清水 健

ABSTRACT The complete genome sequence of Edwardsiella tarda strain GBS0709, isolated from an 81-year-old Japanese patient with the acute motor axonal neuropathy subtype of Guillain–Barré syndrome, was determined. It comprised a 3,632,068 bp circular chromosome and a 5,386 bp plasmid. The overall guanine and cytosine content was 57.3%.

Graphical Abstract

Eighty strains of enterohemorrhagic Escherichia coli O157:H7/H- were analyzed by three single-nucleotide polymorphism (SNP) panels using whole-genome sequencing data. The partial concordance of SNP types among the different SNP panels was observed on minimum spanning trees reconstructed with SNP data. As for lineage I/II strains, some of the clade 7 strains belonged to one unique SNP type as determined by three panels, suggesting that clade 7 should be divided into at least two genotypes, namely, the unique type and the rest. In addition, clade 8 contained two unique genotypes, which was consistent with the previous prediction. Similarly, for lineage II, clade 12 should be divided into three genotype strains. In contrast, many strains of several clades belonging to lineage I were clustered into the same node on each minimum spanning tree upon testing with the three SNP panels. Previous studies reported that lineage I diverged more recently than lineages I/II and II. Such low diversity in lineage I in this study may have arisen because this lineage has not accumulated SNPs because of its relatively recent divergence. Based on the concordance observed in this study, some of the previously published O157 genotype distribution data were successfully interpreted to clarify the clade distribution, which was well supported by previous literature.

Enterohaemorrhagic <italic> <named-content content-type="species"> <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3093" xlink:type="simple">Escherichia coli</ext-link> </named-content> </italic> (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Although <italic>stx1</italic> and <italic>stx2</italic> were found within the late operons of the Stx-encoding phages (Stx-phages), <italic>stx1</italic> could mainly be transcribed from the <italic>stx1</italic> promoter (<italic>P</italic> _Stx1), which represents the functional operator-binding site (Fur box) for the transcriptional regulator Fur (ferric uptake regulator), upstream of <italic>stx1</italic>. In this study, we found that the production of Stx1 by EHEC was affected by oxygen concentration. Increased Stx1 production in the presence of oxygen is dependent on Fur, which is an Fe²⁺-responsive transcription factor. The intracellular Fe²⁺ pool was lower under microaerobic conditions than under anaerobic conditions, suggesting that lower Fe²⁺ availability drove the formation of less Fe²⁺-Fur, less DNA binding to the <italic>P</italic> _Stx1 region, and an increase in Stx1 production.

Sub-MICs of the 14-membered macrolides erythromycin (EM) and clarithromycin (CAM) decreased the growth of Pseudomonas aeruginosa PAO1 and increased its sensitivity to endogenous and exogenous nitrosative stress. However, a 16-membered macrolide, josamycin (JM), was not or less effective. In 9 of 13 non-multidrug-resistant P. aeruginosa (non-MDRP) and 9 of 27 MDRP ST235 strains, the sub-MIC of EM induced significant reductions in bacterial numbers following treatment with a nitric oxide donor.

Enterohemorrhagic Escherichia coli (EHEC) has at least three enzymes, NorV, Hmp, and Hcp, that act independently to lower the toxicity of nitric oxide (NO), a potent antimicrobial molecule. This study aimed to reveal the cooperative roles of these defensive enzymes in EHEC against nitrosative stress. Under anaerobic conditions, combined deletion of all three enzymes significantly increased the NO sensitivity of EHEC determined by the growth at late stationary phase; however, the expression of norV restored the NO resistance of EHEC. On the other hand, the growth of Δhmp mutant EHEC was inhibited after early stationary phase, indicating that NorV and Hmp play a cooperative role in anaerobic growth. Under microaerobic conditions, the growth of Δhmp mutant EHEC was inhibited by NO, indicating that Hmp is the enzyme that protects cells from NO stress under microaerobic conditions. When EHEC cells were exposed to a lower concentration of NO, the NO level in bacterial cells of Δhcp mutant EHEC was higher than those of the other EHEC mutants, suggesting that Hcp is effective at regulating NO levels only at a low concentration. These findings of a low level of NO in bacterial cells with hcp indicate that the NO consumption activity of Hcp was suppressed by Hmp at a low range of NO concentrations. Taken together, these results show that the cooperative effects of NO-metabolizing enzymes are regulated by the range of NO concentrations to which the EHEC cells are exposed.

© 2018, The Author(s). Shiga toxigenic Escherichia coli (STEC) are responsible for a worldwide foodborne disease, which is characterized by severe bloody diarrhea and hemolytic uremic syndrome (HUS). Subtilase cytotoxin (SubAB) is a novel AB5 toxin, which is produced by Locus for Enterocyte Effacement (LEE)-negative STEC. Cleavage of the BiP protein by SubAB induces endoplasmic reticulum (ER) stress, followed by induction of cytotoxicity in vitro or lethal severe hemorrhagic inflammation in mice. Here we found that steroids and diacylglycerol (DAG) analogues (e.g., bryostatin 1, Ingenol-3-angelate) inhibited SubAB cytotoxicity. In addition, steroid-induced Bcl-xL expression was a key step in the inhibition of SubAB cytotoxicity. Bcl-xL knockdown increased SubAB-induced apoptosis in steroid-treated HeLa cells, whereas SubAB-induced cytotoxicity was suppressed in Bcl-xL overexpressing cells. In contrast, DAG analogues suppressed SubAB activity independent of Bcl-xL expression at early time points. Addition of Shiga toxin 2 (Stx2) with SubAB to cells enhanced cytotoxicity even in the presence of steroids. In contrast, DAG analogues suppressed cytotoxicity seen in the presence of both toxins. Here, we show the mechanism by which steroids and DAG analogues protect cells against SubAB toxin produced by LEE-negative STEC.

Enterohemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Nitric oxide (NO), which acts as an antimicrobial defense molecule, was found to enhance the production of Stx1 and Stx2 in EHEC under anaerobic conditions. Although EHEC O157 has two types of anaerobic NO reductase genes, an intact norV and a deleted norV, in the deleted norV-type EHEC, a high concentration of NO (12-29 mu mol/L, maximum steady-state concentration) is required for enhanced Stx1 production and a low concentration of NO (similar to 12 mu mol/L, maximum steady-state concentration) is sufficient for enhanced Stx2 production under anaerobic conditions. These results suggested that different concentration thresholds of NO elicit a discrete set of Stx1 and Stx2 production pathways. Moreover, the enhancement of Shiga toxin production in the intact norV-type EHEC required treatment with a higher concentration of NO than was required for enhancement of Shiga toxin production in the deleted norV-type EHEC, suggesting that the specific NorV type plays an important role in the level of enhancement of Shiga toxin production in response to NO. Finally, Fur derepression and RecA activation in EHEC were shown to participate in the NO-enhanced Stx1 and Stx2 production, respectively.

Although the adhesion of enterohemorrhagic Escherichia coli (EHEC) is central to the EHEC-host interaction during infection, it remains unclear how such adhesion regulates virulence factors. Adhesion to abiotic surfaces by E. coli has been reported to be an outer membrane lipoprotein NlpE-dependent activation cue of the Cpx pathway. Therefore, we investigated the role of NlpE in EHEC on the adhesion-mediated expression of virulence genes. NlpE in EHEC contributed to upregulation of the locus of enterocyte effacement (LEE) genes encoded type III secretion system and to downregulated expression of the flagellin gene by activation of the Cpx pathway during adherence to hydrophobic glass beads and undifferentiated Caco-2 cells. Moreover, LysR homologue A (LrhA) in EHEC was involved in regulating the expression of the LEE genes and flagellin gene in response to adhesion. Gel mobility shift analysis revealed that response regulator CpxR bound to the lrhA promoter region and thereby regulated expressions of the LEE genes and flagellin gene via the transcriptional regulator LrhA in EHEC. Therefore, these results suggest that the sensing of adhesion signals via NlpE is important for regulation of the expression of the type III secretion system and flagella in EHEC during infection.

A novel virulence gene, norV, that encodes nitric oxide (NO) reductase, was examined to investigate the emergence of enterohemorrhagic Escherichia coli (EHEC) O157 subgroup C clusters 2 and 3 from subgroup C cluster 1. Deletion of norV occurred at a point between cluster 1 and cluster 2 just after or at the same time that an stx2 bacteriophage, which retains Shiga toxin 2 gene, was inserted into wrbA, which encodes a novel multimeric flavodoxin-like protein, in EHEC O157. Sensitivity of NO to anaerobic growth was correlated with the deletion of norV in all EHEC O157 individuals tested. The C467A mutation of fimH, which encodes minor component of type 1 fimbriae, occurred within cluster 1, not as a transition from cluster 1 to cluster 2, indicating that there is a cluster 1 minority branch that leads to cluster 2. These data refine the evolutionary history of an emerging EHEC O157. (C) 2015 Elsevier B.V. All rights reserved.

病原細菌は多様なエフェクター分子や毒素分子を産生し、宿主生体内の標的臓器/組織の摩耗・機能障害を引き起こす。さらに一部の細菌エフェクター・毒素は、免疫に代表される生体防御系を鈍化あるいは暴走させ、結果としてより複雑な感染病態を作り上げていく。これら細菌性病原因子は、宿主との相互作用を介した進化圧・淘汰圧の下、標的細胞の構造的・機能的脆弱性を巧妙に利用する姿へと変容し作り上げられてきたのであろう。それゆえ、細菌毒素・エフェクターの標的となる分子やシグナル伝達系を網羅的に解明する作業は、高等真核生物が築いてきた一見精緻な生体システムが持つ予期せぬアキレス腱を暴き出す結果につながる。そこで本稿では、こうした視点に立ち、宿主生体システムに潜む疾病感受性・疾病脆弱性の分子基盤を細菌エフェクター・細菌毒素研究を通して論じた6つの最近の知見を紹介する。(著者抄録)

Pathogenic bacteria produce a variety of effectors and/or toxins, which subvert target cell/tissue functions in the infected hosts. Some of those effectors/toxins also perturb host defense mechanism, thereby making up more complicated pathophysiological conditions. Such bacterial effectors/toxins may have been positively selected during evolution because they directly strike vulnerable points in the host system. In turn, this indicates that systemic exploration of molecules and signaling pathways targeted by bacterial effectors/toxins provides a powerful tool in digging up an unexpected Achilles' heel(s), malfunctioning of which gives rise to disorders not restricted to infectious diseases. Based on this viewpoint, this review shows molecular basis underlying host susceptibility and vulnerability to diseases through the studies of host molecules targeted by bacterial effectors and toxins.

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis, and in more severe cases, a serious clinical complication called hemolytic uremic syndrome (HUS). Shiga toxin (Stx)is one of the factors that cause HUS. Serotypes of Stx produced by EHEC include Stx1 and Stx2. Although some genetically mutated toxoids of Stx have been developed, large-scale preparation of Stx that is practical for vaccine development has not been reported. Therefore, overexpression methods for Stx2 and mutant Stx2 (mStx2) in E. coli were developed. The expression plasmid pBSK-Stx2(His) was constructed by inserting the full-length Stx2 gene, in which a six-histidine tag gene was fused at the end of the B subunit into the lacZa fragment gene of the pBluescript II SK(+) vector. An E. coli MV1184 strain transformed with pBSK-Stx2(His) overexpressed histidine-tagged Stx2 (Stx2-His) in cells cultured in CAYE broth in the presence of lincomycin. Stx2-His was purified using TALON metal affinity resin followed by hydroxyapatite chromatography. From 1 L of culture, 68.8 mg of Stx2-His and 61.1mg of mStx2-His, which was generated by site-directed mutagenesis, were obtained. Stx2-His had a cytotoxic effect on HeLa cells and was lethal to mice. However, the toxicity of mStx2-His was approximately 1000-fold lower than that of Stx2-His. Mice immunized with mStx2-His produced specific antibodies that neutralized the toxicity of Stx2 in HeLa cells. Moreover, these mice survived challenge with high doses of Stx2-His. Therefore, the lincomycin-inducible overexpression method is suitable for large-scale preparation of Stx2 vaccine antigens.

