The effect of the osmotic pressure of the culture medium on cells of Francisella tularensis vaccinal strain 15

The dependence of the growth rate of F. tularensis on the osmotic properties of the medium can be presented as a curve with the maximum in the area of 500-600 mOsm. Under these circumstances the intracellular osmotic pressure exceeds the extracellular one by 50-100 mOsm. With the rise of the osmotic pressure in the medium the increase of the concentration of K+ in the cells occurs. The energy-dependent accumulation of K+ in the cells at rest is activated by the rise of the osmotic pressure in the medium. F. tularensis are probably capable of osmoregulation, ensured by the energy-dependent osmosensitive K(+)-transporting system.     

The dynamics of antibody formation to a vaccinal strain of Francisella tularensis in different immunization regimens

The use of different schemes of albino mice immunization either by living or by killed preparations of the vaccine strain of Francisella tularensis when obtaining monoclonal antibodies to the tularemia microbe made it possible to reveal definite regularities in the dynamics of antibody formation. The highest titres of antibodies in sera of animals-donors of splenocytes were obtained during the daily (for 3 days) intraperitoneal immunization of mice with living vaccine or with its thrice administration to the spleen thrice with the interval of 10 days. Revaccination against a background of high titres of antibodies decreased their quantity in blood serum of mice, while that against a background of low titres increased them.     

The C antigen of Francisella tularensis]

A new envelope antigen C, specific for virulent strains of Francisella tularensis, was revealed by immunodiffusion analysis. In contrast to antigens A and P this antigen is common for Francisella and Brucella. C-antigenic lipid fraction was obtained by chloroform-ethanol (1:1) extraction of bacterial slime. This fraction contained carbohydrates (31.6%) without proteins and detected by TLC glycolipid, which proved glycolipid nature of C-antigen. Introduction of C-fraction or alive F. tularensis resulted in accumulation of C. precipitins in blood serum.     

Comparative characteristics of biological properties of Francisella tularensis strains isolated in the USSR]

With a large collection of the strains of F. tularensis isolated it has been recently shown that cultures belonging to holarctica and mediaasiatica circulate in the endemic foci of the USSR. By their biological and genetic properties the natural strains of F. tularensis were homogeneous and represented type cultures of F. tularensis. Various ecological conditions in the natural environment did not change within the last 20 years the sensitivity of the tularemia microbe to the antibacterial drugs.     

The ultrastructural characteristics of Francisella tularensis interaction with Tetrahymena pyriformis

The electron-microscopic study of the interaction of F. tularensis virulent and attenuated strains with infusoria of the species T. pyriformis was dynamically studied. In this study the structural changes of F. tularensis and T. pyriformis cells, as well as their capacity for survival, were revealed. The data on their ultrastructure correlated with the dynamics of the number of both F. tularensis and T. pyriformis: during the whole term of observation the tendency to a slow decrease in the number of F. tularensis was registered with the concentration of T. pyriformis remaining stable. The interaction of F. tularensis with T. pyriformis may be regarded as a variant of commensal, but not antagonistic interactions.     

The LPS-protein complex from the outer membrane of Francisella tularensis]

LPS-protein complex containing proteins of 15 kD, 17 kD and 19 kD was isolated from F. tularensis outer membrane by solving with sodium deoxycholate with the subsequent gel filtration on Sephacryl S-200. Protein of 17 kD constituted the main protein component of the complex. The LPS-protein ratio of this complex was 1:1. Proteins contained in LPS-protein complex have mainly the alpha-spiral structure. In the absence of detergent these proteins and LPS formed micelles with molecular weight exceeding 10(7) D. LPS-protein complex was shown to have a protective effect in mice infected with F. tularensis virulent strain 503.     

Kinetics of macrotetrolide-induced ion transport across lipid bilayer membranes

Ion transport across lipid bilayer membranes in the presence of macrotetrolide antibiotics has been studied by stationary conductance and nonstationary relaxation methods. The results are discussed on the basis of a carrier model which has already been successfully applied to valinomycin induced ion transport. Again a kinetic analysis has been performed from which the single rate constants of the carrier model could be derived. In addition the equilibrium constant of complex formation in the aqueous phase could be determined. Measurements have been made for 4 macrotetrolides, for several ions and for various chain lengths of the lipids molecules composing the membrane.     

An electron microscopic study of the intercellular contact of a vaccinal strain of Francisella tularensis with gram-negative and gram-positive bacteria

The use of transmission electron microscopy (the negative contrast and ultrathin section techniques) has made it possible to show that F. tularensis vaccine strain is capable, under normal conditions and in mixtures with other gram-negative and gram-positive bacteria, of forming cell aggregations with close contacts between cells, this contact being probably irreversible. The ultrastructure of bacteria taking part in the formation of intercellular contacts remains intact.     

Biomimetic polyesters and their role in ion transport across cell membranes

Syntheses of biomimetic low-molecular weight poly-(R)-3-hydroxybutanoate mediated by three types of supramolecular catalysts are presented. The utility of these synthetic polyesters for preparation of artificial channels in phospholipid bilayers capable of sodium and calcium ion transport across cell membranes, is discussed. Further studies on possible applications of these bio-polymers for manufacturing drugs of prolonged activity are under way.     

