Expression and properties of an aerolysin–Clostridium septicum alpha toxin hybrid protein

Abstract

SummaryAerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosylphosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.

Channel Formation by the Glycosylphosphatidylinositol-anchored Protein Binding Toxin Aerolysin Is Not Promoted by Lipid Rafts

“…Also shown is the sensitivity of untreated rat erythrocytes to aerolysin (छ). The percentage of cell lysis of erythrocytes was determined spectrophotometrically as described previously (28). Lymphocyte cell viability was measured as described under "Experimental Procedures."…”

“…The aerolysin concentration dependence of rat erythrocyte hemolysis was determined as described previously (28).…”

Clostridium septicumAlpha-Toxin Is Active against the Parasitic ProtozoanToxoplasma gondiiand Targets Members of the SAG Family of Glycosylphosphatidylinositol-Anchored Surface Proteins

“…The binding of AT and aerolysin to SAG proteins provides further evidence that the GPI anchor plays a crucial role in receptor recognition, because both toxins can bind to a variety of proteins from different organisms that are unrelated, except for the presence of a GPI anchor (13)(14)(15)22). Consistent with studies on AT binding to mammalian GPI-anchored proteins (22), the glycosyl portion of the parasite GPI anchor harbors the AT binding determinant, because the toxin continued to recognize GPI-anchored proteins after PI-PLC delipidation.…”

“…AT shares homology in structure and function to the large lobe of aerolysin, a pore-forming toxin from Aeromonas hydrophila (3,14). Despite their similarities, AT and aerolysin exhibit differences in binding to specific mammalian GPI-anchored proteins (14,15,22).…”

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

“…The crystal structure of aerolysin reveals it to be predominantly made up of ␤-sheets and to consist of four domains arranged as a large and a small lobe (37). Aerolysin-based homology modeling of alpha-toxin suggests that it only has a single large lobe made up of three domains (16,30). C. septicum alpha-toxin is encoded by the csa gene and is secreted as an inactive 46-kDa monomer (10,25), which is cleaved to its 43-kDa active form by host cell-associated proteases following binding of the toxin to glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface (19,24).…”