In enterohaemorrhagic Escherichia coli (EHEC) O157, there are two types of anaerobic nitric oxide (NO) reductase genes, an intact gene (norV) and a 204 bp deletion gene (norVs). Epidemiological analysis has revealed that norV-type EHEC are more virulent than norVs-type EHEC. Thus, to reveal the role of NO reductase during EHEC infection, we constructed isogenic norV-type and norVs-type EHEC mutant strains. Under anaerobic conditions, the norV-type EHEC was protected from NO-mediated growth inhibition, while the norVs-type EHEC mutant strain was not, suggesting that NorV of EHEC was effective in the anaerobic detoxification. We then investigated the role of NO reductase within macrophages. The norV-type EHEC produced a lower NO level within macrophages compared with the norVs-type EHEC. Moreover, the norV-type EHEC resulted in higher levels of Shiga toxin 2 (Stx2) within macrophages compared with the norVs-type EHEC. Finally, the norV-type EHEC showed a better level of survival than the norVs-type EHEC. These data suggest that the intact norV gene plays an important role for the survival of EHEC within macrophages, and is a direct virulence determinant of EHEC.

Previously, we have shown that chickens immunized with Shiga toxin (Stx) produce Stx-neutralizing egg yolk immunoglobulin V (IgY) antibody. The anti-Stx-1 IgY and anti-Stx-2 IgY exert their neutralizing activity through their antibody activity against the B subunit of the toxin but not the A subunit. In the present study, chickens were immunized with recombinant Stx-1 B subunit (rStx-1B) and recombinant Stx-2 B subunit (rStx-2B). Induced anti-rStx-1B and anti-rStx-2B IgY neutralized the toxicity of Stx-1 and Stx-2 against HeLa 229 cells. The neutralizing activity of anti-rStx-1B IgY on Stx-1 was almost 10 times stronger than that of anti-Stx-1 IgY, and that of anti-rStx-2B IgY was 2.6 times stronger than that of anti-Stx-2 IgY. Anti-rStx-1B and anti-rStx-2B IgY reacted with multimeric and monomeric forms of the B subunits in contrast to anti-Stx-1 and anti-Stx-2 IgY that reacted with only the multimeric form. These results indicated that recombinant B subunits were promising antigens for induction of neutralizing antibodies in chickens.

Generation of an abnormal isoform of the priori protein (PrPSc) is a key aspect of the propagation of prions. Elucidation of the intracellular localization of PrPSc in prion-infected cells facilitates the understanding of the cellular mechanism of prion propagation. However, technical improvement in PrPSc-specific detection is required for precise analysis. Here, we show that the mAb 132, which recognizes the region adjacent to the most amyloidogenic region of PrP, is useful for PrPSc-specific detection by immunofluorescence assay in cells pre-treated with guanidine thiocyanate. Extensive analysis of the intracellular localization of PrPSc in priori-infected cells using mAb 132 revealed the presence of PrPSc throughout endocytic compartments. In particular, some of the granular PrPSc signals that were clustered at pen-nuclear regions appeared to be localized in an endocytic recycling compartment through which exogenously loaded transferrin, shiga and cholera toxin B subunits were transported. The granular PrPSc signals at pen-nuclear regions were dispersed to the peripheral regions including the plasma membrane during incubation at 20 degrees C, at which temperature transport from the plasma membrane to pen-nuclear regions was impaired. Conversely, dispersed PrPSc signals appeared to return to pen-nuclear regions within 30 min during subsequent incubation at 37 degrees C, following which PrPSc at pen-nuclear regions appeared to redisperse again to peripheral regions over the next 30 min incubation. These results suggest that PrPSc is dynamically transported along with the membrane trafficking machinery of cells and that at least some PrPSc circulates between pen-nuclear and peripheral regions including the plasma membrane via an endocytic recycling pathway.

Shiga toxins (Stxs) are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC) infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY) from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection.

A novel chromosome-plasmid hybrid bioluminescent reporter system (C-P reporter system) utilizing Photorhabdus luminescens luxCDABE genes has been constructed to monitor the expression of Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) in enterohemorrhagic Escherichia colt (EHEC) in real time. The luxCDABE genes of P. luminescens have been cloned and divided into a luxCDAB cassette and a luxE gene. A promoter-less luxE gene introduced downstream from stx1 and from stx2 on EHEC chromosomes in single copies, and other luxCDAB genes were expressed on a multicopy number expression plasmid into the same cells. These Stx1- and Stx2-bioluminescent reporter strains expressed bioluminescence into bacteria cells when the expression of the promoter-less luxE gene was expressed in response to the promoter activity of stx1 and stx2, respectively. The expression levels of bioluminescence were identical to the production levels of Stx1 and Stx2 in the Stx1- and Stx2-bioluminescent reporter strains, and these strains produced both Stxs at the same respective levels as those of the parent EHEC strains. Using these reporter strains, we examined the profiles of Stx1 and Stx2 expression in EHEC. We found that production of both Stx1 and Stx2 in EHEC was enhanced upon contact with intestinal epithelial cells and within macrophages. However, the expression profiles between Stx1 and Stx2 in EHEC were different from each other under these conditions. Thus, these results suggested that this C-P reporter system is useful for determining the gene expression profile of bacteria. (C) 2011 Elsevier B.V. All rights reserved.

