Membrane-damaging action of Clostridium perfringens alpha-toxin on phospholipid liposomes

AbstractThe effect of Clostridium perfringens alpha-toxin on multilamellar liposomes prepared from various phospholipids and cholesterol was investigated. The toxin induced carboxyfluorescein leakage from liposomes composed of the choline-containing phospholipids such as egg-yolk phosphatidylcholine and bovine brain sphingomyelin in a dose-dependent manner, but did not induce leakage from those liposomes composed of bovine brain phosphatidylethanol amine, egg-yolk phosphatidylserine or phosphatidylglycerol. The toxin-induced carboxyfluorescein leakage from egg-yolk phosphatidylcholine liposomes was increased by addition of divalent cations. The toxin induced carboxyfluorescein release from liposomes composed of phosphatidylcholine containing unsaturated fatty acyl residues or shorter chain length saturated fatty acyl residues (12 or 14 carbon atoms), but did not induce such release from liposomes composed of phosphatidylcholine containing saturated fatty acyl residues of between 16 and 20 carbon atoms. Furthermore, the toxin-induced carboxyfluorescein release decreased with increasing chain length of the acyl residues of phosphatidylcholine used. The toxin bound to liposomes composed of phospholipids which are hydrolyzed by the toxin, but did not bind to those composed of phospholipids which are not attacked by the toxin. The toxin-induced carboxyfluorescein release from liposomes composed of dipalmitoleoyl-l-α-phosphatidyl-choline and cholesterol and the toxin binding to the liposomes decreased with decreasing cholesterol contents. These observations suggest that the specific binding site formed by the choline-containing phospholipids and cholesterol, and membrane fluidity in liposomes are essential for the membrane-damaging activity of alpha-toxin

FUNDAMENTAL EXPERIMENT FOR CONSTRUCTING IT-TOW

The purpose of this study was to collect some baseline data which may be used for developing an IT-tow system by using a load cell. The pulling forces were measured in 3 tests, hold1 phase, hold2 phase, and drive phase. The pulling force decreased in drive phase defined as the phase the pullers are driven by opponent. This is because the pulling force in this study was defined as tension of the rope, and then pulling force measured in this phase is not produced by subject but produced by examiners side. If the IT-TOW is made by using the load cell, 2 machines have to exchange and reproduce the pulling force of each other. In case the load cell received 2 different pulling forces, pulling force produced by one side may not be measured with accuracy. To put IT-TOW into practice, the pulling force data must be exchanged and not be measured by a load cell, but defined by another system

BACKWARD PULLING DISTANCE IN DROP PHASE FOR JAPANESE ELITE FEMALE TUG-OF-WAR ATHLETES

The purpose of this study was to clarify the backward pulling distance on 1 second of drop phase for Japanese female elite tug of war team. In order to examine the pulling movement, digital video camera was set at the ceiling of gymnasium, the game performed by 5 elite teams (finalists) and 5 normal teams (non finalists) in 2006 All Japan Tug of War Championship tournament were videotaped from right above and analyzed by using two-dimensional motion analysis system. Backward pulling distance was measured and compared between groups. In some pullers, elite team pulled the rope longer significantly. This might be caused by difference of individual or team pulling techniques to synchronize timing and direction

Role of Sphingomyelinase in Infectious Diseases Caused by Bacillus cereus

Bacillus cereus (B. cereus) is a pathogen in opportunistic infections. Here we show that Bacillus cereus sphingomyelinase (Bc-SMase) is a virulence factor for septicemia. Clinical isolates produced large amounts of Bc-SMase, grew in vivo, and caused death among mice, but ATCC strains isolated from soil did not. A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not. Administration of an anti-Bc-SMase antibody and immunization against Bc-SMase prevented death caused by the clinical isolates, showing that Bc-SMase plays an important role in the diseases caused by B. cereus. Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H2O2 and phagocytosis of macrophages induced by peptidoglycan (PGN), but no effect on the release of TNF-α and little release of LDH under our experimental conditions. Confocal laser microscopy showed that the treatment of mouse macrophages with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity. The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis

