Monoclonal AntibodyAgainst the Oligomeric Form of the Large C-Terminal Fragment (Met225–Ile412) of Hemolysin II of Bacillus cereus are Capable of Strain-Specific Suppression of Hemolytic Activity

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Abstract

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 HlyII B. cereus were obtained using hybridoma technology with the use of a recombinant soluble form of HlyIILCTD as an antigen, which was obtained using the chaperone protein SlyD. Monoclonal antibody LCTD-83 inhibited the hemolytic activity of HlyII, the degree of protection depended on the presence/absence of proline at position 324 in the primary sequence of the toxin. The antibody most effectively inhibited erythrocyte hemolysis caused by HlyII B-771, in the sequence of which Pro is present at position 324 instead of Leu. It was shown that the LCTD-83 antibody interacts with the formed pores on the erythrocyte membranes, thereby blocking the possible release of intracellular contents. HlyII and its mutant forms were obtained using recombinant producer strains of Escherichia coli BL21 (DE3). The ability of antibodies to recognize antigens was characterized by enzyme-linked immunosorbent assay (ELISA) and immunoblotting; immunoprecipitation was used to demonstrate interaction with the membrane pores formed by the toxin. LCTD-83 interacted less effectively with the full-length toxin than with HlyIILCTD, which confirmed 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 for suppressing the cytolytic effect of hemolysin II. LCTD-83 has the potential to identify ways to neutralize the toxin.

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About the authors

O. S. Vetrova

Branch of the Federal State Budgetary Institution of Science, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences

Email: nrudkova@mail.ru
Russian Federation, prosp. Nauki 6, Pushchino, 142290

N. V. Rudenko

Branch of the Federal State Budgetary Institution of Science, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences

Author for correspondence.
Email: nrudkova@mail.ru
Russian Federation, prosp. Nauki 6, Pushchino, 142290

A. V. Zamyatina

Branch of the Federal State Budgetary Institution of Science, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences

Email: nrudkova@mail.ru
Russian Federation, prosp. Nauki 6, Pushchino, 142290

A. S. Nagel

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (IBFM RAS) “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”

Email: nrudkova@mail.ru
Russian Federation, prosp. Nauki 5, Pushchino, 142290

Z. I. Andreeva-Kovalevskaya

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (IBFM RAS) “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”

Email: nrudkova@mail.ru
Russian Federation, prosp. Nauki 5, Pushchino, 142290

A. V. Siunov

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (IBFM RAS) “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”

Email: nrudkova@mail.ru
Russian Federation, prosp. Nauki 5, Pushchino, 142290

F. A. Brovko

Branch of the Federal State Budgetary Institution of Science, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences

Email: nrudkova@mail.ru
Russian Federation, prosp. Nauki 6, Pushchino, 142290

A. S. Solonin

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (IBFM RAS) “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”

Email: nrudkova@mail.ru
Russian Federation, prosp. Nauki 5, Pushchino, 142290

A. P. Karatovskaya

Branch of the Federal State Budgetary Institution of Science, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences

Email: nrudkova@mail.ru
Russian Federation, prosp. Nauki 6, Pushchino, 142290

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2. Fig. 1. HlyIILCTD immunoblotting with mAbs. Before electrophoretic separation, the samples were boiled in 1% SDS for 10 minutes. M is a molecular weight marker (Abcam, Great Britain); 1 is stained with LCTD-82; 2 is stained with LCTD-83; 3 is stained with LCTD-84.

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3. 2. Comparison of the effectiveness of the interaction of mAbs against HlyIILCTD (5 micrograms/ml) with immobilized HlyII of various strains and HlyIILCTD.

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4. 3. HlyII immunoblotting: M – marker of molecular weight; 1 – monomeric form; 2 – pores interacting with LCTD-83 immobilized on magnetic particles; 3 – isotype control; 4 – pores after delipidization. The tracks were stained with biotinylated mAbs LCD-71 [15] and streptavidin conjugated with horseradish peroxidase.

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5. Fig. 4. Experimental scheme. Precipitation of LCD-83 and others formed by HlyII B771 in the erythrocyte membrane.

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