries including thermostable direct hemolysin and type III secretion systems (TTSS1 and -2). In this report, we establish that TTSS1-dependent secretion and translocation of a V. parahaemolyticus effector protein VP1686 into the cytosol induces DNA fragmentation in macrophages. We performed yeast two-hybrid screening to identify the molecules involved in VP1686-mediated cell death pathways and showed that nuclear factor RelA p65/NF-kappaB physically interacts with VP1686. To understand the impact of this interaction on the NF-kappaB DNA binding activities in infected macrophages, we analyzed a series of deletion mutants for the TTSS and its secreted proteins. Induction of DNA binding activity of NF-kappaB was significantly suppressed, and increased macrophage apoptosis has been associated with V. parahaemolyticus strain, which contains both VP1686 and TTSS1. Macrophages lacking Toll-like receptor adaptor molecules MyD88 (myeloid differentiation primary response protein 88) or TRIF (TIR domain-containing adapter-inducing interferon beta) showed similar sensitivity to VP1686. As a consequence of NF-kappaB suppression, microarray analysis has revealed that VP1686 translocation alerted the expression of many genes that have known functions in cellular responses to apoptosis, cell growth, and transcriptional regulation. Our results suggest an important role for Vibrio effector protein VP1686 that activate a conserved apoptotic pathway in macrophages through suppression of NF-kappaB activation independent of Toll-like receptor signaling.

Amino Acid Sequence↗
PubMed2006

Cortactin is essential for F-actin assembly in enteropathogenic Escherichia coli (EPEC)- and enterohaemorrhagic E. coli (EHEC)-induced pedestals and the alpha-helical region is involved in the localization of cortactin to bacterial attachment sites.

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are important human pathogens. Upon attachment to host cells, EPEC and EHEC are able to induce actin polymerization, which accumulates, forming a pedestal-like structure beneath the attached bacteria. Using siRNA, we show here that EPEC- and EHEC-induced pedestals are dependent on cortactin, an F-actin-binding protein found in the mammalian cell cortex. Knock-down of cortactin by siRNA resulted in a dramatic reduction of the pedestal formation induced by both pathogens. We also show that disruption of the Src homology 3 (SH3) domain of cortactin, or its downregulation by specific point mutations, negatively affects pedestal formation, suggesting that this domain is important for regulation of F-actin assembly by EPEC and EHEC. Green fluorescent protein (GFP) fused with the SH3 domain (GFP-SH3), proline-rich region (GFP-PRR) or alpha-helical region of cortactin markedly reduced the amount of F-actin at the bacterial attachment sites. Interestingly, neither GFP-SH3 nor GFP-PRR was recruited to the vicinity of the bacterial adherence sites; however, GFP fused to the alpha-helical region was efficiently recruited and colocalized with the attached bacteria. These results demonstrate that cortactin is a requirement for pedestal formation and suggest a novel function for the predicted alpha-helical region of cortactin in actin assembly induced by EPEC and EHEC.

Actins↗
PubMed2006

Two type IV pili of Vibrio parahaemolyticus play different roles in biofilm formation.

Vibrio parahaemolyticus RIMD2210633 has two sets of type IV-A pilus genes. One set is similar to that found in other Gram-negative bacteria, such as Pseudomonas aeruginosa, Vibrio cholerae (chitin-regulated pilus; ChiRP), and Vibrio vulnificus. The other is homologous to the genes for the mannose-sensitive hemagglutinin (MSHA) pilus. In this study, we analyzed the effects of the deletions in the pilin genes for each type IV pilus (the ChiRP and the MSHA pilus) on biofilm formation. Although the MSHA pilin mutant formed aggregates, the number of bacteria that attached directly to the coverslip was reduced, suggesting that this pilus contributes to the bacterial attachment to the surface of the coverslip. In contrast, the ChiRP mutant attached to the surface of the coverslip, but did not form aggregates, suggesting that ChiRP plays a role in bacterial agglutination during biofilm formation. These results suggest that the two type IV pili of V. parahaemolyticus contribute to biofilm formation in different ways. Both mutants showed a lower fitness for adsorption onto chitin particles than that of the wild type. Collectively, these data suggest that the use of two type IV pili is a refined strategy of V. parahaemolyticus for survival in natural environments.

Bacterial Adhesion↗
PubMed2006

Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1.

