Figure 3.

Requirement of MmpL11 for maintaining the Mtb phospholipid composition under stress. (A) Analysis of phospholipid composition of Wt and ΔM11 strains grown in the presence of either 0.1% Tween 80 or Tyloxapol for 24 hr by fluorescence microscopy at em529 nm (G+) and em605 nm (R+) and a merge of the two channels are shown. (B,C,D) -Quantitation of CL levels by densitometry-B) Separation of CL in fractionated lipids of Mtb by TLC (CHCl₃:CH₃OH:H₂0::65:25:4), samples 1 and 3 represent Wt, 2 & 4- ΔM11 in media containing Tween 80 (1, 2) or Tyloxapol (3, 4), Purified PG, PE, CL were used at increasing concentrations as standards for internal reference; (C) Confirmation of purified CL fraction by Tandem ESI- MS analysis. The MS1 profile of m/z 1404.00 corresponding to CL with the MS2 fragmentation profile and a putative structure shown in inset; (D) Estimation of CL concentration by densitometry is represented as mean ± SD for a representative of 3 biological replicate experiments. (E,F) Analysis of ¹⁴C-oleate incorporation into Mtb lipids- Separation of phospholipids by TLC (E) and quantitation by liquid scintillation count is represented as average relative counts of CL ± SD for a representative of 3 biological replicate experiments. (F,G) Analysis of expression of genes involved in CL biosynthesis of Mtb by qPCR. Ratio of expression in the mutant with respect to Wt is depicted as relative transcripts ΔM11/Wt (fold change in mutant relative to Wt).