The aim of this work was to determine the role of periplasmic chaperone SurA in the pathogenesis of bubonic and pneumonic plague. Yersinia pestis surA knockout mutants were generated by RedGam mutagenesis with the use of a suicide vector. The generated strains were characterized by growth rate and susceptibility to antibiotics, bile acid salts, and sodium dodecyl sulfate, as well as to the bactericidal action of normal human serum. The intracellular localization of the SurA protein was determined by SDS-PAGE and immunoblot methods. The virulence of the ΔsurA mutant was studied in subcutaneously or intranasally infected mice or rats. The SurA-negative Y. pestis strain was characterized by an increase in the permeability of the outer membrane and susceptibility to antibiotics, as well as a decrease in virulence in a model of bubonic and pneumonic plague in two laboratory animal species. The SurA protein can be considered as a molecular target for plague therapy. Reducing the virulence of a strain and its resistance to antibiotics by disrupting the biogenesis of outer-membrane proteins may become a new strategy for solving the problem of combating multidrug resistance, and the widespread distribution of the protein among Gram-negative pathogens makes its use promising for the development of virulence inhibitors with a broad spectrum of action.

Designing of new means for the specific prevention of plague, especially protein subunit vaccines, is impossible without studying the role of individual antigens in the manifestation of the pathogenic and immunogenic properties of Yersinia pestis . The aim of the present study was to determine the antibody levels to Y. pestis antigens in guinea pigs that survived infection with sub-lethal doses of virulent plague agent strains using enzyme immunoassay (ELISA). Materials and methods. Guinea pigs were inoculated subcutaneously with 30 CFU of the wild type Y. pestis subsp. Pestis strain 231 or non-capsular Y. pestis subsp. pestis Caf1-negative strain 358/12. Blood samples from sick or recovered guinea pigs were collected on day 15, 30, 60, and 90 after infection. The antibody response was assessed by 18 recombinant Y. pestis proteins in ELISA. Results and discussion. Heterogeneity of the antibody responses to the majority of the antigens with variation of IgG titers from animal to animal has been revealed. We observed increase in antibody titers by day 90 for the most analyzed antigens in the sera of the guinea pigs injected with wild type Y. pestis 231. On the contrary we found reduction in antibody titers by day 90 in case of inoculation with Y. pestis 358/12. The preservation of antibodies to Y. pestis proteins of different localization in the organism of the guinea pigs, as well functional activity, and the degree of representation on the surface of bacterial cell for a prolonged period of time indicates the multiplex nature of the plague immunity formation. Our findings are significant for the future design and development of effective vaccines against plague and the search for new targets for diagnostics of this disease.

The vaccine strain F. tularensis 15 NIIEG induces long-lived cell-mediated immunity but exhibits a certain reactogenicity and genetic instability. Progress in development of a vaccine against tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. The BALB/c mouse is the most commonly used animal to study tularemia due to its relatively low cost, well-characterized genetics, available immunological tools and mouse infection with virulent F. tularensis recapitulates human disease. CD4 + and CD8 + T cells are known to be critical for the formation of protective immunity but the relative roles of memory T cell subpopulations in long lived protection against virulent strains of F. tularensis are not well established. We hypothesized that this immunity depends on central (T CM ) and effector memory (T EM ) T cells and their functional activity. In this study we have dissected the T cell immune response in BALB/c mice 30, 60 and 90 days after subcutaneous vaccination with 15 NIIEG. Multiparametric flow cytometry were used to characterize in vitro recall responses of splenocytes to F. tularensis antigen. T EM cells were identified as CD3 + CD4 + CD44 + CD62L - and CD3 + CD8 + CD44 + CD62L - , T CM cells as CD3 + CD4 + CD44 + CD62L + and CD3 + CD8 + CD44 + CD62L + , respectively. The functional activity of memory T cells was assessed by the following parameters: the level of expression of the activation marker CD69 and cytokine-producing activity by staining with the intracellular cytokines IFNg and TNFa. Thus, development of a long-lived vaccine directed against F. tularensis is dependent on identifying not only the correlates of immunity present early after vaccination, but also those that persist in the host after the effector phase has ended. The maintenance of long-term protective immunity initiated by vaccination with F. tularensis strain 15 NIIEG has been shown to require the presence of antigen-specific CD4 + and CD8 + memory T cells producing IFNg and TNFa and expressing the activation marker CD69. A decrease in count and functional activity of CD8 + T CM and CD8 + T EM was detected in the long term after vaccination. The detected parameters of functional activity of memory T cells can be used as criteria for evaluation of protective immunity against virulent strains of F. tularensis .