腸管出血性大腸菌（EHEC）は病原因子として志賀毒素1（Stx1）と志賀毒素2（Stx2）を産生する。stx1遺伝子とstx2遺伝子は共にEHECに溶原化している志賀毒素転換ファージのゲノム上に存在しているが，stx1遺伝子はstx1遺伝子の上流近傍に存在するStx1プロモーターから，stx2遺伝子はStx2転換ファージの後期ファージプロモーターから転写される。これらのプロモーターの違いがStx1とStx2の発現条件の違い，発現様式の違い，産生された毒素の菌体内外への局在性の違いの主因である。特に，Stx2は自発的なStx2転換ファージのファージ誘導によって産生され，そしてこのファージの溶菌過程にともなって菌体外に放出される。さらにStx2の産生は外部環境からの刺激による宿主大腸菌のSOS応答によって誘発されるStx2転換ファージのファージ誘導によって増強される。また，EHECにはStx2転換ファージの溶菌過程にともなったStx2の菌体外放出機構の他に，もう一つの特異的なStx2の菌体外放出機構が存在する。<br>

Shiga toxin 1 (Stx1) is located in the periplasmic fraction, while Stx2 is found in the extracellular fraction, suggesting that enterohemorrhagic Escherichia coli (EHEC) contains a specific Stx2 release system. Both stx(1) and stx(2) are found within the late operons of Stx-encoding phages. Stx2 production is greatly induced by mitomycin C, suggesting that stx(2) can transcribe from the late phage promoter of the Stx2-encoding phage. However, the Stx1 promoter adjacent to stx(1) is a dominant regulatory element in Stx1 production. With the deletion of phage lysis genes of the Stx2-encoding phage, Stx2 remains in the bacterial cells. Further, we demonstrate that the Stx2-encoding phage, but not the Stx1-encoding phage, is spontaneously induced at extremely low rates. These results indicate that spontaneously specific Stx2-encoding phage induction is involved in specific Stx2 release from bacterial cells. Furthermore, to examine whether another system for specific Stx2 release is present in EHEC, we analyze the stx-replaced mutants. As expected, Stx2 derived from the Stx1 promoter is located in both the extracellular and cell-associated fractions, while mutant Stx2 (B subunit, S31N) derived from the Stx1 promoter is found only in the cell-associated fraction. These results indicate that EHEC has another Stx2 release system that strictly recognizes the serine 31 residue of the B subunit. Overall, we present evidence that specific Stx2 release from bacterial cells is involved in both the Stx2-encoding phage induction system and another Stx2 release system.

Botulinum neurotoxins (BoNTs) are often acquired from the digestive tract and specifically target neuromuscular junctions where they cause an inhibition of acetylcholine release. A transcytotic mechanism has been evidenced in epithelial intestinal cells, which delivers whole BoNTs across the intestinal barrier, whereas BoNTs enter motoneurons through a pathway that permits the translocation of light chain into the cytosol. We used fluorescent BoNT/A C-terminal part of H chain (Hc) that mediates toxin binding to cell receptors to monitor toxin entry into NG108-15 neuronal cells as well as into Caco-2 and m-ICcl2 intestinal cells. BoNT/A Hc receptors were found to be distributed in membrane structures closely associated to cholesterol-enriched microdomains, but distinct from detergent-resistant microdomains in both cell types. BoNT/A Hc was trapped into endocytic vesicles, which progressively migrated to a perinuclear area in NG108-15 cells, and in a more scattered manner in intestinal cells. In both cell types, BoNT/A Hc entered through a dynamin- and intersectin-dependent pathway, reached an early endosomal compartment labelled with early endosome antigen 1. In neuronal cells, BoNT/A Hc entered mainly via a clathrin-dependent pathway, in contrast to intestinal cells where it followed a Cdc42-dependent pathway, supporting a differential toxin routing in both cell types.

Enterohemorrhagic Escherichia coli (EHEC) produces Stx1 and Stx2 causing severe diseases. Their B-subunits (StxBs) exhibit tow immunogenicity and the anti-StxB antibodies neutralizing both Stxs has not been prepared yet. By intranasal vaccination with His-tagged-StxB (Stx1 B-His or Stx2B-His) plus a mutant heat-labile enterotoxin (mLT) in mice, their serum and tung fluid reacted with appropriate StxB. Mice vaccinated with Stx2B-His plus mLT had antibodies reacting Stx1B and showed the resistance to toxemia of Stx1 and Stx2. This is the first demonstration to get anti-Stx2B serum neutralizing both Stxs. These suggest that the nasal vaccination with Stx2B-His and mLT is effective for preventing toxemias by EHEC. (C) 2008 Published by Elsevier Ltd.

Escherichia coli O157:H7 produces Stx1 and Stx2 causing severe diseases. Their B subunits (StxBs) are useful for a vaccine but exhibit low immunogenicity, especially Stx2B. Nasal vaccination with StxBs plus cholera toxin induces only serum anti-Stx1B antibodies in mice. However, nasal administration of a mutant of E. coli enterotoxin and His-tagged Stx2B induced serum antibodies neutralizing Stx2 in vitro or in vivo and mucosal IgA antibodies in lungs. As His-tagged Stx2B showed five or three poLymers in get filtration chromatography, His-tagged Stx2B forms smaller tertiary structure than the native one and is effective for preventing Stx2 toxemia as a nasal vaccine. (C) 2007 Published by Elsevier Ltd.

