Knockout of fatty acid import and metabolism genes restricts Mtb growth in macrophages.

(A) Schematic of lipid import and metabolism genes in macrophages. Genes targeted for CRISPR-Cas9 mediated knockout are highlighted in red. (B) Scramble sgRNA or specific mutant macrophages were infected with the Mtb Erdman strain at MOI 0.4. Intracellular Mtb growth was measured by plating and counting CFUs in lysed macrophages 5 days post infection (PI). (C) CFUs from lysed macrophages were also plated on day 0, 3 hours post infection to demonstrate that there were minimal differences in bacterial uptake between the mutated macrophage lineages. n=6; ****, P < 0.0001.

Mtb infected macrophages with impaired fatty acid import and metabolism display reduced mitochondrial activities and are more glycolytic.

(A) Seahorse flux analyses of scramble or PLIN2-/-, FATP1-/- and CPT2-/- macrophages infected with Mtb Erdman strain at MOI 1 24 hours post infection. Oxygen consumption rates (OCR) were measured using the Cell Mito Stress Test Kit (Agilent). Oligo, oligomycin; FCCP, fluoro-carbonyl cyanide phenylhydrazone; Rot/A, rotenone and antimycin A. (B) Comparison of basal respiration and spare respiratory capacity (SRC) from A. SRC was calculated by subtracting the normalized maximal OCR from basal OCR. n=3 (2 technical repeats per replicate); ****, P < 0.0001. (C) Extracellular acidification rates (ECARs) of scramble or PLIN2-/-, FATP1-/- and CPT2-/- macrophages infected with Mtb as in A. ECARs were measured using the Agilent Seahorse Glycolysis Stress Test kit. 2DG, 2-Deoxy-D-glucose. (D) Comparison of basal glycolysis and spare glycolytic capacity (SGC) in the indicated mutant macrophages. SGC was calculated as SRC above. n=3 (2 technical repeats per replicate); *, P < 0.05, ****, P < 0.0001.

Knockout of fatty acid import and metabolism genes in macrophages activate AMPK and stabilizes HIF-1α.

(A) Western blot analysis of HIF-1α in uninfected and Mtb infected scramble or PLIN2-/-, FATP1-/- and CPT2-/- macrophages. In Mtb infected conditions, cells were infected with the bacteria at MOI 1 for 48 hours before preparation of cell lysates. (B) Western blot analysis of total and phosphorylated AMPK in uninfected and Mtb infected scramble or PLIN2-/-, FATP1-/- and CPT2-/- macrophages. Cell lysates were prepared as in A.

Supplementation with exogenous oleate fails to rescue the Mtb Δicl1 mutant in PLIN2-/-, FATP1-/- and CPT2-/- macrophages.

(A) Scramble or indicated mutant macrophages were infected with the Mtb H37Rv Δicl1 mutant expressing mCherry at MOI 5. Oleate supplementation (400 μM) was commenced 24 hours before infection in the treatment group, removed during Mtb infection and re-added 3-hours post infection for the entire duration of the experiment. Growth kinetics of Mtb were measured by monitoring mCherry fluorescence using a plate reader. n=4; ****, P < 0.0001. (B) Uninfected scramble or FATP1-/- and CPT2-/- macrophages were supplemented with 400 μM oleate for 24 hours. Cells were then fixed for 20 minutes and stained for lipid droplet inclusions using the Bodipy 493/503 dye. DAPI was used as a counterstain to detect nuclei.

Dual RNA sequencing to identify host and bacterial determinants of Mtb restriction in macrophages with fatty acid import and metabolism knockout genes.

(A) Principal component analysis (PCA) of scramble or PLIN2-/-, FATP1-/- and CPT2-/- macrophages transcriptomes infected with the Mtb smyc’::mCherry strain at MOI 0.5 4 days post infection. (B) Venn diagram of DE gene sets (Supplementary file 2) in PLIN2-/-, FATP1-/- and CPT2-/- mutant macrophages as compared to scramble showing overlapping genes. DE genes cutoff; abs (log2 fold change) > 0.3, adjusted p-value < 0.05. (C-D) Tree plot of top 80 enriched gene ontology terms (biological process) in Mtb infected PLIN2-/- and FATP1-/- upregulated genes.

Nutritional and oxidative stress define the core transcriptome response of Mtb inside PLIN2-/- macrophages.

(A) Schematic showing DE genes in Mtb transcriptomes isolated from PLIN2-/-, FATP1-/- and CPT2-/- macrophages in Figure 5A. (B) Heatmaps of nutritional and oxidative stress DE genes in PLIN2-/- macrophages. Arrows show genes which are also DE in CPT2-/- macrophages in a similar trend (Supplementary file 4).

Inhibitors of fatty acid transport and metabolism block intracellular growth of Mtb in macrophages.

(A) Growth of Mtb in liquid broth in the absence of drug (DMSO) or presence of metformin, FATP1 inhibitor (FATP1 In, 10 μM) and the β-oxidation of fatty acid inhibitor, Trimetazidine (TMZ, 500 nM). Mtb Erdman was grown to log phase and diluted to OD600 0.01 in 7H9 media in the presence of the above inhibitors. Growth kinetics were monitored by OD600 measurements using a plate reader. Rifampicin (RIF) at 0.5 μg/ml was used as a total killing control. (B) Scramble macrophages were infected with Mtb Erdman at MOI 0.5. Inhibitors were added 3 hours post infection following which CFUs were plated 4 days post infection. n=5; **, P < 0.01; ****, P < 0.0001.