The causative agent of plague, Yersinia pestis, is classified as pathogenicity (hazard) group I agent, which means that the work with “wild type” strains should be carried out in BSL-3 facilities. Y. pestis EV NIIEG is a Δpgm strain, allowing experimental studies to be carried out in BSL-2 laboratories. However, the disease and its progression elicited by such strain do not entirely mirror the infection observed with fully virulent strains. Residual virulence of Y. pestis EV NIIEG strain for mice can be increased under in vivo iron supplementation. The aim of the study was to optimize methodological approaches to modeling experimental plague in laboratory animals following administration of attenuated Δpgm Y. pestis strains with iron dextran. Materials and methods. Simulation of plague infection in outbred mice was carried out through subcutaneous inoculation of Y. pestis EV NIIEG strain with iron dextran supplementation. The animal condition was assessed on a daily basis. In the course of the experiment, the pathological presentation and bacterial content in organs of mice were evaluated. Results and discussion. Mice inoculated subcutaneously with Y. pestis EV NIIEG strain in the presence of iron dextran developed a bubonic plague that resulted in lethal outcome with pathological changes of internal organs, characteristic of plague infection. In case of daily administration of iron, LD50 of Y. pestis EV NNIEG strain for the mice significantly exceeded the same one with a single injection. Differences in the survival rate among animals in the groups with a single and multiple administration of iron compared to the control group were statistically valid. Thus, attenuated Δpgm Y. pestis strains in the presence of iron dextran can be used to model experimental plague in mice with marked pathological changes and lethality in BSL-2 laboratories.

The purpose of this work was to obtain genus-specific monoclonal antibodies against the Legionella spp. recombinant PAL protein, which will subsequently allow to use them as a basis for the development of new express tests for pathogenic legionella detection. A short three-week immunization protocol for Wistar rats was used to generate rat-mouse heterohybridomas producing antibodies against PAL. Mouse myeloma cell line Sp2/0-Ag14 served as the fusion partner. Hybridization was performed using two methods: PEG-mediated fusion and electrofusion. Subsequent screening was performed by indirect solid-phase ELISA against the target protein rPAL. Specificity analysis was performed by dot-blot using a panel of lysates obtained from 39 pure cultures of different strains, which included closely related and heterologous microorganisms among others. No difference in the efficiency of stable hybridoma clones production by the two indicated cell-fusion methods was detected. Twelve clones producing specific rat monoclonal antibodies were obtained based on the screening results. The obtained rat monoclonal antibodies are highly specific towards the PAL protein of L. pneumophila of different serological groups and other pathogenic legionella and are good candidates to be used as the components of diagnostic test systems for the detection of pathogenic representatives of the Legionella genus.

It is known that the body's defense against infection by the intracellular bacterium Francisella tularensis is provided by the activation of the cellular and humoral immune response. However, their role in long-term protection (25 years and more) against virulent strains of F. tularensis is not well understood. The identification of clear criteria for assessing protective immunity to the tularemia causative agent at different times after vaccination will make it possible to more efficiently develop new genetically determined vaccine strains. The goal of our research was to select and assess immunological parameters reflecting the protective properties of the vaccine strain F. tularensis 15 NIIEG and its derivatives, F. tularensis 15/23-1∆recA and F. tularensis 15/ 23-1/sodB∆recA, in the long term after immunization. To assess the functional activity of T and B cells, flow cytometry was used.The assessment of the production of cytokines IFN-γ, IL-4, IL-10, IL-17A and titers of specific class G immunoglobulins to F. tularensis lipopolysaccharide (LPS)in blood serum was performed by ELISA on days 30, 60, 90 and 180 after immunization. Evaluation of the protective properties of vaccine preparations in the above-mentioned terms was carried out after subcutaneous infection with test-infecting virulent strains, Schu and 503 of tularensis and holarctica subspecies, respectively. It was shown that vaccination with the studied strains in 100% of cases protected from infection with the strain 503 of the holarctica subspecies, analogous to the vaccine strain. When infected with a virulent Schu strain of the hetrologous tularensis subspecies, a decrease in the effectiveness of protection was observed starting from 60 days after immunization. Evaluation of immunological parameters showed that at all studied periods after immunization, IgG antibodies to F. tularensis LPS were detected in the blood sera of immunized mice. In vitro experiments on stimulation of immune response in spleen lymphocytes of vaccinated mice to the F. tularensis antigen showed a significant increase in the level of secreted IFN-γ, a slight increase in secreted IL-10 and an enhanced expression of the CD69 molecule on the surface of T and B cells. Thus, the level of IFN-γ and the expression of the CD69 molecule on the surface of T and B cells in response to restimulation of lymphocytes of immune animals with tularemia antigen can serve as criteria for immune protection in experimental tularemia in a mouse model at different times after vaccination. Key words: vaccine strain, Fransicella tularensis, immunogenicity, protection, memory T cells, IgG, cellular immunity Funding - The work was supported by the Branch Program of the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing.

