A simple method to differentiate three classes of cholesterol-dependent cytolysins

Area of Science:

• Microbiology
• Bacterial Pathogenesis
• Toxinology

Background:

• Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins produced by Gram-positive bacteria.
• CDCs are categorized into three groups based on their interaction with cell membrane receptors: cholesterol, human CD59, or both.
• Intermedilysin, from Streptococcus intermedius, is the sole known Group II CDC, primarily binding to human CD59.

Purpose of the Study:

• To investigate the roles of cholesterol and human CD59 in CDC-mediated cell lysis.
• To develop a method for characterizing the receptor-binding properties of unclassified Group III CDCs.

Main Methods:

• Human erythrocytes were treated with dithiothreitol (DTT) to disrupt disulfide bonds in CD59, thereby inactivating it.
• The hemolytic activity of different CDC groups (I, II, and III) against DTT-treated and mock-treated erythrocytes was compared.
• Recognition of DTT-treated erythrocytes by an anti-human CD59 monoclonal antibody was assessed.

Main Results:

• DTT treatment abolished intermedilysin (Group II CDC) binding and lysis, confirming CD59 dependence.
• Group I CDCs, which recognize cholesterol, showed unaffected lysis of DTT-treated erythrocytes.
• Group III CDCs exhibited reduced lysis of DTT-treated erythrocytes, indicating partial dependence on CD59.

Conclusions:

• Disulfide bond integrity of CD59 is crucial for Group II CDC recognition.
• DTT treatment of erythrocytes provides a straightforward method to differentiate CDCs based on their reliance on CD59 and cholesterol.
• This approach facilitates the characterization of novel Group III CDCs from Mitis group streptococci.