A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis. A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained. The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F. tularensis LVS.

The increasing prevalence of antibiotic-resistant strains of pathogenic bacteria is a major healthcare problem. Antibacterial lysins are enzymes that cleave the peptidoglycan of the bacterial cell wall. These proteins hold potential as a supplement or an alternative to traditional antibiotics since they are active against antibiotic resistant strains. However, antibacterial lysins are rapidly eliminated from the systemic circulation, which limits their application. Dimerization of an anti-pneumococcal lysin Cpl-1 has been demonstrated to decrease the clearance rate of this protein in mice. In the present work, we constructed a dimer of an anti-staphylococcal lysin lysostaphin by fusing it with an anti-parallel α-helical dimerization domain. Lysostaphin dimer had a more favorable pharmacokinetic profile with increased terminal half-life and area under the curve (AUC) values compared to monomeric lysostaphin. However, the staphylolytic activity of dimerized lysostaphin was decreased. This decrease in activity was likely caused by the dimerization; since the catalytic efficacy of lysostaphin dimer towards pentaglycine peptide was unaltered. Our results demonstrate that, although dimerization is indeed beneficial for the pharmacokinetics of antibacterial lysins, this approach might not be suitable for all lysins, as it can negatively affect the lysin activity.

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.

Простая жидкая питательная среда для молекулярно-генетических исследований Francisella tularensis

Получены варианты вакцинного штамма F. tularensis subsp. holarctic a 15 с делетированными генами iglC . Показано, что в геноме штамма 15 содержится две копии гена iglC . Удаление одной копии гена практически не влияет на культурально-морфологические и ростовые свойства микроба. Бактерии без одной копии гена iglC размножаются в мышиных макрофагах медленнее по сравнению с исходным штаммом, в то время как инактивация двух копий гена iglC в штамме 15 приводит к полной потере способности размножаться в макрофагах.

The genomic analysis of all subspecies F. tularensis, as found in Gen Bank NCBI, reveals the presence of genes encoding proteins like to the multifunctional RecBCD enzyme complex in E. coli and other bacteria. To date, the role of the recD gene in F. tularensis, which encodes the alpha chain of exonuclease V, in DNA metabolism processes, has not been studied either in vitro or in vivo. F. tularensis subsp. holarctica 15 NIIEG, a vaccine strain, served as the basis to create the F. tularensis 15D strain with recD deletion. The lack of the recD gene suppresses the integration of suicide plasmids with F. tularensis genome fragments into the chromosome. The modified strain showed reduced growth in vitro and in vivo. This study shows that such deletion significantly reduces the virulence of the strain in BALB/c mice.