The number of bacteria at time 0 was identical for the LVS strain and the ΔpdpC derivatives in all experiments performed, so the distinct phenotypes https://www.selleckchem.com/products/Methazolastone.html of the mutant could not be explained by differences in its uptake by phagocytes. While LVS and the complemented strain replicated approximately three log10 CFU within the first 24 h, the ΔpdpC mutant showed no growth (Figure 8). In additional experiments, there were no significant increases in bacterial numbers during the first 6 h, or at 48 or 72 h (data not shown). The results unambiguously demonstrated that the ΔpdpC mutant had a markedly impaired ability to replicate intracellularly. Replication

was also assessed in BMDM and PEC and the results were similar to those obtained with J774 cells; the mutant showed no replication whereas the complemented strain replicated as well as LVS (data not shown). To further verify the inability of the mutant to grow intracellularly, bacterial RNA was isolated from infected cells and the expression of the F. tularensis 16S rRNA gene was measured. We observed a 1.4 log10 decrease of 16S rRNA in ΔpdpC-infected cells see more during the first 24 h, while LVS infected cells showed

an increase of 2.8 log10. The data demonstrate that, regardless of method and macrophage type utilized, the ΔpdpC mutant showed no significant intracellular replication and the deficient phenotype could be restored by complementation of the mutation in cis. Figure 8 Intracellular growth of F. tularensis in J774 cells. LVS,

the ΔpdpC mutant, the complemented ΔpdpC mutant, or the ΔiglC mutant was used to infect J774 cells for 2 h, after which the monolayers were washed and medium added (corresponds to 0 h). Cells were then incubated for 24 or 48 h before being lysed, serially diluted and plated to estimate Thymidine kinase the number of CFU. The graph presents a representative experiment out of eight performed. Each bar represents the mean value and the error bars represent the standard deviation. The asterisk indicates that the log10 data differs significantly from LVS (***: P < 0.001). Two-sided Student’s t-test was used to compare means. The ΔpdpC mutant shows attenuation in vivo The lack of intracellular replication observed for the ΔpdpC mutant suggested that it is likely attenuated in vivo. To test this, mice were infected by the intradermal route with LVS, the ΔpdpC mutant, or the complemented mutant. The model has been widely used [25, 28–32] and identifies even marginal levels of attenuation since the LD50 for LVS is estimated to be approximately 2 × 107 CFU [33]. With an infection dose of 4 × 107 CFU, LVS caused 80% mortality (mean time to death 4.3 ± 0.5 days) and all mice infected with the complemented strain died within 4 days (mean time to death 3.6 ± 0.5 days).