Takashi Nozawa

The effect of high dose/high temperature irradiation on high purity fibers and their silicon carbide composites

Silicon carbide composites were fabricated by chemical vapor infiltration method using high purity fiber, Hi-Nicalon Type-S and Tyranno SA, and non-high purity fiber Hi-Nicalon. Bare fibers, SiC/SiC composites and CVD SiC were irradiated at 7.7 dpa and 800 °C or 6.0 dpa and 300 °C. The density of fiber and CVD SiC was measured by gradient column technique. Mechanical properties of the composites were evaluated by four-point flexural tests. Fracture surfaces were observed by SEM. Tyranno SA fiber and CVD SiC showed similar swelling behavior following irradiation at 7.7 dpa and 800 °C. Mechanical properties of composites reinforced with Hi-Nicalon Type-S and Tyranno SA fibers were stable even following neutron irradiation at 7.7 dpa and 800 °C. Fracture surfaces of these composites following irradiation were similar to those of non-irradiated composites with relatively short fiber pull-out.

Specific Behavior of Intracellular Streptococcus pyogenes That Has Undergone Autophagic Degradation Is Associated with Bacterial Streptolysin O and Host Small G Proteins Rab5 and Rab7

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLO-deficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells.

Improvement of mechanical properties of SiC/SiC composites by various surface treatments of fibers

Abstract In order to make an interfacial shear strength control layer (ISSCL), such as pyrolytic C layer, effective and improve SiC/SiC composites mechanical properties, heat treatment at 1500°C under Ar flow in quite low O 2 partial pressure or a CVI SiC coating were applied to Hi-Nicalon™ SiC fibers. Interfacial microstructures between fiber and matrix were examined by SEM and TEM. Interfacial shear properties were evaluated by single-fiber push-out tests and compared with the results of three-point bend tests and tensile tests. Fracture surfaces after mechanical tests were examined by SEM with EDS and optical interferometric profilometry. Active oxidation of the fibers decreased fiber mechanical properties and made the composites brittle, although it was successful in making the ISSCL rough. SiC fiber coating induced strong bonding between fiber and ISSCL and turned the crack path from between the fiber and the ISSCL to the inside of the ISSCL. Interfacial frictional stress was increased and the mechanical properties of tensile tests were improved.

Rab17‐mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion

Autophagy plays a crucial role in host defence by facilitating the degradation of invading bacteria such as Group A Streptococcus (GAS). GAS‐containing autophagosome‐like vacuoles (GcAVs) form when GAS‐targeting autophagic membranes entrap invading bacteria. However, the membrane origin and the precise molecular mechanism that underlies GcAV formation remain unclear. In this study, we found that Rab17 mediates the supply of membrane from recycling endosomes (REs) to GcAVs. We showed that GcAVs contain the RE marker transferrin receptor (TfR). Colocalization analyses demonstrated that Rab17 colocalized effectively with GcAV. Rab17 and TfR were visible as punctate structures attached to GcAVs and the Rab17‐positive dots were recruited to the GAS‐capturing membrane. Overexpression of Rab17 increased the TfR‐positive GcAV content, whereas expression of the dominant‐negative Rab17 form (Rab17 N132I) caused a decrease, thereby suggesting the involvement of Rab17 in RE–GcAV fusion. The efficiency of GcAV formation was lower in Rab17 N132I‐overexpressing cells. Furthermore, knockdown of Rabex‐5, the upstream activator of Rab17, reduced the GcAV formation efficiency. These results suggest that Rab17 and Rab17‐mediated REs are involved in GcAV formation. This newly identified function of Rab17 in supplying membrane from REs to GcAVs demonstrates that RE functions as a primary membrane source during antibacterial autophagy.

High-temperature tensile strength of near-stoichiometric SiC/SiC composites

Abstract In an attempt to characterize mechanical properties of near-stoichiometric SiC/SiC composites, tensile tests were conducted at room temperature in air and at elevated temperature under mild oxidizing gases atmosphere. SiC/SiC composites were fabricated by forced-flow chemical vapor infiltration method using two-dimensional fabrics of carbon coated near-stoichiometric Tyranno™SA fibers. Tensile tests were conducted on composites with two types of lay-up schemes using edge-loading small specimens. The effect of lay-up orientation on the mechanical properties and fracture behavior of composites were also examined. Tensile strength of composite was slightly decreased at 1573 K, while it retained approximately 80% of the strength at room temperature. Porosity dependence on elastic modulus was clearly exhibited.

Effects of fibers and fabrication processes on mechanical properties of neutron irradiated SiC/SiC composites

Abstract Radiation effects on flexural properties of SiC/SiC composites fabricated by forced thermal gradient chemical vapor infiltration (F-CVI) process, reaction sintered (RS) process and polymer impregnation and pyrolysis (PIP) process were investigated. In this study, neutron irradiation at 1073 K up to 0.4×1025 n/m2 (E>0.1 MeV) was performed. For F-CVI and RS SiC/SiC, due to the irradiation damage of interphase like pyrolytic carbon and boron nitride, which were sensitive to neutron irradiation, composite stiffness was slightly decreased. On the contrary, for PIP SiC/SiC, there was no significant change in stiffness before and after irradiation. Composite strength, however, was nearly stable against high-temperature irradiation with such a low fluence, except for RS SiC/SiC, since mechanical characteristics of fiber and matrix themselves were still stable to neutron irradiation. However RS SiC/SiC had a slight reduction of flexural strength due to the severe degradation of the interface by neutron irradiation.

