4′-Phosphopantetheinyl Transferase PptT, a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo
Figure 2
Effect of PptT depletion on the growth of M. bovis BCG and of M. tuberculosis in vitro.
A. M. tuberculosis H37Rv wild-type (WT) and the PMM168 mutant strain were grown in 7H9 containing ADC (with Km, Hyg and ATc for PMM168) at 37°C and streaked onto 7H11 plates supplemented with OADC with or without ATc (300 ng/ml). Plates were incubated for 20 days at 37°C. B. The M. tuberculosis PMM168 mutant was grown in 7H9 containing or not containing ATc at 37°C. Numbers of CFU in cultures with ATc (squares) were determined by plating dilutions of theses cultures onto 7H11 plates supplemented with ATc on days 0, 4, 8 and 12. CFU counts in cultures lacking ATc were determined by plating dilutions on 7H11 plates supplemented with ATc (closed circles) or without ATc (open circles) to estimate the number of ATc-independent CFU. C. Bactericidal effect of PptT depletion. The M. bovis BCG PMM99 (left panel) and M. tuberculosis PMM168 (right panel) mutants were grown in 7H9 containing (+) or not containing (−) ATc at 37°C. Numbers of CFU in cultures were determined by plating dilutions of theses cultures onto 7H11 plates supplemented with ATc on days 0 (D0) and 4 (D4). Values are means ± standard deviations (error bars) of CFU counts for three independent experiments. D. PMM99 was grown in media supplemented with Tween-80 with (100, 1, 0.3, 0.1 ng/ml) and without ATc and bacterial growth was monitored by measuring the optical density at 600 nm (OD600) (left panel). M. bovis BCG wild-type strain was grown in 7H9 supplemented with Tween-80. Data are representative of two independent experiments. Western blot visualization of PptT in crude cell lysates of PMM99 (5 µg/lane) cultivated for 6 days in a medium with ATc (100, 1, 0.3 ng/ml) and in a crude cell lysate of M. bovis BCG wild-type strain (5, 2, 1, 0.5 µg/lane) (right panel). The control lane was loaded with 100 ng of recombinant PptT fused to a poly-histidine tag produced in E. coli.