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.

5,7-Diamino-3,5,7,9-tetradeoxynon-2-ulosonic Acids in the Capsular Polysaccharides of Acinetobacter baumannii.

The polysaccharide capsule surrounding bacterial cell plays an important role in pathogenesis of infections caused by the opportunistic pathogen Acinetobacter baumannii by providing protection from external factors. The structures of the capsular polysaccharide (CPS) produced by A. baumannii isolates and the corresponding CPS biosynthesis gene clusters are highly diverse, although many of them are related. Many types of A. baumannii CPSs contain isomers of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acid (DTNA). Three of these isomers, namely acinetaminic acid (l-glycero-l-altro isomer), 8-epiacinetaminic acid (d-glycero-l-altro isomer), and 8-epipseudaminic acid (d-glycero-l-manno isomer), have not been found so far in naturally occurring carbohydrates from other species. In A. baumannii CPSs, DTNAs carry N-acyl substituents at positions 5 and 7; in some CPSs, both N-acetyl and N-(3-hydroxybutanoyl) groups are present. Remarkably, pseudaminic acid carries the (R)-isomer and legionaminic acid carries the (S)-isomer of the 3-hydroxybutanoyl group. The review addresses the structure and genetics of biosynthesis of A. baumannii CPSs containing di-N-acyl derivatives of DTNA.

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.

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.

Antibodies as Biosensors' Key Components: State-of-the-Art in Russia 2020-2021.

The recognition of biomolecules is crucial in key areas such as the timely diagnosis of somatic and infectious diseases, food quality control, and environmental monitoring. This determines the need to develop highly sensitive display devices based on the achievements of modern science and technology, characterized by high selectivity, high speed, low cost, availability, and small size. Such requirements are met by biosensor systems-devices for reagent-free analysis of compounds that consist of a biologically sensitive element (receptor), a transducer, and a working solution. The diversity of biological material and methods for its immobilization on the surface or in the volume of the transducer and the use of nanotechnologies have led to the appearance of an avalanche-like number of different biosensors, which, depending on the type of biologically sensitive element, can be divided into three groups: enzyme, affinity, and cellular/tissue. Affinity biosensors are one of the rapidly developing areas in immunoassay, where the key point is to register the formation of an antigen-antibody complex. This review analyzes the latest work by Russian researchers concerning the production of molecules used in various immunoassay formats as well as new fundamental scientific data obtained as a result of their use.

Magnetic immunoassay for detection of staphylococcal toxins in complex media.

Method of highly sensitive registration of magnetic nanoparticles by their nonlinear magnetization is used in a novel sandwich-type immunoassay for detection of staphylococcal toxins in complex media of virtually any volume, with increasing sensitivity at higher sample volume. The signal is read out from the entire volume of a nontransparent 3D fiber structure employed as a solid phase, which provides large reaction surface, quick reagent mixing, as well as antigen immunofiltration directly in the course of the assay. The method has demonstrated near-linear dose-response curves within a wide range of ~3 decades, while detection of staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST) in neat milk without sample preparation. The limits of detection (LOD) as low as 4 and 10 pg/mL for TSST and SEA, respectively, were obtained in 2-h format using 30-mL samples. The second, 25-min format, showed the LOD of 0.1 and 0.3 ng/mL for the same toxins in a 150 µL sample. The developed immunoassay can be applied in food safety control, in vitro diagnostics, and veterinary for a variety of research from express tests in the field to highly sensitive laboratory tests.

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.

Genomic analysis and assessment of pathogenic (toxicogenic) potential of Staphylococcus haemolyticus and Bacillus paranthracis consortia isolated from bovine mastitis in Russia.

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.

Rapid simultaneous ultrasensitive immunodetection of five bacterial toxins.