Vibrio parahaemolyticus, a gram-negative marine bacterium, is an important pathogen causing food-borne gastroenteritis or septicemia. Recent genome sequencing of the RIMD2210633 strain (a Kanagawa phenomenon-positive clinical isolate of serotype O3:K6) revealed that the strain has two sets of gene clusters that encode the type III secretion system (TTSS) apparatus. The first cluster, TTSS1, is located on the large chromosome, and the second, TTSS2, is on the small chromosome. Previously, we reported that TTSS1 is involved in the cytotoxicity of the RIMD2210633 strain against HeLa cells. Here, we analyzed proteins secreted via the TTSS apparatus encoded by TTSS1 by using two-dimensional gel electrophoresis and identified the proteins encoded by genes VP1680, VP1686, and VPA450. To investigate the roles of those secreted proteins, we constructed and analyzed a series of deletion mutants. Flow cytometry analysis using fluorescence-activated cell sorting with fluorescein isothiocyanate-labeled annexin V demonstrated that the TTSS1-dependent cell death was by apoptosis. The cytotoxicity to HeLa cells was related to one of the newly identified secreted proteins encoded by VP1680. Adenylate cyclase fusion protein studies proved that the newly identified secreted proteins were translocated into HeLa cells. Thus, these appear to be the TTSS effector proteins in V. parahaemolyticus.

Amino Acid Sequence↗
PubMed2005

Escherichia coli verotoxin 1 mediates apoptosis in human HCT116 colon cancer cells by inducing overexpression of the GADD family of genes and S phase arrest.

The Escherichia coli verotoxin 1 (VT1) inhibits protein synthesis, cell proliferation, and damages endothelial cell in the hemolytic uremic syndrome. VT1 can specifically bind and act on endothelial cells as well as on many tumor cells because these cells express its high affinity receptor, globotriaosylceramide. This indicates that VT1 may have both antiangiogenic and antineoplastic activities. We investigated this potential of VT1 by incubating several colon cancer cell lines with VT1 for different time periods and found that HCT116 cells were especially sensitive to VT1. A combination of morphological studies, flow cytometry, DNA laddering and annexin V staining confirmed that VT1 irreversibly arrests these cells in S phase within 24 h and prolonged incubation triggers DNA fragmentation. Concomitant to the activation of the S phase checkpoint, increased levels of mRNA and proteins of growth arrest and DNA damage-inducible gene family that include GADD34, GADD45alpha, and GADD45beta was observed. Interestingly, no significant changes in expression of key cell cycle related proteins such as cdk2, cdk4, p21, p27, and p53 was found during the S phase arrest and apoptosis. We therefore suggest that GADD proteins might play an important role in VT1 induced S phase arrest and programmed cell death in HCT116 cells.

Antigens, Differentiation↗
PubMed2005

Microarray analysis identifies apoptosis regulatory gene expression in HCT116 cells infected with thermostable direct hemolysin-deletion mutant of Vibrio parahaemolyticus.

The thermostable direct hemolysin (TDH) is considered as a major virulence factor of Vibrio parahaemolyticus. We observed this potential in several human cancer cell lines by using the TDH-producing wild-type (RIMD2210633) as well as tdh-deletion mutant of V. parahaemolyticus and found that the deletion of tdh did not affect cytotoxicity to any of the cell lines tested. DNA fragmentation and annexin V staining showed that both wild-type and tdh-mutant trigger apoptosis in these cells. To understand the molecular basis of cell death in the absence of TDH, gene expression profile of human colon cancer cell line HCT116 infected with tdh-deletion mutant was carried out using human cDNA microarrays consisting of 33,000 known genes. In infected cells, differentially expressed genes including genes for early growth response, growth arrest and DNA damage, and activating transcription factor that affect programmed cell death pathways were detected. Interestingly, mutant strains having a deletion in type III secretion system 1 (TTSS1) failed to elicit DNA fragmentation in HCT116 cells. Our results strongly suggest that apoptosis requires functional TTSS1 and TTSS1-dependent translocation factor(s) to be associated with the host cell death, and thus pathogenesis of V. parahaemolyticus.

Annexin A5↗
PubMed2005

Thermostable direct hemolysin of Vibrio parahaemolyticus is a bacterial reversible amyloid toxin.