Direct measurement of ferrous ion transport across membranes using a sensitive fluorometric assay

The fluorophore, Phen Green SK (PGSK), was assessed for its suitability to be used in an assay for ferrous ion transport into membrane vesicles. The long wavelengths of excitation and emission (506 and 520 nm, respectively) enable PGSK fluorescence to be detected in membranes, such as the chloroplast inner envelope, that contain high levels of carotenoids which absorb light at lower wavelengths. At low concentrations of Fe2+, less than 3 microM, the interaction between PGSK and Fe2+ appears to result in both static and dynamic quenching of the PGSK fluorescence. The characteristics of this quenching were used to develop a calibration curve to determine the concentration of free Fe2+ at these low concentrations. Pronounced quenching of PGSK fluorescence entrapped within chloroplast inner envelope membrane vesicles was observed when Fe2+ was added. The extent of quenching of PGSK fluorescence trapped inside asolectin vesicles on Fe2+ addition was much less. The kinetics of the quenching of PGSK fluorescence by Fe2+ in vesicles was quite different from that for PGSK and Fe2+ in solution. Using the calibration curve developed for interaction of PGSK and low Fe2+ concentrations the initial rates of iron transport could be determined for the chloroplast inner envelope membranes.     

Activation energy of sulfate ion transport across methylated human erythrocyte membranes

Activation energy EA of the sulfate ions transport process across human erythrocyte membranes modified by reductive methylation has been measured. It has been found that exhaustive reductive methylation (3 times) with formaldehyde and borohydride inhibits the sulfate-equilibrium exchange, by a maximum of about 40%. However, methylation has no measurable effect on activation energy, since the evaluated EA values for control and methylated cells remain the same within the experimental error range.     

The preventive activity of monoclonal antibodies specific to the lipopolysaccharide of Francisella tularensis]

The preventive activity of five monoclonal antibodies (McAb) in experimental tularemia was evaluated. McAb produced by hybridoma FB11-k (IgG2a), specific to F. tularensis lipopolysaccharide, prevented the death of mice and guinea pigs infected with F. tularensis virulent strain 503 of the holarctic subspecies.     

Features of the interaction of Escherichia coli and Francisella tularensis RNA polymerases with hybrid plasmids bearing fragments of Francisella tularensis chromosomal DNA]

Hybrid plasmids containing the fragments of Francisella tularensis chromosomal DNA and capable of tet-gene expression both in Escherichia coli and Francisella tularensis cells were constructed. The regions of francisella chromosomal DNA binding the RNA-polymerases of Escherichia coli and Francisella tularensis were found by the electron microscopy technique. Interconnection of those regions with the expression of tet-gene of the hybrid plasmids was demonstrated.     

Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis

Glycoconjugate-based vaccines have proved to be effective at producing long-lasting protection against numerous pathogens. Here, we describe the application of bacterial protein glycan coupling technology (PGCT) to generate a novel recombinant glycoconjugate vaccine. We demonstrate the conjugation of the Francisella tularensis O-antigen to the Pseudomonas aeruginosa carrier protein exotoxin A using the Campylobacter jejuni PglB oligosaccharyltransferase. The resultant recombinant F. tularensis glycoconjugate vaccine is expressed in Escherichia coli where yields of 3 mg l⁻¹ of culture were routinely produced in a single-step purification process. Vaccination of BALB/c mice with the purified glycoconjugate boosted IgG levels and significantly increased the time to death upon subsequent challenge with F. tularensis subsp. holarctica. PGCT allows different polysaccharide and protein combinations to be produced recombinantly and could be easily applicable for the production of diverse glycoconjugate vaccines.     

Role of gangliosides in the processes of calcium ion transport across the membranes of nerve cells

It has been demonstrated that the content of extracellular Ca in the nervous system is inversely related to the content of gangliosides. The results obtained on invertebrates, lower and higher vertebrates indicate that the highest content of extracellular Ca is typical of the nervous tissue of invertebrates, whereas the lower one--of the nervous tissue of higher vertebrates (mammals). Ganglioside content, on the contrary, is the highest in the brain tissue of the higher vertebrates (mammals and birds), being significantly lower in lower vertebrates; no gangliosides was found at all in the nervous tissue of protostomian invertebrates. The highest ganglioside content in the organism of vertebrates is characteristic to the surface membranes of the nervous cells, especially in the region of synapses. Functional significance of the inverse relationship between the content of extracellular Ca and gangliosides is discussed from the standpoint of one of the authors (R. Veh) who postulated the existence of calcium--ganglioside buffer in the vicinity of the surface of the nervous cells.     

Characterization and application of a glucose-repressible promoter in Francisella tularensis

Francisella tularensis, the causative agent of tularemia, is a category A biodefense agent. The examination of gene function in this organism is limited due to the lack of available controllable promoters. Here, we identify a promoter element of F. tularensis LVS that is repressed by glucose (termed the Francisella glucose-repressible promoter, or FGRp), allowing the management of downstream gene expression. In bacteria cultured in medium lacking glucose, this promoter induced the expression of a red fluorescent protein allele, tdtomato. FGRp activity was used to produce antisense RNA of iglC, an important virulence factor, which severely reduced IglC protein levels. Cultivation in glucose-containing medium restored IglC levels, indicating the usefulness of this promoter for controlling both exogenous and chromosomal gene expression. Moreover, FGRp was shown to be active during the infection of human macrophages by using the fluorescence reporter. In this environment, the FGRp-mediated expression of antisense iglC by F. tularensis LVS resulted in reduced bacterial fitness, demonstrating the applicability of this promoter. An analysis of the genomic sequence indicated that this promoter region controls a gene, FTL_0580, encoding a hypothetical protein. A deletion analysis determined the critical sites essential for FGRp activity to be located within a 44-bp region. This is the first report of a conditional promoter and the use of antisense constructs in F. tularensis, valuable genetic tools for studying gene function both in vitro and in vivo.