Interactions between residues in staphylococcal alpha-hemolysin revealed by reversion mutagenesis

Area of Science:

• Microbiology
• Molecular Biology
• Protein Structure and Function

Background:

• Alpha-hemolysin (alpha HL) from Staphylococcus aureus is a pore-forming toxin.
• Previous studies identified key residues for alpha HL pore formation via site-directed mutagenesis.
• The alpha HL-H35N mutant is nonlytic and forms a stable prepore.

Purpose of the Study:

• To identify novel amino acid residues involved in alpha HL pore formation and lytic activity.
• To investigate the functional roles of second-site revertants of the alpha HL-H35N mutant.
• To elucidate the cooperative mechanisms in the final steps of alpha HL assembly.

Main Methods:

• Error-prone PCR was used to generate second-site revertants of the alpha HL-H35N mutant.
• Revertant mutants were analyzed for their ability to lyse rabbit red blood cells (rRBC).
• Conformational analysis by limited proteolysis assessed the conversion rate of prepores to fully assembled pores.

Main Results:

• Twenty-two revertants were identified, with mutations clustering in three regions: amino acids 107-111, 144-155, and 217-228.
• Residues Lys-110 and Asp-152, flanking the glycine-rich loop, were found to be crucial for lytic activity, similar to His-35.
• The third cluster of mutations in the 217-228 region, previously unexplored, also contributed to lytic function.
• Revertant prepores showed a more rapid conversion to fully assembled pores, correlating with increased hemolytic activity.

Conclusions:

• The N-terminal residue His-35 cooperates with residues flanking the central loop and C-terminal residues (217-228) in alpha HL pore assembly.
• Reversion mutagenesis provides a complementary approach to direct mutagenesis for mapping functional residues.
• These findings expand the understanding of the molecular mechanisms underlying alpha HL pore formation and its lytic function.