ABSTRACT
PVL (Panton-Valentine leukocidin) and other Staphylococcus aureus ß-stranded pore-forming toxins are important virulence factors involved in various pathologies that are often necrotizing. The present study characterized leukotoxin inhibition by selected SCns (p-sulfonato-calix[n]arenes): SC4, SC6 and SC8. These chemicals have no toxic effects on human erythrocytes or neutrophils, and some are able to inhibit both the activity of and the cell lysis by leukotoxins in a dose-dependent manner. Depending on the type of leukotoxins and SCns, flow cytometry revealed IC50 values of 6-22 µM for Ca2+ activation and of 2-50 µM for cell lysis. SCns were observed to affect membrane binding of class S proteins responsible for cell specificity. Electrospray MS and surface plasmon resonance established supramolecular interactions (1:1 stoichiometry) between SCns and class S proteins in solution, but not class F proteins. The membrane-binding affinity of S proteins was Kd=0.07-6.2 nM. The binding ability was completely abolished by SCns at different concentrations according to the number of benzenes (30-300 µM; SC8>SC6â«SC4). The inhibitory properties of SCns were also observed in vivo in a rabbit model of PVL-induced endophthalmitis. These calixarenes may represent new therapeutic avenues aimed at minimizing inflammatory reactions and necrosis due to certain virulence factors.
Subject(s)
Calixarenes/pharmacology , Exotoxins/antagonists & inhibitors , Exotoxins/metabolism , Staphylococcus aureus/metabolism , Animals , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/metabolism , Calixarenes/metabolism , Down-Regulation/drug effects , Drug Evaluation, Preclinical , Hemolysin Proteins/antagonists & inhibitors , Hemolysin Proteins/metabolism , Humans , Macromolecular Substances/metabolism , Models, Biological , Phenols/metabolism , Phenols/pharmacology , Protein Binding/drug effects , Protein Binding/physiology , Rabbits , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Sphingomyelin Phosphodiesterase/metabolism , Staphylococcus aureus/pathogenicity , Virulence Factors/antagonists & inhibitors , Virulence Factors/metabolismABSTRACT
The binding component C2II of the binary actin ADP-ribosylating C2-toxin from Clostridium botulinum is essential for intoxication of target cells. Activation by a protease leads to channel formation and this is presumably required for the transport of the toxic C2I component into cells. The C2II-channel is cation selective and contains a binding site for fluphenazine and structurally related compounds. Ion transport through C2II and in vivo intoxication is blocked when the sites are occupied by the ligands. C2II was reconstituted into artificial lipid bilayer membranes and formed ion permeable channels. The binding constant of chloroquine, primaquine, quinacrine, chloropromazine and fluphenazine to the C2II-channel was determined using titration experiments, which resulted in its block. The ligand-induced current noise of the C2II-channels was investigated using fast Fourier transformation. The noise of the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz. Upon addition of ligands to the aqueous phase the current through C2II decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically and its frequency dependence was of Lorentzian type, which was caused by the on and off-reactions of the ligand-mediated channel block. The ligand-induced current noise of C2II was used for the evaluation of the binding kinetics for different ligands to the channel. The on-rate constant of ligand binding was between 10(7) and 10(9) M(-1) s(-1) and was dependent on the ionic strength of the aqueous phase. The off-rate varied between about 10 s(-1) and 3900 s(-1) and depended on the structure of the ligand. The role of structural requirements for the effective block of C2II by the different ligands is discussed.
