Fig. 2: Effect of fusion of various PanD derivatives on the level of RFP reporter protein.
From: Pyrazinamide triggers degradation of its target aspartate decarboxylase
a Schematic of derivatives of PanD fused translationally to constitutively expressed red fluorescence protein (RFP) as episomal reporter on plasmid pMV262 and transformed into Mtb. C-terminal = mycobacterium-specific C-terminal tail of PanD. RFP–PanDL136R and RFP–PanDH21R are PZA/POA-resistant mutants harboring missense mutation in the N-terminal His21 or the C-terminal tail Leu136 (indicated in red) and have been described previously4,6. b Fluorescence levels (expressed as RFU, relative fluorescence units) of mid-log phase cultures (OD600 = 0.2) were determined as a measure of intrabacterial fusion protein level in either Mtb wild-type (WT) or PZA/POA-resistant mutant Mtb POAR18 [clpC1: Lys209Glu]7 harboring RFP–PanD reporter constructs shown in a. One-way ANOVA multiple comparisons test (Dunn’s posttest, GraphPad Prism) was used to compare fluorescence levels conferred by the various constructs relative to the native RFP in wild-type background (left cluster 1–6) and in clpC1 mutant background (right cluster 1–4), respectively; *p value < 0.05; nsp value > 0.05. The two-sided Mann–Whitney test (GraphPad Prism) was used to compare fluorescence levels conferred by each RFP construct in the clpC1 mutant versus wild-type background (horizontal bars placed across left and right clusters). *p value < 0.05; nsp value > 0.05. c Effect of bortezomib (BZ) on fluorescence of Mtb cultures carrying different RFP–PanD reporter constructs shown in a. Cultures were treated with bortezomib (Mtb MIC50 = 25 µM) for 3 days. One-way ANOVA multiple comparisons and Dunn’s posttest (GraphPad Prism) were used to compare fluorescence levels in drug-free versus BZ-treated cultures. *p value < 0.05. The dotted line indicates mean normalized culture fluorescence of drug-free RFP controls. Experiments were repeated twice independently with two technical replicates. Error bars were defined as standard deviations. Source data are provided as a Source Data file.
