Clostridium perfringensε‐toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia ...in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringensλ‐protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin–Darby canine kidney (MDCK) cells. The binding of ε‐toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε‐toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin‐2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε‐toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.
Clostridium perfringensε‐toxin is the etiologic agent of enterotoxaemia and appears to target the brain and kidneys. The crystal structure of ε‐toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin‐2 from Bacillus thuringiensis, and a lectin from Laetiporus sulphurous. Like these toxins, ε‐toxin forms pores in target cell membranes, but differs from these toxins because of its high specificity and potency
Lysenin from the coelomic fluid of the earthworm Eisenia fetida belongs to the aerolysin family of small β-pore-forming toxins (β-PFTs), some members of which are pathogenic to humans and animals. ...Despite efforts, a high-resolution structure of a channel for this family of proteins has been elusive and therefore the mechanism of activation and membrane insertion remains unclear. Here we determine the pore structure of lysenin by single particle cryo-EM, to 3.1 Å resolution. The nonameric assembly reveals a long β-barrel channel spanning the length of the complex that, unexpectedly, includes the two pre-insertion strands flanking the hypothetical membrane-insertion loop. Examination of other members of the aerolysin family reveals high structural preservation in this region, indicating that the membrane-insertion pathway in this family is conserved. For some toxins, proteolytic activation and pro-peptide removal will facilitate unfolding of the pre-insertion strands, allowing them to form the β-barrel of the channel.
Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in ...multiple sclerosis in humans. Etx is a member of the aerolysin family of β-PFTs (aβ-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double β-barrel, a common feature of the aβ-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.
Summary
Progress towards understanding the molecular basis of virulence in Clostridium difficile has been hindered by the lack of effective gene transfer systems. We have now, for the first time, ...developed procedures that may be used to introduce autonomously replicating vectors into this organism through their conjugative, oriT‐based mobilization from Escherichia coli donors. Successful transfer was achieved through the use of a plasmid replicon isolated from an indigenous C. difficile plasmid, pCD6, and through the characterization and subsequent circumvention of host restriction/modification (RM) systems. The characterized replicon is the first C. difficile plasmid replicon to be sequenced and encodes a large replication protein (RepA) and a repetitive region composed of a 35 bp iteron sequence repeated seven times. Strain CD6 has two RM systems, CdiCD6I/M.CdiCD6I and CdiCD6II/M. CdiCD6II, with equivalent specificities to Sau96I/M. Sau96I (5′‐GGNMCC‐3′) and MboI/M. MboI (5′‐GMATC‐3′) respectively. A second strain (CD3) possesses a type IIs restriction enzyme, Cdi I, which cleaves the sequence 5′‐CATCG‐3′ between the fourth and fifth nucleotide to give a blunt‐ended fragment. This is the first time that an enzyme with this specificity has been reported. The sequential addition of this site to vectors showed that each site caused between a five‐ and 16‐fold reduction in transfer efficiency. The transfer efficiencies achieved with both strains equated to between 1.0 × 10−6 and 5.5 × 10−5 transconjugants per donor.
epsilon toxin (Etx) is a pore forming toxin that causes enterotoxaemia in ruminants and may be a cause of multiple sclerosis in humans. To date, most in vitro studies of Etx have used the Madin-Darby ...canine kidney (MDCK) cell line. However, studies using Chinese hamster ovary (CHO) cells engineered to express the putative Etx receptor, myelin and lymphocyte protein (MAL), suggest that amino acids important for Etx activity differ between species. In this study, we investigated the role of amino acids Y42, Y43 and H162, previously identified as important in Etx activity towards MDCK cells, in Etx activity towards CHO-human MAL (CHO-hMAL) cells, human red blood cells (hRBCs) and synthetic bilayers using site-directed mutants of Etx. We show that in CHO-hMAL cells Y42 is critical for Etx binding and not Y43 as in MDCK cells, indicating that surface exposed tyrosine residues in the receptor binding domain of Etx impact efficiency of cell binding to MAL-expressing cells in a species-specific manner. We also show that Etx mutant H162A was unable to lyse CHO-hMAL cells, lysed hRBCs, whilst it was able to form pores in synthetic bilayers, providing evidence of the complexity of Etx pore formation in different lipid environments.
