S. A. Ivanov

Flexible, fast and inexpensive vaccine development and production technologies are needed to prevent the spread of future epidemics and meet the need for v

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

INTRODUCTION Flexible, fast and inexpensive vaccine development and production technologies are needed to prevent the spread of future epidemics and meet the need for vaccination. The purpose of this study was a comparative assessment of two methods of isolation and purification of the main component of plague vaccines — capsule antigen F1. MATERIALS AND METHODS To create an expression vector, the caf operon was cloned at the EcoRI site in the pesticinogenicity plasmid pPst of the plague microbe. The constructed plasmid pPKB-F1 was introduced by cryotransformation into the Y. pseudotuberculosis 11M strain. F1 capsule antigen preparations were obtained from the culture liquid by precipitation at the isoelectric point or salting out with ammonium sulfate. RESULTS The recombinant strain Y. pseudotuberculosis 11M/pPKB-F1 had the ability to secrete capsular antigen, and the level of production and serological activity of the protein were higher than that of the vaccine strain Y. pestis EV NIIEG. The safety and immunogenicity of the recombinant capsular antigen when administered subcutaneously to mice, as well as protection against infection with massive doses of a virulent strain of the plague microbe, were shown. CONCLUSION Y. pseudotuberculosis strain 11M/pPKB-F1 is an effective producer of capsular antigen F1. The highly purified F1 antigen preparation surpasses the preparation isolated at the isoelectric point in protective properties and may be a potential component in the development of a subunit vaccine against plague.

Goal. Determination of the role of periplasmic chaperone SurA in the pathogenesis of bubonic and pneumonic plague. MATERIALS AND METHODS Yersinia pestis surA knockout mutants were generated by RedGam mutagenesis with the use of a suicide vector. The generated strains were characterized by their growth rate, sensitivity to antibiotics, bile acid salts and sodium dodecyl sulfate, as well as to the bactericidal action of normal human serum. Intracellular localization of the SurA protein was determined by DSN-PAAG and immunoblot methods. The virulence of the ΔsurA mutant was studied in subcutaneously or intranasally infected mice or rats. RESULTS The SurA-negative Y. pestis strain was characterized by an increase in the permeability of the outer membrane and sensitivity to antibiotics, as well as a decrease in virulence in a model of bubonic and pneumonic plague in two laboratory animal species. CONCLUSION 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.

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.