Showing papers by "Sumio Shinoda published in 1996"

Iron uptake mechanisms of pathogenic bacteria

Abstract: 鉄 (イオン) はほとんどの生物の生存と増殖に不可欠な元素である。しかし, 宿主生体における鉄は大部分が結合型やヘム鉄として存在し, 細菌が自由に利用できる遊離鉄は極めて少ない。これは有害な活性酸素の生成防止と共に細菌感染症に対する非特異的生体防御機構の一つとなっている。それ故, 宿主生体中で増殖し得る病原菌は何らかの巧妙な鉄獲得系を保持している筈である。鉄欠乏下に発現する2つの鉄獲得系が明かにされている: 1) Fe3+に高親和性の輸送キレート剤, シデロフォア (siderophore, siderochrome とも呼ばれる) を産生し, トランスフェリンやラクトフェリンに結合している鉄を奪い取り, そのコンプレックスに特異的なレセプターを介して鉄を取り込む系; 2) トランスフェリン, ラクトフェリン, ヘムに結合している鉄をそれぞれに特異的なレセプターを介して直接利用する系。この能力は細菌の生体内増殖を可能にするので, 病原性 (強化) 因子の一つと考えられている。近年, 分子生物学的あるいは分子遺伝学的手法を用いて, これら鉄獲得系の発現調節機構や病原性強化における役割が個々の病原菌についてより詳細に解明されつつある。さらに, 鉄獲得に関与する遺伝子群と共に鉄獲得に直接関係のない病原因子遺伝子の発現も鉄欠乏に呼応して増加し, これに係わる統括的 (global) 調節因子の存在が明かにされた。病原菌の鉄獲得機構の解明は感染症防御のための新たな手段, 戦略を提供する可能性を秘めている。

Involvement of Vulnibactin and Exocellular Protease in Utilization of Transferrin-and Lactoferrin-Bound Iron by Vibrio vulnificus

Abstract: In vitro growth experiments were conducted to evaluate the ability of vulnibactin, a siderophore produced by Vibrio vulnificus, to sequester transferrin- or lactoferrin-bound iron for growth. Comparative studies with the strain producing vulnibactin and its exocellular protease-deficient mutant revealed the involvement of the protease in addition to vulnibactin in effective utilization of iron ion (Fe3+) bound to transferrin and lactoferrin. It appears that the protease causes cleavage of these proteins, thereby making bound iron more accessible to vulnibactin.

Regulation of sulphate assimilation in saccharomyces cerevisiae

Abstract: We examined how the activity of O-acetylserine and O-acetylhomoserine sulphydrylase (OAS/OAH) SHLase of Saccharomyces cerevisiae is affected by sulphur source added to the growth medium and genetic background of the strain. In a wild-type strain, the activity was repressed if methionine, cysteine or glutathione was added to the growth medium. However, in a strain deficient of cystathionine gamma-lyase, cysteine and glutathione were repressive, but methionine was not. In strains deficient of serine O-acetyltransferase (SATase), OAS/OAH SHLase activity was low regardless of sulphur source and was further lowered by cysteine and glutathione, but not by methionine. From these observations, we concluded that S-adenosylmethionine should be excluded from being the effector for regulation of OAS/OAH SHLase. Instead, we suspected that S. cerevisiae would have the same regulatory system as Escherichia coli for sulphate assimilation; i.e. cysteine inhibits SATase to lower the cellular concentration of OAS which is required for induction of the sulphate assimilation enzymes including OAS/OAH SHLase. Subsequently, we obtained data supporting this speculation.

Expression of virulence-related properties by, and intestinal adhesiveness of, Vibrio mimicus strains isolated from aquatic environments.

Abstract: A study of the major pathogenic characteristics of Vibrio mimicus was carried out with 77 strains isolated from aquatic environments in Okayama, Japan. Of the strains tested, 96% demonstrated in vitro adherence to the rabbit intestinal mucosa, of which 36, 20, and 43% belonged to the strongly, moderately, and weakly adhesive groups, respectively. Of the 27 strains which appeared to be enterotoxigenic in the experiments using rabbit ileal loops, 74% belonged to the strongly adhesive group. All strains of V. mimicus at early log phase showed cell-mediated hemagglutination, and 70% of strongly hemagglutinative strains belonged to the strongly adhesive group, implying a possible correlation between cell-mediated hemagglutination and bacterial adherence. However, no significant correlation could be detected in the production of putative exocellular pathogenic factors and bacterial adherence or enterotoxigenicity.

