Multiple deletions in the polyketide synthase gene repertoire of Mycobacterium tuberculosis reveal functional overlap of cell envelope lipids in host–pathogen interactions
Article first published online: 16 OCT 2013
© 2013 John Wiley & Sons Ltd
Volume 16, Issue 2, pages 195–213, February 2014
How to Cite
Passemar, C., Arbués, A., Malaga, W., Mercier, I., Moreau, F., Lepourry, L., Neyrolles, O., Guilhot, C. and Astarie-Dequeker, C. (2014), Multiple deletions in the polyketide synthase gene repertoire of Mycobacterium tuberculosis reveal functional overlap of cell envelope lipids in host–pathogen interactions. Cellular Microbiology, 16: 195–213. doi: 10.1111/cmi.12214
- Issue published online: 13 JAN 2014
- Article first published online: 16 OCT 2013
- Accepted manuscript online: 13 SEP 2013 04:44AM EST
- Manuscript Accepted: 5 SEP 2013
- Manuscript Revised: 4 SEP 2013
- Manuscript Received: 8 APR 2013
- Fondation pour la Recherche Médicale. Grant Number: DEQ20090515399
- Centre National de la Recherche Scientifique (CNRS)
- ANR. Grant Number: 2010-PATH-007-01/GeMoA
- European Structural Funds (FEDER)
- Région Midi-Pyrénées. Grant Number: CPER 2007–2013
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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