C-Terminal Domain of Bacillus cereus Hemolysin II is Capable of Forming Homo- and Hetero-Oligomeric Forms of Toxin on the Membrane Surface

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Abstract

Hemolysin II (HlyII) is one of the key pathogenic factors of the opportunistic gram-positive bacterium Bacillus cereus. HlyII lyses target cells by forming pores on membranes. HlyII belongs to the group of β-pore-forming toxins. A distinctive feature of HlyII is the presence of a C-terminal domain of 94 amino acid residues (HlyIICTD). It was shown that under slightly acidic conditions (pH 5.0), corresponding to the near-membrane region, the C-terminal domains, both by themselves and as part of the toxin, form stable complexes consisting of full-length and truncated toxin molecules. HlyII, HlyIILCTD (large C-terminal fragment Met225–Ile412) and HlyIICTD were obtained using recombinant producer strains Escherichia coli BL21(DE3). Biotinylation of HlyIICTD was carried out using N-hydroxysuccinimide ester of biotin. The interaction of HlyIICTD with HlyIICTD, HlyIILCTD, and HlyII, as well as the interaction of HlyIICTD with erythrocyte membranes, were studied by enzyme-linked immunosorbent assay and immunoblotting using both horseradish peroxidase-conjugated streptavidin and monoclonal antibodies against HlyII. Under slightly acidic conditions, HlyIICTD interacted with both the HlyIICTD domain within the full-length toxin and with the HlyIICTD protein. The interaction of HlyIICTD with the erythrocyte membrane increased fold in the presence of the toxin. The property of the C-terminal domain to form complexes with each other was revealed, regardless of whether it is part of the full-length toxin, the large C-terminal fragment, or the short HlyIICTD under conditions corresponding to those existing near the cell membrane (pH 5.0). The toxin in the peri membrane region exists in a partially molten globule state, in which the C-terminal domains of the monomers can bind to each other, increasing the local concentration of full-length toxins.

About the authors

O. S Vetrova

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS)

Pushchino, Russia

N. V Rudenko

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS)

Email: nrudkova@mail.ru
Pushchino, Russia

B. S Melnik

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS); Institute of Protein Research of the Russian Academy of Sciences (IPR RAS)

Pushchino, Russia

A. P Karatovskaya

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS)

Pushchino, Russia

A. V Zamyatina

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS)

Pushchino, Russia

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”

Pushchino, Russia

Zh. 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”

Pushchino, Russia

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”

Pushchino, Russia

F. A Brovko

Branch of the Federal State Budgetary Institution of Science, State Scientific Center of the Russian Federation, Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova Russian Academy of Sciences (Branch of the State Research Center IBCh RAS)

Pushchino, Russia

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”

Pushchino, Russia

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