Development of an immunoassay test system based on monoclonal antybodies and immunomagnetic particles for the detection of F. tularensis cells.

Tularemia is an especially dangerous infection caused by the gram-negative bacterium Francisella tularensis. It belongs to natural focal infections, and therefore is under continuous control by quarantine services. When carrying out their activities they use a whole range of diagnostic tools. The objective of this research is to develop an enzyme immunoassay based on highly specific monoclonal antibodies and immunomagnetic particles for monitoring the tularemia pathogen. To produce hybridomas mice were immunized with cells of the vaccine strain F. tularensis subsp. holarctica 15 NIIEG. After cell fusion hybridomas were selected by a solid-phase enzyme immunoassay (ELISA) using lipopolysaccharide (LPS) of the tularemia microbe. As a result, two hybridomas, 1C2 and 3F5, were produced. MABs of the hybridomas were obtained by using BALB / c mice. The MABs were purified by sepharose A affinity chromatography and used for conjugation with magnetic particles, and for biotinylation followed by matching a pair for ELISA. The pair of IMPs and MABs 3F5 as well as biotinylated FB11-x MABs was the best in detecting tularemia cells. The use of this MAB pair in ELISA allowed the identification of 105 microbial cells/ml in a 4 ml sample and 5×103 microbial cells/ml in a 45ml sample. Interaction with F. tularensis subsp. novicida Utah112 cells was absent.

The use of loop-mediated isothermal DNA amplification for the detection and identification of the anthrax pathogen.

The results of detection and identification of Bacillus anthracis strains in loop-mediated isothermal DNA amplification (LAMP) reaction performed under optimized conditions with original primers and thermostable DNA polymerase are presented. Reproducible LAMP-based detection of chromosomal and plasmid DNA targets specific for B. anthracis strains has been demonstrated. No cross reactions with DNA from bacterial strains of other species of the B. cereus group were detected. The development of tests for anthrax-pathogen detection based on the optimized reaction of loop isothermal DNA amplification is planned. These tests will be convenient for clinical studies and field diagnostics due to the absence of requirements for sophisticated equipment.

[CONSTRUCTION AND PROPERTIES OF THE FRANCISELLA TULARENSIS VACCINE STRAIN WITHOUT ONE COPY OF THE IGLC GENE AND WITHOUT RECA GENE].

The live vaccine based on the Francisella tularensis subsp. holarctica vaccine strain 15 NIIEG line is used in Russia against tularemia. This vaccine is highly effective, but fairly unstable. Therefore, development of stable live tularemia vaccine with minimal side effect is rather urgent. The method of allel removal in the F. tularensis vaccine strain was used to remove one copy of the iglC gene, which is required to provide intracellular production of the vaccine strain, as well as removal of the recA gene. The latter is crucial for homological recombination. pGM5 suicide vector based on pHV33 bireplicon plasmid was constructed to provide replacement of intact F. tularensis chromosome segments by modified segments. Modified chromosome segments contain F. Tularensis DNA fragment without iglC structural gene segment 545 p. b. (in pGMΔiglC plasmid), as well as DNA fragment containing no recA structural gene segment 1060 p.b. (pGMΔrecA plasmid). The constructed 15/23-1ΔrecA mutant, in contrast to the vaccine strain 15, was capable of reproducing in the macrophage-like cells J774A.1 line, whereas the efficiency of the reproduction was 8-10 times less. BALB/c mouse responded to immunization by the 15/23-1ΔrecA strain by smaller weight decrease (-2%) as compared to the strain 15 (-14%). Bacteria of the 15/23-1ΔrecA strain were virtually incapable of germinating from the BALB/c murine spleen 14 days after invasion, whereas bacteria of the strain 15 were found in the murine organs even after 21 days. The F. tularensis 15/23-1ΔrecA strain having smaller reaction ability can be used as a basis for construction of stable live safe tularemia vaccine.

[CITRULLINUREIDASE GENE DIVERSITY IN THE GENUS FRANCISELLA].

This work describes the results, of the in silico analysis of the genetic diversity of the citrullinureidase gene (ctu) in two species of bacteria of the genus Francisella: tularensis (ssp. tularensis, holarctica, mediasiatica, novicida) and philomiragia. The strains of the Central Asiatic subspecies possessing the citrullinureidase activity differ in the gene ctu from the ssp tularensis Schu by three nucleotide substitutions leading to two insignificant amino acid substitutions in the encoded polypeptide. In the strain F. tularensis of the ssp. holarctica the gene ctu encodes inactive enzyme, which is probably due to amino acid substitutions: 151 Gly --> Asp, 183 Pro --> Leu, 222 Asp --> Asn. Except for the Japan biovar bacteria, in all strains of the Holarctic subspecies there are two stop codons in the gene ctu. The bacteria of the subspecies novicida contain the ctu gene only in the strain 3523, whereas the other strains contain the gene FTN_0827 encoding the C-N hydrolase, which probably provides the citrullinureidase activity.

[Francisella tularensis strains molecular typing using the multiple-locus variable-number tandem repeat analysis].

Variable-number tandem repeat analysis (VNTR) of 25 loci was used for molecular typing of the Francisella tularensis strains isolated from different regions of Russia and the former Soviet Union. This approach allowed us to subdivide F. tularensis subspecies and determine genotype diversity with regard to the geographical prevalence. All 25 loci were examined for their ability to discriminate subspecies and local geographical group. 42 genotypes among the 58 investigated F. tularensis subsp. holarctica isolates were found using cluster VNTR analysis.

Biological properties and structure of the lipopolysaccharide of a vaccine strain of Francisella tularensis generated by inactivation of a quorum sensing system gene qseC.

