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.     

Immune response in experimental animals immunized with Burkholderia pseudomallei surface antigens

The influence of the chromatographic fractions of B. pseudomallei surface antigenic complex (C, C1, D, H) on immune response in white rats and white mice was under study. These antigenic complexes were noted to produce perceptible stimulating effect on the immune system of white rats, in contrast to that of white mice. The immunization of the mice the above-mentioned fractions suppressed the phagocytic activity of peritoneal macrophages (PM) and slightly enhanced cell-mediated immunity. In experiments on white rats, fraction C induced the growth of specific antibody titers and stimulated the phagocytic activity of PM, as well as the indices of delayed hypersensitivity (DH). Fraction D showed a lower level of the induction of the phagocytic activity of PM and was inactive in the manifestation of cell-mediated immunity, but induced a high level of humoral immunity. Antigenic complexes C1 and H increased the phagocytic activity of PM and DH characteristics with a low level of antibody production. The studied fractions of the causative agent of melioidosis decreased the content of bactericidal cationic proteins (BCP) in rat blood neutrophils, and in mice a decreased content of BCP in phagocytes was registered. The fractions increased the activity of myeloperoxidase in blood neutrophils in mice and rats. As revealed with the use of immunoelectrophoresis, SDS PAAG electrophoresis and immunoblotting, the surface antigenic complex contained proteins of 18, 22, 39 kD and glycoproteins 42, 55, 90 kD. The latter glycoprotein was found in all the fractions under study, having protective properties.     

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.     

Active suppression of the pulmonary immune response by Francisella tularensis Schu4

Francisella tularensis is an obligate, intracellular bacterium that causes acute, lethal disease following inhalation. As an intracellular pathogen F. tularensis must invade cells, replicate, and disseminate while evading host immune responses. The mechanisms by which virulent type A strains of Francisella tularensis accomplish this evasion are not understood. Francisella tularensis has been shown to target multiple cell types in the lung following aerosol infection, including dendritic cells (DC) and macrophages. We demonstrate here that one mechanism used by a virulent type A strain of F. tularensis (Schu4) to evade early detection is by the induction of overwhelming immunosuppression at the site of infection, the lung. Following infection and replication in multiple pulmonary cell types, Schu4 failed to induce the production of proinflammatory cytokines or increase the expression of MHCII or CD86 on the surface of resident DC within the first few days of disease. However, Schu4 did induce early and transient production of TGF-beta, a potent immunosuppressive cytokine. The absence of DC activation following infection could not be attributed to the apoptosis of pulmonary cells, because there were minimal differences in either annexin or cleaved caspase-3 staining in infected mice compared with that in uninfected controls. Rather, we demonstrate that Schu4 actively suppressed in vivo responses to secondary stimuli (LPS), e.g., failure to recruit granulocytes/monocytes and stimulate resident DC. Thus, unlike attenuated strains of F. tularensis, Schu4 induced broad immunosuppression within the first few days after aerosol infection. This difference may explain the increased virulence of type A strains compared with their more attenuated counterparts.     

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.     

iTRAQ quantitative analysis of Francisella tularensis ssp. holarctica live vaccine strain and Francisella tularensis ssp. tularensis SCHU S4 response to different temperatures and stationary phases of growth

Proteomics has been shown to significantly contribute to the investigation of the pathogenicity of the extremely infectious bacteria Francisella tularensis. In this study, the authors employed iTRAQ quantitative proteomic analysis in order to monitor alterations in proteomes of F. tularensis ssp. holarctica live vaccine strain and F. tularensis ssp. tularensis SCHU S4 associated with the cultivation at different temperatures or in the stationary phase. Correlated production of the identified proteins studied by the exploratory statistical analysis revealed novel candidates for virulence factors that were regulated in a similar manner to the genes encoded in the Francisella Pathogenicity Island. Moreover, the assessment of the adaptation of live vaccine strain and SCHU S4 strain to the examined stimuli uncovered differences in their physiological responses to the stationary phase of growth.     

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.     

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.     