The closely related Shiga toxins, Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), can bind to Gb3 receptors. However, Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains are more commonly associated with serious human disease (viz., hemolytic-uremic syndrome) than Stxl-producing strains. To clarify the relationship between properties and toxicity of these toxins, we constructed and analyzed a hybrid holotoxin composed of Stx2A and Stx1B, designated as Stx2A1B, and a B subunit chimeric holotoxin composed of Stx2A and Stx2B (III V), designated as Stx2A2B (III V). The affinity of Stx2A1B to Gb3 was lower than that of Stx1, higher than that of Stx2 and identical to that of Stx2A2B (III V). On the other hand, the 50% lethal dose (LD50) for mice of Stx2A1B was lower than that of Stx1, but higher than that of Stx2. These results suggested that pathogenicity in mice was inversely related to the receptor affinity of the holotoxins. However, LD50 of Stx2A1B was not identical to that of Stx2A2B (III V). Gel filtration analysis indicated that Stx2A2B (III V) was relatively less stable than Stx2A1B. Moreover, cross-linking experiments demonstrated that the modes of cell surface binding of Stx2A2B (III V) and Stx2A1B were different. These results indicated that the receptor affinity, stability and binding mode of Shiga toxins might be important determinants for toxicity in mice. (c) 2007 Elsevier Ltd. All rights reserved.

Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) include Shiga toxin I (Stx1) as well as Shiga toxin 2 (Stx2). Stx1 is cell associated, whereas Stx2 is localized to the culture supernatant. We have analyzed the secretion of Stx2 by generating histidine-tagged StxB (StxB-H). Although neither StxB1-H nor StxB2-H was secreted in StxB-H-overexpressed EHEC, StxB2-H-overexpressed EHEC showed inhibited Stx2 secretion. On the other hand, StxB1-H-overexpressed EHEC showed no alteration of Stx2 secretion. B-subunit chimeras of Stx1 and Stx2 were used to identify the specific residue of StxB2 that the Stx2 secretory system recognizes. Alteration of the serine 31 residue to an asparagine residue (S31N) in StxB2-H enabled the recovery of Stx2 secretion. On the other hand, alteration of the asparagine 32 residue to a serine residue (N32S) in StxB1-H caused the partial secretion of a point-mutated histidine-tagged B subunit in EHEC. Based on the evidence, it appeared possible that this residue might contain secretion-related information for Stx2 secretion. To investigate this hypothesis, we constructed an isogenic mutant EHEC (Stx1B subunit, N32S) strain and an isogenic mutant EHEC (Stx2B subunit, S31N) strain. Although the mutant Stx2 was cell associated in isogenic mutant EHEC, mutant Stx1 was not extracellular. However, when we used plasmids for the expression of the mutant holotoxins, the overexpressed mutant Stx1 was found in the supernatant fraction, and the overexpressed mutant Stx2 was found in the cell-associated fraction in mutant holotoxin gene-transformed EHEC. These results indicate that the serine 31 residue of the B subunit of Stx2 contains secretion-related information.

Completion of the whole genome sequence of Clostridium perfringens strain 13 revealed the presence of an extracellular nuclease gene, cadA. Transcriptional analysis showed that the cadA gene is negatively regulated by the two-component VirR/VirS system and its secondary regulator VR-RNA. The CadA protein possesses an N-terminal signal sequence and a Gram-positive cell wall anchoring motif consisting of a sorting signal (LPXTG motif), a hydrophobic domain, and positively charged residues at the end of C-terminus. By comparing the DNase production between the wild type and the cadA mutant, and DNase activity assay with the recombinant truncated CadA protein, we confirmed that the cadA gene product is one of the DNases produced by C. perfringens. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Background. IgA nephropathy is the most common form of glomerulonephritis worldwide. We previously reported a novel form of glomerulonephritis with glomerular IgA deposits following methicillin-resistant Staphylococcus aureus (S. aureus) infection. We investigated the role of S. aureus related antigens in the immunopathogenesis of IgA nephropathy by producing several monoclonal antibodies against S. aureus surface antigens and determining the epitopes of deposited antigens in patients with IgA nephropathy. Methods. Cell membrane proteins were isolated from cultured S. aureus. Mouse monoclonal antibodies against these proteins were generated, and their target epitopes were determined by antibody affinity chromatography and amino acid sequence analysis, and by monoclonal antibody screening of Escherichia coli clones transfected with plasmids from the Lambda S. aureus Genomic Library. Renal biopsy specimens from 116 patients with IgA nephropathy and 122 patients with other forms of renal disease were examined for glomerular antigen depositions by immunofluorescence microscopy. Results. The major antigen recognized by monoclonal antibodies against S. aureus cell membrane was identified as the S. aureus cell envelope antigen designated 'probable adhesin' (ACCESSION AP003131-77, Protein ID; BAB41819.1). In 68.1% (79/116) of renal biopsy specimens from patients with IgA nephropathy, S. aureus cell envelope antigen was localized in the glomeruli, and the data confirmed that S. aureus cell envelope antigen was co-localized with IgA antibody in the glomeruli. No deposition of this antigen was detected in the glomeruli of patients with non-immune complex deposit forms of glomerulonephritis. Conclusion. S. aureus cell envelope antigen is a new candidate for the induction of IgA nephropathy.

Two Stx-converting phages, designated Stx1phi and Stx2phi-II, were isolated from an Escherichia coli O157:H7 strain, Morioka V526, and their entire nucleotide sequences were determined. The genomes of both phages were similar except for the stx gene-flanking regions. Comparing these phages to other known Stx-converting phages, we concluded that Stx1phi is a novel Stx1-converting phage closely related to Stx2-converting phages so far reported.