Thermostable direct hemolysin (TDH), a major virulence factor of Vibrio parahaemolyticus, is detoxified by heating at approximately 60-70 degrees C but is reactivated by additional heating above 80 degrees C. This paradoxical phenomenon, known as the Arrhenius effect, has remained unexplained for approximately 100 years. We now demonstrate that the effect is related to structural changes in the protein that produce fibrils. The native TDH (TDHn) is transformed into nontoxic fibrils rich in beta-strands by incubation at 60 degrees C (TDHi). The TDHi fibrils are dissociated into unfolded states by further heating above 80 degrees C (TDHu). Rapid cooling of TDHu results in refolding of the protein into toxic TDHn, whereas the protein is trapped in the TDHi structure by slow cooling of TDHu. Transmission electron microscopy indicates the fibrillar structures of TDHi. The fibrils show both the property of the nucleation-dependent elongation and the increase in its thioflavin T fluorescence. Formation of beta-rich structures of TDH was also observed in the presence of lipid vesicles containing ganglioside G(T1b), a putative TDH receptor. Congo red was found to inhibit the hemolytic activity of TDH in a dose-dependent manner. These data reveal that the mechanism of the Arrhenius effect which is tightly related to the fibrillogenicity of TDH.

Amyloid↗
PubMed2005

vpaH, a gene encoding a novel histone-like nucleoid structure-like protein that was possibly horizontally acquired, regulates the biogenesis of lateral flagella in trh-positive Vibrio parahaemolyticus TH3996.

A histone-like nucleoid structure (H-NS) is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Here, we cloned and characterized the gene for the H-NS-like protein VpaH in Vibrio parahaemolyticus. vpaH encodes a protein of 134 amino acids that shows approximately 55%, 54%, and 41% identities with VicH in Vibrio cholerae, H-NS in V. parahaemolyticus, and H-NS in Escherichia coli, respectively. The vpaH gene was found in only trh-positive V. parahaemolyticus strains and not in Kanagawa-positive or in trh-negative environmental strains. Moreover, the G+C content of the vpaH gene was 38.6%, which is lower than the average G+C content of the whole genome of this bacterium (45.4%). These data suggest that vpaH was transmitted to trh-possessing V. parahaemolyticus strains by lateral transfer. The vpaH gene was located about 2.6 kb downstream of the trh gene, in the convergent direction of the trh transcription. An in-frame deletion mutant of vpaH lacked motility on semisolid motility assay plates. Western blot analysis and electron microscopy observations revealed that the mutant was deficient in lateral flagella biogenesis, whereas there was no defect in the expression of polar flagella. Additionally, the vpaH mutant showed a decreased adherence to HeLa cells and a decrease in biofilm formation compared with the wild-type strain. Introduction of the vpaH gene in the vpaH-negative strain increased the expression of lateral flagella compared with the wild-type strain. In conclusion, our findings suggest that VpaH affects lateral flagellum biogenesis in trh-positive V. parahaemolyticus strain TH3996.

Amino Acid Sequence↗
PubMed2005

Vibrios commonly possess two chromosomes.

The prevalence of the two-chromosome configuration was investigated in 34 species of vibrios and closely related species. Pulsed-field gel electrophoresis of undigested genomic DNA suggested that vibrios commonly have two chromosomes. The size of the large chromosome is predominantly within a narrow range (3.0 to 3.3 Mb), whereas the size of the small chromosome varies considerably among the vibrios (0.8 to 2.4 Mb). This fact suggests that the structure of the small chromosome is more flexible than that of the large chromosome during the evolution of vibrios.

Biological Evolution↗
PubMed2005

Antibodies among healthy population of developing countries against enterohaemorrhagic Escherichia coli O157:H7.

In Thailand, no reports are available on Escherichia coli serotype O157:H7, a causative agent of severe bloody diarrhoea, sometimes associated with haemolytic-uraemic syndrome and thrombotic thrombocytopenic purpura. The reason for the non-identification of infection due to E. coli O157 in this country and in other developing countries has not been rigorously discussed. The aim of this study was to determine the humoral response against the infectious organism. The IgM and IgG antibody responses against E. coli O157 lipopolysaccharide were studied using indirect enzyme-linked immunosorbent assay. Three hundred and thirty-two serum samples obtained from healthy blood donors and patients with diseases unrelated to diarrhoea were investigated. With a cut-off value of mean +2 SDs for each age-group, the frequency of the IgM and IgG responses to O157 lipopolysaccharide was 11.74% (39 of 332 samples) and 22.59% (75 of 332 samples) respectively. Furthermore, agglutination test of 173 subjects revealed titres ranging from 10 to 40 in all the samples. The results suggest possible exposure of the Thai population to cross-reacting antigens from other intestinal organisms in addition to infection due to E. coli O157:H7.

