PubMed Health⌕ Search

Biomedical subjects

Asaomi Kuwae

Publications and source records attributed to Asaomi Kuwae.

11 recordsLinked to original sources
PubMed2006

BopC is a novel type III effector secreted by Bordetella bronchiseptica and has a critical role in type III-dependent necrotic cell death.

In Bordetella bronchiseptica, the functional type III secretion system (TTSS) is required for the induction of necrotic cell death in infected mammalian cells. To identify the factor(s) involved in necrotic cell death, type III-secreted proteins from B. bronchiseptica were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry. We identified a 69-kDa secreted protein designated BopC. The gene encoding BopC is located outside of the TTSS locus and is also highly conserved in both Bordetella parapertussis and Bordetella pertussis. The results of a lactate dehydrogenase release assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay demonstrated that BopC is required for necrotic cell death. It has been reported that tyrosine-phosphorylated proteins (PY) of host cells are dephosphorylated during B. bronchiseptica infection in a TTSS-dependent manner. We found that BopC is also involved in PY dephosphorylation in infected host cells. It appears that the necrotic cell death triggered by BopC occurs prior to the PY reduction in host cells, because Bordetella-induced cell death was not affected even in the presence of a dephosphorylation inhibitor. Furthermore, a translocation assay showed that the signal sequence for both secretion into culture supernatant and translocation into the host cell is located in 48 amino acid residues of the BopC N terminus. This report reveals for the first time that a novel type III effector, BopC, is required for the induction of necrotic cell death during Bordetella infection.

Animals↗
PubMed2006

Assembly of the type III secretion apparatus of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.

Blotting, Western↗
PubMed2005

Isolation of a low-molecular-weight, multicopy plasmid, pNHK101, from Thermus sp. TK10 and its use as an expression vector for T. thermophilus HB27.

We isolated a small multicopy cryptic plasmid, pNHK101, from Thermus sp. TK10 for use as a replicon of a Thermus expression vector. The nucleotide sequence of pNHK101 revealed that this plasmid was 1564bp long, with a total G+C content of 66.8%, which was in agreement with that of Thermus genomic DNA. The sequence did not show any significant similarities to any other plasmids; also, the amino acid sequences of four putative open reading frames, found in the plasmid, did not show strong similarities to those in the databases, except the ORF1, which had very slight similarities to several replication proteins of plasmids from other bacteria. pNHK101 was able to replicate in Thermus thermophilus HB27 with copy number about 80, and was stably maintained at 60 degrees C, but became unstable at 70 degrees C. Based on pNHK101, we constructed a plasmid vector, pKMH052, containing the highly thermostable kanamycin resistance gene as a selective marker. The copy number of pKMH052 decreased to about one-fourth of that of pNHK101, but stability at 60 degrees C did not alter under non-selective conditions. pKMH052 was compatible with pTT8, and interestingly, the presence of pTT8 in the same cells improved the stability of pKMH052 at 70 degrees C. Cloning of the crtB gene of T. thermophilus HB27 encoding phytoene synthase into pKMH052, and introduction into T. thermophilus cells resulted in a 2.8-fold production of carotenoids, indicating the potential use of this plasmid for overexpression of genes from thermophiles and hyperthermophiles.

Alkyl and Aryl Transferases↗
PubMed2005

Type-III effectors: sophisticated bacterial virulence factors.

Bacterial pathogens cause a wide spectrum of diseases in human and other animals. Some virulence factors, which are referred to as effectors, are directly translocated into the host cell via an injection apparatus, i.e., the type-III secretion system. Most effectors mimic host molecules, and translocated effectors are thereby able to perturb or modulate host cell signaling, cytoskeletal rearrangement, vesicular traffic, and autophagy, thus eliciting disease. Effectors are roughly classified among exotoxins, but in most cases, their functions are exerted focally when they are translocated into the host cell.

Animals↗
PubMed2005

Enteropathogenic Escherichia coli type III effectors EspG and EspG2 alter epithelial paracellular permeability.

Enteropathogenic Escherichia coli (EPEC) delivers a subset of effectors into host cells via a type III secretion system. Here we show that the type III effector EspG and its homologue EspG2 alter epithelial paracellular permeability. When MDCK cells were infected with wild-type (WT) EPEC, RhoA was activated, and this event was dependent on the delivery of either EspG or EspG2 into host cells. In contrast, a loss of transepithelial electrical resistance and ZO-1 disruption were induced by infection with an espG/espG2 double-knockout mutant, as was the case with the WT EPEC, indicating that EspG/EspG2 is not involved in the disruption of tight junctions during EPEC infection. Although EspG- and EspG2-expressing MDCK cells exhibited normal overall morphology and maintained fully assembled tight junctions, the paracellular permeability to 4-kDa dextran, but not the paracellular permeability to 500-kDa dextran, was greatly increased. This report reveals for the first time that a pathogen can regulate the size-selective paracellular permeability of epithelial cells in order to elicit a disease process.

Animals↗
PubMed2004

Enteropathogenic Escherichia coli activates the RhoA signaling pathway via the stimulation of GEF-H1.

Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease. Here, we show that the type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria. Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro. A recent study showed that microtubule-bound GEF-H1, a RhoA-specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation. Indeed, EspG- and Orf3-induced stress fiber formation was inhibited by the expression of dominant-negative forms of GEF-H1 and RhoA, but not of Rac1 and Cdc42, and by treatment with a ROCK inhibitor. These results indicate that the impact of EspG/Orf3 on microtubule networks triggers the activation of the RhoA-ROCK signaling pathway via GEF-H1 activity. This report reveals for the first time that a pathogen can exploit the host factor GEF-H1.

