Rapid Identification by Polymerase Chain Reaction of Staphylococcal Exfoliative Toxin Serotype A and B Genes

A new system was designed to detect staphylococcal exfoliative toxin A (ETA) and B (ETB) genes by the polymerase chain reaction (PCR). The primer pairs for the ETA gene (eta) were 20 and 20-mer, and its PCR product was a 741-bp eta fragment, while the primer pairs for the ETB gene (etb) were also 20 and 20-mer, and its PCR product was a 629-bp etb fragment. When these primers were simultaneously used in the PCR, the two types of ET were clearly detected as two bands in an ETA and ETB double-producer using only one colony within 3 hr. We examined 66 strains of Staphylococcus aureus isolated from patients with staphylococcal scalded skin syndrome (SSSS) and compared the results obtained by ELISA and PCR. The same results were obtained for 56 of the strains, i.e., 30 strains were ETA producers, 20 strains were ETB producers, and 6 strains were double-producers. However, positive results were obtained for 5 of the 10 non-ET-producing strains. Two of these strains were judged by PCR as ETA producers and three as ETB producers. Thus, PCR is very sensitive and rapid in detecting ETA and ETB gene fragments in colonies isolated from patients with SSSS.     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Solution structure of the potassium channel inhibitor agitoxin 2: caliper for probing channel geometry.

The structure of the potassium channel blocker agitoxin 2 was solved by solution NMR methods. The structure consists of a triple-stranded antiparallel beta-sheet and a single helix covering one face of the beta-sheet. The cysteine side chains connecting the beta-sheet and the helix form the core of the molecule. One edge of the beta-sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface-exposed residues, Arg24, Lys27, and Arg31 of the beta-sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K+ channel. The sequential and spatial locations of Arg24 and Arg31 are not conserved among the homologous toxins. Knowledge on the details of the channel-binding sites of agitoxin 2 formed a basis for site-directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel-toxin interactions.     

Evolution and structure of two ADP-ribosylation enterotoxins, Escherichia coli heat-labile toxin and cholera toxin

Nucleotide sequence comparisons of the heat-labile enterotoxin (LTh) genes of E. coli pathogenic for humans with cholera toxin (CT) genes suggest that the two toxin genes have evolved from a common ancestry by a series of single base changes, while conserving the catalytic fragment A1 (ADP-ribose transferase). Based on the local hydrophilicity profiles of LTh and CT peptides, a transmembrane segment appears to be present in A1 in both toxins.     

The detection of picomolar quantities of GD1b,GT1b and GQ1b on thin-layer chromatograms by the direct binding of125I-labeled tetanus toxin,fragment C

The¹²⁵I-labeled fragment C of tetanus toxin was found to bind specifically to the gangliosides G_D1b, G_T1b, and G_Q1b when applied to thin-layer chromatograms on which a mixture of gangliosides had been resolved. As little as 2.5 pmoles of these gangliosides could be detected by this method. In addition to factors determined by the sample, namely the amount and species of gangliosides present, optimal binding of the¹²⁵I-labeled fragment C also depended upon the iodination procedure used to generate the probe, the toxin concentration, and the concentration, buffer type, pH, and ionic strength of the binding solution. This new technique was shown to be a sensitive method for the detection and identification of specific gangliosides originating from extraneural or neural cells.Nomenclature: The gangliosides follow the nomenclature system of Svennerholm [Eur J Biochem (1977) 79:11–21] G_M3 II³NeuAc-LacCer - G_D3 II³(NeuAc)₂-LacCer - G_M1 II³NeuAc-GgOse₄Cer - G_D1a IV³NeuAc, II³NeuAc-GgOse₄Cer - G_D1b II³(NeuAc)₂-GgOse₄Cer - G_T1b IV³NeuAc, II³(NeuAc)²-GgOse₄Cer - G_Q1b IV³(Neu-Ac)₂, II³(NeuAc)₂-GgOse₄Cer - G_P1b IV³(NeuAc)₃, II³(NeuAc)₂-GgOse₄Cer     

Biological activities of lipopolysaccharides from Pectinatus cerevisiophilus

Abstract A procedure is described in which the protein crystals produced by Bacillus thuringiensis var. israelensis were solubilized in 50 mM NaOH with 10 mM EDTA at pH 11.7. This solubilization procedure gave protein gel profiles identical with those for intact crystals while maintaining full biological activity in the form of erythrocyte lysis capability. Crystals with and without protease activity were equally toxic to Aedes aegypti larvae.     

The rate assay of alpha-toxin assembly in membrane

Abstract A rapid and easy method to determine the 'rate' of the assembly of α-toxin from Staphylococus aureus in erythrocyte membrane was described. Upon addition of a small amount of α-toxins into erythrocyte suspension, absorbance at 700 nm decreased linearly after a short period of lag time. From the linear portion of the record the rate of the assembly of α-toxin was calculated. An optimum temperature and an optimum pH for the assembly of the toxin on erythrocyte membranes were found to be 25–30°C and pH 5.     

Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of M_r=16,000. The amino acid composition of the toxin type K₂ of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.