Figures & data
Figure 1 Alignment of the PanD amino acid sequences of M. tuberculosis (MTB), M. smegmatis (MSG) and E. coli (ECO). The conserved residues are boxed. The C-terminal sequence without crystal structure is highlighted in yellow. The asterisks indicate the mutant residues identified in the POA-resistant M. tuberculosis mutants.
Figure 2 M. tuberculosis became resistant to POA when panD expression was induced by ATc. Each strain was printed in triplicate with a replicator at a density of ∼105 CFU/mL. (A) WT/pUV15tetORs-panDRv (expressing panD from M. tuberculosis H37Rv) without (− sign) and with (+ sign) ATc, (B) WT/pUV15tetORs-panDG351A (expressing panD mutant) without and with ATc, (C) WT/pUV15tetORs-panDT431C (expressing panD mutant) without and with ATc, (D) POAR 156 (M117I) without and with ATc, (E) WT/pUV15tetORs-panDMS (expressing panD from M. smegmatis) without and with ATc, (F) WT/pUV15tetORs-panDEC (expressing panD from E. coli) without and with ATc, (G) WT/pUV15tetORs vector control without and with ATc.
Figure 3 Antagonism of POA's activity against M. tuberculosis by pantothenate and β-alanine. (A) M. tuberculosis parent strain H37Ra on 7H11 agar, (B) a POA-resistant mutant with a PanD mutation (M117I), (C) parent strain on 7H11 agar containing 0.1 mM calcium pantothenate, (D) parent strain with 0.1 mM β-alanine, (E) parent strain with 0.1 mM L-alanine, (F) parent strain with 5 mM calcium L-aspartate, (G) parent strain with 10 mM L-valine, (H) parent strain with 10 mM glutamate.
Figure 4 Effect of β-alanine concentration on POA susceptibility. M. tuberculosis H37Ra on 7H11 agar with no β-alanine (A), 0.1 µM β-alanine (B), 1 µM β-alanine (C), 5 µM β-alanine (D), 10 µM β-alanine (E), and 100 µM β-alanine (F).
Figure 5 Effect of pantothenate concentration on POA susceptibility. M. tuberculosis H37Ra on 7H11 agar with no calcium pantothenate (A), 0.1 µM calcium pantothenate (B), 1 µM calcium pantothenate (C), 5 µM calcium pantothenate (D), 10 µM calcium pantothenate (E), and 100 µM calcium pantothenate (F).
Figure 6 Overexpression and purification of M. tuberculosis PanD in E. coli. Lane 1–4: PanD protein, which appears as a 16-kD band, eluted by 50, 100, 200, 400 mM imidazole, respectively. Lane 5: Protein molecular weight marker (116, 66, 45, 35, 25, 18, 14 kD).
Figure 7 Concentration-dependent inhibition of M. tuberculosis PanD by POA but not by PZA or nicotinamide. The rightmost lane is a 14C-aspartic acid control without PanD or POA addition. (A) POA at 25, 50, or 100 µg/mL significantly inhibited the enzymatic activity of both wild-type and M117I PanD in a concentration-dependent manner as shown by the reduced conversion of aspartate (Asp) to β-alanine (β-Ala). At 200 µg/mL, POA completely inhibited the enzymatic activity of wild-type PanD (panels B and C, first lane from left). In contrast, neither PZA as a prodrug (B) nor nicotinamide (NIC) (C) as a structural analog control at 100 and 200 µg/mL had an apparent effect on PanD's enzymatic activity.
Table 1 panD mutations identified in POA-resistant mutants of M. tuberculosis