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Dive into the research topics where Shigeyuki Hamada is active.

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Featured researches published by Shigeyuki Hamada.


Molecular Microbiology | 2008

Fba, a novel fibronectin‐binding protein from Streptococcus pyogenes, promotes bacterial entry into epithelial cells, and the fba gene is positively transcribed under the Mga regulator

Yutaka Terao; Shigetada Kawabata; Eiji Kunitomo; Jumpei Murakami; Ichiro Nakagawa; Shigeyuki Hamada

In infection by Streptococcus pyogenes, fibronectin (Fn)‐binding proteins play important roles as adhesins and invasins. Here, we present a novel Fn‐binding protein of S. pyogenes that exhibits a low similarity to other Fn‐binding proteins reported. After searching the Oklahoma Streptococcal Genome Sequencing Database for open reading frames (ORFs) with an LPXTG motif, nine ORFs were found among those recognized as putative surface proteins, and one of them was designated as Fba. The fba gene was found in M types 1, 2, 4, 22, 28 and 49 of S. pyogenes, but not in other serotypes or groups of streptococci. Fba, a 37.8u2003kDa protein, possesses three or four proline‐rich repeat domains and exhibits a high homology to FnBPA, the Fn‐binding protein of Staphylococcus aureus. Recombinant Fba exhibited a strong binding ability to Fn. In addition, Fba‐deficient mutants showed diminished invasive capabilities to HEp‐2 cells and low mortality in mice following skin infection. The fba gene was located downstream of the mga regulon and analysis using an mga‐inactivated mutant revealed that it was transcribed under the control of the Mga regulator. These results indicate that Fba is a novel protein and one of the important virulence factors of S. pyogenes.


Molecular Microbiology | 2005

Silkworm pathogenic bacteria infection model for identification of novel virulence genes.

Chikara Kaito; Kenji Kurokawa; Yasuhiko Matsumoto; Yutaka Terao; Shigetada Kawabata; Shigeyuki Hamada; Kazuhisa Sekimizu

Silkworms are killed by injection of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pyogenes, into the haemolymph. Gene disruption mutants of S. aureus whose open reading frames were previously uncharacterized and that are conserved among bacteria were examined for their virulence in silkworms. Of these 100 genes, three genes named cvfA, cvfB, and cvfC were required for full virulence of S. aureus in silkworms. Haemolysin production was decreased in these mutants. The cvfA and cvfC mutants also had attenuated virulence in mice. S. pyogenes cvfA‐disrupted mutants produced less exotoxin and had attenuated virulence in both silkworms and mice. These results indicate that the silkworm‐infection model is useful for identifying bacterial virulence genes.


Journal of Virology | 2003

Influenza A virus-infected hosts boost an invasive type of Streptococcus pyogenes infection in mice.

Shigefumi Okamoto; Shigetada Kawabata; Ichiro Nakagawa; Yoshinobu Okuno; Toshiyuki Goto; Kouichi Sano; Shigeyuki Hamada

ABSTRACT The apparent worldwide resurgence of invasive Streptococcus pyogenes infection in the last two decades remains unexplained. At present, animal models in which toxic shock-like syndrome or necrotizing fasciitis is induced after S. pyogenes infection are not well developed. We demonstrate here that infection with a nonlethal dose of influenza A virus 2 days before intranasal infection with a nonlethal dose of S. pyogenes strains led to a death rate of more than 90% in mice, 10% of which showed necrotizing fasciitis. Infection of lung alveolar epithelial cells by the influenza A virus resulted in viral hemagglutinin expression on the cell surface and promoted internalization of S. pyogenes. However, treatment with monoclonal antibodies to hemagglutinin markedly decreased this internalization. Our results indicate that prior infection with influenza A virus induces a lethal synergism, resulting in the induction of invasive S. pyogenes infection in mice.


Infection and Immunity | 2002

Novel laminin-binding protein of Streptococcus pyogenes, Lbp, is involved in adhesion to epithelial cells.

Yutaka Terao; Shigetada Kawabata; Eiji Kunitomo; Ichiro Nakagawa; Shigeyuki Hamada

ABSTRACT The lbp gene, which encodes a laminin-binding protein (Lbp) of Streptococcus pyogenes, was found in all S. pyogenes M types. An Lbp-deficient mutant showed a significantly lower efficiency of adhesion to HEp-2 cells than did the wild-type strain. These results indicate that Lbp is one of the important S. pyogenes adhesins.


