Extended Data Fig. 1: Mycobacterial D-serine, but not L-serine is induced by hypoxia through Rv0884c.
(a) Quantitative metabolomics approach. (b) Assay of L-serine concentration from culture supernatants of H37Rv incubated under aeration or hypoxia. (c) Quantitative proteomics approach. (d) IB of Rv0884c107-376aa. (e) qPCR analysis of Rv0884c mRNA from H37Rv incubated under aeration or hypoxia. (f) IB of cell lysates derived from Rv0884c_cKDTet strains treated with or without ATc. (g) L-serine concentration in the culture supernatants from Rv0884c_cKDTet strain incubated with or without ATc under aeration or hypoxia. (h-j) Mice infected with GFP-H37Rv for 4 weeks were exposed to either 95% O2 or normal air for 20 hours before sacrifice. Immunofluorescence staining of dLN was performed. MFI of PIMO were quantified with ImageJ (h). Scale bar, 100 μm; L-serine concentration of lung homogenate supernatants (i) and sera (j) were analyzed. (k, l) Mice infected with Rv0884c_cKDTet treated with or without ATc for 4 weeks were exposed to either 95% O2 or normal air for 20 hours before sacrifice. L-serine concentration of lung homogenate supernatant (k) and serum (l) were analyzed. (m-n, p) L-serine concentration of lung homogenate supernatant (m) and serum (n) from indicated Mtb strains infected-mice were analyzed at 4 weeks post-infection; The c.f.u. of lungs were assayed 1 day post-infection (p). (o) Heatmap of metabolites secreted from indicated Mtb strains incubated under aeration or hypoxia. (q) H37Rv infected-mice were administered with or without D-serine. Lung c.f.u. were assayed 1 day post-infection. All data represent one experiment with at least three independent biological replicates. Results are shown as mean ± s.e.m. Two-way ANOVA with Tukey’s multiple comparisons test (b, e, g), one-way ANOVA with Tukey’s multiple comparisons test (i-n, p) and Two-tailed unpaired Student’s t-test (h, q) were used for statistical analysis.
