Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3-4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membraneembedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.Targeting and attachment of proteins to membranes is one of the key steps in many cellular processes (1-3). Protein-membrane interactions have been studied intensively in recent years with many different examples of proteins and membranes. These interactions can be promoted at the lipid-water interface by lipid anchors, electrostatic forces or surface-exposed aromatic and aliphatic residues (1, 2, 4). Compared with protein-protein interactions, details of protein-membrane interactions are poorly defined. Some of the best characterized examples are a phospholipase C pleckstrin homology domain specific for phosphatidylinositol trisphosphate (5) and small protein kinase-C-conserved (C2) domains specific for zwitterionic, particularly phosphatidylcholine membranes (6).Another group of proteins interacting with lipid membranes are pore-forming toxins (PFT) 1 (7-10), which bind to membranes before eliciting their toxic effects via the formation of transmembrane pores. The most studied PFT are bacterial since this group includes important virulence factors. Few examples of eukaryotic PFT have been well characterized, exceptions being the actinoporins, cytolysins found exclusively in sea anemones (10, 11). Members of this family have properties distinct from other PFT: they are composed of 175-179 amino acids, contain no cysteine residues, have pIϾ9.5, and show a preference for sphingomyelin (SM)-containing membranes. Actinoporins act on cellular and model lipid membranes by forming cation-selective pores with a hydrodynamic diameter of ϳ2 nm. The mechanism of pore formation involves at least two steps: binding of the water soluble m...

Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp 112 and Tyr 113 exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins. Sphingomyelin (SM)8 is an important eukaryotic membrane lipid, located for the most part in the outer leaflet of the plasma membrane in the form of specialized cholesterol-rich microdomains, so-called lipid rafts (1, 2). Many pathogens and toxic proteins employ lipid rafts to invade cells (3, 4), but currently little is known about the molecular details of the recognition mechanism of the lipid components present in the rafts. In the particular case of SM, the specific recognition occurs even though SM exposes the same phosphorylcholine headgroup as the other abundant lipid, phosphatidylcholine. SM-binding proteins are currently exploited as specific markers for cellular SM (5) and are used to identify other proteins involved in sphingolipid metabolism (6).Actinoporins are extremely potent cytolysins produced exclusively by sea anemones (7,8). They may be used to capture prey, in intraspecific aggression, or in preventing adhesion of other organisms (7, 9). The two most studied representatives are EqtII, isolated from the sea anemone Actinia equina, and sticholysin II (StII) from Stichodactyla helianthus. Actinoporins constitute a family of conserved proteins that cause hemolysis of red blood cells by colloid-osmotic lysis and exhibit cytolytic activity against various cell lines (7, 10 -12). Even the most distant members of the family share more than 60% sequence identity and the available threedimensional structures of EqtII and StII are nearly superimposable (13-15). The structure is composed of a tightly folded ␤-sandwich flanked b...

The nucleotide sequence of the Bacillus licheniformis bacitracin-resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP-binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydrophobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram-negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide-binding sites, BcrA protein and mammalian multidrug transporter or P-glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide-binding protein to determine the importance of various regions in transport.

Population-wide tolerance and persisters enable susceptible bacterial cells to endure hostile environments, including antimicrobial exposure. The SOS response can play a significant role in the generation of persister cells, population-wide tolerance, and shielding. The SOS pathway is an inducible DNA damage repair system that is also pivotal for bacterial adaptation, pathogenesis, and diversification. In addition to the two key SOS regulators, LexA and RecA, some other stressors and stress responses can control SOS factors. Bacteria are exposed to DNA-damaging agents and other environmental and intracellular factors, including cigarette smoke, that trigger the SOS response at a number of sites within the host. The Escherichia coli TisB/IstR module is as yet the only known SOS-regulated toxin-antitoxin module involved in persister formation. Nevertheless, the SOS response plays a key role in the formation of biofilms that are highly recalcitrant to antimicrobials and can be abundant in persisters. Furthermore, the dynamic biofilm environment generates DNA-damaging factors that trigger the SOS response within the biofilm, fueling bacterial adaptation and diversification. This review highlights the SOS response in relation to antimicrobial recalcitrance to antimicrobials in four clinically significant species, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium tuberculosis.

Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10 -28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the ␤-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the ␤-sandwich and importantly the C-terminal ␣-helix, do not undergo large conformational rearrangements during the pore formation. The role of the ␤-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp 117 . These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable ␤-sandwich are pre-requisite for proper pore formation by the actinoporin family.

Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina. It readily creates pores in membranes containing sphingomyelin. Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis. At approximately every tenth residue, a cysteine was introduced. Nineteen single cysteine mutants were produced in Escherichia coli and purified. The accessibility of the thiol groups in lipid-embedded cysteine mutants was studied by reaction with biotin maleimide. Most of the mutants were modified, except those with cysteines at positions 105 and 114. Mutants R144C and S160C were modified only at high concentrations of the probe. Similar results were obtained if membrane-bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C. Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding. These results were further confirmed by labeling the mutants with acrylodan, a polarity-sensitive fluorescent probe. When labeled mutants were combined with vesicles, the following mutants exhibited blue-shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C. The overall results suggest that at least two regions are embedded within the lipid membrane: the N-terminal 13±20 region, probably forming an amphiphilic helix, and the tryptophan-rich 105±120 region. Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups. The association with the membrane appears to be unique and different from that of bacterial pore-forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel-forming toxins.Keywords: acrylodan; biotin maleimide; cysteine-scanning mutagenesis; pore-forming protein; sea anemone.Pore-forming peptides and proteins are found in a variety of organisms, such as bacteria, plants, fungi, primitive metazoans, insects and humans [1±5]. They are mostly produced as watersoluble molecules destined to form pores in the lipid membranes of the host organism. Despite their diverse biological roles, most of them function by a common mode of action: they interact with cell or artificial lipid membranes, change conformation and oligomerize in the plane of the membrane to build water-filled pores that are permeable to solutes [6]. Insight into the assembly and operation of such pores calls for their three-dimensional structure. However, at present it is extremely hard to obtain the structure of protein complexes associated with lipids, as is true for membrane proteins [7]. So far, only the structure of a pore created by the bacterial cytolysin, Staphylococcus a-toxin Equinatoxin II (EqtII), a 179 amino-acid residue cytolysin from Actinia equina [18], belongs to the family of pore-forming proteins found in sea anemones [2]. Analysis o...

The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations.

BackgroundPhenotypic heterogeneity may ensure that a small fraction of a population survives environmental perturbations or may result in lysis in a subpopulation, to increase the survival of siblings. Genes involved in DNA repair and population dynamics play key roles in rapid responses to environmental conditions. In Escherichia coli the transcriptional repressor LexA controls a coordinated cellular response to DNA damage designated the SOS response. Expression of LexA regulated genes, e.g. colicin encoding genes, recA, lexA and umuDC, was examined utilizing transcription fusions with the promoterless gfp at the single cell level.ResultsThe investigated LexA regulated genes exhibited heterogeneity, as only in a small fraction of the population more intense fluorescence was observed. Unlike recA and lexA, the pore forming and nuclease colicin activity genes as well as umuDC, exhibited no basal level activity. However, in a lexA defective strain high level expression of the gene fusions was observed in the large majority of the cells. All of the investigated genes were expressed in a recA defective strain, albeit at lower levels, revealing expression in the absence of a spontaneous SOS response. In addition, the simultaneous expression of cka, encoding the pore forming colicin K, and lexA, investigated at the single cell level revealed high level expression of only cka in rare individual cells.ConclusionLexA regulated genes exhibit phenotypic heterogeneity as high level expression is observed in only a small subpopulation of cells. Heterogenous expression is established primarily by stochastic factors and the binding affinity of LexA to SOS boxes.