Interaction of Clostridium perfringens Epsilon Toxin with the Plasma Membrane: The Role of Amino Acids Y42, Y43 and H162.

Clostridium perfringens 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 vaccine candidate lacking toxicity to cells expressing myelin and lymphocyte protein.

A variant form of Clostridium perfringens epsilon toxin (Y30A-Y196A) with mutations, which shows reduced binding to Madin-Darby canine kidney (MDCK) cells and reduced toxicity in mice, has been proposed as the next-generation enterotoxaemia vaccine. Here we show that, unexpectedly, the Y30A-Y196A variant does not show a reduction in toxicity towards Chinese hamster ovary (CHO) cells engineered to express the putative receptor for the toxin (myelin and lymphocyte protein; MAL). The further addition of mutations to residues in a second putative receptor binding site of the Y30A-Y196A variant further reduces toxicity, and we selected Y30A-Y196A-A168F for further study. Compared to Y30A-Y196A, Y30A-Y196A-A168F showed more than a 3-fold reduction in toxicity towards MDCK cells, more than a 4-fold reduction in toxicity towards mice and at least 200-fold reduction in toxicity towards CHO cells expressing sheep MAL. The immunisation of rabbits or sheep with Y30A-Y196A-A168F induced high levels of neutralising antibodies against epsilon toxin, which persisted for at least 1 year. Y30A-Y196A-A168F is a candidate for development as a next-generation enterotoxaemia vaccine.

The pore structure of Clostridium perfringens epsilon toxin.

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 ß-PFTs (aß-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 ß-barrel, a common feature of the aß-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.

Clostridium perfringens Epsilon Toxin Compromises the Blood-Brain Barrier in a Humanized Zebrafish Model.

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.

Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis.

BACKGROUND: It was recently reported that, using Western blotting, some multiple sclerosis (MS) patients in the United States had antibodies against epsilon toxin (Etx) from Clostridium perfringens, suggesting that the toxin may play a role in the disease. OBJECTIVE: We investigated for serum antibodies against Etx in UK patients with clinically definite multiple sclerosis (CDMS) or presenting with clinically isolated syndrome (CIS) or optic neuritis (ON) and in age- and gender-matched controls. METHODS: We tested sera from CDMS, CIS or ON patients or controls by Western blotting. We also tested CDMS sera for reactivity with linear overlapping peptides spanning the amino acid sequence (Pepscan) of Etx. RESULTS: Using Western blotting, 24% of sera in the combined CDMS, CIS and ON groups ( n = 125) reacted with Etx. In the control group ( n = 125), 10% of the samples reacted. Using Pepscan, 33% of sera tested reacted with at least one peptide, whereas in the control group only 16% of sera reacted. Out of 61 samples, 21 (43%) were positive to one or other testing methodology. Three samples were positive by Western blotting and Pepscan. CONCLUSION: Our results broadly support the previous findings and the role of Etx in the aetiology of MS warrants further investigation.

Interactions Between Pseudomonas Immunotoxins and the Plasma Membrane: Implications for CAT-8015 Immunotoxin Therapy.

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 Pseudomonas 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 Pseudomonas 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.

Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein.

Lysenin from the coelomic fluid of the earthworm Eisenia fetida belongs to the aerolysin family of small ß-pore-forming toxins (ß-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 ß-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 ß-barrel of the channel.

Red Blood Cell Susceptibility to Pneumolysin: CORRELATION WITH MEMBRANE BIOCHEMICAL AND PHYSICAL PROPERTIES.

This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell.

Variable protection against experimental broiler necrotic enteritis after immunization with the C-terminal fragment of Clostridium perfringens alpha-toxin and a non-toxic NetB variant.

Necrotic enteritis toxin B (NetB) is a pore-forming toxin produced by Clostridium perfringens and has been shown to play a key role in avian necrotic enteritis, a disease causing significant costs to the poultry production industry worldwide. The aim of this work was to determine whether immunization with a non-toxic variant of NetB (NetB W262A) and the C-terminal fragment of C. perfringens alpha-toxin (CPA247-370) would provide protection against experimental necrotic enteritis. Immunized birds with either antigen or a combination of antigens developed serum antibody levels against NetB and CPA. When CPA247-370 and NetB W262A were used in combination as immunogens, an increased protection was observed after oral challenge by individual dosing, but not after in-feed-challenge.

Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia.

Epsilon toxin (Etx) is a ß-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.

Identification of a key residue for oligomerisation and pore-formation of Clostridium perfringens NetB.

Necrotic enteritis toxin B (NetB) is a ß-pore-forming toxin produced by Clostridium perfringens and has been identified as a key virulence factor in the pathogenesis of avian necrotic enteritis, a disease causing significant economic damage to the poultry industry worldwide. In this study, site-directed mutagenesis was used to identify amino acids that play a role in NetB oligomerisation and pore-formation. NetB K41H showed significantly reduced toxicity towards LMH cells and human red blood cells relative to wild type toxin. NetB K41H was unable to oligomerise and form pores in liposomes. These findings suggest that NetB K41H could be developed as a genetic toxoid vaccine to protect against necrotic enteritis.

Protection against avian necrotic enteritis after immunisation with NetB genetic or formaldehyde toxoids.

NetB (necrotic enteritis toxin B) is a recently identified ß-pore-forming toxin produced by Clostridium perfringens. This toxin has been shown to play a major role in avian necrotic enteritis. In recent years, a dramatic increase in necrotic enteritis has been observed, especially in countries where the use of antimicrobial growth promoters in animal feedstuffs has been banned. The aim of this work was to determine whether immunisation with a NetB toxoid would provide protection against necrotic enteritis. The immunisation of poultry with a formaldehyde NetB toxoid or with a NetB genetic toxoid (W262A) resulted in the induction of antibody responses against NetB and provided partial protection against disease.

