Vol 33, No 2 (2018)

Maintaining cellular homeostasis in biological systems is the basic principle that ensures the functioning of organs and systems. At the cellular level, this process is regulated by genetically determined efferent signals. During evolution, biological systems developed hierarchically varying mechanisms of molecular genetic regulation of cell death. Numerous studies of extra- and intracellular mechanisms of programmed cell death (PCD) in eukaryotes have shown the complexity and the physiological selectivity of this phenomenon, which has been greatly important in medicine. In prokaryotes, the mechanisms and regulation of cell death have been studied less, but research of them has the great interest to characterize the existence of microbial communities. The aim of this review is to provide molecular genetic characteristics of cell death in prokaryotes based on available data.

A search for genes involved in the molecular mechanisms of atherogenesis and the atheroprotective role of HDL has been carried out to study these processes on the transcriptome level. Bioinformatic analysis of gene expression in peripheral blood of patients with coronary artery stenosis using data (GSE20129, GSE12288, GSE10195, GSE20686) from the GEO genome-wide studies database revealed 947 differentially expressed genes. Of these, 66 genes associated with atherosclerotic coronary artery disease, underlying coronary heart disease (CHD) and myocardial infarction, have been selected. We added to this list the genes for which the association with coronary artery atherosclerosis was determined by the authors of abovementioned genome-wide researches using bioinformatic algorithms (19 genes) or RT-PCR (67 genes), as well as 21 genes involved in HDL metabolism according to Reactome database. Analysis of the resulting list of genes revealed that 60 of them were took part in lipoprotein and lipid metabolism, reverse cholesterol transport, and inflammation. We suggest that these genes are involved in HDL metabolism and atherogenesis in CHD patients. Evaluation of their differential expression in human peripheral blood cells can be applied for further study of the molecular mechanisms of coronary artery atherosclerosis and the atheroprotective role of HDL.

Apart from strains of Vibrio cholerae biovar El Tor imported from cholera-endemic regions, causing outbreaks or sporadic cases of cholera, a significant number of different variants of nontoxigenic strains that can induce acute enteric infections are annually isolated from surface water bodies and human beings in the Russian Federation. We have carried out analysis of whole genomes that we sequenced (29) and obtained from the GenBank database (11), of 40 toxigenic and nontoxigenic strains isolated in cholera nonendemic (Russia, Ukraine, Turkmenistan, Georgia) and endemic (India, Bangladesh, Vietnam, and others) territories. It is established that they fall into three main groups which differ in the composition of mobile elements with pathogenicity and epidemicity genes, both from toxigenic isolates and among themselves. Nontoxigenic strains of the first group are deprived of all intact virulence prophages (CTXφ, RS1φ, TLCφ), pathogenicity (VPI-1, VPI-2) and pandemicity (VSP-I, VSP-II) islands, which testifies to their epidemic safety. The strains of the second group differ from the first one by the presence of the TLCφ prophage and two pathogenicity islands. It is demonstrated that this population of nontoxigenic strains, which is detected only in the Russian Federation and other cholera nonendemic regions, does not pose a potential epidemic hazard as was assumed earlier, despite the availability of tcpA–F genes in VPI-1. The lack of not only the CTXφ prophage but also VSP-I and VSP-II, which we revealed in these strains, eliminates the possibility of their reversion to epidemically hazardous ones. The strains of the third group have opulent similarity to toxigenic strains, being different in the absence of the CTXφ prophage only. It is this group of nontoxigenic strains, isolated only in cholera-endemic territories and capable of acquiring CTXφ through phage conversion, that is potentially epidemically hazardous. The results of whole genome SNP analysis of 58 different strains confirmed these data. We have also shown with the help of model animals (7- to 8-day-old rabbits) that nontoxigenic strains, irrespective of the structure of their genome, cannot cause typical cholera infection. The results provide insight into genomic diversity of nontoxigenic strains, as well as draw us nearer to an understanding of the mechanisms of reversion of their different variants into epidemically hazardous isolates.