Rapid ultrasensitive detection of gastrointestinal pathogens presents a great interest for medical diagnostics and epidemiologic services. Though conventional immunochemical and polymerase chain reaction (PCR)-based methods are sensitive enough for many applications, they usually require several hours for assay, whereas as sensitive but more rapid methods are needed in many practical cases. Here, we report a new microarray-based analytical technique for simultaneous detection of five bacterial toxins: the cholera toxin, the E. coli heat-labile toxin, and three S. aureus toxins (the enterotoxins A and B and the toxic shock syndrome toxin). The assay involves three major steps: electrophoretic collection of toxins on an antibody microarray, labeling of captured antigens with secondary biotinylated antibodies, and detection of biotin labels by scanning the microarray surface with streptavidin-coated magnetic beads in a shear-flow. All the stages are performed in a single flow cell allowing application of electric and magnetic fields as well as optical detection of microarray-bound beads. Replacement of diffusion with a forced transport at all the recognition steps allows one to dramatically decrease both the limit of detection (LOD) and the assay time. We demonstrate here that application of this "active" assay technique to the detection of bacterial toxins in water samples from natural sources and in food samples (milk and meat extracts) allowed one to perform the assay in less than 10 min and to decrease the LOD to 0.1-1 pg/mL for water and to 1 pg/mL for food samples.

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 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.

Comparative Analysis of Milk Microbiomes and Their Association with Bovine Mastitis in Two Farms in Central Russia.

Bovine mastitis is a widespread infectious disease. In addition to the economic damages associated with reduced milk yield due to mastitis, the problem of food contamination by microorganism metabolites, in particular toxins, is also a concern. Horizontal transfer of microorganisms from animal populations to humans can also be complicated by antibiotic resistance. Therefore, bovine mastitis is relevant to the study of microbiology and veterinary medicine. In this study, we investigated the microbiome of milk samples from healthy cows and cows with different forms of mastitis from individual quarters of the udder of cows during first and second lactation. Total DNA was extracted from milk samples. The V3-V4 regions of the bacterial 16S rRNA genes from each sample were amplified to generate a library via high-throughput sequencing. We revealed significant dominance of several operational taxonomic units (OTUs) corresponding mostly to groups of Staphylococcus aureus, Aerococcus spp., and Streptococcus spp. In addition, we unexpectedly identified Streptococcus thermophilus in samples with high SCC quantities. We found some infectious agents that characterized summer mastitis. We demonstrated that in Central Russia, mastitis is associated with a wide variety of causal organisms. We observed some differences in the diversity of the two investigated farms. However, we did not find any significant difference among healthy, mastitis and subclinical samples according to their SCC status from either farms by principal component analysis. Linear discriminant analysis effect size (LEfSe) confirmed the presence of several indicator genera in farms from Moscow and the Tula Region. These results confirm the complex bacterial etiology of bovine mastitis.

Cytokinin-binding protein (70 kDa) from etioplasts and amyloplasts of etiolated maize seedlings and chloroplasts of green plants and its putative function.

Cytokinins regulate chloroplast differentiation and functioning, but their targets in plastids are not known. In this connection, the plastid localization of the 70 kDa cytokinin-binding protein (CBP70) was studied immunocytochemically in 4-d-old etiolated maize seedlings (Zea mays L., cv. Elbrus) using monoclonal antibodies (mAbs) against CBP70 recognizing this protein not only in nuclei and cytoplasm, but also in plastids. CBP70 was detected in the amyloplasts of the root cap and etioplasts of the mesocotyl, stem apex, and leaves encircling the stem axis in the node. Immunogold electron microscopy demonstrated CBP70 localization in amyloplasts outside starch grains and revealed a dependence of CBP70 content in etioplasts on the degree of their inner membrane differentiation: the low CBP70 amount in etioplasts at the early stages of membrane development, the high content in etioplasts with actively developing membranes, and a considerable decrease in plastids with the formed prolamellar body. This suggests that CBP70 is involved in etioplast structure development. CBP70 was also observed in chloroplasts of the bundle sheath of green maize leaves. CBP70 purified from etioplasts mediated trans-zeatin-dependent activation of transcription elongation in vitro in the transcription systems of maize etioplasts and barley chloroplasts, suggesting that CBP70 is a plastid transcription elongation factor or a modulator of plastid elongation factor activity. CBP70 involvement in the cytokinin-dependent regulation of plastid transcription elongation could be essential for the cytokinin control of the biogenesis of this organelle.

Virulence Factors and Phylogeny of Staphylococcus aureus Associated With Bovine Mastitis in Russia Based on Genome Sequences.