Identification of novel clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene

Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ~65 kb. Complete sequence analysis of the ~65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ~65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains. © 2011 Miyamoto et al

Association of SIX1/SIX6 locus polymorphisms with regional circumpapillary retinal nerve fibre layer thickness: The Nagahama study

SIX1 and SIX6 are glaucoma susceptibility genes. Previous reports indicate that the single nucleotide polymorphism (SNP) rs33912345 in SIX6 is associated with inferior circumpapillary retinal nerve fibre layer (cpRNFL) thickness (cpRNFLT). Although the region of visual field defect in glaucoma patients is directly related to cpRNFL thinning, a detailed sector analysis has not been performed in genetic association studies. In the present study, we evaluated 26 tagging SNPs in the SIX1/SIX6 locus ±50 kb region in a population of 2, 306 Japanese subjects with 4- and 32-sector cpRNFLT analysis. While no SNPs showed a significant association with cpRNFLT in the 4-sectored analysis, the finer 32-sector assessment clearly showed a significant association between rs33912345 in the SIX1/SIX6 locus with inferior cpRNFL thinning at 292.5–303.8° (β = −4.55, P = 3.0 × 10−5). Furthermore, the fine-sectored cpRNFLT analysis indicated that SIX1/SIX6 polymorphisms would affect cpRNFL thinning at 281.3–303.8°, which corresponds to parafoveal scotoma in a visual field test of glaucoma patients

Interaction of Clostridium perfringens Iota Toxin and Lipolysis-Stimulated Lipoprotein Receptor (LSR)

Iota toxin produced by Clostridium perfringens is a binary, actin ADP-ribosylating toxin that is organized into the enzymatically active component Ia and the binding component Ib. Lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a cellular receptor of Ib. Here, we investigated the functional interaction between Ib and LSR, where siRNA for LSR blocked the toxin-mediated cytotoxicity and the binding of Ib. The addition of Ib to LSR-green fluorescence protein (GFP)-transfected cells at 4 °C resulted in colocalization with LSR and Ib on the cell surface. Upon transfer of the cells from 4 °C to 37 °C, LSR and Ib were internalized and observed in cytoplasmic vesicles. When the cells were incubated with Ib at 37 °C and fractionated using the Triton-insoluble membrane, Ib oligomer was localized in insoluble factions that fulfilled the criteria of lipid rafts, and LSR was clustered in lipid rafts. To examine the interaction between N-terminal extracellular region of LSR and Ib, we constructed a series of LSR N-terminal deletions. Ten amino acids residues can be deleted from this end without any reduction of Ib binding. However, deletion of 15 N-terminal residues drastically reduces its ability to bind Ib. These results demonstrate that Ib binds to the LSR N-terminal 10 to 15 residues and endocytoses into trafficking endosomes together with LSR

Cathepsin Release from Lysosomes Promotes Endocytosis of Clostridium perfringens Iota-Toxin

Iota-toxin from Clostridium perfringens type E is a binary toxin composed of two independent proteins: actin-ADP-ribosylating enzyme component, iota-a (Ia), and binding component, iota-b (Ib). Ib binds to target cell receptors and mediates the internalization of Ia into the cytoplasm. Extracellular lysosomal enzyme acid sphingomyelinase (ASMase) was previously shown to facilitate the internalization of iota-toxin. In this study, we investigated how lysosomal cathepsin promotes the internalization of iota-toxin into target cells. Cysteine protease inhibitor E64 prevented the cytotoxicity caused by iota-toxin, but aspartate protease inhibitor pepstatin-A and serine protease inhibitor AEBSF did not. Knockdown of lysosomal cysteine protease cathepsins B and L decreased the toxin-induced cytotoxicity. E64 suppressed the Ib-induced ASMase activity in extracellular fluid, showing that the proteases play a role in ASMase activation. These results indicate that cathepsin B and L facilitate entry of iota-toxin via activation of ASMase