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.

Multifunctional Enzyme with Endoglucanase and Alginase/Glucuronan Lyase Activities from Bacterium Cellulophaga lytica.

Cellulophaga lytica is a Gram-negative aerobic bacterium in the genome of which there are many genes encoding polysaccharide degrading enzymes. One of the enzymes named ClGP contains a glycoside hydrolase domain from the GH5 family and a polysaccharide lyase domain from the PL31 family. The enzyme also contains the TAT signaling peptide and the TIGR04183 domain that indicates extracellular nature of the enzyme. Phylogenetic analysis has shown that the enzymes most closely related to ClGP and containing all four domains (TAT, GH5, PL31, TIGR04183) are widespread among bacterial species belonging to the Flavobacteriaceae family. ClGP produced by the recombinant strain of E. coli was purified and characterized. ClGP exhibited activity of endoglucanase (EC 3.2.1.4) and catalyzed hydrolysis of ß-D-glucan, carboxymethyl cellulose sodium salt (CMC-Na), and amorphous cellulose, but failed to hydrolyze microcrystalline cellulose and xylan. Products of CMC hydrolysis were cellobiose and cellotriose, whereas ß-D-glucan was hydrolyzed to glucose, cellobiose, cellotetraose, and cellopentaose. ClGP was more active against the poly-ß-D-mannuronate blocks than against the poly-α-L-glucuronate blocks of alginic acid. This indicates that the enzyme is a polyM lyase (EC 4.2.2.3). ClGP was active against polyglucuronic acid, so it displayed a glucuronan lyase (EC 4.2.2.14) activity. The enzyme had a neutral pH-optimum, was stable in the pH range 6.0-8.0, and displayed moderate thermal stability. ClGP effectively saccharified two species of brown algae, Saccharina latissima and Laminaria digitata, that suggests its potential for use in the production of biofuel from macroalgae.

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.

Recombinant xylanase from Streptomyces coelicolor Ac-738: characterization and the effect on xylan-containing products.

A xylanase gene was isolated from the genomic DNA of Streptomyces coelicolor Ac-738. The 723-bp full-length gene encoded a 241-amino acid peptide consisting of a 49-residue putative TAT signal peptide and a glycoside hydrolase family-11 domain. The mature enzyme called XSC738 was expressed in Escherichia coli M15[pREP4]. The electrophoretically homogeneous protein with a specific activity of 167 U/mg for beechwood xylan was purified. The pH optimum of XSC738 was at pH 6; a high activity was retained within a pH range of 4.5-8.5. The enzyme was thermostable at 50-60 °C and retained an activity at pH 3.0-7.0. Xylanase XSC738 was activated by Mn²âº, Co²âº and largely inhibited by Cd²âº, SDS and EDTA. The products of xylan hydrolysis were mainly xylobiose, xylotriose, xylopentaose and xylohexose. Xylotetraose appeared as a minor product. Processing of such agricultural xylan-containing products as wheat, oats, soy flour and wheat bran by xylanase resulted in an increased content of sugars.

Iron regulates expression of Bacillus cereus hemolysin II via global regulator Fur.

The capacity of pathogens to respond to environmental signals, such as iron concentration, is key to bacterial survival and establishment of a successful infection. Bacillus cereus is a widely distributed bacterium with distinct pathogenic properties. Hemolysin II (HlyII) is one of its pore-forming cytotoxins and has been shown to be involved in bacterial pathogenicity in a number of cell and animal models. Unlike many other B. cereus pathogenicity factors, HlyII is not regulated by pleiotropic transcriptional regulator PlcR but is controlled by its own regulator, HlyIIR. Using a combination of in vivo and in vitro techniques, we show that hlyII expression is also negatively regulated by iron by the global regulator Fur via direct interaction with the hlyII promoter. DNase I footprinting and in vitro transcription experiments indicate that Fur prevents RNA polymerase binding to the hlyII promoter. HlyII expression profiles demonstrate that both HlyIIR and Fur regulate HlyII expression in a concerted fashion, with the effect of Fur being maximal in the early stages of bacterial growth. In sum, these results show that Fur serves as a transcriptional repressor for hlyII expression.

Bacillus cereus can attack the cell membranes of the alga Chara corallina by means of HlyII.

We studied the influence of Bacillus cereus bacteria on cells of the freshwater alga Chara corallina. These bacteria and recombinant Bacillus subtilis strains are capable of producing the secreted toxin HlyII, which changes the electrophysiological parameters of the algal electrically excitable plasma membrane by forming pores. Cooperative incubation of bacterial cells, which carry active hlyII gene, and Chara corallina cells caused a decrease in the resting potential (V(m)) and plasma membrane resistance (R(m)) of algal cells. The efficiency of each strain was commensurable with its ability to produce HlyII. Purified hemolysin II caused a similar effect on V(m) and R(m) of intact and perfused cells. This protein changed the kinetics and magnitude of transient voltage-dependent calcium and calcium-activated chloride currents owing to the formation of additional Ca(2+)-permeable pores in algal cell membrane. Occurrence of the cellulose cell wall with pores 2.1 to 4.6nm in diameter suggests that HlyII molecules reach the plasma membrane surface strictly as monomers.

Quick assessment of cytotoxins effect on Daphnia magna using in vivo fluorescence microscopy.

A novel approach to contaminant toxicity screening is proposed. The use of fluorescent microscopy with fluorescent dyes allows for assessing intoxication of Daphnia magna tissues, at various stages of exposure, to contaminants present in water. As shown, D. magna may not only be used as a test species in toxicity tests based on its lethality, but due to its translucency and application of fluorescent probes, separate steps of its intoxication and dying can be visualized. Using a variety of fluorescent probes, the present study also contributes to a better understanding of the toxicity mechanisms.

Expression of Bacillus cereus hemolysin II in Bacillus subtilis renders the bacteria pathogenic for the crustacean Daphnia magna.

Hemolysin II (HlyII) is a pore-forming toxin of the opportunistic pathogen Bacillus cereus. Despite our understanding of the mechanism of HlyII cytotoxicity in vitro, many of its characteristics, including potential target cells, conditions of its action and expression, are not known. Here we report that the expression of hlyII in Bacillus subtilis renders the bacteria hemolytic and is able to kill the crustacean Daphnia magna. The hemolytic activity of hlyII-encoded B. subtilis strains in culture media is positively correlated with virulence in D. magna. Fluorescence microscopy reveals postinfection changes in the mitochondrial potential of intestinal tissue, suggesting that the formation of ionic pores leads to cell death. In the presence of the transcriptional regulator HlyIIR, HlyII expression decreases 200-fold, and B. subtilis expressing both hlyII and hlyIIR remains hemolytic, but not pathogenic to the crustacean.