Staphylococcus aureus is a causative agent of different infectious processes, food poisoning, and autoimmune disorders. The horizontal transfer of pathogenic strains can occur from animal to human under both house and farm conditions, and the spread of strains with antibiotic resistance is an existing problem. In addition to the spread of antibiotic-resistant strains in clinics, this problem also exists in veterinary medicine. It is especially important to monitor antibiotic resistance on farms where antibiotics are the standard treatment of animals, which may trigger the spread of antibiotic-resistant strains among animals and to the human population, and these strains can also be distributed in milk products produced by these farms (milk, cheese, and butter). In this work, we investigated 21 S. aureus isolates using whole-genome sequence analysis and tried to establish a relationship between these isolates with the development of bovine mastitis in seven regions of Western Russia. An S. aureus virulence profile was identified. We identified two groups of S. aureus associated with subclinical mastitis, namely, the enterotoxin-positive and enterotoxin-negative groups. The most prevalent factor associated with bovine mastitis in Russia was cytotoxins, including hemolysins and leukocidins. Multidrug resistance strains were investigated, and antibiotic resistance genes were identified. We identified S. aureus ST 97 type as the most common type in the regions in Western Russia. To the best of our knowledge, this is the first in-depth study of a range S. aureus isolates originating from cattle infections in Russia.

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.

Draft Genome Sequences of Five Staphylococcus aureus Strains Isolated from Clinically Healthy Cows in the Russian Federation.

We present here the draft genome sequences of five Staphylococcus aureus strains isolated from milk samples from clinically healthy cows in the Russian Federation. Four of them were determined to be sequence type 97 (ST-97), and one was determined to be ST-22. All the strains are characterized by their genome possessing genes that code for enterotoxins and cytotoxins.

Immunochemical assay with monoclonal antibodies for detection of staphylococcal enterotoxin H.

Staphylococcal enterotoxins cause food poisoning of various degrees of severity. For milk and meat products, there is a high probability of contamination with staphylococcal enterotoxin H (SEH). In this regard specific and sensitive methods are required to be developed for its detection and monitoring. In this work, the gene seh was expressed and a preparation of recombinant toxin was obtained. Using hybridoma technology, a panel of high-affinity monoclonal antibodies (mAbs) to SEH was produced. The antibodies were characterized and shown to have no cross-reactivity towards the main staphylococcal enterotoxins (A, B, C1, D, E, G and I). Based on these mAbs, a method for specific and quantitative detection of SEH was developed in the format of sandwich enzyme immunoassay (linear range, 0.2-3 ng/ml). All the mAbs produced revealed SEH by immunoblotting. Immunochemical analysis of the culture fluids of staphylococcal isolates obtained from the milk of mastitis-infected cows by immunoblotting and sandwich enzyme immunoassay demonstrated the conformity of these methods. Using the developed method, the toxin was revealed in blood serum and liquid food products practically to 100%. From non-liquid foods, it was shown to be extracted to a maximum with a buffer of pH 4.0-4.5.

The neutralizing human recombinant antibodies to pathogenic Orthopoxviruses derived from a phage display immune library.

A panel of recombinant human antibodies to orthopoxviruses was isolated from a combinatorial phage display library of human scFv antibodies constructed from the Vh and Vl genes cloned from the peripheral blood lymphocytes of Vaccinia virus (VACV) immune donors. Plaque-reduction neutralization tests showed that seven selected phage-displaying scFv antibodies (pdAbs) neutralized both CPXV and VACV, and five of them neutralized Monkeypox virus (MPXV). Western blot analysis of VACV and CPXV proteins demonstrated that seven neutralizing antibodies recognized a 35 kDa protein. To identify this target protein, we produced a recombinant J3L protein of CPXV and showed that all the selected neutralizing antibodies recognized this protein. Neutralizing pdAb b9 was converted into fully human mAb b9 (fh b9), and scFv b9 displayed high binding affinities (K(d) of 0.7 and 3.2 nM). The fh b9 reduced VACV plaque formation in a dose-dependent manner.

Generation of scFv phages specific to Staphylococcus enterotoxin C1 by panning on related antigens.

A method for generation of highly specific miniantibodies within the phage particle has been developed, and used to produce antibodies against Staphylococcus enterotoxin type C1. Under successive panning of the non-immune phage miniantibody (scFv) library with enterotoxins SE (types A, B, C1, D, E, G, and I) adsorbed on the plate surface, we generated 11 individual phage clones to Staphylococcus enterotoxin type C1. Five of them interacted specifically only with SEC1 and had no cross-reactions with the other enterotoxins.