Clostridium perfringens epsilon toxin binds to membrane lipids and its cytotoxic action depends on sulfatide

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology

Clostridium perfringens Epsilon Toxin Binds to Membrane Lipids and its Cytotoxic Action Depends on Sulfatide

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology

The Cytotoxicity of Epsilon toxin from Clostridium perfringens on lymphocytes is mediated by MAL protein expression

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein that crosses the blood-brain barrier, binds to myelin, and, hence, has been suggested to be a putative agent for the onset of multiple sclerosis, a demyelinating neuroinflammatory disease. Recently, myelin and lymphocyte (MAL) protein has been identified to be a key protein in the cytotoxic effect of Etx; however, the association of Etx with the immune system remains a central question. Here, we show that Etx selectively recognizes and kills only human cell lines expressing MAL protein through a direct Etx-MAL protein interaction. Experiments on lymphocytic cell lines revealed that MAL protein-expressing T cells, but not B cells, are sensitive to Etx and reveal that the toxin may be used as a molecular tool to distinguish subpopulations of lymphocytes. The overall results open the door to investigation of the role of Etx and Clostridium perfringens on inflammatory and autoimmune diseases like multiple sclerosis

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB

New Mutants of Epsilon Toxin from Clostridium perfringens with an Altered Receptor-Binding Site and Cell-Type Specificity

Epsilon toxin (Etx) from Clostridium perfringens is the third most potent toxin after the botulinum and tetanus toxins. Etx is the main agent of enterotoxemia in ruminants and is produced by Clostridium perfringens toxinotypes B and D, causing great economic losses. Etx selectively binds to target cells, oligomerizes and inserts into the plasma membrane, and forms pores. A series of mutants have been previously generated to understand the cellular and molecular mechanisms of the toxin and to obtain valid molecular tools for effective vaccination protocols. Here, two new non-toxic Etx mutants were generated by selective deletions in the binding (Etx-ΔS188-F196) or insertion (Etx-ΔV108-F135) domains of the toxin. As expected, our results showed that Etx-ΔS188-F196 did not exhibit the usual Etx binding pattern but surprisingly recognized specifically an O-glycoprotein present in the proximal tubules of the kidneys in a wide range of animals, including ruminants. Although diminished, Etx-ΔV108-F135 maintained the capacity for binding and even oligomerization, indicating that the mutation particularly affected the pore-forming ability of the toxin

Estudi de la unió de la toxina èpsilon de "Clostridium perfringens" a diferents teixits i el seu pas a través de la barrera hematoencefàlica

