Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin

Abstract

Monoand polyclonal antibodies were used to construct a sandwich enzyme-linked immunosorbent assay that permitted quantitation of Eschenichia coli hemolysin in soluble and membrane-bound forms. Toxin concentrations of 4 to 14 ,g/ml were measured in culture supernatants of E. coli LE 2001 at times of peak hemolytic activity. Quantitative studies on the binding of E. coli hemolysin to rabbit erythrocytes were conducted at 0 and 37°C. At 37°C, 85 to 95% of bindable toxin was cell bound after 60 min, and no saturability of binding was observed in the studied range of concentrations, which resulted in deposition of approximately 100 to 50,000 toxin molecules per cell. Binding was slower and less effective at 0°C; however, hemolysis did occur at low temperature. The number of cell-bound toxin molecules required to generate a hemolytic lesion within 60 min was estimated to be approximately 100 molecules per cell at 37°C and 800 to 1,000 molecules per cell at 0°C. Upon prolonged incubation (5 to 20 h, 37°C), the number of molecules evoking a functional lesion decreased to approximately 5 to 20 per cell. These results are compatible with the concept that E. coli hemolysin first adsorbs to the cell surface, with membrane insertion and pore formation following in a second step that may be temporally dissociated from that of binding. The data support the pore concept of toxin action by showing that attachment of a low and finite number of toxin molecules to an erythrocyte will ultimately generate a cytolytic lesion.

Characterization of a neutralizing monoclonal antibody to Pasteurella haemolytica leukotoxin

“…However, it remains unclear whether this requirement is important in initial recognition or at a secondary stage. Regardless, binding and pore formation appear to be separable events (9,27), and evidence reported by Cruz et al (8) indicates that the binding functions are distinct from the lytic functions of Al leukotoxin. Mutant toxins with whole or partial deletions of the putative membrane-spanning regions agglutinated BL-3 cells and, in addition, were antagonistic for cell lysis by native leukotoxin.…”

“…The carboxy-terminal amino acids, at least for the alpha-hemolysin, appear to function in secretion (11), while the amino-terminal region appears to facilitate cytolysis, perhaps in the initial stages of host membrane insertion (37). Binding and cytolysis appear to be separable events (9,27), and recent evidence indicates that lysis-defective mutant leukotoxin proteins with whole or partial deletions of the membrane-spanning region retain the capacity to bind to target cells and to competitively inhibit cytolysis by native leukotoxin (8).…”

Effect of lipid A on the deformability, membrane rigidity and geometry of human adult red blood cells

“…Other authors suggest a two step reaction, i.e. adsorption followed by insertion into the cell membrane [21, 22,23]. Studies by Price and Jacobs [24] suggest that in the first step lipid A adheres as an ion to the cell membrane surface.…”

Antibodies to Bordetella pertussis adenylate cyclase toxin in neonatal and maternal sera

“…Pertussis toxin (PT) and filamentous hemagglutinin (FHA) were purchased from Institut Merieux and were demonstrated to be pure by SDS-PAGE. E. coli a-hemolysin was prepared as described by Eberspficher et al [10] from the hemolysin-producing strain 0040. Culture supernate from the nonhemolytic strain 10450 of E. coli was treated identically and was used as a control.…”