A variety of novel lipid A structures obtained from Francisella tularensis live vaccine strain

F. tularensis is a Gram-negative coccobacillus that causes tularemia. Its LPS has nominal biological activity. Currently, there is controversy regarding the structure of the lipid A obtained from F. tularensis live vaccine strain (LVS). Therefore, to resolve this controversy, the purification and structural identification of this LPS was crucial. To achieve this, LPS from F. tularensis LVS was acid hydrolyzed to obtain crude lipid A that was methylated and purified by HPLC and the fractions were analyzed by MALDI-TOF MS. The structure of the major lipid A species was composed of a glucosamine disaccharide backbone substituted with four fatty acyl groups and a phosphate (1-position) with a molecular mass of 1505. The major lipid A component contained 18:0[3-O(16:0)] in the distal subunit and two 18:0(3-OH) fatty acyl chains at the 2- or 3-positions of the reducing subunit. Additional variations in the lipid A species include: heterogeneity in fatty acyl groups, a phosphate or a phosphoryl galactosamine at the 1-position, and a hexose at the 4' or 6' position, some of which have not been previously described for F. tularensis LVS. This analysis revealed that lipid A from F. tularensis LVS is far more complex than originally believed.     

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.     

Proteomics analysis of the Francisella tularensis LVS response to iron restriction: induction of the F. tularensis pathogenicity island proteins IglABC

Francisella tularensis is a highly virulent, facultative intracellular pathogen that causes tularemia in humans and animals. Although it is one of the most infectious bacterial pathogens, little is known about its virulence mechanisms. In this study, the response of F. tularensis live vaccine strain to iron depletion, which simulates the environment within the host, was investigated. In order to detect alterations in protein synthesis, metabolic labeling, followed by 2D-PAGE analysis was used. Globally, 141 protein spots were detected whose levels were significantly altered in the iron-restricted medium. About 65% of the spots were successfully identified using mass spectrometric approaches. Importantly, among the proteins produced at an increased level during iron-limited growth, three proteins were found encoded by the igl operon, located in the F. tularensis pathogenicity island I (FPI). Of these, the IglC and IglA proteins were previously reported to be necessary for full virulence of F. tularensis. These results, obtained at the proteome level, support and confirm recently published data showing that the igl operon genes are transcribed in response to iron limitation.     

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (<10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the gold standard method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.     

The characteristics of potassium ion transport across the bacterial membranes in a vaccinal culture of Francisella tularensis]

The presence of the systems of energy-dependent accumulation of potassium ions and their passive exchange for protons in cytoplasmic bacterial membranes of F. tularensis strain 15 has been demonstrated by means of a K(+)-selective electrode and the turbidimetric method. The kinetic parameters of the energy-dependent K+ transport: Km = 0.83 +/- 0.20 mM K+, Vmax = 0.23 +/- 0.04 mM K+/mg of dry weight. min. Under the conditions of anoxia F. tularensis cannot retain potassium ions in their cytoplasm, and thus they are considered to be incapable of anaerobiosis.     

Mutations in Francisella tularensis, decreasing the virulence of these bacteria and leading to a change in the immune response upon infection of experimental animals with them]

The research was aimed at isolation of Francisella tularensis mutants possessing the decreased virulence for experimental animals and mediating the changes in the animal immune response. A number of spontaneous and induced mutants of the American and European subtypes of Francisella tularensis were selected for antibiotics resistance or detergent sensitivity. All the obtained mutants have the decreased virulence and differ in their ability to induce the protective antitularemia immunity or ability to induce the humoral immune response in the laboratory animals. The dimeric immunoprecipitation in gel as well as immunoblotting have shown the mutations decreasing the virulence to cause the loss by bacteria of a number of antigenic structures (in case the virulence is completely lost) or changes in antigenic structure resulting in inability of bacteria to induce the humoral immune response when immunizing the laboratory animals. The latter occurs in partially virulent mutants of the vaccine mutant type. The concomitant changes in virulence, ability to cause protective immunity or humoral immune response of the mutants is discussed.