Pseudotuberculosis is an acute infectious disease caused by Yersinia pseudotuberculosis. In the 1950 and 1960s, outbreaks of a new, previously unknown disease were registered in the Far East of Russia, called the “Far East scarletlike fever” (FESLF). FESLF is pseudotuberculosis, which has a specific clinical form. The disease is endemic to the Far Eastern, Siberian, and Northwestern regions of Eurasia. At present, there is only one system designed to detect Y. enterocolitis and Y. pseudotuberculosis for diagnostics by polymerase chain reaction. The disadvantage of this system is the lack of the possibility for the specific detection of Y. pseudotuberculosis. In this work, 35 strains of Y. pseudotuberculosis were characterized by the detection of the plasmid gene encoding the adhesin (yadA), MLST typing, and detection of the chromosomal gene of the Y. pseudotuberculosis—the cytotoxic necrotizing factor (cnfY). Detection of the yadA gene correlated with presence of the plasmid (pYV). The strains belonged to five MLST sequence types (STs), one of which, ST240, was described for the first time. All the studied strains of Y. pseudotuberculosis showed the presence of the cnfY gene. Additionally, the results of 68 previously described Y. pseudotuberculosis strains were analyzed. A total of 103 strains showed two allelic variants. Allele 1 was found in all strains isolated from patients with FESLF. The cnfY gene was not specifically associated with the presence of plasmids. The cnfY gene was absent in the Y. enterocolitica strains. Thus, the cnfY gene is a stable and specific marker of Y. pseudotuberculosis. On the basis of the obtained data, a PCR diagnostic system was developed for the detection and intraspecific differentiation of Y. pseudotuberculosis by determining the cytotoxic necrotizing factor gene in the biomaterial.

Live attenuated influenza vaccine (LAIV) is an effective measure to prevent influenza infection. The vaccine strain for LAIV is derived by reassortment of epidemic influenza strain with cold-adapted master donor virus (MDV) in developing chicken embryos. Molecular analysis of genomic composition of generated reassortants to select appropriate candidate for LAIV is a critical stage of vaccine strain development. Method of analysis permitting quick and precise detection of the genome composition of reassortants is required. The purpose of this study was to develop pyrosequencing-based assay using PyroMark Q24 for analysis of the origin of genes in preparing reassortant LAIV candidates for influenza A vaccine strains based on A/Leningrad/134/17/57 master donor virus and influenza B vaccine strains based on B/USSR/60/69 or B/Leningrad/14/77/55 master donor viruses, respectively. Objectives included development of primers set for genotyping of influenza A and B reassortants by pyrosequencing, establishing optimal dispensation order and assessment of effectiveness of protocol application for detecting the source of genes in virus samples. We have shown that a unified genome analysis protocol using PyroMark Q24 system is able to detect the source of internal genes in reassortant influenza strain by pyrosequencing. It allows analyzing the genome composition by a sensitive and specific innovative method in the first stages of vaccine strain development.

This study was aimed at MALDI-TOF identification of protein markers of V. cholerae toxicity for express diagnostics of the causative agent of cholera. The MALTDI-TOF MS electronic passports of 140 V. cholerae strains that were divided into two main groups with the presence or absence of the gene of cholera toxin (ctx– and ctx+) were subjected to computer analysis. For the first time, we have identified a taxon, a specific 3202 Da protein peak, that is a characteristic marker of V. cholerae ctx+, but not of V. cholerae ctx– strains.

Canine parvovirus 2 (CPV-2) is the causative agent of myocarditis and acute hemorrhagic enteritis in dog populations. In this study, vaccinal strains and sixty wild strains (CPV-2a, CPV-2b) of CPV-2 from fecal samples of enteric (30 strains) and healthy (30 strains) dogs detected by Duplex-PCR, were compared together by application of RE mapping based on a partial sequence of VP2 gene (681 bp) by DdeI endonuclease. Our results showed that both CPV-2a and CPV-2b are pathogenic for dogs, but CPV-2a are more prevalent in healthy dogs while CPV-2b strains are more prevalent in enteric cases, significantly (p < 0.05). RE mapping based on DdeI restriction endonuclease demonstrated homology in the nucleotide sequence in amplified part of VP2 gene region in the genome of vaccinal and wild strains of CPV-2 detected in fecal samples of tested animals.