Actins↗
PubMed2004

The type III secreted protein BopD in Bordetella bronchiseptica is complexed with BopB for pore formation on the host plasma membrane.

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although BopB, BopN, BopD, and Bsp22 have been identified as type III secreted proteins, these proteins remain to be characterized. In this study, in order to clarify the function of BopD during Bordetella infection, a BopD mutant was generated. Although secretion of BopD into the culture supernatant was completely abolished by the bopD mutation, the secretion of other type III secreted proteins was not affected by this mutation. It has been reported that severe cytotoxicity, including cell detachment from the substrata, and release of lactate dehydrogenase (LDH) into the supernatant are induced in L2 cells by wild-type B. bronchiseptica infection, and these phenotypes are dependent on the type III secretion system. In contrast, neither cell detachment nor LDH release was induced in L2 cells infected with the BopD mutant. Furthermore, the hemolytic activity of the BopD mutant was greatly impaired compared with that of the wild-type strain. On the basis of the results of coimmunoprecipitation assays with anti-BopB antibodies, we conclude that BopD has the ability to associate with BopB. Finally, we show that the BopD-BopB complex is responsible for the pore formation in the host plasma membrane that functions as the conduit for the transition of effector proteins into host cells.

Animals↗
PubMed2003

BopB is a type III secreted protein in Bordetella bronchiseptica and is required for cytotoxicity against cultured mammalian cells.

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although the precise mechanism of the type III secretion system is unknown, BopN, BopD and Bsp22 have been identified as type III secreted proteins. In order to identify other proteins secreted via the type III secretion machinery in Bordetella, a type III mutant was generated, and its secretion profile was compared with that of the wild-type strain. The results showed that the wild-type strain, but not the type III mutant, secreted a 40-kDa protein into the culture supernatant. This protein was identified as BopB by the analysis of its N-terminal amino acid sequence. Severe cytotoxicity such as necrosis was induced in L2 cells by infection with the wild-type B. bronchiseptica. In contrast, this effect was not observed by the BopB mutant infection. The haemolytic activity of the BopB mutant was greatly impaired compared with that of the wild-type strain. The results of a digitonin assay strongly suggested that BopB was translocated into HeLa cells infected with the wild-type strain. Taken together, our results demonstrate that Bordetella secretes BopB via a type III secretion system during infection. BopB may play a role in the formation of pores in the host plasma membrane which serve as a conduit for the translocation of effector proteins into host cells.

Amino Acid Sequence↗
PubMed2003

Shigella-induced necrosis and apoptosis of U937 cells and J774 macrophages.

It is currently unclear whether Shigella kills its phagocytic host cells by apoptosis or necrosis. This study shows that rapid necrosis ensues in macrophage-like cell lines (U937 cells differentiated by all-trans-retinoic acid and J774 cells) infected with the Shigella flexneri strain YSH6000. The infected cells rapidly lose membrane integrity, a typical feature of necrosis, as indicated by the release of the cytoplasmic lactate dehydrogenase and the exposure of phosphatidylserine (PS) associated with the rapid uptake of propidium iodide (PI). The infected cells exhibit DNA fragmentation without nuclear condensation, and substantial involvement of either caspase-3/-7 or caspase-1 was not detected, which is also contrary to what is normally observed in apoptosis. Cytochalasin D potently inhibited Shigella-induced cell death, indicating that only internalized Shigella can cause necrosis. Osmoprotectants such as polyethylene glycols could suppress cell death, suggesting that insertion of a pore by Shigella into the host cell membrane induces the necrosis. The pore was estimated to be 2.87+/-0.4 nm in diameter. Shigella was also found to be able to induce apoptosis but only in one of the lines tested and under specific conditions, namely U937 cells differentiated with interferon-gamma (U937IFN). Caspase-3/-7 but not caspase-1 activation was observed in these infected cells and the exposure of PS occurred without the uptake of PI. An avirulent Shigella strain, wild-type Shigella killed with gentamicin, and even Escherichia coli strain JM109, could also induce apoptosis in U937IFN cells, and cytochalasin D could not prevent apoptosis. It appears therefore that Shigella-induced apoptosis of U937IFN cells is unrelated to Shigella pathogenicity and does not require bacterial internalization. Thus, Shigella can induce rapid necrosis of macrophage-like cells in a virulence-related manner by forming pores in the host cell membrane while some cells can be killed through apoptosis in a virulence-independent fashion.

Apoptosis↗
PubMed2002

Shigella deliver an effector protein to trigger host microtubule destabilization, which promotes Rac1 activity and efficient bacterial internalization.

Shigella deliver a subset of effectors into the host cell via the type III secretion system, that stimulate host cell signal pathways to modulate the actin dynamics required for invasion of epithelial cells. Here we show that one of the Shigella effectors, called VirA, can interact with tubulin to promote microtubule (MT) destabilization, and elicit protrusions of membrane ruffling. Under in vitro conditions, VirA inhibited polymerization of tubulin and stimulated MT destabilization. Upon microinjection of VirA into HeLa cells, a localized membrane ruffling was induced rapidly. Overexpression of VirA in host cells caused MT destruction and protruding membrane ruffles which were absent when VirA was co-expressed with a dominant-negative Rac1 mutant. Indeed, Shigella but not the virA mutant stimulated Rac1, including the formation of membrane ruffles in infected cells. Importantly, the MT structure beneath the protruding ruffling was destroyed. Furthermore, drug-induced MT growth in HeLa cells greatly enhanced the Shigella entry. These results indicate that VirA is a novel type of bacterial effector capable of inducing membrane ruffling through the stimulation of MT destabilization.

Actins↗