Figure S1

A unstim IFN-ɣ Mtb Mtb+ IFN-ɣ

Hif-1α

actin

B - DMOG + DMOG unstim TB TB/ɣ 0.05 TB/ɣ 0.25 TB/ɣ 1.25 unstim TB TB/ɣ 0.05 TB/ɣ 0.25 TB/ɣ 1.25

HIF-1α

actin

C D Macrophage infection

1.5 6.5 Erd OD600 2 6.0 R = 0.993

) Lux OD600

m 5.5 n 1.0

5.0 600 (

D 0.5 4.5 Log(10) RLU O 4.0 0.0 3.5 0 2 4 6 8 10 2.5 3.0 3.5 4.0 4.5 5.0 Time (days) log(10)CFU

Figure S1. Characterization of DMOG treatment of BMDM and characterization of TB-lux expressing the luxCDABE operon. (A) HIF-1α western blot at 12h post infection. IFN-γ is at 1.25ng/ml (B) HIF-1α western blot 12h post-infection of WT BMDM with IFN-γ dose response (.05-1.25ng/ml) with and without addition of 200uM DMOG at the end of the 4hr phagocytosis (C) wildtype (Erdman) M. tuberculosis was compared to Erdman carrying a plasmid that constitutive- ly expresses the luxCDABE operon. Bacteria were seeded into 7H9 at an OD600 of 0.05. OD600 measurements were taken daily to demonstrate that expression of the luxCDABE operon does not inhibit growth. (D) BMDM were infected with different MOIs of bacteria expressing the luxCDABE operon. After a 4h phagocytosis, the infected macrophages were washed and media was replaced. Luminescence was measured, and subsequently the infected monolayers were lysed and plated for CFU. Figure S2

A B WT Hif Glut1 tb tb/ɣ tb tb/ɣ Glut1 Hk Hk1 Hk2 g6p Hk3 Gpi Gpi Pfkfb3 Pfkfb3 f2,6bp f6p Pfkm Pfk1 Aldo Pfkp f1,6bp Pfkl AldoA Tpi AldoC dap g3p Tpi Gapdh Gapdh 1,3bpg Pgk1 Pgam1 Pgk Eno1 3pg Eno2 Pgam Eno3 2pg Pkm Ldha Eno Ldhb ** pep Ldhd Mct Ldh Pkm Mct4

lactate pyruvate log (4.6) log (-4.6) 2 2

Figure S2. HIF-1α activates expression of glycolytic in M. tuberculosis infected macro- phages. (A) pathway depicting metabolites (grey bubbles) and . (B) Heat map depict- ing relative expression data from RNA-seq. Wild-type (WT) is relative to uninfected; HIF-1α deficient (Hif) is relative to the same condition in WT. glucose 6 phosphate (g6p), fructose 6 phosphate (f6p), fructose 2,6 bisphosphate (f2,6bp), fructose 1,6 bisphosphate (f1,6bp), dihydroxyacetone phosphate (dap), glycer- aldehyde 3 phosphate (g3p) 1,3 bisphosphoglycerate (1,3bpg), 3-phosphoglycerate (3pg), 2-phosphoglyc- erate (2pg), phosphoenoylpyruvate (pep). Figure S3

glucose 6-phosphate fructose-6-phosphate fructose-1,6-bp *** 2e5 p=0.06 3e5 4e5

1.5e5 3e5 2e5 1e5 2e5 1e5 0.5e4 1e5

normalized ion count 0 0 0

Mtb Mtb Mtb uninf uninf uninf IFN-ɣ + IFN-ɣ + IFN-ɣ + Mtb Mtb Mtb

glyceraldehyde-3-phosphate 3-phosphoglycerate Phosphoenolpyruvate p=0.10 4e5 2.5e5 6e4 ** 2e5 3e5 4e4 1.5e5 2e5 1e5 2e4 1e5 0.5e4

normalized ion count 0 0 0

Mtb Mtb Mtb uninf uninf uninf IFN-ɣ + IFN-ɣ + IFN-ɣ + Mtb Mtb Mtb

pyruvate ** 2e5 ** 5e5 4e5 1.5e5 3e5 1e5 2e5 0.5e4 1e5

normalized ion count 0 0

Mtb Mtb uninf uninf IFN-ɣ + IFN-ɣ + Mtb Mtb

Figure S3. Steady state levels of glycolytic intermediates in cells infected with M. tuberculosis. Resting and IFN-γ activated WT BMDM were infected with M. tuberculosis at MOI=1. Metabolite levels were measured using high-resolution tandem mass spectrometry in quintuplicate samples 24h post-in- fection and were normalized to an external control. Dihydroxyacetone phosphate (DHAP) and glyceral- dehyde-3-P are isomers that are indistinguishable by mass spectrometry and are represented as glycer- aldehyde-3-P. Error bars represent SEM. p-values were determined by unpaired t-test ***p≤0.001, **p≤ 0.01. Figure S4

A 0.015 B 0.015 p=0.07

** 0.010 0.010 expression expression 0.005 0.005 Il1a Hk2 0 0 10 15 20 10 15 20

C D 0.6 0.03 * ***

0.4 0.02 expression

expression 0.2 0.01 Il1b Cox2 0 0 10 15 20 10 15 20

Figure S4. HIF-1α target genes are regulated in vivo after the onset of IFN-γ. CD11b+ cells were isolated from of infected mice as in Figure 7 and analyzed by qPCR. (A-D) Timecourse of actin normalized expression of the indicated genes. WT is in black and HIF-1α-/- is in red. p-values were determined using an unpaired t-test, ***p≤ 0.001, **p≤0.01, *p≤0.05.