Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis
- Cornell University, United States
Figures
Figure 1 with 2 supplements
LucA facilitates cholesterol uptake in Mtb.
(A) Complementation with lucA restores cholesterol toxicity in the Δicl1::hyg lucA::TnHimar mutant. (B) The ΔlucA::hyg and ΔyrbE4A::hyg mutants are defective in the catabolic release of 14CO2 from …
Figure 1—figure supplement 1
Rational for genetic screen and summary of mutants selected by the screen.
(A) Schematic detailing cholesterol toxicity in Δicl1 Mtb. (B) Identity of the mutants identified where parentheses denote the number of independent insertions found in the specific gene. Genes …
Figure 1—figure supplement 2
Mutant construction and kinetic analysis of cholesterol uptake.
(A) Southern blot of PvuI-digested genomic DNA that has been probed using the indicated probe confirms allelic exchange in the ΔlucA::hyg mutant. (B) The ΔlucA::hyg mutant has no growth defect in …
Figure 2 with 1 supplement
Transcriptional profile of the ΔlucA::hyg mutant during infection in macrophages.
Bacterial gene expression profiles were determined at day 3-post infection in resting murine macrophages. Gene expression values from the ΔlucA::hyg mutant (Δ) and complement (C) strains were …
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Figure 2—source data 1
Spreadsheet of curated gene expression data.
- https://doi.org/10.7554/eLife.26969.006
Figure 2—figure supplement 1
Validation of cholesterol utilization defect with a GFP based reporter.
(A) Flow cytometery-based analysis of wild type Mtb carrying the prpD promoter reporter (prpD’::GFP smyc’::mCherry) confirms that this strain expresses GFP in response to cholesterol (100 μM) or …
Figure 3 with 3 supplements
LucA facilitates fatty acid uptake during infection and in axenic culture.
(A) Bodipy-C16 does not accumulate in ΔlucA::hyg mutant as cytosolic lipid inclusions. Representative confocal images of infected macrophages (red = mCherry Mtb, green = Bodipy-C16). Scale bar 5.0 …
Figure 3—figure supplement 1
Characterization of the mutant during infection in macrophages.
(A) Bacterial viability determined at day 3-post infection from infected macrophages. Data are means ± SD (n = 3). (B) Three-dimensional reconstructions of infected macrophages labeled with …
Figure 3—figure supplement 2
Kinetic data used used in comparisons.
(A) Representative 14C-palmitate uptake data used to calculate palmitate uptake rates for (Figure 3F). (B) Representative 14C-oleate uptake data used to calculate oleate uptake rates for (Figure 3H).…
Figure 3—figure supplement 3
Deletion of the full Mce1 operon leads to fatty acid uptake defect.
(A and B) the Δmce1::hyg mutant can metabolize and import cholesterol. (C and D) the Δmce1::hyg mutant is defective in oleic acid metabolism and uptake. (E and F) the Δmce1::hyg is defective in …
Figure 4 with 1 supplement
LucA interacts with subunits of Mce1 and Mce4 transporters and is required for their stability.
(A) Schematic of split-DFHR protein constructs, shading indicates proteins that interact with LucA and striped boxes indicate predicted transmembrane domains. (B) Serial dilutions of Msm …
Figure 4—figure supplement 1
Details of interaction clones, gene expression analysis of Mce1 genes, and model of Mce mediated nutrient import.
(A) Protein interaction constructs. ± indicates robustness of growth on trimethoprim. (B) Quantification of RNA transcripts by qPCR from untreated bacterial strains. Fold change values were …
Figure 5
LucA is required for the full fitness of Mtb in macrophages and survival in mouse lungs.
(A) Bacterial replication in resting human monocyte derived macrophages. Data are means ± SD (n = 3). (B) Bacterial replication in resting murine bone-marrow-derived macrophages. Data are means ± SD …
Additional files
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Supplementary file 1
Details of strains.
- https://doi.org/10.7554/eLife.26969.015
