Identification of Functional Domains of Clostridium septicum Alpha Toxin

Abstract

Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin belonging to the aerolysin-like family of toxins. AT is predicted to be a three-domain molecule based on functional and sequence similarity with aerolysin, for which the crystal structure has been solved. In the present study we have substituted the entire primary structure of AT with alanine or cysteine in order to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization and pore formation. These studies revealed that receptor binding is restricted to domain 1 of the AT structure, whereas domains 1 and 3 are involved in oligomerization. These studies also revealed the presence of a putative functional region of AT proximal to the receptor-binding domain, but distal from the pore-forming domain that is proposed to regulate the insertion of the transmembrane β-hairpin of the prepore oligomer.Clostridium septicum is a major cause of non-traumatic gas gangrene in humans, a fulminant form of myonecrosis that can progress to a fatal infection in less than 24 hours with reported mortality rates ranging from 67-100% (1-4). Of its many virulence factors, alpha toxin (AT) is the only lethal factor secreted by this organism (5) and it has recently been shown to be absolutely required for virulence of C. septicum (6). AT is classified as a pore-forming toxin and is a member of the aerolysin-like family of pore-forming toxins. AT and aerolysin share a great deal of structural and sequence similarity (72%) (5), which has allowed for the development of a molecular model of AT using the previously solved crystal structure of aerolysin ( Fig. 1) (7,8). Aerolysin is a two lobed protein in which the small lobe is comprised of domain 1 (D1) and the large lobe is comprised of domains 2-4 (D2-D4, Fig. 1). The primary structure of AT exhibits similarity with the large lobe of aerolysin but it lacks the small lobe structure of about 83 amino acids. Both toxins appear to follow a similar ordered path to form pores in cell membranes (5). Following secretion, they bind to receptors on the cell membrane where they are cleaved into their active form by cell surface proteases, usually furin. Once activated, the toxins oligomerize on the cell surface into a prepore complex followed by insertion of a transmembrane β-barrel into the membrane (8).

Bacillus thuringiensis Cry1Ab Mutants Affecting Oligomer Formation Are Non-toxic to Manduca sexta Larvae

“…In other pore-forming toxins mutations affecting toxin oligomerization correlated with a severe disruption in toxicity, suggesting that oligomerization is an essential step (21)(22)(23)(24)(25). To determine the role of oligomer formation for Cry1Ab toxin action, we isolated and characterized single point mutations that affected toxin oligomerization.…”

Identification of a target protein of Hydra actinoporin-like toxin-1 (HALT-1) using GST affinity purification and SILAC-based quantitative proteomics

“…Therefore, the folate receptor alpha is localized on the outer surface of the cell membrane and could serve as the membrane attachment site for extracellular proteins, possibly including HALT-1. This hypothesis is supported by the fact that the alpha-toxin of Clostridium septicum binds to the GPI-anchored form of folate receptor alpha (Gordon et al 1999) before it is cleaved approximately 4 kDa from the C-terminus to become an active cytolysin (Melton-Witt et al 2006). Similarly, we suggest that folate receptor alpha could serve as a membrane receptor for HALT-1 instead of the lipid sphingomyelin, which has been previously thought to be the solely attachment site for HALT-1 on the cell membrane (Liew et al 2015).…”

Pore-Forming Activity of Alpha-Toxin Is Essential for Clostridium septicum -Mediated Myonecrosis

“…The modified csa genes (30,31) were PCR amplified from expression vectors with the high-fidelity DNA polymerase Pwo (Roche) and primers JRP1075 (5Ј-ATCCCGCGAAATTAATAC-3Ј) and JRP1923 (5Ј-CGT TAATTAATATCAATTTTTTTATCA-3Ј), where JRP1923 incorporates a unique PacI site and the additional 3Ј adenosine native to JIR6086 and other C. septicum alpha-toxin sequences (4). The product was then cleaved with PacI and BbsI and cloned into csa suicide plasmid pJIR2230, which carries an erm(B) gene flanked by 5Ј and 3Ј csa fragments (26), so that the erm(B) gene was replaced with an internal csa fragment that included the required mutation.…”

“…Recent studies (30,31) provided greater insights into the role of the three domains of alpha-toxin. Alanine scanning mutagenesis of the entire protein revealed that residues important in receptor binding were restricted to loops 1 and 3 and to an ␣-helix in domain 1.…”

“…Alanine scanning mutagenesis of the entire protein revealed that residues important in receptor binding were restricted to loops 1 and 3 and to an ␣-helix in domain 1. Residues in loop 2 of domain 1 were found to be important in prepore-to-pore conversion since replacement of these residues with alanine abolished cytotoxicity, even though the toxin was still able to bind and oligomerize on SupT1 cell membranes (31). Cysteine scanning in an alpha-toxin C86A background revealed that several residues throughout the protein are required for effective oligomerization; however, the greatest concentration of residues was found to be in domain 3 (31).…”