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Abstract <p><b>Objective:</b> Hemolysin II (HlyII) is one of the key pathogenic factors of the opportunistic gram-positive bacterium <i>Bacillus cereus</i>. 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 <i>C</i>-terminal domain of 94 amino acid residues (HlyIICTD). It was shown that, under slightly acidic conditions (pH 5.0) corresponding to the perimembrane region, the <i>C</i>-terminal domains, both by themselves and as part of the toxin, form stable complexes consisting of full-length and truncated toxin molecules. <b>Methods:</b> HlyII, HlyIILCTD (large <i>C</i>-terminal fragment Met225–Ile412), and HlyIICTD were obtained using recombinant producer strains <i>Escherichia coli</i> BL21(DE3). Biotinylation of HlyIICTD was carried out using <i>N</i>-hydroxysuccinimide ester of biotin. The interaction of HlyIICTD with HlyIICTD, HlyIILCTD, and HlyII, as well as that 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. <b>Results and Discussion:</b> 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 was enhanced several-fold in the presence of the toxin. <b>Conclusions:</b> The property of the <i>C</i>-terminal domain to form complexes with other HlyIICTDs, regardless of whether it is part of the full-length toxin, the large <i>C</i>-terminal fragment, or the short HlyIICTD under conditions corresponding to those existing near the cell membrane (pH 5.0), was revealed. The toxin in the perimembrane region exists in a partially molten-globule state, in which the <i>C</i>-terminal domains of the monomers can bind to each other, increasing the local concentration of full-length toxins.</p>

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

  • 期刊论文

  • 发表日期：

    2025年7月28日

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  • O. S. Vetrova,
  • N. V. Rudenko,
  • B. S. Mel’nik,
  • A. P. Karatovskaya,
  • A. V. Zamyatina,
  • A. S. Nagel’,
  • Zh. I. Andreeva-Kovalevskaya,
  • A. V. Siunov,
  • F. A. Brovko,
  • A. S. Solonin

摘要

Abstract

Objective: 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 perimembrane 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. Methods: 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 that 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. Results and Discussion: 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 was enhanced several-fold in the presence of the toxin. Conclusions: The property of the C-terminal domain to form complexes with other HlyIICTDs, 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), was revealed. The toxin in the perimembrane 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.