[cat] La toxina-ε, produïda per la soca D de Clostridium perfringens, és la principal responsable de l’enterotoxèmia en animals de granja. Produeix un edema generalitzat, uns efectes citotòxics molt greus al ronyó (mort de les cèl•lules epitelials dels túbuls distals) i també efectes neurològics (mort neuronal provocada per excitotoxicitat). Tots aquests efectes porten finalment a la mort de l’animal. Actualment existeixen tractaments preventius de la toxina mitjançant la vacunació, però encara es desconeix el mecanisme d’acció letal, el seu receptor i els primers passos que porten a la citotoxicitat. Amb la finalitat d’estudiar el comportament d’aquesta toxina, al nostre laboratori vàrem produir una proteïna de fusió formada per la toxina-ε i la proteïna verda fluorescent GFP (toxina-ε-GFP). Aquesta eina va ser de gran utilitat, ja que es comportava igual que la toxina-ε nativa i podia localitzar-se de manera directa utilitzant microscòpia de fluorescència. De fet, injeccions i.v de toxina-ε-GFP a ratolí, varen revelar un edema generalitzat i una mort de les cèl•lules epitelials dels túbuls distals del ronyó de manera idèntica a la toxina nativa. A més, també mostrava citotoxicitat en cultius de la línia cel•lular MDCK, on heptameritzava a la membrana cel•lular, formava porus i provocava la mort cel•lular (Soler-Jover et al., 2004). L’objectiu d’aquesta tesi va ser aprofundir en el coneixement de les primeres etapes de la intoxicació per la toxina-ε, caracteritzant la seva unió i distribució en els diferents òrgans i sistemes, en especial el renal i nerviós. Per dur a terme aquest objectiu general, vàrem realitzar dues aproximacions experimentals: 1. Vàrem incubar la toxina-ε-GFP sobre seccions de teixits, on vàrem observar la seva unió a la mielina tant del SNC com del SNP. També vàrem veure que en el sistema renal, la toxina-ε-GFP reconeixia cèl•lules epitelials dels túbuls distals i col•lectors del ronyó, així com cèl•lules de l’uroteli. Els estudis realitzats en aquest treball (tractaments amb pronasa E, detergents, Nglicosidasa F i beta-eliminació) apunten que el receptor podria tractar-se d’una Oglicoproteïna tant en el sistema nerviós com en el renal, i que un ambient lipídic (o la integritat de la membrana) seria necessari per permetre la unió de la toxina-ε al seu receptor. A més, els estudis realitzats amb cross-linkers en les cèl•lules MDCK han permès identificar una proteïna d’uns 36 kDa (Annexina A2) que podria actuar com a correceptor o intervenir en l’oligomerització i formació de porus a la membrana plasmàtica. Mitjançant un assaig d’ELISA en cèl•lules MDCK, vàrem observar que només hi ha un sol lloc d’unió per a la toxina-ε, i aquest és saturable i d’alta afinitat. 2. Vàrem reproduir a ratolí els efectes de la toxina injectant i.v dosi letals de toxina-ε- GFP, i vàrem estudiar la seva distribució en el sistema nerviós. Vàrem veure que la toxina-ε-GFP a banda d’unir-se a les cèl•lules endotelials també era capaç de travessar la barrera hematoencefàlica (BHE) i arribar així al parènquima del cervell, on provocaria la mort neuronal ja fos d’una manera directa o indirecta (Soler-Jover et al., 2007). Donat l’interés en trobar nous vehicles capaços de travessar la BHE, vàrem analitzar aquesta propietat en diferents mutants de toxina-ε, tot i que encara no hem identificat cap mutant que hagi perdut la capacitat tòxica però no seva capacitat invasiva. La futura identificació del receptor de la toxina-ε, la formació dels heptàmers i la seva inserció a la membrana són, sens dubte, els grans reptes que ajudaran a caracteritzar el mecanisme d’acció de la toxina-ε de Clostridium perfringens.[eng] ε-Toxin, produced by Clostridium perfringens type D, is the main agent responsible for enterotoxaemia in livestock. ε-Toxin accumulates specifically in the renal and nervous system were it produces the death of the epithelial cells from the distal tubules and neurons, respectively. Vaccines are very useful in the prevention of the ε-toxin intoxication, but nothing is known about the receptor and the first intoxication steps. We produced a recombinant ε-toxin protein with the green fluoresence protein GFP (ε- toxin-GFP) which is a useful tool because it behaves as the native ε-toxin and it can be detected by fluorescence (Soler-Jover et al., 2004). The aim of this thesis is to go into detail in the knowledge of the first steps in the intoxication pathway, characterizing the binding and distribution of the ε-toxin in different organs and tissues, especially in the renal and nervous system. To achieve this aim we performed two experimental approaches. On one hand, we incubated the ε-toxin-GFP on tissue sections, were its binding to myelin from CNS and PNS was observed. We also observed binding of ε-toxin to the urothelium and epithelial cells from distal and collecting tubules in the kidney. Our work revealed that the receptor in the nervous and renal system could be an Oglycoprotein, and that a lipidic environment (or the membrane integrity) would be required for the binding of ε-toxin to its receptor. In addition, an ELISA-based binding assay revealed a single high-affinity binding site for ε-toxin in MDCK cells. Moreover, cross-linking experiments identified a 36 kDa protein (Annexin A2) which could be involved in the membrane pore formation step. On the other hand, we reproduced in mice the in vivo effects by injecting i.v ε-toxin- GFP. The toxin crossed the BBB and penetrated the brain parenchyma producing neuronal death (Soler-Jover et al., 2007). We are interested in the finding of new vehicles to cross the BBB. In fact, some ε-toxin mutants have been studied but no one is able to cross the BBB without producing cell damage and animal death

Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology

Lung endothelial cells are sensitive to epsilon toxin from Clostridium perfringens

The pore‐forming protein epsilon toxin (Etx) from Clostridium perfringens produces acute perivascular edema affecting several organs, especially the brain and lungs. Despite the toxin evident effect on microvasculature and endothelial cells, the underlying molecular and cellular mechanisms remain obscure. Moreover, no Etx‐sensitive endothelial cell model has been identified to date. Here, we characterize the mouse lung endothelial cell line 1G11 as an Etx‐sensitive cell line and compare it with the well‐characterized Etx‐sensitive Madin‐Darby canine kidney epithelial cell line. Sev‐ eral experimental approaches, including morphological and cytotoxic assays, clearly demonstrate that the 1G11 cell line is highly sensitive to Etx and show the specific binding, oligomerization, and pore‐forming activity of the toxin in these cells. Recently, the myelin and lymphocyte (MAL) protein has been postulated as a putative receptor for Etx. Here, we show the presence of Mal mRNA in the 1G11 cell line and the presence of the MAL protein in the endothe‐ lium of some mouse lung vessels, supporting the hypothesis that this protein is a key element in the Etx intoxication pathway. The existence of an Etx‐sensitive cell line of endothelial origin would help shed light on the cellular and molecular mechanisms underlying Etx‐induced edema and its consequences

Clostridium perfringens Epsilon Toxin Binds to Membrane Lipids and its Cytotoxic Action Depends on Sulfatide

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology

Clostridium perfringens epsilon toxin binds to membrane lipids and its cytotoxic action depends on sulfatide

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology