Naoko Morinaga

Purification, cloning and characterization of variant LukE-LukD with strong leukocidal activity of staphylococcal bi-component leukotoxin family.

Staphylococcus aureus produces bi‐component leukotoxins composed of non‐associated soluble proteins, S and F. Neither S nor F component alone is cytotoxic, but components together are active. These include Panton‐Valentine leukocidin (PVL), γ‐hemolysin, LukE‐LukD and others. Purification of leukotoxin from Staphylococcus aureus V8 strain (ATCC 27733) which does not have PVL genes, identified an F component with 100% identical to that of PVL in the first twenty‐five N‐terminal amino acids. Molecular cloning of this toxin obtained 2,595 nucleotides sequences containing two novel open reading frames for S and F. Deduced amino acid sequences of the S and F were respectively 91 and 94% identical to those of LukE and LukD. These were named variant of LukE‐LukD (LukEv‐LukDv). The activity of the recombinant LukEv‐LukDv to rabbit leukocytes was similar to that of recombinant PVL. LukEv‐LukDv was hemolytic to rabbit red blood cells although the activity was only 8% of γ‐hemolysin, but PVL was not. These activities were quite different from the LukE‐LukD which was reported no hemolytic and poorly cytotoxic to leukocytes compared to PVL. The lukEv‐lukDv was found in 87% of clinical isolates of Staphylococcus aureus but lukE‐lukD was not detected. These data demonstrate the existence of variant LukE‐LukD in V8 strain (ATCC 27733).

Enhanced polyadenosine diphosphate‐ribosylation in cirrhotic liver and carcinoma tissues in patients with hepatocellular carcinoma

Aim: The aim of this study is to assess the poly ADP‐ribosylation activity in human hepatocellular carcinoma (HCC) and in liver cirrhosis (LC) as compared to the activity in normal livers (NL).

Effects of interferon on cell and virus growth in transformed human cell lines.

The anticellular and antiviral effects of human leukocyte interferons were studied in vitro in the transformed human embryonic cell lines. RSa and RSb. The growth of these cells was inhibited and they began to deteriorate about 48 h after treatment with 500 units/ml of interferon. When interferon was washed out within 48 h, their growth recovered gradually. The effects of interferon on cell growth depended on the amount of interferon added per cell. A subline, named IFr, was isolated which grows in the presence of 2000 units/ml of interferon, whereas growth of vesicular stomatitis virus in these cells is suppressed by 10 units/ml of interferon, just as in the parent cells. The anticellular and antiviral effects of interferon on IFr cells are discussed in relation to cell surface receptors.

Two Distinct Cytotoxic Activities of Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli

ABSTRACT Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 μg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was ∼1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.

Inhibitory Effects of Polyphenols on Gastric Injury by Helicobacter pylori VacA Toxin

Background.  Helicobacter pylori induces gastric damage and may be involved in the pathogenesis of gastric cancer. H. pylori‐vacuolating cytotoxin, VacA, is one of the important virulence factors, and is responsible for H. pylori‐induced gastritis and ulceration. The aim of this study is to assess whether several naturally occurring polyphenols inhibit VacA activities in vitro and in vivo.

Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin

Some shiga toxin‐producing Escherichia coli secrete a novel AB5 cytotoxin, named subtilase cytotoxin (SubAB), which induces vacuole formation in addition to cytotoxicity in susceptible cells. By immunoprecipitation with SubAB from Vero cells, we discovered proteins of 100 kDa, 135 kDa and 155 kDa as potential candidates for its receptor. These proteins were N‐glycosylated in their extracellular domains, a modification that was necessary for interaction with SubAB. Biotinylated receptors were partially purified by Datura stramonium agglutinin affinity chromatography and avidin‐agarose and analysed by TOF mass spectroscopy. The peptide sequences of p135 were identical to β1 integrin, and its identification was confirmed with anti‐integrin β1 antibody. The p155 protein was identified as α2 integrin using anti‐integrin α2 antibody. In addition, treatment of Vero cells with β1 integrin RNAi before exposure to SubAB prevented vacuolating activity. These results suggested that SubAB recognizes α2β1 integrin as a functional receptor; this first interaction may be an important key step leading to the SubAB‐induced morphological changes in Vero cells.

