Figure 2.

Identification of PhoPR as a novel regulator of c-di-AMP synthesis and secretion in M. tuberculosis

(A) Production of c-di-AMP in whole-cell extracts of M. tuberculosis H37Rv, its ΔphoPR mutant, and the mutant strain complemented (comp) with the phoPR genes. (B) Secretion of c-di-AMP into the supernatant of strains depicted in (A). Data are the mean and standard deviation from at least three biological replicates. (C) RNA sequencing (RNA-seq) profiles of three different genetic regions in M. tuberculosis MT103, MTBVAC, H37Rv, and the H37Rv ΔphoPR mutant. The disA, cnpB, and pks2 gene expression is indicated with a dashed box. Note that disA and cnpB show equivalent transcription levels in MTBVAC and the H37Rv ΔphoPR mutant relative to their wild-type strains. In contrast, the pks2 gene, which is a well-known PhoPR-regulated gene, shows downregulation in MTBVAC and the H37Rv ΔphoPR mutant. (D) Quantification of DisA and CnpB protein levels in H37Rv and the H37Rv ΔphoPR mutant by MRM-MS. Chromatograms show the identification of three different transitions from a specific peptide. Bars represent the area under the curve for every transition in H37Rv and the H37Rv ΔphoPR mutant. Equivalent results were obtained for the DisA peptides ANVQLVPDPSIPTDESGTR, HVLTDSATILSR, ANQAIATLER, and VFGYPTTTEAQDSTLSPR and for the CnpB peptides VEVSFAAPATLPESLR, LGALGDLTDSGR, VLGSAQLVSEAVGGR, and TVNLAAVASGFGGGGHR (data not shown).