Cellular Microbiology | 2001

Cytochrome c‐mediated caspase‐9 activation triggers apoptosis in Streptococcus pyogenes‐infected epithelial cells

Ichiro Nakagawa; Masanobu Nakata; Shigetada Kawabata; Shigeyuki Hamada

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria–host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp‐2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6+, F1+) and JRS145 (M6−, F1+ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6+, F1− mutant) or SAM2 (M6−, F1− mutant) infected HEp‐2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4u2003h of infection. Western blot analyses showed that the amounts of Bcl‐2 and Bcl‐xL were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase‐9 inhibitor (Ac‐LEHD‐CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS‐induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.


BMC Genomics | 2009

Comparative genomic analyses of Streptococcus mutans provide insights into chromosomal shuffling and species-specific content

Fumito Maruyama; Mitsuhiko Kobata; Ken Kurokawa; Keishin Nishida; Atsuo Sakurai; Kazuhiko Nakano; Ryota Nomura; Shigetada Kawabata; Takashi Ooshima; Kenta Nakai; Masahira Hattori; Shigeyuki Hamada; Ichiro Nakagawa

BackgroundStreptococcus mutans is the major pathogen of dental caries, and it occasionally causes infective endocarditis. While the pathogenicity of this species is distinct from other human pathogenic streptococci, the species-specific evolution of the genus Streptococcus and its genomic diversity are poorly understood.ResultsWe have sequenced the complete genome of S. mutans serotype c strain NN2025, and compared it with the genome of UA159. The NN2025 genome is composed of 2,013,587 bp, and the two strains show highly conserved core-genome. However, comparison of the two S. mutans strains showed a large genomic inversion across the replication axis producing an X-shaped symmetrical DNA dot plot. This phenomenon was also observed between other streptococcal species, indicating that streptococcal genetic rearrangements across the replication axis play an important role in Streptococcus genetic shuffling. We further confirmed the genomic diversity among 95 clinical isolates using long-PCR analysis. Genomic diversity in S. mutans appears to occur frequently between insertion sequence (IS) elements and transposons, and these diversity regions consist of restriction/modification systems, antimicrobial peptide synthesis systems, and transporters. S. mutans may preferentially reject the phage infection by clustered regularly interspaced short palindromic repeats (CRISPRs). In particular, the CRISPR-2 region, which is highly divergent between strains, in NN2025 has long repeated spacer sequences corresponding to the streptococcal phage genome.ConclusionThese observations suggest that S. mutans strains evolve through chromosomal shuffling and that phage infection is not needed for gene acquisition. In contrast, S. pyogenes tolerates phage infection for acquisition of virulence determinants for niche adaptation.


Journal of Fermentation and Bioengineering | 1996

Ethanol production from Jerusalem artichoke tubers by Aspergillus niger and Saccharomyces cerevisiae

Toyohiko Nakamura; Yasuko Ogata; Shigeyuki Hamada; Kazuyoshi Ohta

Simultaneous saccharification and fermentation of Jerusalem artichoke tubers were conducted batchwise at 30°C using Aspergillus niger 817 and Saccharomyces cerevisiae 1200. Ethanol concentrations obtained were 10.4% (v/v) from the ground tubers after 15 h, 15.0% from the juice concentrate after 72 h, and 20.1% from the flour after 120 h.


Journal of Fermentation and Bioengineering | 1994

Occurrence of two forms of extracellular endoinulinase from Aspergillus niger mutant 817

Toyohiko Nakamura; Yuhi Nagatomo; Shigeyuki Hamada; Yoshihiko Nishino; Kazuyoshi Ohta

