Extracellular membrane vesicles derived from Komagataeibacter oboediens exposed on the International Space Station fuse with artificial eukaryotic membranes in contrast to vesicles of reference bacterium.

Membranous Extracellular Vesicles (EVs) of Gram-negative bacteria are a secretion and delivery system that can disseminate bacterial products and interact with hosts and the environment. EVs of nonpathogenic bacteria deliver their contents by endocytosis into eukaryotic cells, however, no evidence exists for a fusion delivery mechanism. Here, we describe the fusion of exposed to space/Mars-like stressors simulated on the International Space Station vesicles (E-EVs) from Komagataeibacter oboediens to different types of model planar membranes in comparison with the EVs of the ground-based reference strain. The most reliable fusion was achieved with PC:PE:ergosterol or sterol-free PC:PE bilayers. The relative permeability ratio (PK+/PCl-) estimated from the shift of zero current potential according to Goldman-Hodgkin-Katz equation consisted of 4.17 ± 0.48, which coincides with preferential cation selectivity of the EV endogenous channels. The increase in membrane potential from 50 mV to 100 mV induced the fusion of E-EVs with all tested lipid compositions. The fusion of model exosomes with planar bilayer lipid membranes was confirmed by separate step-like increases in its conductance. In contrast, the ground-based reference K. oboediens EVs never induced the fusion event. In our study, we show membrane lipidome perturbations and increased protein aggregation occurred in the exposed samples in the harsh environment when outer membranes of K. oboediens acquired the capability of both homo- and heterotypic fusion possibly by altered membrane fluidity and the pore-forming capability.

Clostridium perfringens beta-toxin forms potential-dependent, cation-selective channels in lipid bilayers.

Recombinant beta-toxin from Clostridium perfringens type C was found to increase the conductance of bilayer lipid membranes (BLMs) by inducing channel activity. The channels exhibited a distribution of conductances within the range of 10 to 380 pS, with the majority of the channels falling into two categories of conductance at 110 and 60 pS. The radii of beta-toxin pores found for the conductance states of 110 and 60 pS were 12.7 and 11.1 A, respectively. The single channels and the steady-state currents induced by beta-toxin across the BLMs exhibited ideal monovalent cation selectivity. Addition of divalent cations (Zn(2+), Cd(2+), or Mg(2+)) at a concentration of 2 mM increased the rate of beta-toxin insertion into BLMs and the single-channel conductance, while application of 5 mM Zn(2+) to a beta-toxin-induced steady-state current decreased the inward current by approximately 45%. The mutation of arginine 212 of beta-toxin to aspartate, previously shown to increase the 50% lethal dose of beta-toxin for mice nearly 13-fold, significantly reduced the ability of beta-toxin to form channels. These data support the hypothesis that the lethal action of beta-toxin is based on the formation of cation-selective pores in susceptible cells.

The mechanism of pore assembly for a cholesterol-dependent cytolysin: formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins.

Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of membrane-penetrating toxins. The CDCs form large homooligomers (estimated to be comprised of up to 50 CDC monomers) that are responsible for generating a large pore in cholesterol-containing membranes of eukaryotic cells. The assembly of the PFO cytolytic complex was examined to determine whether it forms an oligomeric prepore complex on the membrane prior to the insertion of its membrane-spanning beta-sheet. A PFO oligomeric complex was formed on liposomes at both 4 degrees C and 37 degrees C and shown by SDS-agarose gel electrophoresis to be comprised of a large, comparatively homogeneous complex instead of a distribution of oligomer sizes. At low temperature, the processes of oligomerization and membrane insertion could be resolved, and PFO was found to form an oligomer without significant membrane insertion of its beta-hairpins. Furthermore, PFO was found to increase the ion conductivity through a planar bilayer by large and discrete stepwise changes in conductance that are consistent with the insertion of a preassembled pore complex into the bilayer. The combined results of these analyses strongly support the hypothesis that PFO forms a large oligomeric prepore complex on the membrane surface prior to the insertion of its transmembrane beta-sheet.

The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins.

Perfringolysin O (PFO), a water-soluble monomeric cytolysin secreted by pathogenic Clostridium perfringens, oligomerizes and forms large pores upon encountering cholesterol-containing membranes. Whereas all pore-forming bacterial toxins examined previously have been shown to penetrate the membrane using a single amphipathic beta hairpin per polypeptide, cysteine-scanning mutagenesis and multiple independent fluorescence techniques here reveal that each PFO monomer contains a second domain involved in pore formation, and that each of the two amphipathic beta hairpins completely spans the membrane. In the soluble monomer, these transmembrane segments are folded into six alpha helices. The insertion of two transmembrane hairpins per toxin monomer and the major change in secondary structure are striking and define a novel paradigm for the mechanism of membrane insertion by a cytolytic toxin.

Monoclonal antibodies can uncouple the main alpha-latrotoxin effects: toxin-induced Ca2+ influx and stimulated neurotransmitter release.

A panel of monoclonal antibodies has been produced against alpha-latrotoxin using black widow spider venom. Five of them were characterized relative to their affinity for alpha-latrotoxin and ability to modify the main toxin effects--to increase calcium permeability of synaptosomes, to stimulate the neurotransmitter release and to form the ion channels in artificial lipid membrane. The results reported here show that: (i) the monoclonal antibodies do not alter the alpha-latrotoxin affinity for the membrane acceptor; (ii) two monoclonal antibodies, A6 and A24, can simultaneously inhibit the alpha-latrotoxin induced Ca2+ uptake and GABA release; (iii) monoclonal antibodies A4 completely block the toxin-induced Ca2+ uptake, but decrease partially the rate of GABA release; (iv) monoclonal antibodies A15 that do not modify the alpha-latrotoxin ability to stimulate Ca2+ uptake and GABA release are able to alter the properties of channels formed by the toxin in the artificial lipid bilayer. From these data we hypothesize that the alpha-latrotoxin molecule has separate functional sites which provide a high-affinity binding to the membrane acceptor, the toxin-induced Ca2+ uptake and toxin-stimulated neurotransmitter release. A separate part of alpha-latrotoxin molecule is responsible for the formation of cationic channels in the artificial lipid bilayer.