It is well recognized that the Shiga-like toxins (Stxs) preferentially bind to Gb3 glycolipids and the cholera toxin (CT) and heat-labile enterotoxin (LTp) bind to GM1 gangliosides. After binding to the cell surface, A-B bacterial enterotoxins have to be internalized by endocytosis. The transport of the toxin-glycolipid complex has been documented in several manners but the actual mechanisms are yet to be clarified. We applied a heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3' -dithiopropionate (SASD), to detect the membrane proteins involved in the binding and the transport of A-B bacterial enterotoxins in cultured cells. Both Stx1 and Stx2 bound to the detergent-insoluble microdomain (DIM) of Vero cells and Caco-2 cells, which were susceptible to the toxin, but neither was bound to insusceptible CHO-K1 cells. Both CT and LTp bound to the DIM of Vero cells, Caco-2 cells, and CHO-K1 cells. In a cross-linking experiment, Stx 1 cross-linked only with a 27-kDa molecule, while Stx2, which was more potently toxic than Stx 1, cross-linked with 27- and 40-kDa molecules of Vero cells as well as of Caco-2 cells; moreover, no molecules were cross-linked with the insusceptible CHO-K1 cells. LTp was cross-linked only to the 27-kDa molecule of these three cell types but the CT, which was more toxic than Up, was also cross-linked with 27- and 40-kDa molecules of Vero cells, Caco-2 cells, and CHO-K1 cells. The 27- and the 40-kDa molecules might play a role in the endocytosis and retrograde transport of A-B bacterial enterotoxins. (C) 2003 Elsevier Science B.V All rights reserved.

While searching for Escherichia coli O157 in the aquatic environment of Calcutta using an immunodetection procedure, we fortuitously detected five strains of Citrobacter braakii, which cross-reacted with the commercially available O157 polyvalent antiserum. The five C. braakii isolates gave positive results when a sensitive dot-ELISA was performed with E. coli O157 monoclonal antibody. Further, the O157 monoclonal antibody recognized the bands of proteinase K treated whole cells of lipopolysaccharide of all the C. braakii isolates. Apart from weak reactions with two or three of the DNA probes, all the C. braakii strains did not hybridize with the other probes spanning the minimum region required for O157 O-antigen biosynthesis. These strains did not possess any of the virulence genes that are commonly found in the Shiga toxin-producing E. coli (STEC) specially the serotype O157:H7. Therefore, it appears that the serological cross-reaction between C. braakii and E. coli O157 antiserum is based on structural mimicry between the O-polysaccharide of C. braakii and E. coli O157.

Shiga toxin 2-converting phage was isolated from Escherichia coli O157:H7 associated with an outbreak that occurred in Okayama, Japan in 1996 (M. Watarai, T. Sato, M. Kobayashi, T. Shimizu, S. Yamasaki, T. Tobe, C. Sasakawa and Y. Takeda, Infect. Immun. 61 (1998) 3210-3204). In this study, we analyzed the complete nucleotide sequence of Shiga toxin 2-converting phage, designated Stx2phi-I, and compared it with three recently reported Stx2-phage genomes. Stx2phi-I consisted of 61,765 bp, which included 166 open reading frames. When compared to 933W, VT2-Sakai and VT2-Sa phages, six characteristic regions (regions I-VI) were found in the Stx2 phage genomes although overall homology was more than 95% between these phages. Stx2phi-I exhibited remarkable differences in these regions as compared with VT-2 Sakai and VT2-Sa genes but not with 933W phage. Characteristic repeat sequences were found in regions I-IV where the genes responsible for the construction of head and tail are located. Regions V and VI, which are the most distinct portion in the entire phage genome were located in the upstream and downstream regions of the Stx2 operons that are responsible for the immunity and replication, and host lysis. These data indicated that Stx2phi-I is less homologous to VT2-Sakai and VT2-Sa phages, despite these three phages being found in the strains isolated at the almost same time in the same geographic region but closely related to 933W phage which was found in the E. coli O157 strain 933W isolated 14 years ago in a different geographic area. (C) 2003 Elsevier Science B.V. All rights reserved.

The proteins under the control of the two-component system VirR/VirS in Clostridium perfringens were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the virR mutant. Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified. Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system at the transcriptional level, but the remaining proteins were modified with a VirR/VirS-directed protease at the posttranslation and secretion levels. We purified and characterized the VirR/VirS-directed protease from the culture supernatant and identified it as a kind of clostripain. Because this proteolytic activity was strongly inhibited by leupeptin and antipain, it was concluded that this protease was a member of the family of cysteine proteases of C. perfringens.

Several studies have shown that the emergence of the O139 serogroup of Vibrio cholerae is a result of horizontal gene transfer of a fragment of DNA from a serogroup other than O1 into the region responsible for O-antigen biosynthesis of the seventh pandemic V. cholerae O1 biotype El Tor strain. In this study, we show that the gene cluster responsible for O-antigen biosynthesis of the O139 serogroup of V. cholerae is closely related to those of O22. When DNA fragments derived from O139 O-antigen biosynthesis gene region were used as probes, the entire O139 O-antigen biosynthesis gene region could be divided into five classes, designated as I-V based on the reactivity pattern of the probes against reference strains of V. cholerae representing serogroups O1-O193. Class IV was specific to O139 serogroup, while classes I-III and class V were homologous to varying extents to some of the non-Ol, non-O139 serogroups. Interestingly, the regions other than class IV were also conserved in the O22 serogroup. Long and accurate PCR was employed to determine if a simple deletion or substitution was involved to account for the difference in class IV between O139 and O22. A product of approx. 15 kb was amplified when O139 DNA was used as the template, while a product of approx. 12.5 kb was amplified when O22 DNA was used as the template, indicating that substitution but not deletion could account for the difference in the region between O22 and O139 serogroups. In order to precisely compare between the genes responsible for O-antigen biosynthesis of O139 and O22, the region responsible for O-antigen biosynthesis of O22 serogroup was cloned and analyzed. In concurrence with the results of the hybridization test, all regions were well conserved in O22 and O139 serogroups, although wbfA and the five or six genes comprising class IV in O22 and O139 serogroups, respectively, were exceptions. Again the genes in class IV in O22 were confirmed to be specific to O22 among the 155 'O' serogroups of V. cholerae. These data suggest that the gene clusters responsible for O139 O-antigen biosynthesis are most similar to those of O22 and genes within class IV of O139, and O22 defines the unique O antigen of O139 or O22. (C) 1999 Elsevier Science B.V. All rights reserved.

