Transposon sequencing reveals metabolic pathways essential for Mycobacterium tuberculosis infection
Fig 3
Loss of purF causes death of M. tuberculosis in the absence of nutrient supplementation in vitro and in mice.
(A) Pathway for de novo synthesis of purine nucleotides and thiamine in M. tuberculosis. Solid lines indicate a single step; dashed lines indicate multiple steps. Proteins that catalyze the first five steps are indicated. Intermediate abbreviations: PPP, pentose phosphate pathway; PRPP, 5-phosphoribosyl pyrophosphate; PRA, 5-phospho-D-ribosylamine; GAR, 5’-phosphoribosylglycinamide; FGAR, 5’-phosphoribosyl-N-formylglycinamide; FGAM, 5’-phosphoribosyl N-formylglycinamidine; AIR, 5-aminoimidazole ribotide. (B-C) Strains grown in 7H9 supplemented with 60 μM thiamine and 150 μM hypoxanthine were washed in PBS-T and diluted to OD600 = 0.01 in 7H9 or 7H9 supplemented with 60 μM thiamine and 150 μM hypoxanthine. Bacterial growth and survival were measured by (B) optical density at 600 nm or (C) serial dilutions and plating to recover viable CFU. (D-E) C57BL/6J mice were infected by aerosol with the indicated strains. Mice (n = 6) were euthanized at days 1, 7, 21, and 42 post-infection. Lung (D) and spleen (E) homogenates were serially diluted and plated to recover viable CFU. In (C-E), WT and purF::Tn pMV-purF were plated on 7H10 agar; purF::Tn was plated on 7H10 agar supplemented with 60 μM thiamine and 150 μM hypoxanthine. Data represent the mean ± standard error of three biological replicates (B-C) or six animals (D-E). Asterisks indicate P-value < 0.05; n.d. indicates not detected (detection limit = 1 CFU).