Fig. 4: TcrXY-dependent gene expression is activated at acidic pH.
a TcrXY is required for Rv3706c induction at acidic pH. H37Rv (PRv3706c::mCherry; PrpoB::gfp) and ΔtcrXY (PRv3706c::mCherry; PrpoB::gfp) were cultured in pH 7 or pH 5.4 minimal media, then mCherry and GFP fluorescence quantified by flow cytometry. Each bar represents the median fluorescence intensity from three replicates (open circle), representing two experiments. Error bars denote SD. b Exemplar confocal micrographs of resting murine BMDMs infected with H37Rv (PRv3706c:mCherry; PrpoB::gfp) or ΔtcrXY (PRv3706c::mCherry; PrpoB::gfp) for 24 h and stained with LysoTracker. Images depict cell nucleus (blue, Hoechst), H37Rv (WT) or ΔtcrXY (KO) reporter strain (red, mCherry (mCh) and green, GFP) and acidic compartments (cyan, LysoTracker). ‘+’ denotes LysoTracker co-localisation with Mtb, ‘−’ denotes no co-localisation. Scale bar, 2 μm. c Rv3706c transcription is activated in acidic intracellular compartments in a TcrXY-dependent manner. Resting or activated BMDMs were infected with H37Rv (PRv3706c:mCherry; PrpoB::gfp) or ΔtcrXY (PRv3706c::mCherry; PrpoB::gfp) for 24 h and fluorescence quantified by microscopy. Data is presented as sum Mtb reporter signal (mCherry signal in GFP-positive bacteria) per voxel obtained from two biological replicates, filtered by LysoTracker co-localisation in Imaris (v9.6.1). For H37Rv-infected BMDMs, n = 140 (red), n = 175 (blue), n = 78 (green), n = 93 (purple). For ΔtcrXY-infected BMDMs, n = 104 (red), n = 133 (blue), n = 101 (green), n = 101 (purple). Horizontal bars denote the median. Statistical analysis was performed using a one-way ANOVA with Turkey’s multiple comparisons test. d TcrXY signalling is required for induction of Rv3706c during acid stress. A wild-type tcrXY allele, phospho-mutant tcrXD54AY or tcrXYH256Q allele, were expressed in a ΔtcrXY genetic background. Strains were cultured at pH 7 or pH 5.4 for 24 h and Rv3706c mRNA levels were compared by qRT-PCR. H37Rv (WT) mRNA levels were used as a control. Each bar represents the mean fold-change from three biological replicates (open circle). Statistical analysis was performed using an unpaired two-tailed t-test. Error bars denote SD. Source data are provided as a Source Data file.
