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.

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.

Objective: The β-pore-forming toxins hemolysin II (HlyII) and cytotoxin K2 (CytK2) are important pathogenic factors of the opportunistic bacterium Bacillus cereus and are secreted as monomers that can oligomerize in the presence of a target cell to form transmembrane channels. Analysis of the nucleotide sequence of HlyII and CytK2 suggests the possibility of truncated forms of these proteins that was confirmed by immunochemical, and chromatography-mass spectroscopic analyzes. Methods: HlyII and CytK2 were expressed in recombinant strains of E. coli BL21(DE3) carrying plasmids pET29 which produced the intracellular proteins HlyII and CytK2 from B. cereus ATCC14579T. In the case of HlyII, fractions containing soluble intracellular proteins, periplasmic proteins, and cellular debris were obtained and analyzed using monoclonal antibodies. The truncated protein forms HlyII and CytK2 were analyzed by chromatography-mass spectrometry analysis. Results and Discussion: Analysis of the primary amino acid sequences of HlyII and CytK2 revealed the presence of potential internal translation initiation sites within the sequence. The presence of the truncated forms of HlyII was confirmed by immunochemical analysis using monoclonal antibodies and by chromatography-mass spectrometry. During expression of the CytK2 protein in E. coli cells, a protein fragment was identified and excised from a polyacrylamide gel, the identity of which with the CytK2 protein was confirmed by chromatography-mass spectrometry analysis. Conclusions: The results obtained confirm the general concept that the bacterial genome is capable of encoding a wider range of proteins than the number of genes it contains. The opportunistic bacterium B. cereus is often the cause of nosocomial infections, one of its key virulent factors are hemolysin II and cytotoxin K2. Using monoclonal antibodies and mass spectrometric analysis it has been shown that these hemolytic enzymes are capable of forming shortened forms.

The pathogenicity of many bacteria, including Bacillus cereus and Staphylococcus aureus, depends on pore-forming toxins (PFTs), which cause the lysis of host cells by forming pores in the membranes of eukaryotic cells. Bioinformatic analysis revealed a region homologous to the Lys171-Gly250 sequence in hemolysin II (HlyII) from B. cereus in over 600 PFTs, which we designated as a “homologous peptide”. Three β-barrel PFTs were used for a detailed comparative analysis. Two of them—HlyII and cytotoxin K2 (CytK2)—are synthesized in Bacillus cereus sensu lato; the third, S. aureus α-toxin (Hla), is the most investigated representative of the family. Protein modeling showed certain amino acids of the homologous peptide to be located on the surface of the monomeric forms of these β-barrel PFTs. We obtained monoclonal antibodies against both a cloned homologous peptide and a 14-membered synthetic peptide, DSFNTFYGNQLFMK, as part of the homologous peptide. The HlyII, CytK2, and Hla regions recognized by the obtained antibodies, as well as an antibody capable of suppressing the hemolytic activity of CytK2, were identified in the course of this work. Antibodies capable of recognizing PFTs of various origins can be useful tools for both identification and suppression of the cytolytic activity of PFTs.

Pathogenicity of many bacteria including Bacillus cereus and Staphylococcus aureus depends on pore-forming toxins (PFTs), which cause lysis of host cells by forming pores in membranes of eukaryotic cells. Bioinformatic analysis revealed in over 600 PFTs a region homologous to the Lys171-Gly250 sequence in hemolysin II (HlyII) from B. cereus, which we designated as “homologous peptide”. Three β-barrel PFTs were used for a detailed comparative analysis. Two of them –HlyII and cytotoxin K2 (CytK2), – are synthesized in Bacillus cereus sensu lato; the third – S. aureus α-toxin (Hla) – is the most investigated representative of the family. Protein modeling showed certain amino acids of homologous peptide to be located on the surface of the monomeric forms of these β-barrel PFTs. We obtained monoclonal antibodies against both a cloned homologous peptide and a 14-membered synthetic peptide DSFNTFYGNQLFMK as part of homologous peptide. The HlyII, CytK2 and Hla regions recognized by the obtained antibodies, as well as an antibody capable of suppressing the hemolytic activity of CytK2, were identified in the course of the work. Antibodies capable of recognizing PFTs of various origins can be useful tools for both identification and suppression of the cytolytic activity of PFTs.

Hemolysin II (HlyII) is one of the virulence factors of the opportunistic bacterium Bacillus cereus belonging to the group of β-pore-forming toxins. This work created a genetic construct encoding a large C-terminal fragment of the toxin (HlyIILCTD, M225–I412 according to the numbering of amino acid residues in HlyII). A soluble form of HlyIILCTD was obtained using the SlyD chaperone protein. HlyIILCTD was first shown to be capable of agglutinating rabbit erythrocytes. Monoclonal antibodies against HlyIILCTD were obtained by hybridoma technology. We also proposed a mode of rabbit erythrocyte agglutination by HlyIILCTD and selected three anti-HlyIILCTD monoclonal antibodies that inhibited the agglutination.