High efficiency of a combined preparation including synergistic polymyxin B and 4-hexylresorcinol was shown for treatment of experimental sepsis caused by an antibiotic-resistant highly virulent hypermucoid Klebsiella pneumoniae strain KPM9Pmr in mice. Complex therapy with polymyxin B (1 mg/kg) and 4-hexylresorcinol (30 mg/kg) led to cure in 80%; in 20% of these mice, no bacterial cells were found. After treatment with polymyxin B alone, only 50% animals survived and all of them contained bacterial cells. Comparative analysis of the results of monotherapy and combined treatment indicates that 4-hexylresorcinol not only increases the efficiency of antibiotic, but also minimizes persistence of the infection agent and therefore, the risk of development of antibiotic resistance.

In recent years, bacteriophages have been widely used to prevent and to treat various bacterial infections in humans. Lytic phages are often the only effective tool to combat multi-antibiotic resistant microorganisms. The high specificity of the bacteriophages allows avoiding negative effects on the human microbiome, making them more therapeutically attractive compared to broad-spectrum antibacterials. In our work, we evaluated therapeutic and prophylactic effects of bacteriophage Pm3 by using two models of Proteus-associated infection in mice, lethal sepsis and skin burn wound infection. Our results showed that a single preventive intraperitoneal administration of the phage at 108 PFU saved 90% of animals infected with a lethal dose of Proteus mirabilis M32. The course treatment of generalized Proteus infection initiated 1.5 hours after infection protected 80% of mice from death and was comparable to antibiotic therapy (90% therapeutic effect). Parenteral and external use of bacteriophage Pm3 for treating Proteus infection of burns showed a weak therapeutic effect manifested as decreased contamination of the wound surface with P. mirabilis M32 cells and prevention of infection invasion in some animals. Ciprofloxacin applied as a wound irrigation solution or subcutaneously gave a pronounced therapeutic effect: the wound surface and deep skin layers were sanitized in 100% of cases, and Proteus culture in the spleen and kidneys of the animals was absent. Thus, in vivo antibacterial activity of bacteriophage Pm3 was close to that of ciprofloxacin, but in preventing and treating Proteus-associated sepsis only. The phage is low effective in treating burn Proteus infection. Ciprofloxacin is highly effective in treating both Proteus-associated sepsis and skin thermal wound infections caused by P. mirabilis M32 in mice. Keywords: Proteus mirabilis, Proteus infection, sepsis, burn infection, mouse model, bacteriophage, phage therapy, ciprofloxacin

HtpG (high-temperature protein G) is a bacterial homologue of the highly conserved molecular chaperone Hsp90 of eukaryotes, which plays an important role in protection against stress in many bacterial species. The role of the htpG gene encoding the synthesis of high-temperature prokaryotic G protein in the pathogenesis of bacterial infections is still unclear.The aim of this work is to study the functional importance of HtpG in the pathogenesis of plague.Materials and methods. Isogenic Yersinia pestis sets based on attenuated and virulent strains differing in the presence of the functional htpG gene (YPO3119) were generated with the help of site-directed mutagenesis. The HtpG amino acid sequence was analyzed using the BLAST program. The properties of the resulting mutant strains were evaluated using microbiological and biological methods.Results and discussion. The bioinformatics analysis showed high conservativeness of the HtpG protein within the Y. pestis species (100% identity), as well as 99 % identity with the Y. pseudotuberculosis protein and 96 % identity – Y. enterocolitica protein. Y. pestis htpG knock-out mutants showed increase of susceptibility to temperature and oxidative stress like mutants of the other bacterial species. However, the mutant was not sensitive to osmotic stress and human serum complement. The loss of the ability to synthesize HtpG by plague microbe did not affect the virulence and average life duration of mice and guinea pigs challenged subcutaneously. It means that htpG gene is not a good molecular target for the treatment and/or immunoprophylaxis of plague.