The genes responsible for O157O-antigen synthesis were cloned from Shiga toxin 1 (Stx1)-producing enterohaemorrhagic Escherichia coli O157:H-. Based on the published sequence of the rfbE(EcoO157:H7) gene, the rfbE gene was amplified by PCR and used as the probe. One clone (1-4-1) out of ten agglutinated using the slide agglutination test with O157 antiserum. The DNA sequence of a 31.5 kb fragment in p1-4-1 was determined and found to contain 26 open reading frames (ORFs). These ORFs were organized as two clusters of genes, comprising orf2 to orf16 and orf17 to orf24 regions, which were aligned in opposite directions. The GC contents of orf1, orf12 and orf14 to orf26 were similar to the overall GC content of the E. coli chromosome (51%). However, orf2 to orf11 and orf13 showed a much lower GC content of 30.0 to 39.4%. The minimum region essential for O157 O-antigen expression in E. coli K-12 lay between orf2 and orf13, which corresponded to the region of low GC content in E. coli O157. A colony hybridization test was carried out against reference strains of E. coli representing serogroups O1 to O173 using O157 O-antigen synthesis genes (orf2-orf13) of E. coli O157 as probes. With the exception of orf12, all probes from E. coli O157 O-antigen synthesis gene cluster did not hybridize with DNA from E. coli O1 to O173, except O157 reference strains, under high stringency conditions. These data suggest that the region from orf2 to orf13 has originated from a non-E. coli species with a low GC content. (C) 1999 Academic Press.

Shiga toxins 1 (Stx1) and 2 (Stx2) are encoded hv toxin-converting bacteriophages of Stx-producing Escherichia coli (STEC), and so far two Stx1 and one Stx2-converting phages have been isolated from two STEC strains (A, D, O'Brien, J, W, Newlands, S, F. Miller, R. K, Holmes, H. W. Smith, and S. E. Formal, Science 226: 694-696, 1984), In this study, we isolated two Stx2-converting phages, designated Stx2 Phi-I and Stx2 Phi-II, from two clinical strains of STEC associated with the outbreaks in Japan in 1996 and found that Stx2 Phi-I resembled 933W, the previously reported Stx2-converting phage, in its infective properties for E. coli K-12 strain C600 while Stx2 Phi-II was distinct from them. The sizes of the plaques of Stx2 Phi-I and Stx2 Phi-II in C600 were different; the former was larger than the latter. The restriction maps of Stx2 Phi-I and Stx2 Phi-II were not identical; rather, Stx2 Phi-II DNA was approximately 3 kb larger than Stx2 Phi-I DNA. Furthermore, Stx2 Phi-I and Stx2 Phi-II showed different phage immunity, with Stx2 Phi-I and 933W belonging to the same group. Infection of C600 by Stx2 Phi-I or 933W was affected by environmental osmolarity differently from that by Stx2 Phi-II. When C600 was grown under conditions of high osmolarity, the infectivity of Stx2 Phi-I and 933W was greatly decreased compared with that of Stx2 Phi-II. Examination of the plating efficiency of the three phages for the defined mutations inn C600 revealed that the efficiency of Stx2 Phi-I and 933W for the fadL mutant decreased to less than 10(-7) compared with that for C600 whereas the efficiency of Stx2 Phi-II decreased to 0.1% of that for C600. In contrast, while the plating efficiency of Stx2 Phi-II for the lamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2 Phi-I and 933W were not changed. This was confirmed by the phage neutralization experiments with isolated outer membrane fractions from C600,fadL mutant, or lamB mutant or the purified His(6)-tagged FadL and LamB proteins, Based on the data, we concluded that FadL acts as the receptor for Stx2 Phi-I and Stx2 Phi-II whereas LamB acts as the receptor only for Stx2 Phi-II.

We have developed a large-scale purification method of the phosphorylated form (131 kDa) of membrane-bound guanylyl cyclase (mGC) from Hemicentrotus pulcherrimus spermatozoa. The purified mGC contained 26.0 +/- 1.3 moles of phosphate/mol enzyme (mean +/- S.D., n = 6). Phosphorylated peptides were isolated from the trypsin digest of the carboxymethylated Hi. pulcherrimus sperm mGC by affinity chromatography on a Chelating Sepharose Fast Flow column, and the peptides were then subjected to mass spectrometric analysis and determination of phosphoserines, after the conversion of phosphoserines to S-ethylcysteines by amino acid analysis. Based on the observed mass number and the content of phosphoserine, serine residues at positions 561, 565, 652, 722, 740, 755, 894, 897, 914, 918, 927, 930, 951, and 985, in addition to two residues among those at positions 666, 670, and 671, were shown to be phosphorylated. They are all located in the intracellular region (kinase-like and catalytic domains). Notably, serine residues at positions 894, 918, 927, and 930, that are conserved in the sequence of mammalian mGCs and medaka fish-eye-specific mGCs, are phosphorylated in the sea urchin sperm mGC.

