Fig. 3: A ∆lamH strain retains LAM in the cytoplasmic membrane and does not produce capsular AM.
a LM/LAM was harvested from the indicated M. bovis BCG strains grown in 7H9 broth with 0.05% Tween-80 at OD600nm = 0.6 and analysed by SDS-PAGE followed by Pro-Q Emerald staining (Supplementary Fig. 1). Total fluorescence of LAM (a) and LM (b) for three biological replicates is presented with error bars indicating standard deviation and the centre of measure defined as the mean. a **P = 0.0018; ***P = 0.0006, b ****P < 0.0001. c The capsular material from the indicated strains was normalised based on wet mass, labelled with 2-AB and separated by size-exclusion chromatography with fluorescence detection (Ex = 320 nm, Em = 420 nm). Peaks were identified by analysing samples pre-digested with appropriate enzymes. d The α-glucan and AM peaks from (c) from three biological replicates were integrated, and the relative amount of AM was computed by dividing the total AM peak area by the sum of the α-glucan and AM peak areas. Error bars indicate standard deviation. **P = 0.003; ****P < 0.0001. e The indicated M. bovis BCG strains were grown in 7H9 in the presence of (1-14C) acetic acid sodium salt until OD600 = 0.6. Polar lipid extracts were analysed from three biological replicates by two-dimensional TLC and annotated as per ref. 80. The relative intensity of each PIM species is reported with error bars representing standard deviation. *P = 0.0306; ***P = 0.001. f Schematic summary of phenotypic changes due to loss of lamH. WT—M. bovis BCG Danish 1331, Mutant—M. bovis BCG Danish 1331 lamH::Himar1, Complement—M. bovis BCG Danish 1331 lamH::Himar1, L5::pMV306-lamH, Catalytic Null—M. bovis BCG Danish 1331 lamH::Himar1, L5::pMV306-lamHD96A. Significance was determined with a one-way ANOVA with Tuckey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.