Clostridium perfringens epsilon toxin (Etx) is a pore‐forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent ...by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx‐H149A), previously reported to have reduced, but not abolished, toxicity. The three‐dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx‐H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx‐H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx‐H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx‐H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx‐H149A identified a glycan (β‐octyl‐glucoside) binding site in domain III of Etx‐H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.
NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the ...removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.
Background:Clostridium perfringens toxin NetB is a key factor in avian necrotic enteritis.
Results: NetB forms heptameric pores structurally similar to Staphylococcus aureus toxins but lacks a phosphocholine binding pocket. NetB activity is enhanced by cholesterol.
Conclusion: NetB has distinct binding specificity, and cholesterol may act as a receptor.
Significance: The structure of NetB will facilitate development of control measures against necrotic enteritis.
Highlights • Etx mutant Y30A-Y196A showed markedly reduced cytotoxicity towards MDCK.2 cells. • Y30A-Y196A is inactive in mice after intraperitoneal administration. • Y30A-Y196A is able to induce a ...specific antibody response in rabbits. • Y30A-Y196A polyclonal antibody is able to induce protective immunity in vitro. • Y30A-Y196A could form the basis of a recombinant vaccine against enterotoxemia.
Acute Lymphoblastic Leukemia (ALL) remains the most frequent cause of cancer-related mortality in children and novel therapies are needed for the treatment of relapsed/refractory childhood ALL. One ...approach is the targeting of ALL blasts with the
immunotoxin CAT-8015. Although CAT-8015 has potent anti-leukemia activity, with a 32% objective response rate in a phase 1 study of childhood ALL, haemolytic-uremic syndrome (HUS) and vascular leak syndrome (VLS), major dose-limiting toxicities, have limited the use of this therapeutic approach in children. Investigations into the pathogenesis of CAT-8015-induced HUS/VLS are hindered by the lack of an adequate model system that replicates clinical manifestations, but damage to vascular endothelial cells (ECs) and blood cells are believed to be major initiating factors in both syndromes. Since there is little evidence that murine models replicate human HUS/VLS, and CAT-8015-induced HUS/VLS predominantly affects children, we developed human models and used novel methodologies to investigate CAT-8015 interactions with red blood cells (RBCs) from pediatric ALL patients and ECs of excised human mesenteric arteries. We provide evidence that CAT-8015 directly interacts with RBCs, mediated by
toxin. We also show correlation between the electrical properties of the RBC membrane and RBC susceptibility to CAT-8015-induced lysis, which may have clinical implication. Finally, we provide evidence that CAT-8015 is directly cytototoxic to ECs of excised human mesenteric arteries. In conclusion, the human models we developed constitutes the first, and very important, step in understanding the origins of HUS/VLS in immunotoxin therapy and will allow further investigations of HUS/VLS pathogenesis.
Clostridium perfringens epsilon toxin (ETX) is hypothesized to mediate blood-brain barrier (BBB) permeability by binding to the myelin and lymphocyte protein (MAL) on the luminal surface of ...endothelial cells (ECs). However, the kinetics of this interaction and a general understanding of ETX's behavior in a live organism have yet to be appreciated. Here we investigate ETX binding and BBB breakdown in living Danio rerio (zebrafish). Wild-type zebrafish ECs do not bind ETX. When zebrafish ECs are engineered to express human MAL (hMAL), proETX binding occurs in a time-dependent manner. Injection of activated toxin in hMAL zebrafish initiates BBB leakage, hMAL downregulation, blood vessel stenosis, perivascular edema, and blood stasis. We propose a kinetic model of MAL-dependent ETX binding and neurovascular pathology. By generating a humanized zebrafish BBB model, this study contributes to our understanding of ETX-induced BBB permeability and strengthens the proposal that MAL is the ETX receptor.
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•ProETX binds specifically to hMAL in live humanized zebrafish•hMAL expression in zebrafish confers susceptibility to ETX-mediated BBB breakdown•Live imaging reveals ETX-mediated edema, hMAL downregulation, stenosis, and stasis•Antibody neutralization abrogates ETX-mediated vascular pathology
Pathogenic Organism; Vascular Remodeling; Molecular Mechanism of Behavior; Model Organism
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