To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. A similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with maximally hydrolyzed peptidoglycan had a greater protectivity compared to BGs with a preserved peptidoglycan skeleton.

Background/Objectives: Yersinia pestis is an important zoonotic pathogen responsible for the rare but deadly disease of people with bubonic, septic, or pneumonic forms of plague. The emergence of multidrug-resistant Y. pestis strains has attracted more and more researchers’ attention to the search for molecular targets for antivirulence therapy, including anti-nutritional-virulence therapy. The glnALG operon plays a crucial role in regulating the nitrogen content within a bacterial cell. This operon codes for three genes: the structural gene glnA and the two regulatory genes glnL and glnG. In this study, we tested the effect of the deletion of glnA and glnALG on the pathogenic properties of Y. pestis. Methods: To assess the contribution of nitrogen metabolism to Y. pestis virulence, knockout mutants ΔglnA and ΔglnALG were constructed. The former was unable to synthesize glutamine, while the latter was not only defective in glutamine synthesis but also lacked the two-component sensor–transcriptional activator pair GlnL and GlnG, which could partially compensate for the decrease in intracellular glutamine concentrations by transporting it from the host or by catabolic reactions. For vaccine studies, immunized mice and guinea pigs were injected s.c. with 200 LD₁₀₀ of the wild-type Y. pestis strain. Results: A single knockout mutation in the glnA gene did not affect the virulence of Y. pestis in mice and guinea pigs. Knockout of the entire glnALG gene cluster was required for attenuation in these animals. The ΔglnALG strain of Y. pestis did not cause death in mice (LD₅₀ > 10⁵ CFU) and guinea pigs (LD₅₀ > 10⁷ CFU) when administered subcutaneously and provided 100% protection of animals when subsequently infected with 200 LD₁₀₀ of the Y. pestis virulent wild-type strain 231. Conclusions: Y. pestis, defective in both the glutamine synthetase GlnA and the two-component sensor–transcriptional activator pair GlnL-GlnG, completely lost virulence and provided potent protective immunity to mice and guinea pigs subsequently challenged with a wild-type Y. pestis strain, demonstrating the potential use of the glnALG operon as a new molecular target for developing a safe and efficient live plague vaccine.

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 knoc

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.

The goal of this study is to find out the effect of deletion of some genes encoding biogenesis of components of the outer membrane protein complex on the virulence of Yersinia pseudotuberculosis mutants. Materials and methods. Y. pseudotuberculosis surA, degP, bamB, and ompH mutants were generated by RedGam mutagenesis using suicide vectors. The constructed mutants were characterized by growth rate, sensitivity to antibiotics, bile salts, and sodium dodecyl sulfate, as well as to the bactericidal action of normal human serum. Total composition of the outer membrane proteins of the knockout mutants was determined by SDS-PAGE. The virulence of DsurA, DdegP, DbamB and DompH was studied after subcutaneous, intraperitoneal, or oral infection of mice. Results. It was found that BamB, OmpH and SurA proteins were necessary for maintaining the integrity of the Y. pseudotuberculosis cell membrane, SurA and BamB proteins are important for resistance to the bactericidal action of serum complement and a high degree of attenuation of Y. pseudotuberculosis strains with deleted surA, degP, bamB genes for outbred mice was revealed. Conclusion. The surA, degP, and bamB genes can be used as potential molecular targets for construction of Y. pseudotuberculosis vaccine strains.

The goal of this study is to identify the effect of deletion of some genes encoding biogenesis of outer membrane protein complex components on the virulenc