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies.

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 (ß-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.

Galleria mellonella as a model system to test the pharmacokinetics and efficacy of antibiotics against Burkholderia pseudomallei.

Mammalian models of infection are paramount to elucidating the mechanisms of bacterial pathogenesis and are also used for evaluating the efficacy of novel antimicrobials before the commencement of human trials. In this study, Galleria mellonella was used to determine the efficacy of antibiotics towards a Burkholderia thailandensis infection in G. mellonella larvae. Kanamycin, imipenem, ceftazidime, doxycycline and ciprofloxacin could all provide some protection when given 1 h before challenge with B. thailandensis; however, at 2 h or 6 h post challenge, imipenem and kanamycin were unable to rescue larvae. The most effective antibiotic for the prevention or treatment of disease was ceftazidime. Pharmacokinetic properties of a single dose of these antibiotics in G. mellonella larvae were also determined, and it was demonstrated that this model is useful for approximating the antibiotic response in humans. The G. mellonella model was used to screen a panel of novel antimicrobials for activity towards B. thailandensis and Burkholderia pseudomallei, and three novel compounds with antibiotic activity were identified. These results support the hypothesis that G. mellonella can be used to screen antimicrobial efficacy. This is the first study to determine the pharmacokinetic parameters of clinically relevant antibiotics in this model system.

Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens.

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.

A Burkholderia pseudomallei toxin inhibits helicase activity of translation factor eIF4A.

The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei, reveals a similarity to Escherichia coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity. BPSL1549 promotes deamidation of glutamine-339 of the translation initiation factor eIF4A, abolishing its helicase activity and inhibiting translation. We propose to name BPSL1549 Burkholderia lethal factor 1.

Molecular basis of toxicity of Clostridium perfringens epsilon toxin.

Clostridium perfringens ε-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. perfringens λ-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.

The conserved translocase Tim17 prevents mitochondrial DNA loss.

Maintenance of an intact mitochondrial genome is essential for oxidative phosphorylation in all eukaryotes. Depletion of mitochondrial genome copy number can have severe pathological consequences due to loss of respiratory capacity. In Saccharomyces cerevisiae, several bifunctional metabolic enzymes have been shown to be required for mitochondrial DNA (mtDNA) maintenance. For example, Ilv5 is required for branched chain amino acid biosynthesis and mtDNA stability. We have identified OXA1 and TIM17 as novel multicopy suppressors of mtDNA instability in ilv5 cells. In addition, overexpression of TIM17, but not OXA1, prevents the complete loss of mtDNA in cells lacking the TFAM homologue Abf2. Introduction of the disease-associated A3243G mutant mtDNA into human NT2 teratocarcinoma cells frequently causes mtDNA loss. Yet when human TIM17A is overexpressed in NT2 cybrids carrying A3243G mtDNA, the proportion of cybrid clones maintaining mtDNA increases significantly. TIM17A overexpression results in long-term mtDNA stabilization, since NT2 cybrids overexpressing TIM17A maintain mtDNA at levels similar to controls for several months. Tim17 is a conserved suppressor of mtDNA instability and is the first factor to be identified that can prevent mtDNA loss in a human cellular model of mitochondrial disease.

The yeast Holliday junction resolvase, CCE1, can restore wild-type mitochondrial DNA to human cells carrying rearranged mitochondrial DNA.

Rearrangements of mitochondrial DNA (mtDNA) are a well-recognized cause of human disease; deletions are more frequent, but duplications are more readily transmitted to offspring. In theory, partial duplications of mtDNA can be resolved to partially deleted and wild-type (WT) molecules, via homologous recombination. Therefore, the yeast CCE1 gene, encoding a Holliday junction resolvase, was introduced into cells carrying partially duplicated or partially triplicated mtDNA. Some cell lines carrying the CCE1 gene had substantial amounts of WT mtDNA suggesting that the enzyme can mediate intramolecular recombination in human mitochondria. However, high levels of expression of CCE1 frequently led to mtDNA loss, and so it is necessary to strictly regulate the expression of CCE1 in human cells to ensure the selection and maintenance of WT mtDNA.

Expression of algal nuclear ATP synthase subunit 6 in human cells results in protein targeting to mitochondria but no assembly into ATP synthase.

Artificial transfer of mitochondrial genes to the nucleus has implications for the understanding of mitochondrial function, evolution, and human health. Therefore, we created nuclear compatible versions of human subunit a (A6) of ATP synthase, linked to a mitochondrial targeting signal. Expression and targeting of human nuclear subunit a were compared to subunit a of Chlamydomonas reinhardtii, which naturally occurs in the nucleus. Algal subunit a was targeted to mitochondria more efficiently than human nuclear subunit a variants. However, there was no evidence of improved mitochondrial function in cultured cells; on the contrary, long-term expression of algal subunit a was associated with poor survival and intolerance of growth conditions that demand heavy reliance on oxidative phosphorylation. Analysis of enriched mitochondrial membrane fractions on native gels revealed a high-molecular- weight complex containing FLAG-tagged subunit a; however, this complex did not colocalize with ATP synthase. Thus, there was no evidence of assembly of algal subunit a into holoenzyme, nor did human nuclear subunit a colocalize with ATP synthase holoenzyme. In conclusion, obstacles remain to functional expression of mitochondrial genes transferred to the nucleus.