The causative agent of plague, Yersinia pestis, is a highly virulent bacterial pathogen and a potential bioweapon. Depending on the route of infection, two prevalent forms of the disease – bubonic and pneumonic, are known. The latter is featured by a high fatality rate. Mortality in untreated bubonic plague patients reaches up to 40-60%, whereas untreated pneumonic plague is always lethal. The development of the infectious process in susceptible host is accounted for by a whole set of pathogenicity factors in plaque pathogen displaying various functional modalities being expressed depending on stage of infectious process, providing their coordinated expression. Knocking out any of such factors, in turn, may not either affect microbe virulence or lead to its attenuation. A search for new Yersinia pestis pathogenicity factors and subsequent development of highly effective subunit and live attenuated plague vaccines inducing development of pronounced cellular and humoral immune reactions, and/or assessment of their potential use as molecular targets for plague therapy still remain a pressing issue, as both currently licensed plague vaccines do not meet the WHO requirements, whereas strains of plague microbe isolated in Madagascar are resistant to all drugs recommended for plague antibacterial therapy. Here we summarize an impact of described and newly discovered pathogenicity factors into the virulence of Y. pestis strains and their protective anti-plague activity. An effect of loss of genes encoding regulatory proteins as well as mutations in the genes for various transport systems of Y. pestis on attenuation of virulent strains is described as well. Perspectives for introducing characterized antigens into prototype subunit vaccine as well as some other obtained mutants into prototypes of living attenuating vaccines were assessed. The use of antibiotics for plague treatment has been embraced by the World Health Organization Expert Committee on Plague as the ‘gold standard’ treatment. However, concerns regarding development of antibiotic-resistant Y. pestis strains accounted for further exploring alternatives to plaque therapy. Several research groups continue working on seeking for other alternative approaches, e.g. treatment with inhibitors of pathogenicity factors. Preliminary data attempting to treat plague patients with pathogenicity factor inhibitors are summarized. Аnti-virulence drugs targeting key microbial factors represent new promising therapeutic options in fight against antibiotic-resistant bacteria.
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.
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. 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 peptidoglycan maximally hydrolyzed had a greater protectivity compared to BGs with preserved peptidoglycan skeleton.
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.
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