A Monoclonal Antibody Against the Oligomeric Form of the Large C-Terminal Fragment (Met225–Ile412) of Bacillus cereus Hemolysin II is Capable of Strain-Specific Inhibition of the Hemolytic Activity

Abstract

Objective: The pore-forming toxin hemolysin II (HlyII) secreted by the gram-positive bacterium Bacillus cereus is one of the main pathogenic factors of this microorganism. The action of HlyII leads to cell lysis due to pore formation on membranes. Monoclonal antibodies against the large C-terminal fragment (Met225–Ile412, HlyIILCTD) of B. cereus HlyII were obtained by the hybridoma technology using the recombinant soluble form of HlyIILCTD as an antigen. The monoclonal antibody LCTD-83 inhibited the hemolytic activity of HlyII, and the degree of protection depended on the presence or absence of proline at position 324 in the primary sequence of the toxin. The antibodies most effectively inhibited the hemolysis of erythrocytes, induced B-771 HlyII, the sequence of which contains Pro324. It was shown that the antibody interacts with the pores formed in the RBC membranes, thereby blocking the possible release of intracellular contents. Methods: HlyII and its mutant forms were obtained using recombinant E. coli BL21(DE3) producer strains. The soluble form of HlyIILCTD was obtained using the chaperone protein SlyD. Monoclonal antibodies were obtained by the hybridoma technology. The ability of antibodies to recognize antigens was characterized by the enzyme-linked immunosorbent assay and immunoblotting; immunoprecipitation was used to demonstrate interaction with the membrane pores formed by the toxin. Results and Discussion: A monoclonal antibody against the oligomeric form of the LCTD-83 antigen inhibited the hemolytic activity of B. cereus B-771 HlyII by blocking the transmembrane channels formed by the toxin. Inhibition of the cytolytic activity of the toxin by LCTD-83 depended on the presence of the Pro324 residue in the primary sequence of HlyII. Conclusions: The neutralizing monoclonal antibody LCTD-83 recognized the formed HlyII transmembrane channel and was sensitive to conformational changes during its formation. The substitution of Pro324 by Leu in the primary sequence of HlyII affected the neutralizing ability of the antibody. The LCTD-83 antibody less effectively interacts with the full-length toxin than with HlyIILCTD, which is evidenced by the fact that pore formation is accompanied by a change in the toxin conformation. In this regard, antibodies interacting with its oligomeric form are promising candidates for inhibiting the cytolytic effect of hemolysin II, and LCTD-83 has the potential to identify ways to neutralize the toxin.