Selected mutants were attenuated greater than 16-fold in the CNS and less than https://www.selleckchem.com/products/dorsomorphin-2hcl.html 4-fold in the lung tissue (P < 0.05). Mutants annotated as ""ND"" were not detected in the brain in quantities detectable by PCR, and were therefore likely highly attenuated in CNS tissue. To verify our results from the pooled infections, we tested the M. learn more tuberculosis pknD mutant individually. Mice were intravenously infected with M. tuberculosis wild-type

or pknD mutant strains and sacrificed at days 1 and 49 following infection. Equal numbers of the M. tuberculosis wild-type and pknD mutant strains were implanted at day 1 in the brain (2.58 ± 0.07 and 2.52 ± 0.07 log10 CFU; P = 0.61) and lungs (4.98 ± 0.14 and 5.06 ± 0.15 log10 CFU; P = 0.50) LY2606368 nmr respectively

(Figure 1A). Note that even though a modest invasion defect is expected for the pknD mutant, the in vivo models are not powered to reliably observe these modest differences at day 1, which, however, are amplified by day 49. The M. tuberculosis pknD mutant was significantly attenuated for survival in the brain (18.7 fold), compared to the wild-type strain (P = 0.004), but not in the lung tissue (Figure 1A). Taken together with our observations during pooled infection in both mice and guinea pigs, these data indicate a CNS-associated defect for the M. tuberculosis pknD mutant. Figure 1 Invasion and survival of M. tuberculosis pknD mutant in host-derived cells. A. BALB/c mice were infected with M. tuberculosis CDC1551 or pknD mutant, and sacrificed at days 1 and 49 after infection. The mutant

Protirelin for M. tuberculosis pknD was significantly attenuated (P = 0.004) in mouse brain, but not lung tissue, 49 days after infection. No defect was observed in the lungs at either time point. Bacterial burden is represented as log10 CFU/organ for all animal experiments. B. Invasion of host-cell monolayers by wild-type CDC1551, wild-type intergenic transposon control, pknD transposon mutant (pknD:Tn), and pknD genetic complement (pknD:Comp) was examined and normalized to the wild-type control. Invasion assays were performed in brain microvascular endothelial cells (HBMEC), epithelial A549 cells, and umbilical vein endothelia (HUVEC). No difference in invasion was observed in A549 cells (P = 0.31) or HUVEC (P = 0.41). A significant reduction in invasive capacity, however, was observed in the CNS-derived HBMEC (P = 0.02). This defect was restored by genetic complementation with the native pknD/pstS2 operon. N.S. = not significantly different. C. Intracellular survival of each of the above M. tuberculosis strains was examined in HBMEC at days 1, 3, 5, and 7 after infection. The pknD:Tn mutant demonstrated an invasion and intracellular survival defect in HBMEC relative to wild-type over the course of the seven day infection. D. Survival was also examined by infection of activated J774 macrophages.