Central residues of the amphipathic ß-hairpin loop control the properties of Clostridium perfringens epsilon-toxin channel.

Clostridium perfringens epsilon toxin (ETX) is a heptameric pore-forming toxin of the aerolysin toxin family. ETX is the most potent toxin of this toxin family and the third most potent bacterial toxin with high cytotoxic and lethal activities in animals. In addition, ETX shows a demyelinating activity in nervous tissue leading to devastating multifocal central nervous system white matter disease in ruminant animals. Pore formation in target cell membrane is most likely the initial critical step in ETX biological activity. Eight single to quadruple ETX mutants were generated by replacement of polar residues (serine, threonine, glutamine) in middle positions of the ß-strands forming the ß-barrel and facing the channel lumen with charged glutamic residues. Channel activity and ion selectivity were monitored in artificial lipid monolayer membranes and cytotoxicity was investigated in MDCK cells by the viability MTT test and propidium iodide entry. All the mutants formed channels with similar conductance in artificial lipid membranes and increasing cation selectivity for increasing number of mutations. Here, we show that residues in the central position of each ß-strand of the amphipathic ß-hairpin loop that forms the transmembrane pore, control the size and ion selectivity of the channel. While the highest cationic ETX mutants were not cytotoxic, no strict correlation was observed between ion selectivity and cytotoxicity.

Membrane Activity and Channel Formation of the Adenylate Cyclase Toxin (CyaA) of Bordetella pertussis in Lipid Bilayer Membranes.

The Gram-negative bacterium Bordetella pertussis is the cause of whooping cough. One of its pathogenicity factors is the adenylate cyclase toxin (CyaA) secreted by a Type I export system. The 1706 amino acid long CyaA (177 kDa) belongs to the continuously increasing family of repeat in toxin (RTX) toxins because it contains in its C-terminal half a high number of nine-residue tandem repeats. The protein exhibits cytotoxic and hemolytic activities that target primarily myeloid phagocytic cells expressing the αMß2 integrin receptor (CD11b/CD18). CyaA represents an exception among RTX cytolysins because the first 400 amino acids from its N-terminal end possess a calmodulin-activated adenylate cyclase (AC) activity. The entry of the AC into target cells is not dependent on the receptor-mediated endocytosis pathway and penetrates directly across the cytoplasmic membrane of a variety of epithelial and immune effector cells. The hemolytic activity of CyaA is rather low, which may have to do with its rather low induced permeability change of target cells and its low conductance in lipid bilayer membranes. CyaA forms highly cation-selective channels in lipid bilayers that show a strong dependence on aqueous pH. The pore-forming activity of CyaA but not its single channel conductance is highly dependent on Ca2+ concentration with a half saturation constant of about 2 to 4 mM.

The major outer membrane protein of Legionella pneumophila Lpg1974 shows pore-forming characteristics similar to the human mitochondrial outer membrane pore, hVDAC1.

Legionella pneumophila is an aerobic and nonspore-forming pathogenic Gram-negative bacterium of the genus Legionella. It is the causative agent of Legionnaires' disease, also known as Legionellosis. The hosts of this organism are diverse, ranging from simple water borne protozoans such as amoebae to more complex hosts such as macrophages in humans. Genome analyses have shown the presence of genes coding for eukaryotic like proteins in several Legionella species. The presence of these proteins may assist L. pneumophila in its adaptation to the eukaryotic host. We studied the characteristics of a protein (Lpg1974) of L. pneumophila that shows remarkable homologies in length of the primary sequence and for the identity/homology of many amino acids to the voltage dependent anion channel (human VDAC1, Porin 31HL) of human mitochondria. Two different forms of Lpg1974 were overexpressed in Escherichia coli and purified to homogeneity: the one containing a putative N-terminal signal sequence and one without it. Reconstituted protein containing the signal sequence formed ion-permeable pores in lipid bilayer membranes with a conductance of approximately 5.4 nS in 1 M KCl. When the predicted N-terminal signal peptide of Lpg1974 comprising an α-helical structure similar to that at the N-terminus of hVDAC1 was removed, the channels formed in reconstitution experiments had a conductance of 7.6 nS in 1 M KCl. Both Lpg1974 proteins formed pores that were voltage-dependent and anion-selective similar to the pores formed by hVDAC1. These results suggest that Lpg1974 of L. pneumophila is indeed a structural and functional homologue to hVDAC1.

