Abstract
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 GM2 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 GM2, 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 GM2 as receptor and forms anion-selective channels.
Highlights
Clostridium perfringens produces numerous toxins and is responsible for severe diseases in humans and animals including intestinal or foodborne diseases as well as gangrenes
Oligonucleotide P723, deduced from internal sequence of peak 18, was synthesized according to the Clostridium codon usage and with inosine at the most degenerated positions (Table 1). This probe hybridized with total DNA as well as with plasmid preparations of C. perfringens strain CP24-03 and NCTC8131, suggesting that Delta toxin gene is located on plasmid DNA of these Clostridium strains
Delta toxin is one of the major toxins produced by C. perfringens which is lethal for mice and cytotoxic for various red and white blood cells [2,4,31]
Summary
Clostridium perfringens produces numerous toxins and is responsible for severe diseases in humans and animals including intestinal or foodborne diseases as well as gangrenes. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains [2]. This toxin was purified from a C. perfringens type C strain and characterized as a basic (pI 9.1) 42 kDa protein which hemolyzes erythrocytes from even-toed ungulates (sheep, goats and pigs) [2]. The selective cytotoxicity of Delta toxin was correlated to a specific binding to the ganglioside GM2. Iodinated Delta toxin was shown to bind to ganglioside GM2 extracted from membrane of sensitive cells and to liposome containing GM2 [7]. The mechanism of cytotoxicity remains unclear, since Delta toxin was reported to not insert into cell membrane and to induce membrane lysis by an unknown process [6,7]
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