Purification and characterization of novel hemagglutinins from Vibrio mimicus: A 39-kilodalton major outer membrane protein and lipopolysaccharide

Abstract: Two hemagglutinins (HAs) mediating the agglutinability to rabbit erythrocytes were isolated from 32-h culture supernatant of enterotoxigenic strain E-33 of Vibrio mimicus by ultrafiltration followed by gel filtration and anion-exchange column chromatography. The HAs were designated R-HA and C-HA on the basis of specific hemagglutinating activity towards rabbit erythrocytes only (R-HA) and towards chicken and rabbit erythrocytes (C-HA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent staining with Coomassie brilliant blue revealed no detectable protein band and a single band of Mr 39,000 in the case of R-HA and C-HA, respectively. However, silver staining of the gel containing R-HA revealed the appearance of low-molecular-weight material. These two HAs differed from each other and from previously reported HA/protease in receptor specificity, molecular composition, and biochemical and immunochemical properties. No simple sugar other than glycoproteins, including mucin, inhibited hemagglutinating activities of both C-HA and R-HA. Rabbit antibody against R-HA or C-HA could agglutinate E-33 whole cells, implying a possible cell surface origin of the two HAs. The isolated E-33 lipopolysaccharide (LPS) or its polysaccharide moiety conferred biochemical and immunochemical properties identical to those of R-HA, confirming that the R-HA represents polysaccharide of LPS. The LPS preparations from heterologous strains of Vibrio mimicus and Vibrio cholerae non-O1 confirmed that the hemagglutinating ability is a common function of LPS. On the other hand, the antibody against C-HA specifically recognized a major outer membrane protein (OMP) with an Mr of around 39,000 in both homologous and heterologous strains of V. mimicus, suggesting an OMP origin of C-HA. Furthermore, the antibody recognized a major OMP with an Mr of around 37,000 in V. cholerae. Although the immunogenicity of LPS and OMP is well documented for important intestinal pathogens, the hemagglutinating properties of such attractive cell surface components are hitherto unrecognized and will definitely contribute towards understanding their role in bacterial adherence.

Bacterial Proteases as Pathogenic Factors, with Special Emphasis on Vibrio Proteases

Abstract: Bacterial proteases play various pathogenic roles in infection. Pathogenic species of the genus Vibrio, such as V. cholerae, V. parahaemolyticus or V. vulnificus also produce exocellular proteases, and almost of them are metalloproteases having a zinc atom. V. vulnificus is an opportunistic human pathogen causing septicemia or wound infections characterized by the formation of edema, necrotic ulcer and cellulitis on the skin. The protease produced by the vibrio (VVP) enhanced vascular permeability through activation of the Hageman factor-plasma kallikrein-kinin cascade and/or exocytotic histamine release from mast cells, and formed hemorrhagic lesions which subsequently provoke severe dermonecrosis. Thus, VVP is the most probable candidate for edema formation in the infection. Other pathogenic roles of VVP were also demonstrated.The VVP gene was cloned, and the amino acid sequence was deduced from the nucleotide sequence. The mature protein was demonstrated to be composed of 413 amino acid residue...

Comparison of a fluorogenic assay with a conventional method for rapid detection of Vibrio parahaemolyticus in seafoods.

Abstract: A conventional method and a fluorogenic assay for the detection of Vibrio parahaemolyticus were compared. Among 29 seafood samples examined for the presence of V. parahaemolyticus, 17 samples harbored V. parahaemolyticus, and trypsinlike activity was noticed in 19 seafoods. The added fluorogenic substrate was cleaved in single samples of shrimp, turbo, and cuttlefish from which V. parahaemolyticus could not be isolated by the conventional method. Vibrio alginolyticus, in addition to V. parahaemolyticus, was found to exhibit intracellular trypsinlike activity. Trypsinlike activity in seafoods was observed after the most probable number for the initial density of V. parahaemolyticus-like organisms was found to have reached > 10(2) per g. A V. parahaemolyticus inoculum at 10(4) CFU/ml in arabinose-glucuronate medium was required to attain growth to 10(6) CFU/ml, which is the level necessary for the release of detectable amounts of fluorescent compound from the added substrate.

Determination of selenium (IV) in water by graphite furnace atomic absorption spectrometry after preconcentration with microorganism

Abstract: 凍結乾燥した細菌を用いて排水中からセレン (IV) を濃縮し, 黒鉛炉原子吸光法で測定する方法を開発した.排水処理に利用されている微生物製剤から分離したBacillus sp.を用いた場合にセレン (IV) の濃縮率が最も高かった.セレン含有排水50mlに6%硝酸5mlを添加した後, 凍結乾燥した細菌10mgを加え, 2時間攪拌して菌体にセレン (IV) を濃縮させた.菌体を遠心分離した後, 菌体分解用の硝酸 (30%) 0.7mlと原子吸光測定におけるマトリックスモディファイアとしての硝酸パラジウム (1000mg/l) 0.3mlを添加して菌体を分散した.この分散液20μ1を直接黒鉛炉原子吸光装置に導入しセレン (IV) を測定した.各種金属イオンの影響を検討した結果, 各イオンともセレン10μg/lに対して100μg/l程度までは影響しなかったが, クロム酸イオン, 金イオン等は1000μg/lでは負の影響を与えた.検量線は0から30μg/lの範囲で直線となった.セレン (IV) の定量下限は0.5μg/lであり, 相対標準偏差率は2.2%であった.本方法で排水中セレン (IV) を測定したところ, 一部の排水については銅―DDTC共沈濃縮/黒鉛炉原子吸光法による測定結果とよく一致した.しかし, 本方法は塩濃度の高い排水には適用できなかった.