Akemi Takade

Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin

The ClyA protein is a pore-forming cytotoxin expressed by Escherichia coli and some other enterobacteria. It confers cytotoxic activity toward mammalian cells, but it has remained unknown how ClyA is surface exposed and exported from bacterial cells. Outer-membrane vesicles (OMVs) released from the bacteria were shown to contain ClyA protein. ClyA formed oligomeric pore assemblies in the OMVs, and the cytotoxic activity toward mammalian cells was considerably higher than that of ClyA protein purified from the bacterial periplasm. The redox status of ClyA correlated with its ability to form the oligomeric pore assemblies. In bacterial cells with a defective periplasmic disulphide oxidoreductase system, the ClyA protein was phenotypically expressed in a constitutive manner. The results define a vesicle-mediated transport mechanism in bacteria, and our findings show that the localization of proteins to OMVs directly may contribute to the activation and delivery of pathogenic effector proteins.

Restoration of culturability of starvation-stressed and low-temperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds

Abstract Late-exponential-phase cells of Escherichia coli O157:H- strain E32511/HSC became nonculturable in sterilized distilled water microcosms at 4 °C. Plate counts declined from 3 × 106 to less than 0.1 CFU/ml in about 21 days. However, when samples of microcosms at 21 days were inoculated onto an agar medium amended with catalase or nonenzyme peroxide-degrading compounds such as sodium pyruvate or α-ketoglutaric acid, plate counts increased to 104–105 CFU/ml within 48 h. The proposed mode of action of the catalase or pyruvate is via the degradation of the metabolic by-product H2O2, rather than through supplementation of a required nutrient in the recovery of nonculturable cells. Our studies were based on the assumption that E32511/HSC strain responds to starvation and a low temperature by entering a nonculturable state and that the correction of oxidative stress upon the inoculation of bacteria on agar plates promotes recovery of nonculturable cells.

Curli Fibers Are Required for Development of Biofilm Architecture in Escherichia coli K-12 and Enhance Bacterial Adherence to Human Uroepithelial Cells

Sessile bacteria show phenotypical, biochemical, and morphological differences from their planktonic counterparts. Curli, extracellular structures important for biofilm formation, are only produced at temperatures below 30 C in Escherichia coli K‐12 strains. In this report, we show that E. coli K‐12 can produce curli at 37 C when grown as a biofilm community. The curli‐expressing strain formed more biofilms on polyurethane sheets than the curli‐deficient strain under growth temperatures of both 25 C and 37 C. Curli are required for the formation of a three‐dimensional mature biofilm, with characteristic water channels and pillars of bacteria. Observations by electron microscopy revealed the presence at the surfaces of the curli‐deficient mutant in biofilm of flagella and type I pili. A wild‐type curli‐expressing E. coli strain significantly adhered to several lines of human uroepithelial cells, more so than an isogenic curli‐deficient strain. The finding that curli are expressed at 37 C in biofilm and enhance bacterial adherence to mammalian host cells suggests an important role for curli in pathogenesis.

The Iron-Binding Protein Dps Confers Hydrogen Peroxide Stress Resistance to Campylobacter jejuni

We identified and characterized the iron-binding protein Dps from Campylobacter jejuni. Electron microscopic analysis of this protein revealed a spherical structure of 8.5 nm in diameter, with an electron-dense core similar to those of other proteins of the Dps (DNA-binding protein from starved cells) family. Cloning and sequencing of the Dps-encoding gene (dps) revealed that a 450-bp open reading frame (ORF) encoded a protein of 150 amino acids with a calculated molecular mass of 17,332 Da. Amino acid sequence comparison indicated a high similarity between C. jejuni Dps and other Dps family proteins. In C. jejuni Dps, there are iron-binding motifs, as reported in other Dps family proteins. C. jejuni Dps bound up to 40 atoms of iron per monomer, whereas it did not appear to bind DNA. An isogenic dps-deficient mutant was more vulnerable to hydrogen peroxide than its parental strain, as judged by growth inhibition tests. The iron chelator Desferal restored the resistance of the Dps-deficient mutant to hydrogen peroxide, suggesting that this iron-binding protein prevented generation of hydroxyl radicals via the Fenton reaction. Dps was constitutively expressed during both exponential and stationary phase, and no induction was observed when the cells were exposed to H(2)O(2) or grown under iron-supplemented or iron-restricted conditions. On the basis of these data, we propose that this iron-binding protein in C. jejuni plays an important role in protection against hydrogen peroxide stress by sequestering intracellular free iron and is expressed constitutively to cope with the harmful effect of hydrogen peroxide stress on this microaerophilic organism without delay.

The Release of Outer Membrane Vesicles from the Strains of Enterotoxigenic Escherichia coli

The clinically isolated heat labile enterotoxin (LT)‐producing strains of Escherichia coli can be separated into two groups, namely spontaneous LT‐releasing strain and non‐spontaneous LT‐releasing strain, based on their phenotypes of spontaneous release of LT into the culture medium. The phenotype of spontaneous LT release was observed to correlate closely with the phenotype of the release of numerous small vesicles into the culture medium. Both morphological and biological examinations of the vesicle showed that the vesicle was released from the outer membrane. It can, therefore, be assumed that LT may be released from the cell at the time the vesicles form.