Aspergillus niger mutant 817 was generated from A. niger 12, a strain that produces high levels of inulinases irrespective of the carbon source, by γ-irradiation with 60Co. A. niger 817 showed 4-fold higher inulinase activity than the wild-type strain 12 in the filtrate of a submerged culture with fructose. An extremely high ratio of inulinase to invertase activity (I/S ratio) of 880 was found in the culture filtrate. Two inulinases, P-IA and P-IB, were purified from the culture filtrate of A. niger 817 grown on sucrose by DEAE-Cellulofine A-500 and Q-Sepharose HP chromatographies. The enzymes were homogeneous as judged by SDS-polyacrylamide gel electrophoresis with apparent molecular weights of 70,000 for P-IA and 68,000 for P-IB. The specific activities were 350 U/mg for P-IA and 340 U/mg for P-IB, and about 3.5-fold higher than that reported for the wild-type endoinulinase P-III. The enzymes were active only toward inulin, and lacked activity toward sucrose, raffinose or levan. The main products of inulin hydrolysis were inulotriose and -tetraose. Thus, both inulinases exhibited the endo-type mode of action on inulin. The apparent Km values for inulin were 0.48 mM for P-IA and 0.50 mM for P-IB at 40°C and pH 5.0, and lower than the 1.25 mM reported for the wild-type endoinulinase P-III.


Applied and Environmental Microbiology | 2013

Genetic Analysis of Capsular Polysaccharide Synthesis Gene Clusters from All Serotypes of Streptococcus suis: Potential Mechanisms for Generation of Capsular Variation

Masatoshi Okura; Daisuke Takamatsu; Fumito Maruyama; Takashi Nozawa; Ichiro Nakagawa; Makoto Osaki; Tsutomu Sekizaki; Marcelo Gottschalk; Yumi Kumagai; Shigeyuki Hamada

ABSTRACT Streptococcus suis strains are classified into 35 serotypes on the basis of the antigenicity of their capsular polysaccharides (CPs). CP synthesis genes are known to be clustered on the chromosome (cps gene cluster). The entire cps gene clusters of S. suis have so far been sequenced in 15 serotypes and found to be located between orfZ and aroA. In this study, to provide comprehensive information about S. suis CPs, we sequenced the entire cps gene clusters of the remaining serotypes and analyzed the complete set of S. suis cps gene clusters. Among the 35 cps gene clusters, 22 were located between orfZ and aroA, whereas the other 13 were flanked by other gene(s) on the chromosomes, and the chromosomal locus was classified into five patterns. By clustering analysis, the predicted products of cps genes found in the 35 serotypes were assigned into 291 homology groups, and all serotypes possessed a serotype-specific gene, except for serotypes 1, 2, 1/2, and 14. Because of the presence of genes encoding flippase (wzx) and polymerase (wzy), CPs of all serotypes were thought to be synthesized by the Wzx/Wzy pathway. Our data also implied the possibility of the transfer of the entire or partial cps gene clusters among S. suis strains, as well as the influence of spontaneous mutations in a single gene or a few genes on the antigenicity of some serotypes. Accumulation of these gene transfers and small-scale mutations may have generated the antigenic diversity of S. suis CPs.


Cellular Microbiology | 2012

The small GTPases Rab9A and Rab23 function at distinct steps in autophagy during Group A Streptococcus infection

Takashi Nozawa; Chihiro Aikawa; Akira Goda; Fumito Maruyama; Shigeyuki Hamada; Ichiro Nakagawa

Autophagy mediates the degradation of cytoplasmic contents in the lysosome and plays a significant role in immunity. Here we identified the small GTPases Rab9A and Rab23 as novel autophagy regulators during Group A streptococcus (GAS) infection. Rab9A was recruited to GAS‐containing autophagosome‐like vacuoles (GcAVs) after autophagosomal maturation and its activity was required for GcAV enlargement and eventual lysosomal fusion. GcAV enlargement appeared to be related to homotypic fusion of GcAVs with Rab9A. Rab23 was recruited to GAS‐capturing forming autophagosomes. Knockdown of Rab23 expression decreased both LC3‐ and Atg5‐positive GAS formation and caused the accumulation of LC3‐positive structures that did not associate with intracellular GAS. It was suggested, therefore, that Rab23 is required for GcAV formation and is involved in GAS targeting of autophagic vacuoles. Furthermore, knockdown of Rab9A or Rab23 expression impaired the degradation of intracellular GAS. Therefore, our data reveal that the Rab9A and Rab23 GTPases play crucial roles in autophagy of GAS. However, neither Rab9A nor Rab23 were localized to starvation‐induced autophagosomes. Not only Rab9A but also Rab23 was dispensable for starvation‐induced autophagosome formation. These findings demonstrate that specific Rab proteins function at distinct steps during autophagy in response to GAS infection.

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