Evaluation of segmentation methods on head and neck CT: Auto-segmentation challenge 2015.

PURPOSE: Automated delineation of structures and organs is a key step in medical imaging. However, due to the large number and diversity of structures and the large variety of segmentation algorithms, a consensus is lacking as to which automated segmentation method works best for certain applications. Segmentation challenges are a good approach for unbiased evaluation and comparison of segmentation algorithms. METHODS: In this work, we describe and present the results of the Head and Neck Auto-Segmentation Challenge 2015, a satellite event at the Medical Image Computing and Computer Assisted Interventions (MICCAI) 2015 conference. Six teams participated in a challenge to segment nine structures in the head and neck region of CT images: brainstem, mandible, chiasm, bilateral optic nerves, bilateral parotid glands, and bilateral submandibular glands. RESULTS: This paper presents the quantitative results of this challenge using multiple established error metrics and a well-defined ranking system. The strengths and weaknesses of the different auto-segmentation approaches are analyzed and discussed. CONCLUSIONS: The Head and Neck Auto-Segmentation Challenge 2015 was a good opportunity to assess the current state-of-the-art in segmentation of organs at risk for radiotherapy treatment. Participating teams had the possibility to compare their approaches to other methods under unbiased and standardized circumstances. The results demonstrate a clear tendency toward more general purpose and fewer structure-specific segmentation algorithms.

Residues involved in the pore-forming activity of the Clostridium perfringens iota toxin.

Clostridium perfringens iota toxin is a binary toxin that is organized into enzyme (Ia) and binding (Ib) components. Ib forms channels in lipid bilayers and mediates the transport of Ia into the target cells. Here we show that Ib residues 334-359 contain a conserved pattern of alternating hydrophobic and hydrophilic residues forming two amphipathic ß-strands involved in membrane insertion and channel formation. This stretch of amino acids shows remarkable structural and functional analogies with the ß-pore-forming domain of C. perfringens epsilon toxin. Several mutations within the two amphipathic ß-strands affected pore formation, single-channel conductance and ion selectivity (S339E-S341E, Q345H N346E) confirming their involvement in channel formation. F454 of Ib corresponds to the Φ-clamp F427 of anthrax protective antigen and F428 of C2II binary toxins. The mutation F454A resulted in a loss of cytotoxicity and strong increase in single-channel conductance (500 pS as compared with 85 pS in 1 M KCl) with a slight decrease in cation selectivity, indicating that the Φ-clamp is highly conserved and crucial for binary toxin activity. In contrast, the mutants Q367D, N430D, L443E had no or only minor effects on Ib properties, while T360I, T360A and T360W caused a dramatic effect on ion selectivity and single-channel conductance, indicating gross disturbance of the oligomer structure. This suggests that, at least in the iota toxin family, T360 has a structural role in the pore organization. Moreover, introduction of charged residues within the channel (S339E-S341E) or in the vestibule (Q367D, N430D and L443E) had virtually no effect on chloroquine or Ia binding, whereas F454A, T360I, T360A and T360W strongly decreased the chloroquine and Ia affinity to Ib. These results support that distinct residues within the vestibule interact with chloroquine and Ia or are responsible for channel structure, while the channel lining amino acids play a less important role.

Isolation, synthesis and characterization of ω-TRTX-Cc1a, a novel tarantula venom peptide that selectively targets L-type Cav channels.

Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (CaV) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of CaV2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba(2+) currents (IBa) mediated by L-type (CaV1.2 and CaV1.3) CaV channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825nM and 2.24µM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited IBa mediated by high voltage-activated CaV channels but did not affect low voltage-activated T-type CaV channels. Cc1a exhibited weak activity at NaV1.5 and NaV1.7 voltage-gated sodium (NaV) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1-E5) or C-terminus (ΔW35-V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type CaV channels.

Chemical engineering and structural and pharmacological characterization of the α-scorpion toxin OD1.

Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure-function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical ßαßß α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Nav1.7, as well as Nav1.4 and Nav1.6. In addition, OD1 also produced potent ß-toxin activity at Nav1.4 and Nav1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Nav1.4 and Nav1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Nav1.7 over Nav1.6, while OD1 K11V was 5-fold more selective for Nav1.6 than Nav1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Nav subtypes.

Conotoxins targeting neuronal voltage-gated sodium channel subtypes: potential analgesics?

