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Isolation of a tryptic fragment from Clostridium perfringens theta-toxin that contains sites for membrane binding and
R K Tweten1, R W Harris, P J Sims
1Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Abstract:
Trypsin cleaves Clostridium perfringens theta-toxin (perfringolysin O or PFO) at a single site between residues 303 and 304 (Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Kawasaki, H., and Ando, S. (1986) Biochemistry 25, 6048-6053; Tweten, R. K. (1988b) Infect. Immun. 56, 3228-3234) and yields an amino-terminal fragment of 30,208 Da (T1) and a carboxyl-terminal fragment of 22,268 Da (T2). Both peptides were purified by reverse phase chromatography of trypsin-nicked PFO. Neither peptide retained hemolytic activity. Peptide T1 had no apparent effect on the hemolytic activity of PFO, whereas T2 was found to inhibit the hemolytic activity of PFO and was analyzed further. The order of binding of T2 and PFO to membranes did not alter the inhibitory effect of T2 on PFO-induced hemolysis, indicating that competitive binding by T2 for PFO membrane binding sites was not the basis for the observed inhibition. Further analysis showed that T2 could inhibit membrane-dependent fluorescence energy transfer (FET) between PFO molecules labeled with fluorescein (fluorescent donor) or tetramethylrhodamine (fluorescent acceptor). This provided evidence that T2 could complex with PFO. T2 was also found to be incapable of self-aggregation (as opposed to PFO), since preincubation of T2 with either erythrocytes or erythrocyte ghost membranes did not affect the T2-dependent inhibition of hemolysis or FET. These data indicate that T2 inhibits PFO-dependent hemolysis by forming a complex with PFO, which inhibits aggregation and that the membrane binding site and a single aggregation site remain intact on T2.
Insights
Trypsin cleavage of Clostridium perfringens theta-toxin (perfringolysin O or PFO) yields two fragments. The carboxyl-terminal fragment (T2) inhibits PFO
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Clostridium perfringens theta-toxin (perfringolysin O or PFO) is a pore-forming toxin.
- PFO mediates hemolysis by binding to cell membranes and forming aggregates.
- Understanding PFO's mechanism is crucial for developing therapeutic interventions.
Purpose of the Study:
- To investigate the functional properties of PFO fragments generated by trypsin cleavage.
- To determine the mechanism by which the carboxyl-terminal fragment (T2) inhibits PFO activity.
Main Methods:
- Purification of trypsin-generated PFO fragments (T1 and T2) using reverse-phase chromatography.
- Assays for hemolytic activity of PFO and its fragments.
- Analysis of PFO-membrane interactions using fluorescence energy transfer (FET).
- Investigation of T2 self-aggregation potential.
Main Results:
- Trypsin cleavage produced N-terminal (T1) and C-terminal (T2) fragments.
- Neither T1 nor T2 retained hemolytic activity independently.
- T2 inhibited PFO-induced hemolysis and FET, independent of binding order.
- T2 inhibited PFO aggregation and did not self-aggregate.
Conclusions:
- The carboxyl-terminal fragment (T2) of PFO inhibits hemolysis by complexing with PFO.
- T2 binding to PFO prevents PFO aggregation, a key step in pore formation.
- T2 retains PFO's membrane binding site and a single aggregation site.