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.

Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin.

Caveolin-1 is a cholesterol-binding scaffold protein, which is localized in detergent-resistant membrane (DRM) rafts and interacts with components of signal transduction systems, including visual cascade. Among these components are neuronal calcium sensors (NCSs), some of which are redox-sensitive proteins that respond to calcium signals by modulating the activity of multiple intracellular targets. Here, we report that the formation of the caveolin-1 complex with recoverin, a photoreceptor NCS serving as the membrane-binding regulator of rhodopsin kinase (GRK1), is a redox-dependent process. Biochemical and biophysical in vitro experiments revealed a two-fold decreased affinity of recoverin to caveolin-1 mutant Y14E mimicking its oxidative stress-induced phosphorylation of the scaffold protein. At the same time, wild-type caveolin-1 demonstrated a 5-10-fold increased affinity to disulfide dimer of recoverin (dRec) or its thiol oxidation mimicking the C39D mutant. The formation of dRec in vitro was not affected by caveolin-1 but was significantly potentiated by zinc, the well-known mediator of redox homeostasis. In the MDCK cell model, oxidative stress indeed triggered Y14 phosphorylation of caveolin-1 and disulfide dimerization of recoverin. Notably, oxidative conditions promoted the accumulation of phosphorylated caveolin-1 in the plasma membrane and the recruitment of recoverin to the same sites. Co-localization of these proteins was preserved upon depletion of intracellular calcium, i.e., under conditions reducing membrane affinity of recoverin but favoring its interaction with caveolin-1. Taken together, these data suggest redox regulation of the signaling complex between recoverin and caveolin-1. During oxidative stress, the high-affinity interaction of thiol-oxidized recoverin with caveolin-1/DRMs may disturb the light-induced translocation of the former within photoreceptors and affect rhodopsin desensitization.

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.

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.

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.

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.