Subject(s)
Botulinum Toxins/antagonists & inhibitors , Botulinum Toxins/metabolism , Electrochemistry/methods , Fluphenazine/metabolism , Fluphenazine/pharmacology , Binding Sites , Biological Transport , Chloroquine/chemistry , Chloroquine/metabolism , Chloroquine/pharmacology , Clostridium botulinum , Electric Conductivity , Escherichia coli , Fluphenazine/chemistry , Kinetics , Ligands , Lipid Bilayers/chemistry , Osmolar Concentration , Primaquine/chemistry , Primaquine/metabolism , Primaquine/pharmacology , Protein Binding/drug effectsABSTRACT
The binary Clostridium botulinum C2 toxin consists of two individual proteins, the transport component C2II (80 kDa) and the enzyme component C2I, which ADP-ribosylates G-actin in the cytosol of cells. Trypsin-activated C2II (C2IIa) forms heptamers that bind to the cell receptor and mediate translocation of C2I from acidic endosomes into the cytosol of target cells. Here, we report that translocation of C2I across cell membranes is accompanied by pore formation of C2IIa. We used a radioactive rubidium release assay to detect C2IIa pores in the membranes of Chinese hamster ovary cells. Pore formation by C2IIa was dependent on the cellular C2 toxin receptor and an acidic pulse. Pores were formed when C2IIa was bound to cells at neutral pH and when cells were subsequently shifted to acidic medium (pH < 5.5), but no pores were detected when C2IIa was added to cells directly in acidic medium. Most likely, acidification induces a change from "pre-pore" to "pore" conformation of C2IIa, and formation of the pore conformation before membrane binding precludes insertion into membranes. When C2I was present during binding of C2IIa to cells prior to the acidification step, C2IIa-mediated rubidium release was decreased, suggesting that C2I interacted with the lumen of the C2IIa pore. A decrease of rubidium efflux was also detected when C2I was added to C2IIa-treated cells after the acidification step, suggesting that C2I interacted with C2IIa in its pore conformation. Moreover, C2I also interacted with C2IIa channels in artificial lipid membranes and blocked them partially. C2I was only translocated across the cell membrane when C2IIa plus C2I were bound to cells at neutral pH and subsequently shifted to acidic pH. When cell-bound C2IIa was exposed to acidic pH prior to C2I addition, only residual intoxication of cells was observed at high toxin concentrations, and binding of C2I to C2IIa was slightly decreased. Overall, C2IIa pores were essential but not sufficient for translocation of C2I. Intoxication of target cells with C2 toxin requires a strictly coordinated pH-dependent sequence of binding, pore formation by C2IIa, and translocation of C2I.
Subject(s)
Botulinum Toxins/metabolism , Cytosol/metabolism , Animals , CHO Cells , Chlorocebus aethiops , Cricetinae , Hydrogen-Ion Concentration , Protein Transport , Vero CellsABSTRACT
The binding component (C2II) of the binary Clostridium botulinum C2 toxin mediates transport of the actin ADP-ribosylating enzyme component (C2I) into the cytosol of target cells. C2II (80 kDa) is activated by trypsin cleavage, and proteolytically activated C2II (60 kDa) oligomerizes to heptamers in solution. Activated C2II forms channels in lipid bilayer membranes which are highly cation selective and voltage-gated. A role for this channel in C2I translocation across the cell membrane into the cytosol is discussed. Amino acid residues 303-331 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which likely facilitates membrane insertion and channel formation by creating two antiparallel beta-strands. Some of the residues are in strategic positions within the putative C2II channel, in particular, glutamate 307 (E307) localized in its center and glycine 316 (G316) localized on the trans side of the membrane. Here, single-lysine substitutions of these amino acids and the double mutant E307K/G316K of C2II were analyzed in vivo and in artificial lipid bilayer experiments. The pH dependence of C2I transport across cellular membranes was altered, and a pH of Subject(s)
ADP Ribose Transferases/metabolism
, Botulinum Toxins/metabolism
, Clostridium botulinum/chemistry
, Glutamic Acid/metabolism
, Ion Channel Gating/drug effects
, Ion Channels/physiology
, Lipid Bilayers
, Macrolides
, Animals
, Anti-Bacterial Agents/pharmacology
, Binding Sites
, Botulinum Toxins/genetics
, Botulinum Toxins/toxicity
, CHO Cells/cytology
, CHO Cells/drug effects
, CHO Cells/metabolism
, Chlorocebus aethiops
, Chloroquine/pharmacology
, Cricetinae
, Electric Conductivity
, Escherichia coli/genetics
, Glutamic Acid/genetics
, Humans
, Hydrogen-Ion Concentration
, In Vitro Techniques
, Ion Channel Gating/physiology
, Ion Channels/drug effects
, Mutagenesis, Site-Directed
, Mutation/genetics
, Poly(ADP-ribose) Polymerases/metabolism
, Protein Binding
, Recombinant Fusion Proteins/metabolism
, Recombinant Fusion Proteins/toxicity
, Sequence Deletion
, Vero Cells/cytology
, Vero Cells/drug effects
, Vero Cells/metabolism