Subtilase cytotoxin, produced by Shiga-toxigenic Escherichia coli, transiently inhibits protein synthesis of Vero cells via degradation of BiP and induces cell cycle arrest at G1 by downregulation of cyclin D1

Subtilase cytotoxin (SubAB) is a AB5 type toxin produced by Shiga‐toxigenic Escherichia coli, which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a subtilase‐like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double‐stranded RNA‐activated protein kinase‐like ER kinase (PERK) and eukaryotic initiation factor‐2α (eIF2α). The phosphorylation of PERK and eIF2α was maximal at 30–60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress‐inducible C/EBP‐homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB‐treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB‐induced translational inhibition and continuous prolonged proteasomal degradation.

Essential domain of receptor tyrosine phosphatase β (RPTPβ) for interaction with Helicobacter pylori vacuolating cytotoxin

Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPβ and RPTPα, RPTPβ was found to be responsible for gastric damage caused by VacA. To define the region of RPTPβ involved in VacA binding, we made mutants of human cDNA RPTPβ-B, a short receptor form of RPTPβ. Immunoprecipitation experiments to assess VacA binding to RPTPβ-B mutants indicated that five residues (QTTQP) at positions 747–751 of the extracellular domain of RPTPβ-B (which is commonly retained in RPTPβ-A, a long form of RPTPβ) play a crucial role in its interaction with VacA, resulting in vacuolation as well as Git-1 phosphorylation. Transfected cells expressing deletion mutant Δ752, which lacks QTTQP, or the double point mutant Δ747 (T748A,T749A) had diminished vacuolation in response to VacA. Treatment of RPTPβ-B and Δ747 (which have QTTQP at 747–751) with neuraminidase and O-glycosidase diminished their VacA binding, whereas chondroitinase ABC did not have an effect. No inhibitory effect of pleiotrophin, a natural RPTPβ ligand, on VacA binding to RPTPβ-B or Δ747 was observed, supporting the conclusion that the extracellular region of RPTPβ-B responsible for VacA binding is different from that involved in binding pleiotrophin. These data define the region in the RPTPβ extracellular domain critical for VacA binding, in particular the sequence QTTQP at positions 747–751 with crucial threonines at positions 748 and 749 and are consistent with a role for terminal sialic acids possibly because of threonine glycosylation.

Effect of Gb3 in lipid rafts in resistance to Shiga-like toxin of mutant Vero cells.

Shiga-like toxin 1 (Stx1), produced by enterohemorrhagic Escherichia coli, binds to its receptor, globotriaosylceramide (Gb3), on target cell membranes, as a prerequisite for inducing host cell intoxication. To examine further toxin-receptor interactions, we established an Stx1-resistant clone of Vero cells by chemical mutagenesis. The mutant cells, expressed Gb3, but did not bind Stx1. These mutant cells were larger and had more Gb3 per cell than wild-type cells. Gb3 from both wild-type and mutant Vero cells was recovered in lipid rafts, isolated from cell lysates as detergent resistant membranes (DRMs); the DRMs derived from mutant cells had a lower density of Gb3 than did those from wild-type cells. Stx1 did not bind to the DRMs of mutant cells, both by ELISA and surface plasmon resonance. However, Stx1 bound to Gb3 separated by thin-layer chromatograms from the DRMs of mutant cells. The results indicate that not only presence of Gb3 but also Gb3 density on lipid rafts were important for Stx binding.

Novel Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli Induces Apoptosis in Vero Cells via Mitochondrial Membrane Damage

ABSTRACT Subtilase cytotoxin (SubAB) is an AB5 cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G1 phase. Here we show that SubAB, but not the catalytically inactive mutant SubAB(S272A), induced apoptosis in Vero cells, as detected by DNA fragmentation and annexin V binding. SubAB induced activation of caspase-3, -7, and -8. Caspase-3 appeared earlier than caspase-8, and by use of specific caspase inhibitors, it was determined that caspase-3 may be upstream of caspase-8. A general caspase inhibitor blocked SubAB-induced apoptosis, detected by annexin V binding. SubAB also stimulated cytochrome c release from mitochondria, which was not suppressed by caspase inhibitors. In HeLa cells, Apaf-1 small interfering RNA inhibited caspase-3 activation, suggesting that cytochrome c might form an apoptosome, leading to activation of caspase-3. These data suggested that SubAB induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage.