<i>FTL_0304</i>, <i>FTL_0291</i>, <i>FTL_0325</i> and <i>FTL_0057</i> mutants are highly attenuated for virulence in BALB/c and C57BL/6 mice.

a<p>6–8 weeks old mice were infected i.n. with either <i>F. tularensis</i> LVS or the indicated mutants and monitored for mortality for 28 days. Data are shown as number of mice survived/total number of mice infected.</p

Immunization with the <i>FTL_0325</i> mutant induces a higher pro-inflammatory cytokine response in the spleen.

<p>BALB/c mice (n = 4) were infected with 1×10⁷ CFU of the indicated mutants and wild type <i>F. tularensis</i> LVS. At the indicated times, the levels of pro-inflammatory cytokines were measured in spleen homogenates using a Cytometric Bead Array assay. The data are representative of two independent experiments conducted and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values were recorded. *<i>P<</i>0.05<i>; **P</i><0.01; ***<i>P<</i>0.001. Ψ = Mice infected with 1×10⁷ CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.</p

Immunization with the <i>FTL_0325</i> mutant induces an early inflammatory response.

<p>BALB/c mice were infected with 1×10⁷ CFU of the <i>F. tularensis</i> LVS, <i>FTL_0325</i> or <i>FTL_0304</i> mutant and the lungs were harvested at the indicated times, sectioned and stained with H & E. Representatives of H & E stained lung sections are shown. Infiltration of neutrophils is shown in the inset (Magnification 10×; Inset 100×). Ψ = Mice infected with 1×10⁷ CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison. Green arrows indicate the sites of cellular infiltration. Yellow circles in inset show neutrophilic infiltration in the lungs of <i>FTL_0325</i> mutant immunized mice.</p

Immunization with the <i>FTL_0325</i> mutant induces an early pro-inflammatory cytokine response in lungs.

<p>BALB/c mice (n = 4) were infected with 1×10⁷ CFU of the indicated mutants and wild type <i>F. tularensis</i> LVS. At the indicated times, the levels of pro-inflammatory cytokines were measured in lung homogenates using a Cytometric Bead Array assay. The data are representative of two independent experiments conducted and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values were recorded. <i>**P</i><0.01; ***<i>P<</i>0.001. Ψ = Mice infected with 1×10⁷ CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.</p

Recall memory response results in an enhanced killing of <i>F. tularensis</i> SchuS4.

<p>The BMDMs were infected with <i>F. tularensis</i> SchuS4 and co-incubated with the splenocytes from naïve, <i>FTL_0325</i> or <i>FTL_0304</i> immunized mice. The BMDMs were lysed 72 hrs PI, diluted 10-fold and plated on MH-chocolate agar plates to determine the intracellular bacterial replication. The results were expressed as log₁₀ CFU/ml and are representative of two independent experiments. The data were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values were recorded. ***<i>P<</i>0.001.</p

The <i>FTL_0325</i> and <i>FTL_0304</i> mutants of <i>F. tularensis</i> LVS exhibit no growth defect under acellular growth conditions.

<p>Growth curves for bacteria grown in MHB were generated in a 96-well plate using 200 µl culture volumes while the growth curves for bacteria grown in BHI and CDM were generated using a 25 ml culture volume. Results shown are representative of two independent experiments.</p

Immunization with the <i>FTL_0325</i> mutant results in a potent memory recall response.

<p>Cell culture supernatants collected from BMDMs infected with either the <i>F. tularensis</i> LVS (A–D) or <i>F. tularensis</i> SchuS4 (E and F) and co-cultured with the splenocytes prepared from immunized mice at day 45 post-immunization (A and C) or at day 60 (B–F) were analyzed for IFN-γ (A, B and E) and IL-17a (C,D and F) cytokines by Cytometric Bead Flex sets. The data are representative of two independent experiments and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values recorded. *<i>P<</i>0.05<i>; **P</i><0.01; ***<i>P<</i>0.001.</p

Correlation analysis of bacterial burden and pro-inflammatory cytokine levels in mice infected with <i>F. tularensis</i> LVS, the <i>FTL_0325</i> and <i>FTL_0304</i> mutants.

*<p>r² value of 0.6 and above was considered as a strong correlation. Values in bold are strongly correlated.</p>**<p>P-value.</p