A High-Homology Region Provides the Possibility of Detecting ß-Barrel Pore-Forming Toxins from Various Bacterial Species.

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 ß-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 ß-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.

Utilizing Extraepitopic Amino Acid Substitutions to Define Changes in the Accessibility of Conformational Epitopes of the Bacillus cereus HlyII C-Terminal Domain.

Hemolysin II (HlyII)-one of the pathogenic factors of Bacillus cereus, a pore-forming ß-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.

Region Met225 to Ile412 of Bacillus cereus Hemolysin II Is Capable to Agglutinate Red Blood Cells.

Hemolysin II (HlyII) is one of the virulence factors of the opportunistic bacterium Bacillus cereus belonging to the group of ß-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.

Protective Capacity of Monoclonal Antibodies against Acinetobacter baumannii K9 Capsular Polysaccharide.

Acinetobacter baumannii is an antibiotic-resistant opportunistic pathogen, one of the main causes of hospital infections. There is an urgent need for the development of therapy strategies which are not based on antibiotics. Hybridoma technology was used to obtain monoclonal antibodies. The antibodies were characterized by enzyme immunoassay and fluorescence microscopy according to their ability to opsonize A. baumannii and to protect model animals from infection upon intraperitoneal and pulmonary injection. Monoclonal antibodies (MAbs), IgG, against the K9 capsular polysaccharide (CPS) of A. baumannii were prepared using a glycoconjugate, synthesized by squaric-acid chemistry, consisting of two CPS K9 monomer units and a carrier protein. The MAbs were highly specific, stained the bacterial surface, allowed detection of A. baumannii in infected lung tissue, effectively opsonized the bacteria at nanogram concentrations (up to 1.5 ng/mL for CPS-407), and demonstrated a high ability to protect an organism against bacterial infection upon intraperitoneal and lung injection. In intraperitoneal infection of a mouse model with A. baumannii K9, the CPS-407 antibody protected at a dose of 25 µg/mouse. When bacteria were injected into the lung, MAb therapy prevented infection of the body and led to a significant reduction of the bacterial load in infected tissues. IMPORTANCE MAbs detected A. baumannii in infected lung tissue, effectively opsonized bacteria, and protected model animals from infection.

Immune Response to Conjugates of Fragments of the Type K9 Capsular Polysaccharide of Acinetobacter baumannii with Carrier Proteins.

The clonal bacterial species Acinetobacter baumannii is an emerging multidrug-resistant pathogen which causes high-lethality infections. Cells of A. baumannii are surrounded by the type-specific capsular polysaccharide (CPS), which provides resistance to the protective mechanisms of the host and is considered a target for immunization. The conjugates of three inert carrier proteins and A. baumannii type K9 CPS fragments, which contained various numbers of oligosaccharide repeats (K-units), were synthesized by periodate oxidation and squaric acid chemistry. The conjugates were applied to immunize mice, and chemical synthesis by squaric acid was shown to significantly improve the immunogenic properties of glycoconjugate. In BALB/c mice, IgG antibodies were predominant among type K9 CPS reactive antibodies, and their total content was several times higher than that of IgM. Immune sera were characterized by their opsonization ability during practically the entire lives of the experimental mice. The sera were cross-reactive, but the highest specificity was observed against the antigen (type K9 CPS) used for immunization. The immunization of BALB/c and ICR-1 mice with a glycoconjugate without adjuvants led to varying degrees of stimulation of IL-10, IL-17A, and TNF-α production, but not IL-4 production in the ICR-1 mice. This is in contrast to the BALB/c mice, in which γ-IFN production was also activated. The protective effectiveness of the glycoconjugates obtained by squaric acid chemistry was demonstrated by experiments that involved challenging immunized and nonimmunized animals with a lethal dose of A. baumannii K9. IMPORTANCE Immunization by glycoconjugates with A. baumannii type K9 CPS fragments induced a high level of antibodies (predominantly IgG) in sera, which reacted specifically with the CPS of A. baumannii type K9, as well as a long immunological memory. The sera of immunized animals efficiently opsonized A. baumannii type K9. Immunization resulted in the balanced production of pro/anti-inflammatory lymphokines and protective antibodies to ensure the survival of the mice infected with A. baumannii. The level of specific antibodies was sufficient to provide protective immunity against the challenge by A. baumannii, making this approach applicable in the development of vaccine preparations.

The zinc ions stabilize the three-dimensional structure and are required for the binding of staphylococcal enterotoxin-like protein P (SEIP) with MHC-II receptors.

Staphylococcus aureus is a common human and animal pathogen. These bacteria have various pathogenicity factors, including enterotoxin-like proteins. SElP (staphylococcal enterotoxin-like protein P) has potential zinc ion-binding sites and is able to interact with major histocompatibility complex class II (MHCII) and T-cell receptor (TCR). A method for the expression and isolation of the enterotoxin-like protein of Staphylococcus aureus (SElP) was developed. The expression was carried out in E. coli cells, and the protein was isolated by affinity chromatography on a NiNTA column. The endotoxins were separated by affinity chromatography on Affi-Prep® polymyxin. It was shown by gel filtration that the resulting protein had a monomeric form. The protein in zinc-bound and zinc-free forms was characterized by protein melting using fluorescence method and it was shown that zinc stabilizes the spatial structure of SElP. The functional activity of SElP was investigated by the ability to interact with the histocompatibility antigen class II receptor (MHC-II) exposed on the B cell line Raji by flow cytofluorometry. The zinc-bound and zinc-free forms were shown to differ in their interaction with MHC-II. The localization of the zinc-binding site was confirmed by the introduction of the H225 and D227 mutations. The mutant protein was characterized by melting, and its propensity to form aggregates was shown.

The C-terminal domain of Bacillus cereus hemolysin II oligomerizes by itself in the presence of cell membranes to form ion channels.

Bacillus cereus hemolysin II, a pore-forming ß-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.

A Monoclonal Antibody against the C-Terminal Domain of Bacillus cereus Hemolysin II Inhibits HlyII Cytolytic Activity.

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 ß-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.