Fig. S1. Strategy for construction and analysis of marked and unmarked pks3/4 mutants.

A. Schematic representation of the genetic structures obtained during the various step of the construction procedure. The black box indicates the region amplified with primers A and B and used for the allelic exchange. The white box indicates the pks3/4 gene. The light grey region shows the pks3/4 fragment deleted and replaced by the res-Ωkm-res cassette during the production of the allelic exchange substrate. The dark grey box represents the res-Ωkm-res cassette. Locations and generic names of primers used for cloning of the pks3/4 internal fragment (primers A and B) and for the PCR analyses of the marked and unmarked mutants are indicated by the arrow heads. The expected PCR products for the parental strain and pks3/4 mutants with various primer couples are indicated below each genetic structures.

B. Result of the PCR analysis of parental strain (WT), marked pks3/4 mutant (PMM150) and unmarked pks3/4 mutant (PMM169).


Fig. S2. Analysis of total lipid content of mutants. TLC analysis of the total lipid content of H37Rv ATCC, H37Rv Pasteur (DAT/PAT-) and PMM127 (DIM-, DAT/PAT-, SL-). Bacteria were incubated with [1-14C] acetate or [1-14C] propionate for 24 h to label either the total lipids or preferentially those containing methyl-branched fatty acids, notably DIM, SL and DAT/PAT. Spots corresponding to the main lipids from the outer layers of the mycobacterial cell envelope were quantified relative to total lipid and compared (inserted table gives the mean ± SD of two independent experiments).


Fig. S3. Polyketide-derived lipids contribute to cell death induction by Mtb. hMDMs were infected for 1 h with GFP-expressing DIM-less mutants deficient in one or more lipids at an moi of 2:1. At 144 h post infection, cells were stained with 7-AAD and percentage of permeabilized (7-AAD+) cells was analysed by flow cytometry.

A. Dot-plot diagrams of a representative experiment show the percentage of 7-AAD+ total (upper panels) or GFP-positive cells (lower panels).

B and C. Graphs represent the mean ± SEM of the percentage of 7-AAD+ cells analysed in both total (B) and infected (GFP-positive) cells (C), evaluated in four independent experiments. The significance of differences between strains was evaluated. *P < 0.05.


Fig. S4. DIM-deficiency induces acidification of Mtb containing phagosomes. hMDMs were infected for 1 h with GFP-expressing H37Rv ATCC or Pasteur and DIM-less mutants, PMM135 and PMM56. At various times after infection, cells were incubated with LysoTracker for 1 h, fixed and processed for microscopy. The values reported are means ± SEM of six experiments. The significance of differences between strains was evaluated. *P < 0.05; ***P < 0.005.


Table S1. Names and main features of strains and plasmids used in this study.

Table S2. Oligonucleotides used in this study for the construction of recombinant strains.

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