The hatching enzyme (envelysin) of the sea urchin Hemicentrotus pulcherrimus was purified from the medium of hatched blastulae. By cDNA cloning its deduced amino acid sequence and molecular architecture were revealed. The 591-residue precursor with calculated M-r of 66 123 consists of an 18-residue signal sequence, a 151-residue propeptide, and a 422-residue mature enzyme with N-terminal catalytic and C-terminal hemopexin-like domains. As compared with that of Paracentrotus lividus, its amino acid sequence is 69% identical and 10% similar. They share typical structural features with the mammalian MMP gene family members: cysteine switch, zinc-binding signature, methionine-turn, Cys residues near both ends of hemopexin-like domain, etc. However, its propeptide has a 70-residue extra sequence with an Asp- and Glu-rich stretch, supposedly involved in the proenzyme activation by binding Ca2+ ions in seawater. The hinge region is also longer than those of most MMPs, with an extra sequence rich in Thr and Arg residues. Mature 50K enzyme is highly susceptible to autolytic cleavage at Gln(503)-Leu(504), producing the 38K form retaining catalytic activity and substrate specificity against fertilization envelope. The 38K form and 15K fragment were coeluted from a gel-filtration column, suggesting that these two fragments are disulfide-bridged and that the tertiary structure is not much deviated. The 38K form further autolyzed to 32K form by cleaving Tyr(450)-Tyr(451) bond with the loss of protein-substrate specificity, retaining only nonspecific protease activity. Thus, the autolytic release of 2/3 of the C-terminal domain reduced the highly specific enzyme to a common nonspecific protease, implying that the size and structure of almost the entire hemopexin-like domain is essential for the protein substrate specificity. Moreover, autolytic degradation of envelysins from the two species follow quite different pathways despite their high homology in structure. The 38K and 32K forms were inhibited by bovine TIMP-1 with different IC50 values, indicating that its inhibitory activity depends on the extent of the interaction with the C-terminal domain of the enzyme.

Intact sea urchin spermatozoa were successfully biotinylated with NHS-LC-Biotin and the biotinylated spermatozoa retained the viability. Analysis of the membrane prepared from the biotinylated spermatozoa of the sea urchin Hemicentrotus pulcherrimus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that several proteins such as wheat-germ agglutinin (WGA)-binding protein (220 kDa), guanylyl cyclase (131 kDa), sperm-activating peptide-I (SAP-I)-crosslinking protein (71 kDa), GPI-anchored protein (63 kDa) and functionally unknown proteins (50 kDa and 30 kDa) were specifically biotinylated. Experiments using spermatozoa of sea urchins, Anthocidaris crassispina and Clypeasfer japonicus showed that several proteins similar to those of H. pulcherrimus spermatozoa were also labeled with NHS-LC-Biotin. Fucose sulfate glycoconjugate (FSG) isolated from the jelly coat of H. pulcherrimus was mixed with solubilized biotinylated sperm membrane proteins of H. pulcherrimus, A. crassispina or C. japonicus and then subjected to gel filtration chromatography on a Sepharose 2B column, indicating that only two biotinylated H. pulcherrimus sperm proteins were coeluted with H. pulcherrimus FSG.

A cDNA clone encoding the membrane form of guanylyl cyclase was isolated from a Hemicentrotus pulcherrimus testis cDNA library and its nucleotide sequence was determined. The cDNA was 4123 bp long and an open reading frame predicted a protein of 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids which divides the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl-teninal, intracellular domain of 594 amino acids. Three potential N-linked glycosylation sites were present in the extracellular domain. Northern blot analysis of poly(A)(+)RNA from testes, ovaries, eggs and embryos at various developmental stages showed that the cDNA encoding guanylyl cyclase hybridized to a mRNA of 4.4 kb from the testes. We developed a large scale purification method of the phosphorylated (131 kDa) and dephosphorylated (128 kDa) forms of the membrane-bound guanylyl cyclase from H. pulcherrimus spermatozoa. The purified 131 kDa and 128 kDa forms of the guanylyl cyclase contained 26.0 +/- 1.3 and 4.3 +/- 0.7 moles of phosphate per mot protein (mean +/- S.D.; n=6), respectively. The amino-terminal amino acids of both the 131 kDa and 128 kDa forms of the guanylyl cyclase could not be detected, suggesting that they were blocked.

A cDNA clone encoding egg-jelly peptide, SAP-I (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), was isolated from a Hemicentrotus pulcherrimus ovary cDNA library and its nucleotide sequence was determined. The cDNA was 1282 bp long and an open reading frame predicted a protein of 334 amino acids containing 5 SAP-I and 7 SAP-I-like decapeptides, each separated by a single lysine residue. The cDNA hybridized to two species of mRNA (1.3 kb and 2.0 kb) from H. pulcherrimus ovaries. Northern blot analysis showed that the 1.3 kb transcripts appeared in ovaries collected from November to April and the 2.0 kb transcripts were detected only in ovaries collected in January. An expression study of the SAP-I precursor gene, by in situ hybridization with a non-radioactive RNA probe synthesized using the 1.3 kb cDNA as template, demonstrated that abundant SAP-I precursor transcripts were expressed in the accessory cells, but not in the growing oocytes.

A cDNA clone encoding egg-jelly peptide, SAP-I (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), was isolated from a Hemicentrotus pulcherrimus ovary cDNA library and its nucleotide sequence was determined. The cDNA was 1282 bp long and an open reading frame predicted a protein of 334 amino acids containing 5 SAP-I and 7 SAP-I-like decapeptides, each separated by a single lysine residue. The cDNA hybridized to two species of mRNA (1.3 kb and 2.0 kb) from H. pulcherrimus ovaries. Northern blot analysis showed that the 1.3 kb transcripts appeared in ovaries collected from November to April and the 2.0 kb transcripts were detected only in ovaries collected in January. An expression study of the SAP-I precursor gene, by in situ hybridization with a non-radioactive RNA probe synthesized using the 1.3 kb cDNA as template, demonstrated that abundant SAP-I precursor transcripts were expressed in the accessory cells, but not in the growing oocytes.