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

Abstract

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 175 pS in 0.1 M 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.

Introduction

Septicum alpha-toxin is secreted by the highly virulent pathogen Clostridium septicum, the major cause of fulminate traumatic and non-traumatic gas gangrene as well as necrotizing enterocolitis in humans (Stevens et al., 1990). This toxin is the only lethal virulence factor of C. septicum and is absolutely necessary for the pathogenesis of this bacterium (Kennedy et al., 2005, Kennedy et al., 2009). Septicum alpha-toxin is lethal for mice (Tweten, 2001) and cytotoxic for a wide diversity of different cell types (Gordon et al., 1997, Wichroski et al., 2002).

The inactive protoxin is encoded by the csa gene (Ballard et al., 1995, Imagawa et al., 1994) and secreted via the type II secretion pathway as a 46.55 kDa protein with a signal peptide of 31 amino acids (Ballard et al., 1992). Secreted monomers bind to GPI-anchored membrane proteins on target cells (Gordon et al., 1999). Cell bound protoxin is activated by host cell proteases, such as furin, that cleave a C-terminal 45 amino acid peptide (Ballard et al., 1993, Gordon et al., 1997). The cleaved propeptide functions as an intramolecular chaperon that hinders the toxin to undergo incorrect folding or oligomerization in solution (Sellman et al., 1997). Activated monomers interact with each other to initiate an oligomeric prepore complex (Sellman and Tweten, 1997). It can be assumed that through a conformational change of the membrane-spanning domain (between residues 203 and 232; (Melton et al., 2004)), the bound complex forms an amphipathic transmembrane β-barrel, which inserts into the membrane resulting in pores with a diameter of about 1.3–1.6 nm. Because septicum alpha-toxin shares sequence and structure similarities with aerolysin that heptamerizes (Moniatte et al., 1996, Wilmsen et al., 1992) and forms pores with a diameter of about 1.0 nm (van der Goot et al., 1993), it has been assumed that heptamerization is also the case for septicum alpha-toxin. Indeed, septicum alpha-toxin is related to aerolysin of Aeromonas species, which is a bi-lobed protein consisting of four distinct domains (1–4) (Melton and Tweten, 2006, Melton-Witt et al., 2006). Septicum alpha-toxin shows a high sequence accordance of aerolysin domains 2–4 (72% similarity, 27% identity) (Ballard et al., 1995, Howard et al., 1987, Melton et al., 2004) and is predicted to share a similar structure (Melton-Witt et al., 2006, Parker et al., 1994). It is noteworthy that, despite a low amino acid sequence relatedness, Clostridium perfringens epsilon-toxin retains a similar structure with areolysin (Cole et al., 2004). Additionally these toxins share even more common characteristics such as C-terminal activation upon cleavage of a propeptide and formation of oligomeric channels. However, one clear difference between aerolysin versus septicum alpha-toxin and epsilon-toxin is the presence of an additional domain (domain I) in aerolysin, which seems to be involved in receptor binding (Diep et al., 1999, Rossjohn et al., 1997).

In the present work, we investigated the properties of pores formed by recombinant (r-septicum) alpha-toxin in artificial lipid bilayers and the consequence of pore formation on ion diffusion and cell death in cultured cell systems. We show that r-septicum alpha-toxin induces rapid cell depolarization, ATP depletion, and caspase-3 independent cell death. We also show that incubation of the cells in high external K⁺ medium delayed cell death, suggesting that the rapid loss of intracellular K⁺ represents an important early event in the mechanism of cell death caused by these pore-forming toxins.