Abstract Bovine mastitis is one of the most economically important diseases in cattle. Infectious mastitis has a viral or bacterial origin. The most common bacterial agents are Staphylococcus , Streptococcus , coliforms, and Bacillus species. These are opportunistic microorganisms that can switch to a pathogenic form, using various virulence factors with follow various pathologies, both animal and human. Three stable microbial consortia, each composed of Bacillus paranthracis and Staphylococcus haemolyticus strains, were isolated from milk of cows diagnosed with mastitis in three geographically remote regions of Russia. The composition of these consortia remained stable following multiple passages on culture media. Apparently, this stability is due to the structure of the microbial biofilms formed by the communities. The virulence of the consortia depended on the B. paranthracis strains. It seems plausible that the ability of the consortia to cause mastitis in cattle was affected by mutations of the cytK gene of B. paranthracis .

Three stable microbial consortia, each composed of Bacillus paranthracis and Staphylococcus haemolyticus strains, were isolated from milk of cows diagnosed with mastitis in three geographically remote regions of Russia. The composition of these consortia remained stable following multiple passages on culture media. Apparently, this stability is due to the structure of the microbial biofilms formed by the communities. The virulence of the consortia depended on the B. paranthracis strains. It seems plausible that the ability of the consortia to cause mastitis in cattle was affected by mutations of the cytK gene of B. paranthracis.

Hemolysin II (HlyII)-one of the pathogenic factors of Bacillus cereus, a pore-forming β-barrel toxin-possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibition of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemolysis on the presence of proline at position 324 in HlyII outside the conformational antigenic determinant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, obtained via multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interactions between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled the identification of the amino acid residues N339 and K340, included in the conformational antigenic determinant recognized by HlyIIC-20.

Bacillus cereus hemolysin II, a pore-forming β-barrel toxin (HlyII), has a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD). HlyIICTD is capable of forming oligomers in aqueous solutions. Oligomerization of HlyIICTD significantly increased in the presence of erythrocytes and liposomes. Its affinity for erythrocytes of various origins differed insignificantly but was noticeably higher for T-cells. HlyIICTD destroyed THP-1 monocytes and J774 macrophages, acted most effectively on Jurkat T-lymphocytes and had virtually no impact on B-cell lines. HlyIICTD was able to form ion-conducting channels on an artificial bilayer membrane.

The pore-forming activity of SsoHel308 helicase from extreme thermophilic archaea Saccharolobus solfataricus has been demonstrated for the first time. This protein embedded in rabbit erythrocyte membranes may cause erythrocyte hemolysis. It has been shown that this enzyme forms pores in a planar artificial bilayer membrane and acts as a transformer. After embedding this enzyme into biolayer lipid membranes, the membrane conductivity is altered. Taken together, our results show that SsoHel308 helicase is able to form pores in artificial bilayer membranes and, in some cases, the current that flows across the membranes shares features typical of ion channels. The short lifetime of the pores in the membrane significantly reduces the toxicity of helicase for a living cell. The possibility of directed translocation of single-stranded DNA in the presence of ATP will enable the use of this enzyme as a molecular syringe for injecting single-stranded DNA into living cells.

Hemolysin II of Bacillus cereus sensu lato is synthesized in a bacterial cell in the form of a water-soluble secreted monomer and penetrates into eukaryotic membranes. The HlyII protein has a C-terminal extension (HlyIICTD), includes 94 amino acid residues [1]. Removal of HlyIICTD from HlyII significantly complicates transfer of the deletion variant HlyIIDCTD to E. coli cells, possibly due to the attack of the bacterial membrane. Additional deletion of the signal peptide, which excludes the penetration of the protein into the periplasm, provides E. coli cells survival carrying this gene with two deletions. Using monoclonal antibodies against recombinant HlyIICTD [2], showed a similar the binding effectively to red blood cells of various origins and noticeably differ for cells of the J774 and Jurkat lines. HlyIICTD in water solution is able to form oligomeric structures. In the presence of membrane HlyIICTD exits in oligomeric form while monomeric forms are almost completely absent. HlyIICTD trimerized in the presence of 4M urea, forming a possibly some structure that can be integrated into the artificial bilayer membrane with the formation of pores. The current-voltage characteristic of these channels was determined. Such protein structures are characteristic of trimeric autotransporter proteins [3]. In this case, the secreted full-sized monomeric form of hemolysin II acts as a passenger, and HlyIICTD acts as an element involved in adhesion to membrane and secretion from bacterial cells. The materials presented in this paper demonstrate suggests that hemolysin II may belong to trimeric autotransporter proteins – the first case of the description of this family of molecules among Gram positive microorganisms. Acknowledgments. The study was supported by a grant from the Russian foundation for basic research (no. 14-04-00592) and by the Ministry of Science and Higher Education of the Russian Federation (unique project number RFMEFI60419X0218). Baida G., Budarina Z. I., Kuzmin N.P., Solonin A.S. Complete nucleotide sequence and molecular characterization of hemolysin II gene from Bacillus cereus, FEMS Microbiol. 1999, V.180. pp. 7–14. Rudenko, N.V., Karatovskaya, A.P., Zamyatina, A.V., Siunov, A.V., Andreeva-Kovalevskaya, Zh.I., Nagel, A.S., Brovko, F.A., and Solonin, A.S., Russ. J. Bioorg. Chem., 2020, V. 46, pp. 321–326. doi.org/ https://doi.org/10.1134/S1068162020030188 Kiessling A.R., Malik A., Goldman A. Recent advances in the understanding of trimeric autotransporter adhesins. Medical Microbiology and Immunology 2020, V. 209, pp 233–242 https://doi.org/10.1007/s00430-019-00652-3