Golgi-Resident GTPase Rab30 Promotes the Biogenesis of Pathogen-Containing Autophagosomes

Autophagy acts as a host-defense system against pathogenic microorganisms such as Group A Streptococcus (GAS). Autophagy is a membrane-mediated degradation system that is regulated by intracellular membrane trafficking regulators, including small GTPase Rab proteins. Here, we identified Rab30 as a novel regulator of GAS-containing autophagosome-like vacuoles (GcAVs). We found that Rab30, a Golgi-resident Rab, was recruited to GcAVs in response to autophagy induction by GAS infection in epithelial cells. Rab30 recruitment was dependent upon its GTPase activity. In addition, the knockdown of Rab30 expression significantly reduced GcAV formation efficiency and impaired intracellular GAS degradation. Rab30 normally functions to maintain the structural integrity of the Golgi complex, but GcAV formation occurred even when the Golgi apparatus was disrupted. Although Rab30 also colocalized with a starvation-induced autophagosome, Rab30 was not required for autophagosome formation during starvation. These results suggest that Rab30 mediates autophagy against GAS independently of its normal cellular role in the structural maintenance of the Golgi apparatus, and autophagosome biogenesis during bacterial infection involves specific Rab GTPases.

Comparative genome analysis and identification of competitive and cooperative interactions in a polymicrobial disease.

Polymicrobial diseases are caused by combinations of multiple bacteria, which can lead to not only mild but also life-threatening illnesses. Periodontitis represents a polymicrobial disease; Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, called ‘the red complex’, have been recognized as the causative agents of periodontitis. Although molecular interactions among the three species could be responsible for progression of periodontitis, the relevant genetic mechanisms are unknown. In this study, we uncovered novel interactions in comparative genome analysis among the red complex species. Clustered regularly interspaced short palindromic repeats (CRISPRs) of T. forsythia might attack the restriction modification system of P. gingivalis, and possibly work as a defense system against DNA invasion from P. gingivalis. On the other hand, gene deficiencies were mutually compensated in metabolic pathways when the genes of all the three species were taken into account, suggesting that there are cooperative relationships among the three species. This notion was supported by the observation that each of the three species had its own virulence factors, which might facilitate persistence and manifestations of virulence of the three species. Here, we propose new mechanisms of bacterial symbiosis in periodontitis; these mechanisms consist of competitive and cooperative interactions. Our results might shed light on the pathogenesis of periodontitis and of other polymicrobial diseases.

The STX6-VTI1B-VAMP3 complex facilitates xenophagy by regulating the fusion between recycling endosomes and autophagosomes

ABSTRACT Macroautophagy/autophagy plays a critical role in immunity by directly degrading invading pathogens such as Group A Streptococcus (GAS), through a process that has been named xenophagy. We previously demonstrated that autophagic vacuoles directed against GAS, termed GAS-containing autophagosome-like vacuoles (GcAVs), use recycling endosomes (REs) as a membrane source. However, the precise molecular mechanism that facilitates the fusion between GcAVs and REs remains unclear. Here, we demonstrate that STX6 (syntaxin 6) is recruited to GcAVs and forms a complex with VTI1B and VAMP3 to regulate the GcAV-RE fusion that is required for xenophagy. STX6 targets the GcAV membrane through its tyrosine-based sorting motif and transmembrane domain, and localizes to TFRC (transferrin receptor)-positive punctate structures on GcAVs through its H2 SNARE domain. Knockdown and knockout experiments revealed that STX6 is required for the fusion between GcAVs and REs to promote clearance of intracellular GAS by autophagy. Moreover, VAMP3 and VTI1B interact with STX6 and localize on the TFRC-positive puncta on GcAVs, and are also involved in the RE-GcAV fusion. Furthermore, knockout of RABGEF1 impairs the RE-GcAV fusion and STX6-VAMP3 interaction. These findings demonstrate that RABGEF1 mediates RE fusion with GcAVs through the STX6-VAMP3-VTI1B complex, and reveal the SNARE dynamics involved in autophagosome formation in response to bacterial infection.

A Locus Encoding Variable Defense Systems against Invading DNA Identified in Streptococcus suis

Streptococcus suis, an important zoonotic pathogen, is known to have an open pan-genome and to develop a competent state. In S. suis, limited genetic lineages are suggested to be associated with zoonosis. However, little is known about the evolution of diversified lineages and their respective phenotypic or ecological characteristics. In this study, we performed comparative genome analyses of S. suis, with a focus on the competence genes, mobile genetic elements, and genetic elements related to various defense systems against exogenous DNAs (defense elements) that are associated with gene gain/loss/exchange mediated by horizontal DNA movements and their restrictions. Our genome analyses revealed a conserved competence-inducing peptide type (pherotype) of the competence system and large-scale genome rearrangements in certain clusters based on the genome phylogeny of 58 S. suis strains. Moreover, the profiles of the defense elements were similar or identical to each other among the strains belonging to the same genomic clusters. Our findings suggest that these genetic characteristics of each cluster might exert specific effects on the phenotypic or ecological differences between the clusters. We also found certain loci that shift several types of defense elements in S. suis. Of note, one of these loci is a previously unrecognized variable region in bacteria, at which strains of distinct clusters code for different and various defense elements. This locus might represent a novel defense mechanism that has evolved through an arms race between bacteria and invading DNAs, mediated by mobile genetic elements and genetic competence.