Fig 6.

Rel_Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel_Mtb^H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel_Mtb^H80A is M. tuberculosis Δrel_Mtb. Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel_Mtb^H80A, carries the rel_Mtb^H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel_Mtb^H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of <0.01, and three asterisks indicate significance with a P value of <0.005. (D) M. tuberculosis Tet-Rel_Mtb^H80A, Δrel_Mtb, and Δrel_Mtb complemented with WT rel_Mtb were normalized to the same ODλ₆₀₀ and grown in Sauton's media under static, biofilm-forming conditions in a 24-well dish. Biofilms were incubated for 4 weeks at 37°C.