Francisella tularensis is an intracellular bacterium that causes tularemia. Progress in creating a safe and effective vaccine for the prevention of tularemia is challenging due to a lack of knowledge about immunological parameters indicative of protective adaptive immunity. Objective of the research was to assess the effect of modifications of the F. tularensis 15 NIIEG genome on the immunogenic and protective properties of F. tularensis 15/23-1ΔrecA and F. tularensis 15/23-1/sodBΔrecA strains. Materials and methods. Multi-parameter flow cytometry and the measurement of secreted cytokines were used to characterize the responses of mouse spleen lymphocytes in response to re-stimulation of F. tularensis with acid-insoluble complex (AIC) in vitro. Also, the titers of specific antibodies to F. tularensis lipopolysaccharide in blood serum were analyzed by enzyme-linked immunosorbent assay. Results and discussion. It has been shown that immunization with the studied strains led to a significant increase in CD4+ and/or CD8+ T cells capable of expressing functional markers: CD69, CD25 and/or CD28; an increase in the subpopulation of T-helpers synthesizing IFN-γ. In the body of immune mice, a pool of B-lymphocytes was formed, capable of secreting IFN-γ in response to their stimulation with AIC. Immunization with the strain 15/23-1/sodBΔrecA provided 70% protection in mice from intranasal infection with a virulent strain of F. tularensis SchuS4. More pronounced protective properties were associated with the activation of not only B-lymphocytes and T-helpers, but also with the simultaneous activation of cytotoxic T-lymphocytes.

Tularemia is an anthropozoonotic infection caused by Francisella tularensis. In clinical and sanitary-epidemiological practice, traditional diagnostics methods in tularemia are based on serological assays for detecting specific antibodies, allowing to diagnose it and estimate durability of patients’ immunity after vaccination. Previously, it was shown that specific serum antibodies in patients recovered after tularemia, unlike to those vaccinated with the live tularemia vaccine F. tularensis 15 NIIEG, can interact with specific epitopes on lipopolysaccharides isolated from strains of various subspecies — F. tularensis (Ft) and F. novicida (LPS Fn), while LPS Fn-specific immunoglobulins are lacked in the blood of vaccinated individuals. A set of experiments on identifying antibodies with similar specificity in laboratory animals of various species — mice, guinea pigs and rats with differed sensitivity to tularemia, administered with live tularemia vaccine strain as well as virulent F. tularensis strains to simulate vaccine-mediated and infectious processes, respectively was conducted. A methodical approach has been developed that allows to analyze humoral response in modelled infectious process in animals highly sensitive to tularemia such as BALB/c mice and guinea pigs that consisted of preliminary immunization with live tularemia vaccine followed by infection with virulent F. tularensis strains. It was shown that induction of specific anti-LPS Ft antibodies occured in these animal species, both after vaccination and infection with virulent strains. It was noted that, unlike guinea pigs and rats, mice both during vaccination and infection were characterized by significantly lower titers of LPS Ft-specific antibodies. However, no specific interaction between mouse serum and LPS Fn might be detected. Moreover, two types of immunoglobulins with different antigen specificities to the LPS Ft and LPS Fn epitopes were detected by dot-blot analysis in guinea pigs immunized with live tularemia vaccine, followed by infection with a virulent strain. In addition, antibodies to LPS Fn were also detected in the serum of rats infected with virulent, but not vaccine-based, F. tularensis strains. Thus, previous experimental data on the production of immunoglobulins with different antigenic specificity were confirmed in an experimental tularemia modelled in rats and guinea pigs that demonstrated a diagnostic significance and feasibility of using LPS Fn to confirm tularemia infection in humans.

Electrophoretic, homogeneous, highly purified preparations of various isoforms of the immunodominant antigen V (LcrV protein) of Yersinia pestis have been obtained via chromatographic purification. A significant similarity in the chromatographic profiles of the LcrV antigen isoforms of different strains with tryptophan (W113) and glutamic acid (E113) residues at position 113, which differed from the isoform profile containing glycine residue LcrV (G113), has been recorded. The latter is characterized by the highest antigenic and protective activity as compared with other protein isoforms. This isoform almost completely protected the immunized group of animals and had the greatest ability to induce the production of antibodies, the titers of which reached 1 : 250000.