Arresting the Activity of Bacterial β-Barrel Pore-Forming Toxins by Cysteine Insertion Mutagenesis in the Homologous Region

Bacterial β-barrel pore-forming toxins, including Staphylococcus aureus α-toxin (Hla) and Bacillus cereus toxins hemolysin II (HlyII) and cytolytic toxin K2 (CytK-2), are secreted by bacterial cells as water-soluble monomers. These monomers assemble within lipid bilayers to form cylindrical pores, leading to lysis of target eukaryotic cells. We created mutant forms of these toxins that, based on the results of X-ray structural analysis of Hla and the prediction of the 3D structure of HlyII and CytK2, can form intramolecular disulfide bonds in monomers. The substitutions were made in the region responsible for toxin insertion into the target membrane. The mutant forms reversibly altered their hemolytic activity depending on the presence of reducing reagents and were non-toxic when injected into experimental animals. The immune response to injection of the mutant forms of Hla and CytK-2 toxins resulted in higher antibody titers against the wild-type toxins and a higher level of immunological memory than with injection of the HlyII mutant. The mutant form of CytK-2 demonstrates the properties of a prototype vaccine, as immunization with this protein protects animals against the effects of the wild-type toxin.