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.

Two ß-glucanases from bacterium Cellulomonas flavigena: expression in Pichia pastoris, properties, biotechnological potential.

In the genome of Cellulomonas flavigena, two genes that potentially encode endoglucanases - Cfla_2912 and Cfla_2913 were identified. We cloned the genes and created Pichia pastoris-based recombinant producers of two proteins that were expressed from the AOX1 promoter. Each of the endoglucanase molecules contains a GH6 catalytic domain, CBM2 carbohydrate-binding module, and TAT signal peptide. The fermentation of the producers was carried out in a 10 L fermenter; Cfla_2912 and Cfla_2913 were purified using affinity chromatography. The yield comprised 10.3 mg/ml (430 U/ml) for Cfla_2913 and 9 mg/ml (370 U/ml) for Cfla_2912. Cfla_2912 and Cfla_2913 were found to have a high activity against barley ß-glucan and lichenan, a weak activity against carboxymethyl cellulose (CMC), phosphoric-acid treated cellulose, and no activity against laminarin, xylan, soluble starch, microcrystalline cellulose, cellobiose, and cellotriose. Thus, the proteins exhibited ß-glucanase activity. Both proteins had a neutral pH optimum of about 7.0 and were more stable at neutral and slightly alkaline pH ranging from 7.0 to 9.0. Cfla_2912 and Cfla_2913 showed a moderate thermal stability. The products of barley ß-glucan hydrolysis by Cfla_2912 and Cfla_2913 were trisaccharide, tetrasaccharide, and cellobiose. Cfla_2912 and Cfla_2913 efficiently hydrolyzed cereal polysaccharides, which indicate that they may have biotechnological potential.

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 simple method to purify recombinant HCV core protein expressed in Pichia pastoris for obtaining virus-like particles and producing monoclonal antibodies.

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.

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.

Xylanases of Cellulomonas flavigena: expression, biochemical characterization, and biotechnological potential.

Four xylanases of Cellulomonas flavigena were cloned, expressed in Escherichia coli and purified. Three enzymes (CFXyl1, CFXyl2, and CFXyl4) were from the GH10 family, while CFXyl3 was from the GH11 family. The enzymes possessed moderate temperature stability and a neutral pH optimum. The enzymes were more stable at alkaline pH values. CFXyl1 and CFXyl2 hydrolyzed xylan to form xylobiose, xylotriose, xylohexaose, xylopentaose, and xylose, which is typical for GH10. CFXyl3 (GH11) and CFXyl4 (GH10) formed the same xylooligosaccharides, but xylose was formed in small amounts. The xylanases made efficient saccharification of rye, wheat and oat, common components of animal feed, which indicates their high biotechnological potential.