Voltage-gated sodium channels (VGSC) are the primary mediators of electrical signal amplification and propagation in excitable cells. VGSC subtypes are diverse, with different biophysical and pharmacological properties, and varied tissue distribution. Altered VGSC expression and/or increased VGSC activity in sensory neurons is characteristic of inflammatory and neuropathic pain states. Therefore, VGSC modulators could be used in prospective analgesic compounds. VGSCs have specific binding sites for four conotoxin families: µ-, µO-, δ- and ί-conotoxins. Various studies have identified that the binding site of these peptide toxins is restricted to well-defined areas or domains. To date, only the µ- and µO-family exhibit analgesic properties in animal pain models. This review will focus on conotoxins from the µ- and µO-families that act on neuronal VGSCs. Examples of how these conotoxins target various pharmacologically important neuronal ion channels, as well as potential problems with the development of drugs from conotoxins, will be discussed.

Mediators of change in psychosocial interventions for cancer patients: a systematic review.

Careful reviews and meta-analyses have made valuable contributions to understanding the efficacy of psychosocial interventions for cancer patients. An important next step is to determine the mediators that explain the influence of efficacious interventions on outcomes. This systematic review summarizes tests of mediating variables from twenty-two projects conducted from 1989-2010. Although all authors provided some type of rationale for considering particular mediating relationships, the investigations varied widely with respect to the extent to which formal theoretical constructs were tested, the type and goals of the interventions studied, and the broad types of outcomes and potential mediators examined. Although there was some evidence supporting selected mediating relationships, with positive findings often found when mediating variables represented behaviors targeted by an intervention, the findings were mixed. Expanding the focus of research to include mechanisms in psychosocial oncology intervention research is necessary for providing a unified picture of how mediating relationships may be operating in this field.

Localization of Nav 1.7 in the normal and injured rodent olfactory system indicates a critical role in olfaction, pheromone sensing and immune function.

Loss-of-function mutations in the pore-forming α subunit of the voltage-gated sodium channel 1.7 (Nav 1.7) cause congenital indifference to pain and anosmia. We used immunohistochemical techniques to study Nav 1.7 localization in the rat olfactory system in order to better understand its role in olfaction. We confirm that Nav 1.7 is expressed on olfactory sensory axons and report its presence on vomeronasal axons, indicating an important role for Nav 1.7 in transmission of pheromonal cues. Following neuroepithelial injury, Nav 1.7 was transiently expressed by cells of monocytic lineage. These findings support an emerging role for Nav 1.7 in immune function. This sodium channel may provide an important pharmacological target for treatment of inflammatory injury and inflammatory pain syndromes.

Isolation, characterization and total regioselective synthesis of the novel µO-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels.

The µO-conotoxins are notable for their unique selectivity for Na(v)1.8 over other sodium channel isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the discovery of a novel µO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to µO-conotoxin MrVIB. To overcome the synthetic challenges posed by µO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of µO-conotoxins. Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully protected peptide allowed manipulations in organic solvents with no chromatography required between steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields. Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysiological recording techniques. MfVIA preferentially inhibits Na(v)1.8 (IC50 95.9±74.3 nM) and Na(v)1.4 (IC50 81±16 nM), with significantly lower affinity for other Na(v) subtypes (IC50 431-6203 nM; Na(v)1.5>1.6∼1.7∼1.3∼1.1∼1.2). This improved approach to µO-conotoxin synthesis will facilitate the optimization of µO-conotoxins as novel analgesic molecules to improve pain management.

Why are Optimists Optimistic?

This study examined what is brought to mind when responding to the items comprising a measure of dispositional optimism. Participants (N = 113) completed the Life Orientation Test and the COPE, a measure of coping style, and described why they responded the way they did to the items assessing optimism. Participants' explanations comprised eight types of reasoning: (1) faith in a higher power; (2) belief in fate or a just world; (3) personal fortune; (4) belief in the role of one's own ability; (5) reliance on idioms; (6) beliefs about the usefulness of thinking optimistically; (7) matter-of-fact statements; and (8) a feeling, intuition, or hope. These types were also related to coping styles. Responses to positively-worded items were explained with respect to external forces and responses to negatively-worded items were explained with respect to internal forces. Understanding how people explain their optimism may be the first step in fostering this outlook.