The transcription of the hlyIIR gene of Bacillus cereus , which is a negative transcriptional regulator of the hlyII gene of hemolysin II, the virulence factor of B. cereus , has been studied. The sequence of the promoter upstream of the hlyIIR gene was identified and the start point of transcription was determined. The effect of HlyIIR on the transcription of its own promoter was analyzed in vitro and in vivo. The constitutive synthesis of the final product was revealed in B. cereus . The results indicate the absence of autoregulation of the hlyIIR gene and the absence of involvement of the hemolysin II gene promoter in regulation of the hlyIIR gene expression.

In this study, we describe an optimized method of obtaining virus-like particles (VLPs) of the recombinant hepatitis C virus (HCV) core protein (HCcAg) expressed in yeast cells (Pichia pastoris), which can be used for the construction of diagnostic test systems and vaccine engineering. The described simplified procedure was developed to enable in vitro self-assembly of HCcAg molecules into VLPs during protein purification. In brief, the HCcAg protein was precipitated from yeast cell lysates with ammonium sulfate and renatured by gel filtration on Sephadex G-25 under reducing conditions. VLPs were self-assembled after the removal of the reducing agent by gel filtration on Sephadex G-25. Protein purity and specificity were evaluated by SDS-PAGE and immunoblotting analysis. The molecular mass of VLPs and their relative quantity were measured by HPLC, followed by confirmation of VLPs production and estimation of their shape and size by transmission electron microscopy. As a result, we obtained recombinant HCcAg preparation (with ~90% purity) in the form of VLPs and monomers, which has been used to produce hybridomas secreting monoclonal antibodies (mAbs) against HCcAg.

— Hemolysin II (HlyII) is one of the pathogenic factors of Bacillus cereus . With respect to the prototype of β-barrel toxins, the α-toxin of S. aureus , this pore-forming protein has a C-terminal domain (CTD) of 94 amino acids. The role of CTD in membrane pore formation and cell lysis is not clear, although removal of this portion of the protein is known to significantly reduce hemolytic activity. A representative panel of monoclonal antibodies against recombinant CTD recognizing full-length HlyII was obtained. Using the obtained monoclonal antibodies, CTD was shown to bind to rabbit red blood cells.

The pore-forming activity of SsoHel308 helicase from extreme thermophilic archaea Saccharolobus solfataricus has been demonstrated for the first time. This protein, embedded into rabbit erythrocyte membranes, may cause erythrocyte hemolysis. It has been shown that this enzyme forms pores in a flat artificial bilayer membrane and acts as the transformer. After embedding this enzyme into bilayer lipid membranes, the membrane conductivity alters. Taken together, our results show that SsoHel308 helicase is able to form pores in artificial bilayer membranes and, in some cases, the current that flows across the membranes shares features typical of ion channels. The short lifetime of the pores in the membrane significantly reduces the toxicity of helicase for a living cell, and the possibility of directed translocation of single-stranded DNA in the presence of ATP will enable the use of this enzyme as a molecular syringe for injecting single-stranded DNA into living cells.

Bacillus cereus is the fourth most common cause of foodborne illnesses that produces a variety of pore-forming proteins as the main pathogenic factors. B. cereus hemolysin II (HlyII), belonging to pore-forming β-barrel toxins, has a C-terminal extension of 94 amino acid residues designated as HlyIICTD. An analysis of a panel of monoclonal antibodies to the recombinant HlyIICTD protein revealed the ability of the antibody HlyIIC-20 to inhibit HlyII hemolysis. A conformational epitope recognized by HlyIIC-20 was found. by the method of peptide phage display and found that it is localized in the N-terminal part of HlyIICTD. The HlyIIC-20 interacted with a monomeric form of HlyII, thus suppressing maturation of the HlyII toxin. Protection efficiencies of various B. cereus strains against HlyII were different and depended on the epitope amino acid composition, as well as, insignificantly, on downstream amino acids. Substitution of L324P and P324L in the hemolysins ATCC14579T and B771, respectively, determined the role of leucine localized to the epitope in suppressing the hemolysis by the antibody. Pre-incubation of HlyIIC-20 with HlyII prevented the death of mice up to an equimolar ratio. A strategy of detecting and neutralizing the toxic activity of HlyII could provide a tool for monitoring and reducing B. cereus pathogenicity.