Adolescent↗
PubMed2004

Single channel evidence for innate pore-formation by Vibrio parahaemolyticus thermostable direct haemolysin (TDH) in phospholipid bilayers.

Vibrio parahaemolyticus thermostable direct haemolysin (TDH) is widely considered to be a pore-forming toxin. The protein has no significant homology to other known pore-forming toxins and its mechanism of action in vivo remains undefined. We demonstrate single channel pore-forming activity of V. parahaemolyticus TDH in planar lipid bilayers. Channel conductance ranged between 30-450 pS in 0.5 M KCl with a calculated cation selectivity (P(K)/P(Cl)) of 2.7. Channels were formed in NaCl and choline-Cl with and without cholesterol present and in the presence of neutral or negatively charged phospholipids. Zinc ions did not block pore formation. Whilst various techniques have previously suggested that TDH is a pore-forming toxin, the data in this study provide direct single channel evidence and indicate several features of pore formation in synthetic phospholipid membranes.

Amino Acid Sequence↗
PubMed2004

Urease activity of enterohaemorrhagic Escherichia coli depends on a specific one-base substitution in ureD.

The authors previously reported that most enterohaemorrhagic Escherichia coli (EHEC) strains do not express urease activity, despite having the urease gene. This study compared the nucleotide sequences of the urease gene clusters of a urease-activity-positive and a urease-activity-negative strain. The results showed that in the urease-activity-negative strain, ureD, a gene encoding a chaperone protein, had a single base substitution that encoded a premature stop codon resulting in a short ORF. The premature stop codon in ureD was commonly found in urease-activity-negative EHEC strains, but not in urease-activity-positive strains. Urease activity was detected after complementing the urease-activity-negative strain with ureD from the urease-activity-positive strain. Furthermore, introduction of the urease gene cluster from the urease-activity-negative strain into an amber suppressor phenotype Escherichia coli strain, DH5alpha, conferred the ability to produce the active urease. These results suggest that the lack of urease activity in most EHEC strains is due to a premature stop codon in ureD.

Amino Acid Substitution↗
PubMed2004

Cytotoxicity and enterotoxicity of the thermostable direct hemolysin-deletion mutants of Vibrio parahaemolyticus.

The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of Vibrio parahaemolyticus. We have recently completed the genome sequence of a TDH-producing V. parahaemolyticus strain, RIMD2210633. In this study, we constructed tdh-deletion mutants from the sequenced strain by homologous recombination and analyzed their phenotypes. Although the deletion of both copies of tdh completely abolished the hemolytic activity of the wild-type strain, the deletion did not affect the cytotoxicity to HeLa cells. Enterotoxicity, assayed by the rabbit ileal loop test, was lowered by tdh deletion, but the mutant still showed partial fluid accumulation in rabbit intestine. These results indicate that the cytotoxicity and enterotoxicity of TDH-producing V. parahaemolyticus are not explained by TDH alone, and suggest that an unknown virulence factor(s) could be involved in these pathogenic activities.

Animals↗
PubMed2004

Functional characterization of two type III secretion systems of Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. Recent genome sequencing of the clinical V. parahaemolyticus strain RIMD2210633 identified two sets of genes for the type III secretion system (TTSS), TTSS1 and TTSS2. Here, we constructed a series of mutant strains from RIMD2210633 to determine whether the two putative TTSS apparatus are functional. The cytotoxic activity of mutant strains having a deletion in one of the TTSS1 genes was significantly decreased compared with that of the parent and TTSS2-related mutant strains. In an enterotoxicity assay with the rabbit ileal loop test, intestinal fluid accumulation was diminished by deletion of the TTSS2-related genes while TTSS1-related mutants caused a level of fluid accumulation similar to that of the parent. VopD, a protein encoded in the proximity of the TTSS1 region and a homologue of the Yersinia YopD, was secreted in a TTSS1-dependent manner. In contrast, VopP, which is encoded by a pathogenicity island on chromosome 2 and is homologous to the Yersinia YopP, was secreted via the TTSS2 pathway. These results provide evidence that V. parahaemolyticus TTSSs function as secretion systems and may have a role in the pathogenicity of the organism. This is the first report of functional TTSSs in Vibrio species. The presence of TTSS apparatus gene homologues was demonstrated in other vibrios, such as Vibrio alginolyticus, Vibrio harveyi, and Vibrio tubiashii, suggesting that some other vibrios also contain TTSS and that the TTSS has a role in protein secretion in those organisms during interaction with eukaryotic cells.

Animals↗