Fig. 1. Flagellin-deficient B. melitensis mutants infect macrophages in vitro with the same kinetics as wt bacteria but show enhanced persistence in mice.

(A) Western blot analysis of the production of flagellin (FliC, upper panel) by B. melitensis strains harvested at the early log phase and the log phase of growth in 2YT rich medium. Anti-Omp89 detection was used as a loading control (lower panel). Data are representative of two independent experiments. ΔfliC pfliC is the complemented strain. (B) Intracellular replication of B. melitensis 16M wt and ΔfliC strains in RAW264.7 murine macrophages. Error bars represent the standard deviation of triplicates in one representative experiment out of three. (C-D) Infection kinetics in the spleens of wt BALB/c mice (n=5) inoculated intraperitoneally (i.p.) with 4 × 10⁴ CFUs of B. melitensis 16M wt, ΔfliC, complemented ΔfliC pfliC, ΔflbT, or ΔfliF strains. (E) Infection kinetics in the spleens of wt C57BL/6 mice (n=5) inoculated i.p. with 4 × 10⁴ CFUs of B. melitensis 16M wt or ΔfliC strains. Data represent the mean CFUs per organ and error bars represent standard deviation. Results have been analyzed by ANOVA I after testing the homogeneity of variance (Bartlett). ** and *** denote highly significant (p<0.01 and p<0.001 respectively) differences in relation to wt infection. These results are representative of at least two independent experiments.