Photoactivated Ethidium Monoazide Directly Cleaves Bacterial DNA and Is Applied to PCR for Discrimination of Live and Dead Bacteria

Ethidium monoazide (EMA) is a DNA intercalating agent and a eukaryotic topoisomerase II poison. We found that EMA treatment and subsequent visible light irradiation (photoactivation or photolysis) shows a bactericidal effect, hence the mechanism was analyzed. When bacterial cells were treated with more than 10 μg/ml of EMA for 1 hr plus photoactivation for 20 min, cleavage of bacterial DNA was confirmed by agarose gel electrophoresis and electron microscopic studies. The cleavage of chromosomal DNA was seen when it was treated in vitro with EMA and photolysis, which showed that the cleavage directly took place without the assistance of DNA gyrase/topoisomerase IV and the DNA repair enzymes of bacteria. It was also verified, by using negatively supercoiled pBR322 DNA, that medium/high concentrations of EMA (1 to 100 μg/ml) led to breaks of double‐stranded DNA and that low concentrations of EMA (10 to 100 ng/ml) generated a single‐stranded break. EMA is known to easily penetrate dead but not live bacteria. After treatment of 10 μg/ml of EMA for 30 min and photoactivation for 5 min, EMA cleaved the DNA of dead but not live Klebsiella oxytoca. When the cleaved DNA was used for templates in PCR targeting 16S rDNA, PCR product from the dead bacteria was completely suppressed. We demonstrated that EMA and photolysis directly cleaved bacterial DNA and are effective tools for discriminating live from dead bacteria by PCR.

Release of Shiga Toxin by Membrane Vesicles in Shigella dysenteriae Serotype 1 Strains and In Vitro Effects of Antimicrobials on Toxin Production and Release

Effects of various antimicrobials on in vitro Shiga toxin production and release by Shigella dysenteriae serotype 1 was investigated in this study with particular reference to the role of outer membrane vesicles in toxin release by the organism. Five antimicrobials, namely nalidixic acid, ciprofloxacin, norfloxacin, fosfomycin and mitomycin C, were chosen for the study and the toxin titre was measured by the reverse passive latex agglutination (RPLA) method using an available kit. Only mitomycin C was found to induce production of Shiga toxin in the bacteria and its release by outer membrane vesicles. The highest titre of toxin was obtained in vesicle fraction suggesting that the vesicles play an important role in the release of Shiga toxin from periplasmic space by the organism.

Release of the Outer Membrane Vesicles from Vibrio cholerae and Vibrio parahaemolyticus

We found numerous small vesicles released from the cell by thin sectioning of the plate culture of Vibrio cholerae and V. parahaemolyticus fixed with the freeze‐substitution technique. From the broth media of exponentially growing bacteria we could collect the vesicles by the centrifugation but not enough without fixation. The vesicles are encompassed with a membrane structure similar to the outer membrane of these bacteria. The anti‐O (Inaba) serum reacted with the surface of the vesicles and the inside of the vesicle are generally filled with an electron‐dense mass.

Biogenesis of Salmonella enterica Serovar Typhimurium Membrane Vesicles Provoked by Induction of PagC

Gram-negative bacteria ubiquitously release membrane vesicles (MVs) into the extracellular milieu. Although MVs are the product of growing bacteria, not of cell lysis or death, the regulatory mechanisms underlying MV formation remained unknown. We have found that MV biogenesis is provoked by the induction of PagC, a Salmonella-specific protein whose expression is activated by conditions that mimic acidified macrophage phagosomes. PagC is a major constituent of Salmonella MVs, and increased expression accelerates vesiculation. Expression of PagC is regulated at the posttranscriptional and/or posttranslational level in a sigmaS (RpoS)-dependent manner. Serial quantitative analysis has demonstrated that MV formation can accelerate when the quantity of the MV constituents, OmpX and PagC, rises. Overproduction of PagC dramatically impacts the difference in the relative amount of vesiculation, but the corresponding overproduction of OmpX was less pronounced. Quantitative examination of the ratios of PagC and OmpX in the periplasm, outer membrane, and MVs demonstrates that PagC is preferentially enriched in MVs released from Salmonella cells. This suggests that specific protein sorting mechanisms operate when MVs are formed. The possible role(s) of PagC-MV in host cells is discussed.

Purification and characterization of ferritin from Campylobacter jejuni

We purified an iron-containing protein fromCampylobacter jejuni using ultracentrifugation and ionexchange chromatography. Electron microscopy of this protein revealed circular particles with a diameter of 11.5 nm and a central core with a diameter of 5.5 nm. The protein was composed of a single peptide of 21 kDa and did not serologically cross-react with horse spleen ferritin. The UV-visible spectrum of the protein showed no absorption peaks in the visible region, indicating that little or no heme is bound. The ratio of Fe:phosphate ofC. jejuni ferritin was 1.5∶1. From these morphological and chemical examinations, we concluded that theC. jejuni purified protein is a ferritin of the same class as that ofHelicobacter pylori andBacteroides fragilis and differs from the heme-containing bacterioferritin ofEscherichia coli. The 30 N-terminal amino acids were sequenced and were found to resemble the sequences of other ferritins strongly (H. pylori ferritin, 73% identity;B. fragilis ferritin, 50% identity;E. coli gene-165 product, 50% identity), and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 26% identity;Azotobacter vinelandii, 26% identity; horse spleen ferritin 30% identity). Proteins that cross-reacted with antiserum against the ferritin ofC. jejuni were found in otherCampylobacter species and inH. pylori, but not inVibrio, E. coli, orPseudomonas aeruginosa.