A knockout mutant with a deletion in a quorum sensing system gene qseC was generated from the vaccine strain Francisella tularensis 15 by site-directed mutagenesis. The variant with the inactivated gene qseC differed from the parental strain in growth rate on solid nutrient medium but had the same growth dynamics in liquid nutrient medium. The mutation abolished almost completely the resistance of the vaccine strain to normal rabbit serum and its ability to survive in macrophages; in addition, the strain lost the residual virulence. A significant phenotypic alteration was observed in the lipopolysaccharide of F. tularensis. Particularly, the mutant strain synthesized no noticeable amount of the lipopolysaccharide with the high-molecular-mass O-polysaccharide, presumably as a result of impairing biosynthesis of the repeating unit, namely, a loss of the ability to incorporate a formyl group, an N-acyl substituent of 4-amino-4,6-dideoxy-D-glucose.

Cloning and expression of protective antigens of Mycobacterium tuberculosis Ag85B and ESAT-6 in Francisella tularensis 15/10.

The possibility of expression of genes encoding mycobacterial antigens in Francisella tularensis 15/10 vaccine strain cells has been shown for the first time. To obtain stable and effective expression of mycobacterial antigens in the F. tularensis cells, the plasmid vector pPMC1 and hybrid genes consisting of the leader part FL of the F. tularensis membrane protein FopA and structural moieties of the mature protein Ag85B or the fused protein Ag85B-ESAT-6 were constructed. Recombinant strains F. tularensis RVp17 and RVp18 expressing protective mycobacterial antigens in the fused proteins FL-Ag85B and FL-Ag85B-ESAT-6, respectively, were obtained. Expression of the protective mycobacterial antigens in F. tularensis was analyzed using specific antisera to the recombinant proteins Ag85-(His)6 and ESAT-6-(His)6 isolated from Escherichia coli producer strains created on the basis of the pET23b(+) and pET24b(+) vectors. The expression of heterologous protective antigens in F. tularensis 15/10 is promising for creation of live recombinant anti-tuberculosis vaccines on the basis of the tularemia vaccine strain.

Genetic organization of the Francisella plasmid pFNL10.

We report here the molecular characterization of pFNL10, a 3990-bp cryptic plasmid of Francisella novicida-like F6168. The plasmid was maintained in F. novicida Utah 112 and F. tularensis LVS strains. We sequenced the entire plasmid and found six open reading frames (ORFs)-ORF1, ORF2, ORF3, ORF4, ORF5, and ORFm. ORF3, ORF4, ORF5, and ORFm are located on the same strand, and we designated it the plus strand. ORF1 and ORF2 are on the complementary strand. The ORFs appear to be arranged in two operons, one comprising ORF5 and ORF4 and the other ORF1 and ORF2. There exist two distinct promoters similar to the Escherichia coli sigma(70) promoter, one 5' to ORF1-ORF2 operon and the other 5' to ORF5-ORF4 operon. We found that in both promoters the transcriptional start is an adenosine. ORF3 is positioned in tandem with ORF5-ORF4, but has its own transcriptional start, a thymidine. However, sequence analysis revealed no recognizable promoter in physical proximity to ORF3. Sequence analysis revealed transcriptional terminators immediately downstream of the two operons. Experimental results showed that the ORF1-ORF2 terminator is authentic. But we could not definitively confirm the ORF5-ORF4 terminator. Two sets of direct repeats, one 31 and the other 13 bp, characteristic of ori are positioned between the two promoters. ORF1 encodes a protein that bears homology to the replication initiation protein RepA of various bacteria, and disruption of this ORF indeed blocked pFNL10 replication. In contrast, ORF2 disruption caused formation of plasmid multimers, suggesting aberrant replication. Our analysis also suggests that pFNL10 replicates by the theta mode. The ORF5-ORF4 operon resembles the phd-doc operon of Escherichia coli bacteriophage P1, but the significance of this similarity is unclear.

[Tularemia vaccine strain is a potential vector]. / Tuliaremiínyí vaktsinnyí shtamm-potentsial'nyí bakterial'nyí vektor.

The immunological and genetic properties of Francisella tularensis vaccine strain are discussion with regard to its use to produce recombinant vaccines. This bacterium-based vector is supposed to be an excellent object for investigating the role of protective antigens in the development of immunity against intracellular bacteria.

Cryptic plasmid pFNL10 from Francisella novicida-like F6168: the base of plasmid vectors for Francisella tularensis.

The plasmid pFNL1OO was created by ligation of Escherichia coli plasmid pBR328 and plasmid pFNL1O from Francisella novicida-like strain F6168. This plasmid was able to replicate and to express the genes for chloramphenicol and tetracycline resistance in both E. coli and F. tularensis. The origin of replication of pFNL1O, needed for the replication of pFNL1OO in F. tularensis, was mapped. A Sau3A-deletion derivative of pFNL1OO, designated pFNL2OO, was constructed. This plasmid could replicate only in F. tularensis and was found to be stably inherited during cultivation both on solid medium and in liquid cultures.

[Isolation and molecular-genetic characteristic of a cryptic plasmid from the Francisella novicida like F6168 strain]. / Vydelenie i molekuliarno-geneticheskaia kharakteristika kripticheskoi plazmidy iz shtamma Francisella novicida like F6168.

A 4 Kb plasmid DNA has been isolated from Francisella novicida like strain F6168. Restriction map of the plasmid was constructed for restriction endonucleases HindIII, XbaI, EcoRV, BgIII. The plasmid pFN10 has been shown to be stably inherited by F. tularensis. The use of pFN10 for the construction of plasmid vectors for microorganisms of the genus Francisella is discussed.