Pre-intervention distress moderates the efficacy of psychosocial treatment for cancer patients: a meta-analysis.

This meta-analysis examined whether effects of psychosocial interventions on psychological distress in cancer patients are conditional upon pre-intervention distress levels. Published articles and unpublished dissertations between 1980 and 2005 were searched for interventions reporting the Hospital Anxiety and Depression Scale (HADS) or the Spielberger State-Trait Anxiety Inventory (STAI). Multilevel mixed-effects modeling was used to meta-analyze effect-sizes separately for the HADS (27 trials, 2,424 patients) and STAI (34 trials, 2,029 patients). Pre-intervention distress significantly moderated intervention effects, explaining up to 50% of the between-study effect-size variance: effects on anxiety and depression were generally negligible when pre-intervention distress was low and pronounced when it was high. These results could not be explained by differences in intervention type, setting, dose, and whether intervention was targeted at distressed patients. Psychosocial interventions may be most beneficial for cancer patients with elevated distress. Future research should identify which treatment components are most effective for these patients to facilitate optimal treatment tailoring and cost-effective health care.

Clostridium septicum alpha-toxin forms pores and induces rapid cell necrosis.

Alpha-toxin is the unique lethal virulent factor produced by Clostridium septicum, which causes traumatic or non-traumatic gas gangrene and necrotizing enterocolitis in humans. Here, we analyzed channel formation of the recombinant septicum alpha-toxin and characterized its activity on living cells. Recombinant septicum alpha-toxin induces the formation of ion-permeable channels with a single-channel conductance of about 175pS in 0.1M KCl in lipid bilayer membranes, which is typical for a large diffusion pore. Septicum alpha-toxin channels remained mostly in the open configuration, displayed no lipid specificity, and exhibited slight anion selectivity. Septicum alpha-toxin caused a rapid decrease in the transepithelial electrical resistance of MDCK cell monolayers grown on filters, and induced a rapid cell necrosis in a variety of cell lines, characterized by cell permeabilization to propidium iodide without DNA fragmentation and activation of caspase-3. Septicum alpha-toxin also induced a rapid K(+) efflux and ATP depletion. Incubation of the cells in K(+)-enriched medium delayed cell death caused by septicum alpha-toxin or epsilon-toxin, another potent pore-forming toxin, suggesting that the rapid loss of intracellular K(+) represents an early signal of pore-forming toxins-mediated cell necrosis.

Published versus unpublished dissertations in psycho-oncology intervention research.

OBJECTIVE: There are conflicting views regarding whether gray literature, including unpublished doctoral dissertations, should be included in systematic reviews and meta-analyses. Although publication status frequently is used as a proxy for study quality, some research suggests that dissertations are often of superior quality to published studies. METHODS: We examined 107 projects involving doctoral dissertations (42 published, 65 unpublished) that studied psychosocial interventions for cancer patients. RESULTS: Published dissertations were more likely to be supported by research funding but were not more likely than unpublished dissertations to examine specific types of interventions. Across several indices of methodological quality there were minimal differences. Dissertations with significant findings tended to be more likely to be published than those without significant findings. CONCLUSIONS: Unpublished dissertations focusing on psychosocial interventions for cancer patients are not necessarily of vastly inferior quality to those that eventually are published. Because doctoral dissertations are easy to access relative to other forms of gray literature, are free from some types of bias, and are reported thoroughly, they merit inclusion in comprehensive literature reviews.

Lessons to be learned from 25 years of research investigating psychosocial interventions for cancer patients.

Conducting rigorous psychosocial intervention research with cancer patients has many challenges including encouraging them to join studies, asking them to engage in interventions or be part of control conditions, and to provide data over follow-up assessments. Here, we highlight valuable insights regarding such challenges provided by investigators studying psychosocial interventions for cancer patients. Handling these skillfully has important implications for the internal and external validity of this research and the ethical treatment of participants. Challenges noted in research reports included in a systematic review of 25 years of research (comprising 488 unique projects) investigating interventions designed to enhance cancer patients' quality of life were compiled. Among the difficulties mentioned was the fact that patients may not feel the need for psychosocial interventions and thus may not be interested in joining an intervention study. Patients who do feel the need for such interventions may be deterred from joining trials by the prospect of being randomized to a nonpreferred group; if they do join a trial, participants may be disappointed, drop out, or seek compensatory additional assistance when they are assigned to a control group. Apart from randomization, other aspects of research may be off-putting to participants or potential participants, such as the language of consent forms or the intrusiveness of questions being asked. Potential remedies, such as research awareness interventions, monetary incentives, partnering with cancer support organizations, and using designs that take preferences into account merit consideration and further research inquiry.

Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX).

Epsilon-toxin (ETX) is a potent toxin produced by Clostridium perfringens strains B and D. The bacteria are important pathogens in domestic animals and cause edema mediated by ETX. This toxin acts most likely by heptamer formation and rapid permeabilization of target cell membranes for monovalent anions and cations followed by a later entry of calcium. In this study, we compared the primary structure of ETX with that of the channel-forming stretches of a variety of binding components of A-B-types of toxins such as Anthrax protective antigen (PA), C2II of C2-toxin and Ib of Iota-toxin and found a remarkable homology to amino acids 151-180 of ETX. Site-directed mutagenesis of amino acids within the putative channel-forming domain resulted in changes of cytotoxicity and effects on channel characteristics in lipid bilayer experiments including changes of selectivity and partial channel block by methanethiosulfonate (MTS) reagents and antibodies against His(6)-tags from the trans-side of the lipid bilayer membranes.

Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin.

The Bordetella adenylate cyclase-hemolysin (CyaA, ACT, or AC-Hly) is a multifunctional toxin. Simultaneously with promoting calcium ion entry, CyaA delivers into host cells an adenylate cyclase enzyme (AC) and permeabilizes cell membrane by forming small cation-selective pores. Indirect evidence suggested that these two activities were accomplished by different membrane-inserted CyaA conformers, one acting as an AC-delivering monomer and the other as an uncharacterized pore-forming oligomer. We tested this model by directly detecting toxin oligomers in cell membrane and by assessing oligomerization of specific mutants with altered pore-forming properties. CyaA oligomers were revealed in sheep erythrocyte membranes by immunogold labeling and directly demonstrated by pulldown of membrane-inserted CyaA together with biotinylated CyaA-AC(-) toxoid. Membrane oligomers of CyaA could also be resolved by nondenaturing electrophoresis of mild detergent extracts of erythrocytes. Furthermore, CyaA mutants exhibiting enhanced (E581K) or reduced (E570K+E581P) specific hemolytic and pore-forming activity were found to exhibit also a correspondingly enhanced or reduced propensity to form oligomers in erythrocyte membranes. On the other hand, processed CyaA, with the AC domain cleaved off by erythrocyte proteases, was detected only in a monomeric form excluded from the oligomers of unprocessed CyaA. These results provide the first direct evidence that oligomerization is involved in formation of CyaA pores in target membranes and that translocation of the AC domain across cell membrane may be accomplished by monomeric CyaA.

Characteristics and methodological quality of 25 years of research investigating psychosocial interventions for cancer patients.

The considerable amount of research examining psychosocial interventions for cancer patients makes it important to examine its scope and methodological quality. This comprehensive overview characterizes the field with as few exclusions as possible. A systematic search strategy identified 673 reports comprising 488 unique projects conducted over a 25 year time span. Although the literature on this topic has grown over time, the research was predominantly conducted in the United States (57.0%), largely with breast cancer patients (included in 70.5% of the studies). The intervention approach used most frequently was cognitive behavioral (32.4%), the treatment goal was often improving quality of life generally (69.5%), and the professionals delivering the interventions were typically nurses (29.1%) or psychologists (22.7%). Overall, there was some discrepancy between the types of interventions studied and the types of supportive services available to and sought by cancer patients. Strengths of this research include using randomized designs (62.9%), testing for baseline group equivalence (84.5%), and monitoring treatment, which rose significantly from being used in 48.1-64.4% of projects over time. However, deficiencies in such areas as examining treatment mechanisms and the adequacy of reporting of methodology, essential for useful syntheses of research on these interventions, remain to be addressed. Methodological challenges related to the complexity of this applied research, such as participants seeking treatment outside of research, contamination, and reactions to randomization, also were apparent. Future research could benefit from closer interactions between academic and voluntary sectors and expanding the diversity of participants.

Clostridium perfringens delta toxin is sequence related to beta toxin, NetB, and Staphylococcus pore-forming toxins, but shows functional differences.

Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside G(M2) in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to G(M2), in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes G(M2) as receptor and forms anion-selective channels.